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The alpha omega trial is a multicenter, double - blind, placebo - controlled trial with a 2 2 factorial design, which has been described in detail elsewhere (6,7). In collaboration with cardiologists from 32 hospitals in the netherlands, we recruited 4,837 patients aged 6080 years with a clinically diagnosed mi up to 10 years before randomization . For the current study, the results of 1,014 patients with diabetes were analyzed (supplementary fig . These patients were randomly assigned to daily consumption of four different trial margarines for a period of 40 months . The patients were randomly allocated to four groups and received either no additional n-3 fatty acids or 400 mg / day epa+dha, 2 g / day ala, or the combination . The doses corresponded to recommended dietary allowances of those fatty acids . In trial margarines for active treatment groups, the margarines were developed by unilever research and development and could not be distinguished from each other in taste, odor, texture, and color . The trial was conducted in accordance with the helsinki declaration and approved by one central medical ethics committee and by the committees of all participating hospitals . Written informed consent was obtained from all patients . For logistical reasons, all patients used placebo margarine during the first 46 weeks after randomization, after which actual treatment started . After randomization, the patients received eight blinded margarine tubs of 250 g every 12 weeks . Based on this information, the daily intakes of margarine and n-3 fatty acids were calculated . An objective measure of compliance was obtained by determining fatty acids in plasma cholesteryl esters in random subgroups of patients at baseline and after 20 and 40 months of intervention . Our definition of diabetes was based on a physician s diagnosis, use of antidiabetic drugs, and/or elevated blood glucose, defined as a casual plasma glucose level of 7.0 mmol / l (126 mg / dl) for patients who had fasted> 4 h or a level 11.1 mmol / l (200.0 mg / dl) in patients who did not fast . Demographic factors, lifestyle, drug use, and medical history were assessed by questionnaire (6). Medication was coded according to the anatomical therapeutic chemical (atc) classification system (world health organization, 2009), including atc code a10 for antidiabetic medication . Physical activity was assessed by the validated physical activity scale for the elderly (pase) questionnaire . A patient was called physically active when he or she had at least 5 days per week of> 30 min / day of physical activities of at least three metabolic equivalents of task . Anthropometric measures, blood pressure, and heart rate were taken, and nonfasting blood was collected for determining serum lipids, plasma cholesteryl esters, and glucose (6). Examinations were repeated in a random sample of 159 (15.7%) patients after 20 months and after 40 months in 476 patients (46.9%) who completed the trial before 1 january 2009 . Due to budget constraints, the remainder of the cohort filled out mailed questionnaires only . All patients were followed for clinical events, including those who discontinued margarine use during the trial . We monitored vital status of all patients using a computerized link with municipal registers with 100% coverage . For fatal cases, death certificates were obtained from statistics netherlands and general practitioners filled out a standard form on primary and contributing causes of death . Additional information on fatal events was obtained from hospitals, nursing homes, and family members . Three members of the endpoint adjudication committee who were blinded for patient identity, treating physician, and allocated treatment coded the fatal events independently according to the 10th revision of the international classification of diseases . In the cases of disagreement, information about the underlying causes of death was discussed until consensus was reached . In the original trial protocol, the primary end point was fatal chd, and one of the secondary end points was sudden death, a proxy for fatal arrhythmias (6). Due to the low mortality rate, the steering committee approved the switch from primary to secondary end point for fatal chd, and the extension with placement of cardioverter defibrillators for the definition of sudden death, as described in the statistical analysis plan (7). Fatal chd was defined as mortality from mi (i20i25), cardiac arrest (i46), and sudden death undefined (r96) and the composite end - point ventricular arrhythmia related events as nonfatal or fatal cardiac arrest (i46), sudden death (r96), and implantable cardioverter defibrillator placement (6,7). Sudden death was extended with placement of cardioverter defibrillators because ventricular arrhythmias are the main cause of sudden death and cardioverter defibrillators are implanted in patients who are at high risk for developing ventricular arrhythmias (16,17). Self - reported data on implantable cardioverter defibrillator placement were verified against hospital records by trained research nurses or the research physician . All indications for implantable cardioverter defibrillator placement could be retrieved and verified from hospital discharge letters or by telephone with the departments of cardiology where the placements had taken place . The definitions of fatal chd and of the composite end - point ventricular arrhythmia related events include both fatal cardiac arrest (i46) and sudden death undefined (r96) (7). To prevent overlap in these definitions, we limited in the present analysis fatal chd to death from mi (i20i25). Baseline characteristics were compared among the four treatment groups of patients using anova or tests, when appropriate . Time - to - event data were analyzed with the kaplan - meier method and the log - rank test . The three treatment groups (i.e., epa - dha, ala, and epa - dha plus ala groups) were compared with the placebo group for each outcome . Hazard ratios (hrs) and 95% cis were computed for ventricular arrhythmia related events, fatal mi, and the combination of these two end points, using cox proportional hazard models . No adjustments were made for multiple comparisons because only a priori formulated hypotheses were tested based on animal experiments of the effects of n-3 fatty acids on atherosclerosis and cardiac arrhythmias (5). The alpha omega trial is a multicenter, double - blind, placebo - controlled trial with a 2 2 factorial design, which has been described in detail elsewhere (6,7). In collaboration with cardiologists from 32 hospitals in the netherlands, we recruited 4,837 patients aged 6080 years with a clinically diagnosed mi up to 10 years before randomization . For the current study, the results of 1,014 patients with diabetes were analyzed (supplementary fig . These patients were randomly assigned to daily consumption of four different trial margarines for a period of 40 months . The patients were randomly allocated to four groups and received either no additional n-3 fatty acids or 400 mg / day epa+dha, 2 g / day ala, or the combination . The doses corresponded to recommended dietary allowances of those fatty acids . In trial margarines for active treatment groups, the margarines were developed by unilever research and development and could not be distinguished from each other in taste, odor, texture, and color . The trial was conducted in accordance with the helsinki declaration and approved by one central medical ethics committee and by the committees of all participating hospitals . Written informed consent was obtained from all patients . For logistical reasons, all patients used placebo margarine during the first 46 weeks after randomization, after which actual treatment started . After randomization, the patients received eight blinded margarine tubs of 250 g every 12 weeks . Based on this information, the daily intakes of margarine and n-3 fatty acids were calculated . An objective measure of compliance was obtained by determining fatty acids in plasma cholesteryl esters in random subgroups of patients at baseline and after 20 and 40 months of intervention . Our definition of diabetes was based on a physician s diagnosis, use of antidiabetic drugs, and/or elevated blood glucose, defined as a casual plasma glucose level of 7.0 mmol / l (126 mg / dl) for patients who had fasted> 4 h or a level 11.1 mmol / l (200.0 mg / dl) in patients who did not fast . Demographic factors, lifestyle, drug use, and medical history were assessed by questionnaire (6). Medication was coded according to the anatomical therapeutic chemical (atc) classification system (world health organization, 2009), including atc code a10 for antidiabetic medication . Physical activity was assessed by the validated physical activity scale for the elderly (pase) questionnaire . A patient was called physically active when he or she had at least 5 days per week of> 30 min / day of physical activities of at least three metabolic equivalents of task . Anthropometric measures, blood pressure, and heart rate were taken, and nonfasting blood was collected for determining serum lipids, plasma cholesteryl esters, and glucose (6). Examinations were repeated in a random sample of 159 (15.7%) patients after 20 months and after 40 months in 476 patients (46.9%) who completed the trial before 1 january 2009 . Due to budget constraints, all patients were followed for clinical events, including those who discontinued margarine use during the trial . We monitored vital status of all patients using a computerized link with municipal registers with 100% coverage . For fatal cases, death certificates were obtained from statistics netherlands and general practitioners filled out a standard form on primary and contributing causes of death . Additional information on fatal events was obtained from hospitals, nursing homes, and family members . Three members of the endpoint adjudication committee who were blinded for patient identity, treating physician, and allocated treatment coded the fatal events independently according to the 10th revision of the international classification of diseases . In the cases of disagreement, information about the underlying causes of death was discussed until consensus was reached . In the original trial protocol, the primary end point was fatal chd, and one of the secondary end points was sudden death, a proxy for fatal arrhythmias (6). Due to the low mortality rate, the steering committee approved the switch from primary to secondary end point for fatal chd, and the extension with placement of cardioverter defibrillators for the definition of sudden death, as described in the statistical analysis plan (7). Fatal chd was defined as mortality from mi (i20i25), cardiac arrest (i46), and sudden death undefined (r96) and the composite end - point ventricular arrhythmia related events as nonfatal or fatal cardiac arrest (i46), sudden death (r96), and implantable cardioverter defibrillator placement (6,7). Sudden death was extended with placement of cardioverter defibrillators because ventricular arrhythmias are the main cause of sudden death and cardioverter defibrillators are implanted in patients who are at high risk for developing ventricular arrhythmias (16,17). Self - reported data on implantable cardioverter defibrillator placement were verified against hospital records by trained research nurses or the research physician . All indications for implantable cardioverter defibrillator placement could be retrieved and verified from hospital discharge letters or by telephone with the departments of cardiology where the placements had taken place . The definitions of fatal chd and of the composite end - point ventricular arrhythmia related events include both fatal cardiac arrest (i46) and sudden death undefined (r96) (7). To prevent overlap in these definitions, we limited in the present analysis fatal chd to death from mi (i20i25). Baseline characteristics were compared among the four treatment groups of patients using anova or tests, when appropriate . Time - to - event data were analyzed with the kaplan - meier method and the log - rank test . The three treatment groups (i.e., epa - dha, ala, and epa - dha plus ala groups) were compared with the placebo group for each outcome . Hazard ratios (hrs) and 95% cis were computed for ventricular arrhythmia related events, fatal mi, and the combination of these two end points, using cox proportional hazard models . No adjustments were made for multiple comparisons because only a priori formulated hypotheses were tested based on animal experiments of the effects of n-3 fatty acids on atherosclerosis and cardiac arrhythmias (5). Data were analyzed with spss 17.0 statistical software (spss inc ., chicago, il). The definition of diabetes was made in 72.4% on the combination of elevated blood glucose levels, physician - diagnosed self - report, and drug treatment, in 14.4% on elevated glucose levels only, in 9.9% on self - report only, and in 3.4% on drug treatment only . Insulin was used by 273 (26.9%) patients and 577 (56.9%) used other antidiabetic medication, of whom 110 used both insulin and other antidiabetic medication . The proportion of women was lowest in the placebo group and highest in the ala group, and 5 (1.9%) of the 267 women used hormone replacement therapy . The percentage of smokers was highest in the placebo group and lowest in the epa - dha plus ala group . Baseline characteristics of the 1,014 patients with an mi and diabetes, * according to n-3 fatty acid supplementation group data are means sd unless otherwise indicated . As data for a number of patients were missing from several variables (bmi, 4 patients; alcohol use, 1 patient; and physical activity, 9 patients), some percentages are based on a smaller number than the column total . * diabetes was considered to be present if a patient reported having received the diagnosis from a physician, was taking antidiabetic drugs, or had an elevated plasma glucose level (7.0 mmol / l [126 mg / dl] in the case of patients who had fasted more than 4 h or 11.1 mmol / l [200.0 mg / dl] in the case of nonfasting patients). To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586 . To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129 . The bmi is the weight in kilograms divided by the square of the height in meters . * * greater than or equal to three metabolic equivalent of task (indicating at least moderate intensity for at least 30 min / day) during 6 or 7 days / week . The mean intake (sd) of the trial margarine was 18.6 (5.2) g / day; 86.7% of the patients adhered fully to the protocol and consumed a mean of 20.7 (4.2) g / day . Patients in the two epa - dha groups received, on average, an additional amount of 223 (62) mg epa and 149 (42) mg of dha per day and those in the two groups of ala 1.9 (0.5) g / day . Plasma cholesteryl esters were determined as a measure of compliance . At the final examination, the effect of n-3 fatty acid supplementation on fatty acids in serum cholesteryl esters was compared with placebo (supplementary fig . 2). Ala supplementation increased ala by 64.2% and epa by 40.8%; epa - dha supplementation increased plasma epa by 49.8% and dha by 30.9%; and epa - dha plus ala supplementation increased plasma ala by 67.4%, epa by 118.4%, and dha by 44.0% . The median follow - up period was 40.7 months (iqr 36.641.6 months), during which 3,195 person - years of follow - up were accumulated . During follow - up, 29 patients developed a ventricular arrhythmia related event, 2 died suddenly, 1 had a nonfatal cardiac arrest, 11 had a fatal cardiac arrest, and 15 had a cardioverter defibrillator implanted . Indications for these implantable cardioverter defibrillator placements were a left ventricular ejection fraction 35% (n = 13; 87%), resuscitated ventricular fibrillation (n = 1), and ventricular tachycardia with a left ventricular ejection fraction of 45% (n = 1). In addition, 27 patients died of mi, 23 from other vascular diseases, 34 from cancer, and 26 from other causes . The kaplan - meier curves showed that both epa - dha and ala reduced ventricular arrhythmia related events compared with placebo (fig . The lowest incidence was observed in patients who were randomized to the combined supplementation of epa - dha plus ala . After adjustment for imbalances in age, sex, and current smoking, the combined supplementation of n-3 fatty acids reduced the ventricular arrhythmia related events by 84% compared with placebo (cumulative incidence at median follow - up 0.9 vs. 5.6%, fig . Similar effects were observed for the combined end - point cardiac arrest and sudden death (0.13; ci 0.021.09) and for placement of cardioverter defibrillators (0.19: 0.021.55). Kaplan - meier curves of ventricular arrhythmia related events (a), death from mi (b), or both end points combined (c). The cumulative incidence of end points is shown in 1,014 patients with an mi and diabetes . Patients were randomly assigned to receive a margarine containing supplemental epa combined with dha, a margarine containing supplemental ala, a margarine containing both epa - dha and ala, or a placebo margarine . Unadjusted and adjusted hrs of n-3 fatty acid supplementation on end points in 1,014 patients with diabetes, according to n-3 fatty acid supplementation group * data are hrs with 95% cis and p values, with the use of cox proportional hazards models . Adjusted for age, sex, and smoking status . For fatal mi, the kaplan - meier curves did not differ significantly among the four groups (fig . After adjustment for confounders, the strongest, although not significant, effect was observed for epa - dha plus ala (hr 0.53; 95% ci 0.151.81; table 2). The kaplan - meier curves for the composite end - point ventricular arrhythmia related events and fatal mi showed the strongest effect for epa - dha plus ala (fig . After adjustment, these three n-3 fatty acids together reduced this combined end point by 72% (cumulative incidence at median follow - up 2.6 vs. 8.5%, fig . The definition of diabetes was made in 72.4% on the combination of elevated blood glucose levels, physician - diagnosed self - report, and drug treatment, in 14.4% on elevated glucose levels only, in 9.9% on self - report only, and in 3.4% on drug treatment only . Insulin was used by 273 (26.9%) patients and 577 (56.9%) used other antidiabetic medication, of whom 110 used both insulin and other antidiabetic medication . The proportion of women was lowest in the placebo group and highest in the ala group, and 5 (1.9%) of the 267 women used hormone replacement therapy . The percentage of smokers was highest in the placebo group and lowest in the epa - dha plus ala group . Baseline characteristics of the 1,014 patients with an mi and diabetes, * according to n-3 fatty acid supplementation group data are means sd unless otherwise indicated . As data for a number of patients were missing from several variables (bmi, 4 patients; alcohol use, 1 patient; and physical activity, 9 patients), some percentages are based on a smaller number than the column total . * diabetes was considered to be present if a patient reported having received the diagnosis from a physician, was taking antidiabetic drugs, or had an elevated plasma glucose level (7.0 mmol / l [126 mg / dl] in the case of patients who had fasted more than 4 h or 11.1 mmol / l [200.0 mg / dl] in the case of nonfasting patients). To convert the values for cholesterol to milligrams per deciliter, divide by 0.02586 . To convert the values for triglycerides to milligrams per deciliter, divide by 0.01129 . The bmi is the weight in kilograms divided by the square of the height in meters . * * greater than or equal to three metabolic equivalent of task (indicating at least moderate intensity for at least 30 min / day) during 6 or 7 days / week . The mean intake (sd) of the trial margarine was 18.6 (5.2) g / day; 86.7% of the patients adhered fully to the protocol and consumed a mean of 20.7 (4.2) g / day . Patients in the two epa - dha groups received, on average, an additional amount of 223 (62) mg epa and 149 (42) mg of dha per day and those in the two groups of ala 1.9 (0.5) g / day . Plasma cholesteryl esters were determined as a measure of compliance . At the final examination, the effect of n-3 fatty acid supplementation on fatty acids in serum cholesteryl esters was compared with placebo (supplementary fig . 2). Ala supplementation increased ala by 64.2% and epa by 40.8%; epa - dha supplementation increased plasma epa by 49.8% and dha by 30.9%; and epa - dha plus ala supplementation increased plasma ala by 67.4%, epa by 118.4%, and dha by 44.0% . The median follow - up period was 40.7 months (iqr 36.641.6 months), during which 3,195 person - years of follow - up were accumulated . During follow - up, 29 patients developed a ventricular arrhythmia related event, 2 died suddenly, 1 had a nonfatal cardiac arrest, 11 had a fatal cardiac arrest, and 15 had a cardioverter defibrillator implanted . Indications for these implantable cardioverter defibrillator placements were a left ventricular ejection fraction 35% (n = 13; 87%), resuscitated ventricular fibrillation (n = 1), and ventricular tachycardia with a left ventricular ejection fraction of 45% (n = 1). In addition, 27 patients died of mi, 23 from other vascular diseases, 34 from cancer, and 26 from other causes . The kaplan - meier curves showed that both epa - dha and ala reduced ventricular arrhythmia related events compared with placebo (fig . The lowest incidence was observed in patients who were randomized to the combined supplementation of epa - dha plus ala . After adjustment for imbalances in age, sex, and current smoking, the combined supplementation of n-3 fatty acids reduced the ventricular arrhythmia related events by 84% compared with placebo (cumulative incidence at median follow - up 0.9 vs. 5.6%, fig . Similar effects were observed for the combined end - point cardiac arrest and sudden death (0.13; ci 0.021.09) and for placement of cardioverter defibrillators (0.19: 0.021.55). Kaplan - meier curves of ventricular arrhythmia related events (a), death from mi (b), or both end points combined (c). The cumulative incidence of end points is shown in 1,014 patients with an mi and diabetes . Patients were randomly assigned to receive a margarine containing supplemental epa combined with dha, a margarine containing supplemental ala, a margarine containing both epa - dha and ala, or a placebo margarine . Unadjusted and adjusted hrs of n-3 fatty acid supplementation on end points in 1,014 patients with diabetes, according to n-3 fatty acid supplementation group * data are hrs with 95% cis and p values, with the use of cox proportional hazards models . Adjusted for age, sex, and smoking status . For fatal mi, the kaplan - meier curves did not differ significantly among the four groups (fig . After adjustment for confounders, the strongest, although not significant, effect was observed for epa - dha plus ala (hr 0.53; 95% ci 0.151.81; table 2). The kaplan - meier curves for the composite end - point ventricular arrhythmia related events and fatal mi showed the strongest effect for epa - dha plus ala (fig . After adjustment, these three n-3 fatty acids together reduced this combined end point by 72% (cumulative incidence at median follow - up 2.6 vs. 8.5%, fig . Low - dose supplementation of n-3 fatty acids epa - dha plus ala significantly reduced ventricular arrhythmia related events in post - mi patients with diabetes . The combined supplementation of these three fatty acids also reduced the composite end - point ventricular arrhythmia related events plus fatal mi but not fatal mi alone . A limitation of the current study is the small number of patients who developed ventricular arrhythmias (n = 29) or died of mi (n = 27). The study had enough power to detect significant effects of combined supplementation of epa - dha and ala on the end - point ventricular arrhythmia related events whether or not in combination with fatal mi . Significant effects on these end points were not observed for either epa - dha or ala supplementation . The combined effects of epa - dha and ala on the two composite end points are compatible with additive effects of either epa - dha or ala . Assuming an additive model, an hr of 0.47 0.58 = 0.27 is expected for ventricular arrhythmia related events and of 0.81 0.60 = 0.49 for the combination of ventricular arrhythmias and fatal mi . These results fit well with the 95% ci of these end points and are compatible with the hypothesis that all three n-3 fatty acids reduce the risk of ventricular arrhythmias . This is also in accord with the results of animal experiments suggesting that ala, epa, and dha have similar antiarrhythmic effects by influencing the na and l - type ca channels of cardiomyocytes (5). In the gissi - prevenzione trial, patients with a recent mi (<3 months ago) were supplemented with 0.9 g of epa - dha per day, which reduced their risk of sudden death by 45% (8). In later trials, recent meta - analyses quantified the effect of epa - dha supplementation on sudden death at a 19% (hr 0.81; 95% ci 0.521.25) (2) and 13% (0.87; 0.760.99) reduction (3). An explanation for the smaller effect of epa - dha in the more recent trials could be improved drug treatment of cardiovascular risk factors (18). This is supported by the recently published results of the omega trial in which patients were randomized 314 days after acute mi . In these high - risk patients, a supplement of 0.8 g epa - dha per day did not reduce sudden cardiac death (19). In this trial and in the alpha omega trial, almost all patients received antithrombotic drugs, antihypertensive drugs, and statins (7). The patients in the gissi - prevenzione trial were also well treated with antithrombotic and antihypertensive drugs but only 29% received statins (8). The alpha omega trial started in 2002, the omega trial in 2003, and the gissi - prevenzione in 1993 . One of the secondary end points in the alpha omega trial was ventricular arrhythmia related events . Ventricular arrhythmias are the major cause of sudden death and cardiac arrest and cardioverter defibrillators were generally implanted in post - mi patients with a low ejection fraction who are at high risk for these arrhythmias (16,17). The protective effect of the combined supplementation of the three n-3 fatty acids on this end point (hr 0.16; 95% ci 0.040.69) contrasts with the absence of an effect of epa - dha in cardiac patients with implanted cardioverter defibrillators (who all had had a severe arrhythmic event before placement) (2). These discrepant results suggest that n-3 fatty acids may reduce primary arrhythmias in post - mi patients with diabetes with a low ejection fraction but not secondary arrhythmias in cardiac patients with cardioverter defibrillators . Epa - dha supplementation reduced fatal mi by 47% but this reduction was not significant, possibly due to the small number of events . A possible protective effect of epa - dha on fatal mi would be in accord with the results of a prospective study carried out in postmenopausal women (20). In that study, smaller increases in stenosis were observed in diabetic women who consumed greater than two servings of fish or greater than one serving of fatty fish compared with diabetic women who consumed less fish . An intervention study carried out in japan showed that diabetic patients who were supplemented with 1.8 g epa per day in contrast with the control group did not show an increase in intima - media thickness during 2.1 years of follow - up (21). These results suggest that in diabetic patients, fish and epa supplementation may reduce progression of atherosclerosis and the eventual risk of fatal mi . Diabetes alters smooth muscle function through atherosclerotic lesion formation, plaque instability, and cardiovascular events . Basic research shows that peroxisome proliferator activated receptor (ppar-) plays a critical role in the regulation of insulin sensitivity . To become functional, ppar- should bind to an appropriate ligand . Cell culture studies provide evidence that epa and dha are potential ligands for ppar- activation (22). Recently published results of an in vivo experiment suggested that fish oil increases the expression of glucose uptake genes, leading to reduced glucose levels (23). Experiments in obese mice showed that activation of the g protein coupled receptor 120 (gpr120) by epa and dha inhibited multiple inflammation cascades and reversed insulin resistance caused by a high - fat diet (24). These mechanistic findings support an important role for n-3 fatty acids in the etiology of diabetes, a major risk factor of fatal mi . In conclusion, low doses of n-3 fatty acids reduced the risk of ventricular arrhythmia related events in post - mi patients who also have diabetes . Ongoing trials in diabetic patients, e.g., origin (outcome reduction with an initial glargine intervention; clinical trial reg . Nct00069784) (25) and ascend (a study of cardiovascular events in diabetes; nct00135226; http://www.ctsu.ox.ac.uk/ascend/), evaluate the effect of epa - dha on sudden death and fatal chd . The results of more trials on the effect of different n-3 fatty acids are needed before definitive conclusions can be drawn on their role in the etiology of ventricular arrhythmias and fatal chd.
Cardiovascular diseases (cvd) constitute one class of common contributors to morbidity and mortality worldwide . Of the many risk factors for the development of cvd, age, gender, family history and genetic inheritance are un - modifiable, while smoking, physical inactivity, poor diet, obesity, and dyslipidemia are modifiable . Prevalence of overweight and obesity has dramatically increased in developing countries and is related to cardiovascular risk factors . According to world health organization, in 2008 studies show excess adipose tissue, mostly central, is associated with high cardiovascular morbidity and mortality . Diverse methods have been used to assess the amount and the distribution of body fat and its relationship to cvd . Anthropometric parameters, such as body mass index (bmi), waist circumference (wc) and waist - to - hip ratio (whr), have the advantages in the daily clinical practice of being simple to measure with good reproducibility, especially in a developing country like india . Though bmi reflects lean mass and fat, it scarcely identifies the distribution of the latter . Rather, other anthropometric indices, such as wc, waist - to - height ratio, and whr, have been used as alternatives to bmi . Wc is being increasingly accepted as the best anthropometric indicator of abdominal adiposity and metabolic risk . It is less known, however, which one of these anthropometric variables (bmi or whr or wc) is a better link to lipid profile . Some of the previous studies reported internationally show there is a significant correlation between afore - mentioned anthropometric variables and lipid parameters, while others opine there are no such statistically significant correlations . Many indian studies have been reported relating anthropometric parameters with lipid profile in type 2 diabetes and also in hypothyroid patients . Not many studies have been reported in this regard involving healthy subjects in india, especially in eastern india . The present study attempts to correlate some anthropometric variables with lipid parameters in apparently healthy subjects, as also to assess the anthropometric variable which best reflects the altered lipid profile . This is a hospital based, single centered, cross - sectional study conducted at a private hospital after permission from institutional ethical committee during the period from september 2012 to august 2013 . Consenting participants, with apparent cardio - metabolic healthy disposition, between 18 and 65 years of age were included in the study . All subjects with history of diabetes mellitus, cvd, carcinoma, liver diseases, renal diseases, and subjects on lipid - lowering agents were excluded from the study . Of the 2500 consenting participants, 1313 were excluded due to the presence of one or more exclusion criteria after a detailed history and physical examination . The included participants (1187) were subjected to anthropometric measurements such as height (ht), weight (wt), wc, and hip circumference (hc) using standard procedures on the same morning of the day as the blood sample was collected . Height was measured in centimeters, without shoes, with a standard height measuring rod; weight was measured in kilograms (kg), without shoes, using digital scales and was recorded to the accuracy of 0.1 kg . Wc was measured using a measuring tape for each participant with minimal clothing and with feet about 25 - 30 cm apart; measurements were taken in a plane perpendicular to the long axis of the body at the level of umbilicus without compressing the skin . Those with bmi of <25 were considered normal . Those with a bmi of 25.0 - 29.9 kg / m were classified as overweight, whereas those with a bmi 30 kg / m were defined as obese . Whr was derived from the ratio of wc / hc . Blood samples were drawn by a trained phlebotomist in the morning from all study participants after 12 h of overnight fasting . Blood samples were taken in a sitting position according to the standard protocol and were centrifuged within 30 - 45 min of collection . Lipid parameters such as (total cholesterol [tc], triglyceride [tg], low - density lipoprotein [ldl] cholesterol, very ldl [vldl] and high - density lipoprotein [hdl] cholesterol) were estimated by a standard enzymatic method using hitachi 902 auto - analyser . Statistical analysis was done using statistical package for the social sciences (version 16, spss inc . All participants were grouped into two groups according to bmi <25 and bmi 25 . Pearson correlation of anthropometric variables with each lipid parameter was done and represented by the correlation coefficient (r); p <0.05 was considered statistically significant . Of the 1187 participants (637 males and 550 females), two groups were made according to the above - mentioned bmi cut - off points . The mean bmi of both the groups is 22.36 2.02 and 28.05 3.2, respectively . Table 1 shows that the mean values of lipid parameters were not significantly different in both the groups . Comparative data on the mean values of lipid parameters of both the groups with their significance table 2 shows the weak correlation between bmi and lipid parameters . Furthermore, the correlation of bmi with lipid parameters in the bmi <25 group was weak and insignificant; in the bmi 25 group bmi showed a significantly negative correlation with hdl (r = 0.3, p = 0.03). Correlation of anthropometric variables with lipid parameters a glance at table 3 shows that none of the lipid parameters was significantly different in both bmi groups among the males . Comparative data on the mean values of lipid parameters for both the groups in males and females though the bmi was not significantly different in both the age groups, there was a significantly greater wc in the older group than the younger group . Each of tg and vldl was significantly greater in the older age group than the younger group [table 4]. Comparison of anthropometric and lipid variables between the younger and the older age groups table 5 makes a comparison of anthropometric and lipid variables between the genders of younger and older age groups chosen . In the younger age group, the mean values of lipid and anthropometric variables were generally higher for the males than the females; mean values of tc, tg and ldl were significantly higher . For both the genders, the mean values of all variables were higher in the older age group than the younger age group . However, in the older age group the difference in the mean values of vldl, wc and whr were significant between the genders . Comparison of anthropometric and lipid variables between males and females of younger and the older age groups anthropometric parameters are commonly used as research tools to assess the risk factors for noncommunicable diseases in a population . For several decades, dyslipidemia has been found as one of the risk factors for various noncommunicable diseases such as diabetes, hypertension, and many other cvd . The present study makes an attempt to evaluate the correlation between anthropometric variables with lipid parameters, in order to determine which of the former can commonly be used in clinical practice and epidemiological studies as the best link to the altered lipid profile . While a south indian pilot study reports a strong correlation between anthropometric parameters and lipid profile in healthy adults, the present study shows no correlation between bmi and lipid profile; in the bmi 25 group, bmi showed significant negative correlation with hdl . Results of a few other studies support our findings . In the present study, among the anthropometric variables, wc was best correlated with tg (positively) and hdl (negatively). There was no significant difference in lipid profile among the two bmi groups in males and females; in contrast, the brazilian study reports gender differences influencing the association between lipid parameters and anthropometric variables . The main nonmodifiable risk factor for cvd is age; vascular endothelial damage becomes more apparent from the third decade of life and its clinical consequences begin around the age of about 40 years . So in the present study we have grouped all participants into two age groups with a cut - off value of age 40 years . The mean values of wc, tg, and vldl are significantly higher in the older age group . A previous study reports abdominal obesity has a greater effect on vldl and tg levels than on other lipid parameters . In this study, as the older age group reports to have a higher mean wc, that may have affected the mean vldl and tg levels . Variations of lipid and of anthropometric parameters in males and females, for younger and older age groups, give us an insight into interesting observations . Among the lipid variables, there was a significant difference in tc, tg, and ldl values between the genders (viz ., males having higher values than the females) of the younger age group which was not observed in the older age group . On the other aspect, anthropometric variables such as wc and whr were found to be significantly higher for females than for males in the older age group . In the present study, the mean age of females in the older age group is 57 years indicating most of them would have attained menopause . In the older age group, the gender differences in lipid parameters were not seen which may be due to increase in lipid parameters in menopausal females due to loss of protective estrogen effect and age - related changes in anthropometric variables such as wc and whr . Moreover, participants from one single center have been considered in this study which is also a limitation . Although it is difficult to conclusively interpret the present set of data, several meaningful inferences may however be drawn . Not all studied anthropometric variables correlate with the lipid status of the body, but wc remains one of the simple and reliable variables which best reflects the lipid profile . In a developing country like india, where measurement of cardiovascular risk factors such as body fat saturation and lipid profile remains difficult in the rural population, wc may be used as an effective tool, without being used as a substitute . Thus, both genders, especially in older age group, may be at equal risk of developing dyslipidemia.
Reservation - based native americans live in pervasively adverse social and physical environments that place them at increased risk of exposure to a myriad of stressors during childhood which impact their psychological and physical health over their lifetimes . About 1 of 2.9 million native americans that identify as native american alone resides on reservations . Indian reservations were established by treaty during the removal and relocation (18271887) period and are lands set aside for tribes in exchange for ceded land and resources . Of the ten poorest counties in america, five are home to an indian reservation . Concentrated poverty results in higher crime rates, underperforming public schools, poor housing, and poor health and limits access to many services and job opportunities . Adverse childhood experiences (aces) that are substantial contributors to health disparities include childhood physical and sexual abuse, witnessing violence, poverty, and racism . The concept that these experiences become biologically embedded has gained substantial support and provides an explanatory mechanism for health disparities . Aces are linked to differences in the function of the stress - response system including the neuroendocrine system, the parasympathetic nervous system, and the immune system . These changes likely have substantial long and short term impacts on health and well - being . It is likely that these changes are shaped by epigenetic modifications which alter the function but not the structure of the gene . Epigenetic modifications are considered to be an individual's molecular response to the environment and occur in an effort to preserve the health of the individual by increasing the accessibility of genes for transcription and translation that relate to immediate survival . These genes code for proteins that prepare the individual to be able to respond to the stressor through a fight or flight response; yet, in native americans living on reservations, the stressors most encountered are chronic, not acute . Thus, this adaptive response likely results in overactivation of this stress - response system, and this excessive activity has substantial negative consequences on the health and well - being of native americans, individually and across generations . Here we provide a conceptual review of how nurses and other health care professionals can examine health disparities in native americans through epigenetic modifications that likely result from aces (see figure 1), including historical trauma, the residual of which is assumed to be historical loss associated symptoms . We expect this conceptual framework to have implications for or be relevant to the mechanisms of health disparities in other racial or ethnic groups . A neighborhood's safety and access to quality health care, economic opportunities, social connections, and social capital are all key determinants of the health of its residents over time [912]. Reservations are often characterized by low economic status and segregation, both of which limit access and are risk factors for higher rates of morbidity and mortality [13, 14]. Chronic stress such as that which accompanies experiences of racism and poverty over a lifetime places individuals at risk for posttraumatic stress disorder (ptsd). Unique to native americans is the race - based stress associated with historical trauma, as well as discrimination [1720]. Historical trauma is defined here as the collective experience of violence perpetrated against indigenous peoples in the process of colonizing the americas resulting in an unresolved humanitarian crisis for reservation communities . The effects of historical trauma are proposed as being transmitted across generations with historical loss associated symptoms currently exhibited [2123] and include symptoms of complicated bereavement and complex ptsd . This type of trauma has been linked to impaired individual and collective tribal identity [16, 24], which likely also relates to stress and morbidity risk . Over 50% of native americans indicate that they think about loss related to historical trauma, such as loss of language, loss of culture, and loss of land, at least occasionally, and which caused them psychological distress [17, 25]. Discrimination has been associated with early substance use among native american children, and suicidal behavior, and anger, and aggression among adolescents [18, 20, 26]. Thus, this stress combined with other aces may be a significant contributor to health disparities . Native americans are disproportionally affected by trauma in childhood, including abuse, neglect, and exposure to intimate partner violence (ipv) [2729]. Approximately half of native american adolescents and young adults have been exposed to one or more severe traumatic events, and 98% have experienced a traumatic event of any severity . Native american adolescents are more likely than other adolescents to witness violence or to have been physically abused, sexually abused, or neglected as a child, resulting in rates of ptsd that are twice that of the estimated rates in the general u.s . Population . Assaultive trauma in childhood is linked to the highest risk for ptsd, suggesting that this ace is specifically linked to this high risk for psychiatric disorders . Specifically, trauma that involves physical or sexual assault prior to adolescence places an individual at five to ten times the risk for ptsd onset compared to an individual without this experience [32, 33]. Witnessing abuse during childhood, as well as residing in a high - crime area, is also linked to a far greater risk for ptsd [34, 35]; however, assaultive trauma at an early age is the ace most linked to ptsd onset . These studies link physical abuse, witnessing domestic violence, and parental alcohol and drug abuse to a vulnerability for depression symptom onset [36, 37]. In addition, residing in an urban, socioeconomically disadvantaged area has also been linked to risk of depression onset as well as drug use . Exposure to trauma also increases the risk for the early onset of substance use and the onset of substance use disorder . Other studies have found similar results, including that aces increase the risk for drug use and early alcohol abuse and increased the rates of initiating these behaviors during adolescence by a factor of two to four [41, 42]. Thus, aces in general are linked to psychiatric disorder vulnerability, with high degree of comorbidity among these disorders . Reservation - based native americans die at higher rates than other americans from tuberculosis (750% higher), alcoholism (524% higher), diabetes (293% higher), unintentional injuries (153% higher), homicide (103.3% higher), and suicide (66% higher) (20022004, rates adjusted for misreporting of race on state death certificates). Not only do native americans bear a disproportionate burden of disease, but they also experience a lower life expectancy . Life expectancy is an overall measure of quality of life and is one of the indicators used to measure the magnitude of the burden of health disparities . In general, native americans born in 20002002 have a life expectancy that is about 2.4 years less than the overall us population rate: 76.9 years compared to 74.5 years for native americans . However, when this average is disaggregated by ihs area, the life expectancy ranges from 64.8 years (11 years less than for the u.s .) In the aberdeen area to 76.4 years (greater than the u.s . Average of 75.8 years) in the california area (adjusted for race miscoding) using 19941996 data; thus highlighting the within group differences . Additionally, the indian health service, using 2000 census data, found 25.7% of all native americans were living below the poverty level, compared to 12.4% of the u.s . Population overall . The bureau of justice, in the first comprehensive statistical analysis of american indians and crime, reports native american are the victims of violent crimes at two times the rate of the u.s . Population overall, and about 7 in 10 violent victimizations involved an offender who was reported by the victim to be a person of another race . However, this may not apply to all communities, especially those that are more remote and isolated where few non - native american people live . Another report by the department of justice, disclosed native americans sustain rates of violent victimization (rape, sexual assault, robbery, aggravated assault, and simple assault) at rates that are 2 times higher than african americans, 2.5 times that of hispanics, 3 times that of caucasians, and 6.5 times that of asians . Ptsd is the anxiety disorder most linked to trauma and its prevalence in native americans adults is 4.4 times the national average [25, 49]. There is little research regarding the impact that adversity has on tribal communities, so it remains poorly understood . The adverse childhood experiences (ace) study suggests that certain adversities are major risk factors for morbidity and mortality . The study established a relationship between adversity in childhood and suicide attempt, prescription drug use, alcoholism and alcohol abuse [53, 54], illicit drug use, obesity, and depressive disorders . Among adolescents and young adults, childhood adversity was also associated with a greater risk for interpersonal violence perpetration, poor perceived health, more medical care visits, and additional somatic concerns . Therefore, current studies link aces to risks to health and well - being; however, the mechanisms underlying these risks have not yet been well described . In some cases, genetic predisposition may explain some of the enduring effects of aces; however, the evidence for this link remains poorly understood . Genetic inheritance provides information encoded in dna which is transcribed to various types of rna molecules which likely shape the response of the individual to stressors such as aces . One important concept related to phenotypic variation is heritability, which estimates the extent of which genetic inheritance contributes to the phenotypic variance in a population . Heritability is the percent of variation in the genome responsible for the difference in the phenotype . Another parameter used to estimate the contribution of genomic factors in phenotypes is relative risk, which refers to an individual's risk of developing a condition with a family history compared to those without a history . When the heritability estimate or relative risk of a phenotype is low, the influence of the genome sequence is considered to be relatively smaller than the influence of other factors such as environment, and the genomic influence can be easily masked or have a negligible impact . Since most human diseases involve many genes, their interactions, and nongenetic factors, an approach termed endophenotypes is used to characterize the disease in a molecular or genetic manner, rather than using a clinical diagnosis to define the phenotype . Polymorphisms in native americans have been linked to a greater vulnerability for alcohol abuse, as well as obesity . In general, samples of trauma exposed participants link endocrine gene (fkbp5) polymorphisms to a greater risk for ptsd development [59, 61]; yet, these are small and do not include native americans . Thus, it is essential to consider unique genetic inheritance features in native americans which interact with epigenetic modifications and likely contribute to health disparities . The stress - response system provides the individual protection from acute stressors through an activation of interactive biological systems . One biological system that is central to this response and is linked to aces is the hypothalamic - pituitary - adrenal (hpa) axis, with the end result of activation of this system being the production of cortisol . In addition to playing a pivotal role in activating the stress response, the hpa axis also influences biological functions related to mood, growth, immune function, metabolism, and regulation of biological systems on circadian rhythm . The sympathetic nervous system (sns) also is activated by stress providing neuronal focus and energy to muscles in order to escape the stressor . Although these systems are effective in adapting to acute stress, chronic activation is linked to negative consequences . Both the hpa axis and sns impact immune function, and chronic stress is linked to a risk for inflammation . Overactivation of the hpa axis results in disruptions of functioning at rest and following stressors, and these changes have been linked to aces . Hpa axis alterations are linked to health disparities through mechanisms that include impaired neuronal growth and survival, inflammation, reductions in neuropeptide activity, and accelerated cellular aging [6365]. Sns function changes have also been linked to health disparities, with one of the most pivotal mechanisms being a lack of circadian variation in blood pressure, a key risk factor for myocardial infarctions . Evidence is accumulating that environmental influences early in development remain pervasive into adulthood, a relationship that is attributed to an interaction of gene function and environment . Both genetic and environmental factors are critical to developmental processes and even minor changes in either type of factor can result in trajectories of resilience or vulnerability; however, it is the interaction between these factors that may provide the most vital information to understand the heterogeneous response to trauma . This leads us and others to question how future research can address this critical issue . These changes occur through mechanisms such as histone modification, methylation, acetylation, and noncoding ribonucleic acids which alter the accessibility of genes for transcription . The resulting transcription modifications and protein production result from factors such as environmental challenges including, but not limited to, aces [68, 69]. An individual's genome interacts with internal and external factors to create phenotypes such as height, physical appearance, personality, and alterations in the stress - response system . Preclinical models illustrate how aces result in epigenetic modifications in neurons, thereby increasing the risk for psychiatric symptoms . To illustrate this link, a study reports that the offspring of high - licking canine mothers exhibit reduced methylation of the glucocorticoid receptor gene and endocrine regulation of a subsequent stressor . In contrast, offspring that face early adversity exhibit endocrine dysregulation, as well as reductions in neuronal plasticity in the prefrontal cortex (pfc) that persist into adulthood . In studies of rats who exhibit ptsd - like behavior, there is evidence of increased methylation of stress - response genes including brain - derived neurotrophic factor and nuclear protein phosphate-1 in neurons . Although these studies provide additional evidence linking aces to methylation changes in neurons, these studies are not able to clearly determine psychiatric symptoms . Therefore, these studies are limited by not being able to determine the comprehensive risks that relate to aces . The ace most linked to epigenetic differences and vulnerability for health disparities is that of child abuse . To illustrate this link, in a hallmark study by labont in suicide completers, aces were linked to increased dna methylation of the glucocorticoid receptor in the hippocampus, and this differential methylation was particularly linked to childhood abuse . Study, whose subject group was also suicide completers, which reported that childhood abuse was associated with greater methylation levels at cpg sites in the exon1f of the promoter region of the glucocorticoid receptor gene . These studies had the distinct advantage of examining epigenetic modifications in neurons, which is not available in other studies . Epigenetic patterns differ among cell types, even differing among brain regions [79, 80]. Thus, an additional challenge to understanding the impact of aces on health disparities is to determine how epigenetic alterations in the brain differ from those in peripheral tissues and how to advance despite this methodological challenge . In addition, these few studies are not able to determine the role of preexisting methylation in this risk or to measure other factors that may contribute to methylation changes . Epigenetic changes resulting from aces can also be observed in studies that use peripheral blood in living participants, which show that hpa - regulating genes are often impacted . A study by klengel et al . Linked aces to reduced methylation of the fkbp5 gene, an essential regulator of the stress response, as well as to changes in the function of the hpa axis under stress, and to reduced cognitive ability . Direct physical abuse and observing the abuse of a mother have also been associated with greater methylation levels at cpg sites in the exon1f of the promoter region of the glucocorticoid receptor gene in leukocytes . Similar methylation profiles are also reported in the peripheral blood of babies whose mothers were depressed during the third trimester of pregnancy, and these methylation changes were related to salivary cortisol elevations at three months of age . Although glucocorticoid receptors in peripheral tissue may differ from those on the hpa axis, the link between methylation of the glucocorticoid gene in the periphery and the function of the hpa axis has been demonstrated in multiple studies in addition to those of oberlander et al ., 2008 . Additional studies that include analysis of blood samples collected closer to the time of the ace may provide additional insights into the individual variation in response to aces . Other studies link aces to hypomethylation of inflammatory genes, suggesting that these experiences result in a greater inflammation later in life . In a recent study of children who were removed from their parents due to abuse or neglect, a reduction in methylation of nr3c1, an inflammatory regulation gene, as well as differential methylation of cancer related pathways was found in children with aces compared to controls . A study of adults linked child abuse to reduced methylation of igf2as, an antisense transcript of the insulin - like growth factor gene, which encodes for the inflammatory cytokine family of growth factor beta . Borghol et al . Linked childhood poverty to differential methylation of genes related to metabolism and inflammation, and these changes were different from those in participants who experience poverty only during adulthood . Together these studies provide evidence that a variety of aces result in methylation changes, suggesting that these molecular changes likely contribute to health disparities; however, additional, larger, and more representative studies are needed to determine relationships . Altered serotonergic neurotransmission is also postulated to result from aces and provides a mechanistic link to increased vulnerability for psychiatric disorders . The iowa adoption study demonstrated a link between hypermethylation of the serotonin gene slc64a to childhood sex abuse, and this molecular change mediated the development of antisocial personality disorder . This group was also able to relate differences in gene expression of serotonin related genes to methylation, and that genotype influenced methylation at cg22584138 . Additional studies are needed to determine the role of other aces in serotonergic gene methylation and to determine how aces contribute to psychiatric risks . Clinical studies are restricted to examining differential methylation in samples of peripheral fluids, but these studies do provide some key insights into how these molecular changes relate to ptsd, depression, and drug abuse risk . For instance, ptsd is associated with changes in the methylation of inflammatory (toll - like receptors 1 & 3, il-8, chemokine ligand 1, and others) and endocrine genes fk506 binding protein-5 (fkbp5). Another study that measured dna methylation reported that postdeployment hypomethylation of line-1 was associated with ptsd onset following deployment . One study determined that serotonin transporter gene (slc6a4) methylation levels were modified by the effect of the number of traumatic events on ptsd after controlling for slc6a4 genotype, such that persons with more traumatic events were at increased risk for ptsd, but only at lower methylation levels . The other study found that the candidate gene man2c1 showed a significant methylation trauma experience interaction, such that those with both higher man2c1 methylation and greater exposure to traumatic events showed an increase in risk of lifetime ptsd . Thus, there is evidence that ptsd is associated with similar methylation differences in immune, endocrine, and neurotransmitter genes to those linked to aces; suggesting that these chronic differences may be a result of aces, yet additional prospective studies are needed to better describe these relationships . Carried out the first genome - wide dna methylation scan in major depressive disorder patients . The study pinpointed 224 candidate regions, primarily involved in neuronal growth and development genes, which showed differential methylation; prima1 showed the greatest differences . Specific genes that have been shown to be differentially methylated in individuals with depression include those that code for angiotensin converting enzyme, brain - derived neurotrophic factor, orexin a, and gamma - aminobutyric acid receptor alpha1 . In addition to observing differential dna methylation in ptsd and depression, many studies have observed methylation differences in those that suffer from drug abuse as compared to healthy controls . Increases in dna methylation of the oprm1 gene that codes for opioid receptors have been reported in individuals with chronic opioid use [99102] and alcohol dependence, and global methylation differences have also been reported for these two populations [99, 104]. The proopiomelanocortin gene promoter, dopamine transporter gene promoter, homocysteine - induced endoplasmic reticulum protein promoter, and alpha synuclein promoter were found to be differentially methylated in individuals with alcoholism compared to healthy controls . Methylation at the monoamine oxidate a locus was also significantly associated with nicotine and alcohol dependence in women, but not in men . Together these studies show that psychiatric disorders related to aces are associated with methylation changes that may be reflective of aces or psychiatric symptoms; however, there are no prospective studies to elucidate the possible mediating role of methylation on these psychiatric risks in individuals that experience aces . Reservation - based native americans disproportionately experience aces and health disparities, significantly impacting long - term physical and psychological health . In addition to these experiences, the persistence of stress associated with discrimination and historical trauma converges to add immeasurably to these challenges . Here we provide evidence to suggest that aces result in methylation differences in genes that regulate the stress response and that these changes may contribute to an increased vulnerability for developing psychiatric disorders, as depicted in figure 1 . Although we postulate these relationships, the lack of prospective studies in this at - risk group prevents us and others from concluding this causality, as well as more studies that include native americans . Thus, additional studies are needed to better understand the mechanisms through which aces contribute to health and well - being . These studies may inform future interventions to address these serious risks and promote the health and well - being of native americans.
He presented on multiple occasions with hematemesis, melena and/or hematochezia, associated with hemodynamic instability, and required a cumulative transfusion of more than 100 units packed red blood cells over a period of 8 years . The bleeding was typically preceded by cramping abdominal pain localized in the left upper quadrant . Multiple gastrointestinal evaluations were performed, and were unrevealing for a source of the intermittent bleeding . Prior to his evaluation at our institution, the patient had undergone 2 esophagoduodenoscopies, a colonoscopy, a tagged nuclear red blood cell (rbc) scan and an angiogram, which were within normal limits . An intra - operative enteroscopy was performed in 2000, which was significant for blood in the 3rd and 4th portions of the duodenum, multiple arteriovenous malformations (avms) and a single jejunal diverticulum . The patient presented to our institution in 2001 for further evaluation . Over time, repeat endoscopic and radiologic evaluations were performed including 3 upper endoscopies, a colonoscopy and 2 tagged nuclear rbc scans, which were within normal limits . Catheter angiograms were performed on three different occasions, including a provocative visceral angiogram with infusion of tissue plasminogen activator, but failed to identify the site of hemorrhage . Wedged hepatic vein pressure (whvp) was measured by the transjugular approach, and revealed an elevated portosystemic gradient of 18 mm hg, suggestive of portal hypertension . A ct angiogram was performed that showed evidence of a tortuous and prominent splenic artery, otherwise within normal limits . Endoscopic retrograde cholangiopancreatography (ercp) showed mild dilation of the pancreatic duct, but no other significant findings were appreciated . An intra - operative enteroscopy was then performed, which revealed blood in the distal duodenum, avms and a single diverticulum in the proximal jejunum . This led to a segmental resection of 100 cm of proximal jejunum . Despite the surgery however, follow - up push enteroscopy to the mid - jejunum did not reveal angioectasia . Nonspecific red spots in the proximal small bowel were seen on two additional video capsule endoscopies, but no definite source for bleeding was identified . Argon plasma coagulation of 23 non - bleeding avms in the gastric cardia was performed . Double balloon enteroscopy (dbe) was performed in 2005, with normal appearing 205 cm of the distal small bowel and 230 cm of proximal small bowel visualized on retrograde and antegrade dbe, respectively . In july 2005, the patient developed fever, and multiple blood cultures grew mycobacterium chelonae / abscessus group . Antibiotic treatment was initiated with amikacin and clarithromycin, and the porta - cath was explanted . U / l, alt 50 u / l and alkaline phosphatase 229 u / l), and splenomegaly (22.3 superior - inferior length) was found on adominal ultrasound . A ct scan of the abdomen revealed multiple large splenic lesions, consistent with subacute or chronic infarcts, a hypodensity at the tail of the pancreas, and inflammatory changes near the splenic hilum at the tail of the pancreas, suggestive of resolving pancreatitis . An endoscopic ultrasound was performed in august 2005, to evaluate the ct findings, and revealed a 10 mm ill - defined hyperechoic lesion at the tail of the pancreas, suggestive of a benign pancreatic neoplasm . The patient presented with recurrent hematochezia, prompting a repeat provocative visceral angiogram with heparin ., the patient's fever recurred, prompting a fever reevaluation . A ct scan abdomen demonstrated a 1.5 1.5 1.0 cm saccular aneurysm arising from the distal splenic artery, invaginating the tail of the pancreas (fig . 1, fig . The main pancreatic duct was mildly enlarged to 0.6 cm within the head of the pancreas, with gradual taper towards the tail . Multiple small infarcts were seen involving the lower third of the spleen . Upon retrospective review, the prior angiogram was noted to show a small pseudoaneurysm of the distal third of the splenic artery, which was camouflaged by the tortuousity of the splenic artery (fig ., images from the prior ercp showed blunting of the main pancreatic duct at the tail of the pancreas, likely due to mass effect or thrombus from the adjacent splenic artery pseudoaneurysm eroding into the pancreatic duct (fig . The benign neoplasm noted on prior eus, was in fact the pseudoaneurysm containing mural thrombus with focal surrounding pancreatitis . Fortunately, needle biopsy of this suspected pancreatic neoplasm was not attempted at the time of eus . The central venous access was explanted, and antibiotic treatment re - initiated with amikacin, clarithromycin, and gatifloxacin . Despite 4 weeks of antimicrobial therapy, he experienced progressive unilateral knee, neck and lumbar pains . Magnetic resonance imaging identified enhancement and fluid at the l4 - 5 and c4 - 5 interspaces . Percutaneous aspiration of the fluid and open knee debridement were performed, with cultures positive for m. chelonae / abscessus . Progressive clinical improvement was noted at monthly follow - up appointments, and his knee and spine symptoms resolved . Laparotomy was performed, which confirmed the presence of a splenic artery pseudoaneurysm at the tail of the pancreas . Pathology was consistent with a 2.5 cm splenic artery pseudoaneurysm, with organized blood clot in the lumen, extending into an epithelial - lined cavity consistent with the pancreatic duct adjacent to the splenic arterial wall, suggestive of previous rupture into the tail of the pancreas (fig . Avvg (voerhoff vangeesen) stain showed disruption of the internal elastic lamina of the aneurysm, confirming a fistula from the splenic aneurysm into a dilated section of pancreatic duct . The patient's post - operative course was complicated by a minor pancreatic duct leak, successfully treated by ercp with a 7 cm, 7 f stent placement . Follow - up ercp after 2 months showed resolution of leak and the stent was removed . His hemoglobin, which had been in the range of 89 g / dl, was noted to be improved to 12.7 g / dl, with mcv increased from 75 fl to 89 fl at 6 months follow - up . Hemosuccus pancreaticus is defined as bleeding from the pancreatic duct, and was first reported by lower and farrell in 1931 . The term . There are at least 160 cases of hemosuccus pancreatitis reported in the english literature since 1931 . The most common etiology of this relatively rare entity is communication of a splanchnic artery aneurysm with the pancreatic duct, either due to erosion or fistula formation . Aneurysms originating from the splenic, hepatic and pancreaticoduodenal arteries have been described in association with hemosuccus pancreaticus . These are usually pseudoaneurysms that occur as a result of pancreatitis or abdominal trauma to the left upper quadrant [4, 6]. Pseudoaneurysms of the splanchnic arteries presenting as hemosuccus have been reported to involve the splenic artery most commonly (6065%), followed by the gastroduodenal (2025%), pancreaticoduodenal (1015%) and hepatic artery (510%). Splanchnic artery pseudoaneurysms have been found to be present in about 10% of patients with chronic pancreatitis . In addition to fistulization into the pancreatic duct, they can present with intraperitoneal hemorrhage and hypotensive shock . Clay et al . Postulated that hemosuccus pancreaticus is usually preceded by an episode of pancreatitis, which may occur several years prior to the episode of bleeding . The rapid ductal distension that occurs as blood enters the pancreatic duct leads to herald pain prior to onset of clinical bleeding . This increased pressure in the pancreatic duct allows the leak to seal by clot formation . Hence, in order to diagnose hemosuccus pancreaticus, visceral angiogram should ideally be performed at the onset of epigastric pain, when extravasation of contrast from the splanchnic artery into the pancreatic duct can be observed . If the angiogram is delayed until onset of clinical bleeding, the diagnosis may be missed . Other etiologies for hemosuccus pancreaticus include erosion of a pseudocyst into a peripancreatic artery in patients with chronic pancreatitis, direct bleeding from the pancreatic duct as a result of pancreatic duct stones, primary aneurysm of a splenic artery, blunt or penetrating abdominal trauma, mycotic aneurysms, and rarely pancreatic malignancy . Clinical presentation includes recurrent episodes of herald pain localized in the epigastric region, followed by gastrointestinal bleeding, presenting as hematemesis, melena or hematochezia [4, 12]. Patients can present with hemodynamic instability and require multiple units of packed red cell transfusion . The diagnosis may often be delayed several years after initial presentation due to the intermittent nature of bleeding and failure to consider hemosuccus pancreaticus in the differential diagnosis of obscure gastrointestinal bleeding [4, 10]. The overall mortality rate has been reported to be as high as 28% . In the era of video capsule endoscopy and double balloon enteroscopy, it is common to identify incidental findings in the small bowel, which in turn may lead to unnecessary endoscopic and surgical interventions, with their associated risk . Video capsule endoscopy has been shown to identify abnormalities in up to 23% of healthy subjects . Data from studies evaluating double balloon enteroscopy have noted inconsistent verification of video capsule findings in 1935% of patients and detection of incidental findings [17, 18]. Similarly, laparotomy without a precise diagnosis can lead to unnecessary small bowel resections, with a high rate of recurrent bleeding in up to 52% of patients . It is hence extremely important to consider extraluminal etiologies in the differential diagnosis of obscure gastrointestinal bleeding, and to maintain a high index of suspicion in order to establish a diagnosis of hemosuccus pancreaticus . Visceral angiography is useful to confirm the site of bleeding and demonstrate a fistula between the pancreatic duct and peripancreatic vessels, with a sensitivity of 67100% [20, 21]. Sentinel clot sign, characterized by presence of blood clots in the pancreatic duct on ct scan, may also provide a clue to the diagnosis . Three - dimensional ct angiography in addition to establishing the diagnosis allows evaluation of details including size of the aneurysm and surrounding viscera, and is hence useful in determining the appropriate therapeutic intervention [12, 23]. However, ct interpretation by a radiologist with expertise in vascular radiology is extremely important to avoid missing subtle vascular findings . There have been isolated case reports of the diagnosis being made on endoscopy or endoscopic retrograde cholangiopancreaticography, when blood is seen emanating from the ampulla of vater . In addition, the diagnosis may be established in hemodynamically unstable patients at the time of exploratory laparotomy . Conventional treatment of hemosuccus pancreaticus secondary to a splenic artery pseudoaneurysm involves distal pancreatectomy and splenectomy, with no reported failure rate . In comparison, ligation of the splenic artery alone provides adequate treatment without vascular compromise, but has a reported failure rate of 43% [9, 12]. Transcatheter embolization by interventional radiology is a less invasive technique that involves embolization of the aneurysm using gelfoam or platinum spirals . This method of treatment is highly operator dependent, and may rarely be complicated by rebleed, splenic infarction, splenic or pancreatic abscess [9, 15]. Surgical resection may be more effective in the management of splenic artery pseudoaneurysms with associated pseudocysts, due to difficulty in embolization of large pseudocyst cavities . Mycobacteria chelonae / abscessus group are ubiquitous, rapidly growing mycobacteria, and are associated with infections in both the immunocompromised and the immunocompetent patient . Skin and soft tissue infections, infections complicating intravascular catheters, medical procedures or trauma, and less commonly this patient likely relapsed due to persistent intravascular infection, as suggested by a rapid relapse of the catheter - associated bacteremia and dissemination to multiple musculoskeletal sites, the negative echocardiogram notwithstanding . Optimal treatment of m. chelonae / abscessus infections is difficult and ill defined, since these organisms are resistant to first line antituberculous and other antibacterial medications . Successful treatment may require the administration of multiple antimicrobial agents to which the organism is susceptible, since single drug therapy has been associated with the emergence of resistance . Tigecycline is a new glycylcycline agent with good activity against the rapidly growing mycobacteria, especially m. chelonae / abscessus group, and may be a useful component of a multiple drug regimen . Although mycotic aneurysms have been reported to occur secondary to mycobacterium tuberculosis, there are no reports of aneurysms secondary to the rapid growing mycobacteria, including mycobacterium chelonae / abscessus . In our patient, fite stain of the splenic artery aneurysm was negative for acid fast organisms, confirming that the aneurysm was not related to the co - existent mycobacterial infection . It is important to consider the diagnosis of hemosuccus pancreaticus in patients with obscure gastrointestinal bleeding . Angiography should ideally be performed at the onset of herald pain in order to detect extravasation of contrast from the splanchnic vessel into the pancreatic duct . A high index of suspicion should be maintained for extraluminal etiologies of obscure bleeding in order to avoid misdiagnosis and unnecessary endoscopic and surgical interventions in the era of video capsule endoscopy and double balloon enteroscopy.
Acute appendicitis is the most common diagnosis suspected in patients presenting in emergency rooms with acute abdominal pain, and is the most common indication for an urgent abdominal intervention . Yet, it is difficult to diagnose based solely on the patient's medical history, physical examination, and laboratory findings . Both delayed and unnecessary interventions may lead to adverse fetus outcomes. [13] a mean negative appendectomy rate of 26% (1647%) have been reported when the diagnosis is based only on clinical and laboratory findings, dropping to 610% when imaging is performed . Therefore, the use of imaging modalities is critical to confirm the diagnosis, when facing a clinical suspicion of appendicitis . Currently, there is no uniform protocol regarding the use of imaging tests, with some centers starting with ultrasound (us) and others with computed tomography (ct), according to their availability and local experience . Hence, there is a need for guidelines regarding the optimal patient - tailored imaging modality to begin with, and an algorithm for determining further imaging procedures . Meta - analyses of ct and us in the diagnosis of appendicitis were published by researchers from the university of amsterdam, based on articles published between january 1994 and february 2006, and from the university of toronto, based on publications from 1986 to 2004 . In the present review, data from relevant articles on appendicitis imaging published from february 2006 to march 2011 was retrieved from a computerized database (medline). The aim of this review is to analyze the diagnostic capabilities of us, ct, and mri in patients suspected of suffering from acute appendicitis, based on articles published in the last five years . High - resolution linear array transducers with harmonics and compound capabilities in modern us systems allow optimal definition of the structures at the right lower quadrant . In obese patients, a convex array lower frequency transducer may be needed to achieve better penetration . Bowel wall and peristalsis, iliopsoas muscle, iliac vessels, mesenteric fat, and mesenteric lymph nodes are clearly defined . The normal appendix can be visualized from the base of the cecum as a blind - ended, gut - pattern aperistaltic tubular structure, with a wall thickness of 2 mm or less and a 67 mm or less diameter [figure 1]. A 31-year - old - female with right lower quadrant abdominal pain for two days . Normal appendix on us . (b) short axis view . A blind - ended, gut - pattern tubular structure (arrows), with 2 mm wall thickness and 4.5 mm lumen width (cursors) is seen anterior to the right common iliac vessels on gray - scale us graded compression . The inflamed appendix is visualized as an incompressible, blind - ended, gut - pattern fluid - filled tubular structure, with a thickened wall, and a diameter greater than 67 mm . Applying graded compression with the transducer, differentiation between normal displaceable loops and color and power doppler may aid in the diagnosis by showing a hyperemic wall [figure 2]. Manual compression of the lumbar fossa improves visualization of appendicitis, in particular when it is retrocecal . Visualization of the whole appendix is needed to detect if inflammation is localized just at the end of the appendix, known as tip appendicitis . When there is clinical suspicion of appendicitis, the presence of a fecalith in the appendix confirms the diagnosis [figure 3]. Additional findings are pelvic or inter loop peritoneal fluid and hyperechoic inflamed mesenteric fat surrounding the appendix [figure 4]. Perforated appendicitis may be seen as an abscess in the right lower quadrant [figure 5]. Appendicitis - mimicking conditions of gastrointestinal and urogenital origin may be revealed on us images of the right lower quadrant [figure 7]. An incompressible, blind - ended, gut - pattern fluid - filled tubular structure, with thickened wall and diameter greater than 7 mm is seen in the right lower quadrant (arrows). Following diagnosis of acute appendicitis on us examination, patient proceeded to surgery, without further imaging tests . A 28-year - old - female with pelvic pains and fever . A normal proximal appendix, 4.8 mm width followed by enlarged, 12.7 mm width fluid - filled distal appendix is seen on compression us . A hyperechogenic focus with posterior acoustic shadow compatible with a fecalith (b) short axis view of the appendix, showing the normal proximal appendix and the inflamed distal part (measurements), the last containing an appendicolith . A 21-year - old - male with right pelvic pain and fever . Hyperechoic thickened mesenteric fat (arrows) surrounding 9.5 mm inflamed appendix (cursors) is seen on compression us . A 10- year - old- female with abdominal pain for three days and rebound in right lower quadrant . Dual gray scale compression us image (right plot before compression; left plot during compression) showing an uncompressible distended appendix (cursors) surrounded by a fluid collection (arrows). A 45- year - old - female with fever and abdominal pains, 10 days after laparoscopic appendectomy . A) contrast enhanced mdct showing a retrocecal collection displacing the ascending colon (arrows). (b) us of the right lower quadrant demonstrated fluid collection (long arrows) and in the bottom, an echogenic structure with posterior acoustic shadowing (short arrows), compatible with a dropped appendicolith . (c) a 7f pig tail catheter (arrow at the tip) is introduced in the collection under us guidance . (d) the catheter is shown in the partially drained collection (arrow). A six - year - old - female with pain in right lower abdomen and pelvis . (a) longitudinal us scan of the pelvis showing a hyperechoic linear structure with a twinkle artifact on color doppler compatible with a stone, in the right ureter at the uretero vesical junction . They were distinguished by factors including the use of oral or rectal contrast administration of either positive, neutral, or water contrast agents and whether non - contrast or contrast exams after intravenous injection were performed . They also varied in the anatomical extension of the scan, some including the entire abdomen and pelvis and others focusing on the anatomical area, from the level of the xyphoid to the ramus pubis . The normal appendix is visualized as a blind - ended tubular structure exiting from the cecum, filled with contrast media or gas [figure 8]. The normal diameter ranges between 6 and 10 mm, although more than 7 mm is generally considered as pathological . A 10-year - old - male with persistent right lower quadrant abdominal pain and fever, after two negative us exams for appendicitis . A retrocecal subhepatic blind - ended gas filled tubular structure of normal wall thickness and normal diameter is seen (large arrow). The inflamed appendix is seen as a blind - ended tubular structure exiting from the cecum, without contrast or air filling, more than 7 mm in diameter [figure 9]. The presence of contrast or air from gas - forming organisms in the proximal part of the lumen does not exclude appendicitis . Additional findings such as the presence of an appendicolith, interloop peritoneal fluid, cecal wall thickening, and periappendicular fat stranding are especially useful in an indeterminate appendix [figure 10]. Administration of intravenous contrast media, as a bolus injection of 80 - 100 cc non - ionic contrast media, allows evaluation of the appendix wall enhancement, which can be useful in borderline cases . A three - year - old - male with abdominal pains and rebound in right lower abdomen . (a) gray scale us performed by the resident on - duty showed a structure interpreted as mimicking intussusception in the subhepatic area (arrows). Due to the patient's clinical condition and (b) contrast enhanced coronal oblique reformatted mdct showing a widened fluid filled tubular structure, without intraluminal air, of oral contrast media (arrows), surrounded by infiltrated mesenteric fat, compatible with acute appendicitis . A 47-year - old - male with a history of inflammatory bowel disease, presenting with abdominal pain and fever . (a) a blind - ended tubular structure exiting from the cecum, without intraluminal oral contrast media or air, 12 mm in diameter, is seen (large arrow). Mesenteric fat stranding (short arrow) is seen in the proximity of the inflamed appendix . (b) mesenteric fat edema and reactive lymph nodes are seen in a scan proximal to (a). Ct followed a us exam positive for appendicitis, to rule - out signs of active crohn's disease . Alternative diagnosis of right lower quadrant pain may be achieved using ct, to determine if it is of gastrointestinal origin, such as mesenteric adenitis, intussusception, terminal ileitis, diverticulitis, epiploic appendagitis, and typhlitis, or of urogenital origin, such as ureteral stone and urinary tract infection, tubo ovarian abscess, ovarian torsion, ectopic pregnancy, hemorrhagic ovarian cyst or corpus luteum remnants [figures 11 and 12]. Cecum and terminal ileum with thickened walls (arrows) and adjacent peritoneal fluid (f) confirm crohn's disease . An appendicolith (lateral right arrow) is seen at the base of the inflamed appendix, (shown in figure 9). Mri is an alternative method to ct for pregnant patients, offering high soft tissue contrast without ionizing radiation . The imaging protocol for mri evaluation of acute appendicitis in the pregnant population includes t1- and t2- weighted images, and has been extensively described in the literature . The normal appendix is seen as a tubular structure exiting from the cecum, greater than 7 mm in diameter, filled with air or contrast media [figure 13]. A 37- year - old - pregnant patient with lower abdominal pain and fever . A normal sized appendix with air into the lumen is seen in the right lower quadrant in this axial t1 weighted with fat suppression image . A previously performed us was negative for appendicitis, but the normal appendix could not be demonstrated . Acute appendicitis is seen as an enlarged appendix, greater than 7 mm in diameter, and void of air or contrast media . Signs of peri - appendicular inflammation, seen as band - like areas of high signal intensity on t2-weighted images, single - shot fast spin - echo images or fat saturation images, or the presence of an appendicolith, visualized as an intraluminal low signal intensity focus, confirm the diagnosis, especially in the borderline widened appendix [figure 14]. [1620] according to the american college of radiology guidelines for safe mri practices, contrast agents should not be routinely administered to pregnant patients . Routine use of gadolinium - based contrast agents in pregnancy is not approved . A 28-year - old - female of 26 weeks pregnancy, presenting with lower abdominal pain and fever . (a) coronal t2 weighted image showing an enlarged (10 mm in diameter) fluid filled appendix surrounded by a band - like area of high signal intensity (arrow) compatible with appendicitis with periappendicular inflammation . The inflamed appendix is seen as a tubular fluid filled structure (arrow) in the right lower quadrant, behind the pregnant uterus . On surgery, mri is also useful in identifying alternative sources of right lower quadrant pain in patients suspected of acute appendicitis [figure 15]. A 13- year - old - male with a history of crohn's disease, presenting with right lower abdominal pain . Axial t2 weighted image after diluted oral contrast media administration, showing the thickened - wall terminal ileum (long arrows) compatible with crohn's disease . A normal retrocecal appendix is seen (short arrow). In two previous review studies, us and ct performance for the diagnosis of acute appendicitis the respective mean specificities for ct and graded compression us were 90% and 83% . In a head - to - head comparison, ct offered a better test performance than graded compression us in diagnosing appendicitis . Therefore, ct is recommended in patients suspected of acute appendicitis . However, taking into consideration the drawbacks of radiation exposure in young, female and slender patients, graded compression us is recommended as the primary diagnostic test in these patients . Based on 26 studies in children and 31 studies in adults from 1988 to 2004, doria et al found the pooled sensitivity and specificity for us in the diagnosis of appendicitis in children to be 88% and 94% respectively, and for ct studies, 94% and 95% respectively . The pooled sensitivity and specificity for us studies in the diagnosis of appendicitis in adults were 83% and 93% respectively, and for ct studies, 94% and 94% respectively . However, the drawbacks of radiation exposure associated with ct should be considered, especially in children . Methodological shortcomings in both of these studies, however, could influence reported diagnostic test accuracy . In our present review, we selected studies performed from february 2006 to march 2011 comparing graded compression us and ct performance, using surgery or clinical follow - up as the standard reference . The results are presented in table 1 . Us, ct, and mri performance for the diagnosis of acute appendicitis poortman et al developed an imaging diagnostic pathway using us as the primary test . Of a total population of 151 adult patients suspected of appendicitis, 79 patients diagnosed by us as positive for appendicitis were directed to appendectomy and 72 negative or inconclusive patients diagnosed by us proceeded to mdct . An alternative diagnosis was made in 12 patients and no source of abdominal pain was found in 39 patients . In this study, the sensitivity and specificity for ct were 100%, and for us, 77% and 86% respectively . Although less accurate than ct, us can be used as a primary diagnostic modality avoiding the disadvantages of ct . Gaitini et al conducted a retrospective study to evaluate the diagnostic accuracy of color doppler us and contrast - enhanced mdct in 420 adult patients referred from the emergency room with clinical suspicion of appendicitis . All the patients underwent graded compression us and color doppler of the right lower quadrant . Ct was performed in 132 patients due to inconclusive sonographic findings or a discrepancy between clinical and sonographic diagnoses . Sonography and ct correctly diagnosed acute appendicitis in 66 of 75 patients and in 38 of 39 patients, respectively, and correctly ruled out acute appendicitis in 312 of 326 and in 92 of 92 patients . Sonography was inconclusive in 17 of 418 cases and ct, in one of 132 cases . The sensitivity and specificity of us were 74% and 97% respectively, with a 93% negative predictive value, while ct had a sensitivity and specificity of 100% and 99% respectively, with a 100% negative predictive value . The positive predictive value and accuracy for sonography were 88% and 92% and for ct, 97% and 99% . Due to its high negative predictive value (93%), us should be used as the first imaging test in adult patients for the diagnosis of appendicitis and triage of acute abdominal pain, reserving ct as a complementary study for selected cases . Cuschieri et al, of the surgical care and outcomes assessment program (scoap) collaborative group evaluated the relationship between negative appendectomies (na) and negative ct / us over a two year period (20062007). There was a significant increase in the use of ct / us and decrease in na over the study period (p <.0001). Variation between hospitals was linked closely to ct / us accuracy, suggesting that ct / us accuracy should be considered a measure of quality in the care of patients with presumed appendicitis . Us is widely used for the diagnosis of appendicitis in pediatric patients due to concerns regarding risks from exposure to ionizing radiation . In a recent paper, wan et al analyzed cost - effectiveness of us versus ct in young children based on a decision analytic model using costs, utilities and probabilities . The most cost - effective method was to start with a us study and follow negative us studies with a ct examination . This strategy is in concordance with the previously published study of garcia pena et al . Pregnant patients present a special population, and clinical and laboratory findings lack specificity for the diagnosis of appendicitis in pregnancy . An unnecessary laparotomy increases the risk of pre - term contractions, while delay in the treatment of appendicitis leading to perforation increases fetal mortality to 637% . Pedrosa et al investigated the role of mri in 148 pregnant patients suspected of appendicitis in lowering the negative laparotomy rate (nlr) and the perforation rate (pr). An oral contrast media was administrated to facilitate identification of the appendix in mri examinations . Us had a low sensitivity (36%) but an excellent specificity (99%). The sensitivity and specificity of mri were 100% and 99% respectively . Among the patients with a negative diagnosis for appendicitis, the normal appendix could be visualized on us in less than 2% (2 of 126) of cases and on mr in 87% (116 of 134) of cases (p <.0001). The use of mr imaging yields favorable combinations of nlr and pr compared with values previously reported in the literature . Comparison of methods for imaging appendicitis the main advantage of us over ct is the lack of ionizing radiation, which is most important in the pediatric and young adult populations, among which appendicitis occurs more frequently, and who are most vulnerable to radiation's detrimental effects. [2731] in the pediatric population, us also obviates the need for sedation or general anesthesia . Us may be considered as an extension of the physical examination: patients can point to the region of tenderness and graded compression us may elicit a rebound with an increased sensation of pain . Contrast media ingestion, with a consequent delay in surgery, or contrast injection, carrying risks of allergic reaction and nephrotoxicity, are not required for us examinations . Us may not only diagnose an abscess or phlegmon in perforated appendicitis but can also guide percutaneous drainage . Several alternative conditions, especially in the female pelvis, may be diagnosed on us examination. [24] the main disadvantage of us is its operator dependence . Us has a lower sensitivity compared to ct for the diagnosis of appendicitis. [67222433] however, with a high prevalence of the disease and a strict protocol, a sensitivity and specificity of 98.5% and 98.2% respectively was achieved for the us diagnosis of appendicitis in children . The inability of us to properly scan a gas distended bowel or an obese patient, and lack of demonstration of a normal appendix, especially when retrocecal or deeply situated in the pelvis, may lead to indeterminate or false negative exams . In pregnant patients, the main advantage of ct for the diagnosis of appendicitis is its high sensitivity and specificity [table 1]. [3234] marked advancements in ct technology over the last decade have led to excellent image quality . The main disadvantage of ct is the radiation exposure, especially important in the radiosensitive pediatric and young adult appendicitis - suspected population . Allergic reaction and nephrotoxicity risks of iodinated contrast media injection, delay in the diagnosis and therapy due to time invested in ingestion of contrast media, higher cost and lower availability, particularly in small or peripheral centers, are further disadvantages of ct . Operator dependence related to ct protocols and radiologist skills for correct performance and interpretation are also limitations of ct exams . The main advantage of mri in pregnant patients is in avoiding radiation exposure to the fetus . Mri is less operator - dependent than us, lacks exposure to intravenous iodinated contrast media, and may afford alternative diagnoses, such as ovarian torsion or renal obstruction. [203739] mri disadvantages reside in a longer examination time, limitations related to claustrophobia and metal devices, low availability, and higher cost . Guidelines for the diagnosis of appendicitis are needed, related to choosing which is the optimal patient - tailored imaging modality to begin with, and an algorithm for further imaging procedures, if required . According to the present review, imaging of a patient of any age suspected of appendicitis should start with a graded compression us examination . A positive, good - quality us when there is a strong clinical suspicion is enough to proceed to surgery . A negative us exam for appendicitis, where the normal appendix is clearly visualized, or alternatively, when the appendix is not seen but the clinical suspicion for appendicitis is low, may end the investigation, leading to patient discharge or follow - up . Furthermore, if an alternative diagnosis for the clinical presentation is reached, treatment of the alternative condition will be followed . A negative or indeterminate us exam in a non - pregnant adult with a high clinical suspicion of appendicitis a negative us with a high suspicion for appendicitis should lead to an mri examination. [3840] [figure 16]. In the pediatric patient, a second us examination, especially when the first was performed by a less experienced professional, may render definitive results, while avoiding ct radiation risks . A positive us is enough to proceed to surgery or percutaneous drainage of a peri - appendicular abscess a negative us showing the normal appendix may end the investigation and lead to patient discharge . An indeterminate us, whether neither the normal nor the inflamed appendix is seen may lead to follow up or a repeated us if the clinical suspicion is low . This approach seems to offer excellent accuracy, with reported sensitivities of 94% to 99% and specificities of 94% to 95% . High - resolution linear array transducers with harmonics and compound capabilities in modern us systems allow optimal definition of the structures at the right lower quadrant . In obese patients, bowel wall and peristalsis, iliopsoas muscle, iliac vessels, mesenteric fat, and mesenteric lymph nodes are clearly defined . The normal appendix can be visualized from the base of the cecum as a blind - ended, gut - pattern aperistaltic tubular structure, with a wall thickness of 2 mm or less and a 67 mm or less diameter [figure 1]. A 31-year - old - female with right lower quadrant abdominal pain for two days . A blind - ended, gut - pattern tubular structure (arrows), with 2 mm wall thickness and 4.5 mm lumen width (cursors) is seen anterior to the right common iliac vessels on gray - scale us graded compression . The inflamed appendix is visualized as an incompressible, blind - ended, gut - pattern fluid - filled tubular structure, with a thickened wall, and a diameter greater than 67 mm . Applying graded compression with the transducer, differentiation between normal displaceable loops and color and power doppler may aid in the diagnosis by showing a hyperemic wall [figure 2]. Manual compression of the lumbar fossa improves visualization of appendicitis, in particular when it is retrocecal . Visualization of the whole appendix is needed to detect if inflammation is localized just at the end of the appendix, known as tip appendicitis . When there is clinical suspicion of appendicitis, the presence of a fecalith in the appendix confirms the diagnosis [figure 3]. Additional findings are pelvic or inter loop peritoneal fluid and hyperechoic inflamed mesenteric fat surrounding the appendix [figure 4]. Perforated appendicitis may be seen as an abscess in the right lower quadrant [figure 5]. Appendicitis - mimicking conditions of gastrointestinal and urogenital origin may be revealed on us images of the right lower quadrant [figure 7]. An incompressible, blind - ended, gut - pattern fluid - filled tubular structure, with thickened wall and diameter greater than 7 mm is seen in the right lower quadrant (arrows). Following diagnosis of acute appendicitis on us examination, patient proceeded to surgery, without further imaging tests . A normal proximal appendix, 4.8 mm width followed by enlarged, 12.7 mm width fluid - filled distal appendix is seen on compression us . A hyperechogenic focus with posterior acoustic shadow compatible with a fecalith (b) short axis view of the appendix, showing the normal proximal appendix and the inflamed distal part (measurements), the last containing an appendicolith . A 21-year - old - male with right pelvic pain and fever . Hyperechoic thickened mesenteric fat (arrows) surrounding 9.5 mm inflamed appendix (cursors) is seen on compression us . A 10- year - old- female with abdominal pain for three days and rebound in right lower quadrant . Dual gray scale compression us image (right plot before compression; left plot during compression) showing an uncompressible distended appendix (cursors) surrounded by a fluid collection (arrows). A 45- year - old - female with fever and abdominal pains, 10 days after laparoscopic appendectomy . (a) contrast enhanced mdct showing a retrocecal collection displacing the ascending colon (arrows). Lower quadrant demonstrated fluid collection (long arrows) and in the bottom, an echogenic structure with posterior acoustic shadowing (short arrows), compatible with a dropped appendicolith . (c) a 7f pig tail catheter (arrow at the tip) is introduced in the collection under us guidance . (d) the catheter is shown in the partially drained collection (arrow). A six - year - old - female with pain in right lower abdomen and pelvis . (a) longitudinal us scan of the pelvis showing a hyperechoic linear structure with a twinkle artifact on color doppler compatible with a stone, in the right ureter at the uretero vesical junction . They were distinguished by factors including the use of oral or rectal contrast administration of either positive, neutral, or water contrast agents and whether non - contrast or contrast exams after intravenous injection were performed . They also varied in the anatomical extension of the scan, some including the entire abdomen and pelvis and others focusing on the anatomical area, from the level of the xyphoid to the ramus pubis . The normal appendix is visualized as a blind - ended tubular structure exiting from the cecum, filled with contrast media or gas [figure 8]. The normal diameter ranges between 6 and 10 mm, although more than 7 mm is generally considered as pathological . A 10-year - old - male with persistent right lower quadrant abdominal pain and fever, after two negative us exams for appendicitis . A retrocecal subhepatic blind - ended gas filled tubular structure of normal wall thickness and normal diameter is seen (large arrow). The inflamed appendix is seen as a blind - ended tubular structure exiting from the cecum, without contrast or air filling, more than 7 mm in diameter [figure 9]. The presence of contrast or air from gas - forming organisms in the proximal part of the lumen does not exclude appendicitis . Additional findings such as the presence of an appendicolith, interloop peritoneal fluid, cecal wall thickening, and periappendicular fat stranding are especially useful in an indeterminate appendix [figure 10]. Administration of intravenous contrast media, as a bolus injection of 80 - 100 cc non - ionic contrast media, allows evaluation of the appendix wall enhancement, which can be useful in borderline cases . A three - year - old - male with abdominal pains and rebound in right lower abdomen . (a) gray scale us performed by the resident on - duty showed a structure interpreted as mimicking intussusception in the subhepatic area (arrows). Due to the patient's clinical condition and indeterminate us diagnosis, a ct was performed . (b) contrast enhanced coronal oblique reformatted mdct showing a widened fluid filled tubular structure, without intraluminal air, of oral contrast media (arrows), surrounded by infiltrated mesenteric fat, compatible with acute appendicitis . A 47-year - old - male with a history of inflammatory bowel disease, presenting with abdominal pain and fever . Acute appendicitis with mesenteric stranding on ct . (a) a blind - ended tubular structure exiting from the cecum, without intraluminal oral contrast media or air, 12 mm in diameter, is seen (large arrow). Mesenteric fat stranding (short arrow) is seen in the proximity of the inflamed appendix . (b) mesenteric fat edema and reactive lymph nodes are seen in a scan proximal to (a). Ct followed a us exam positive for appendicitis, to rule - out signs of active crohn's disease . Alternative diagnosis of right lower quadrant pain may be achieved using ct, to determine if it is of gastrointestinal origin, such as mesenteric adenitis, intussusception, terminal ileitis, diverticulitis, epiploic appendagitis, and typhlitis, or of urogenital origin, such as ureteral stone and urinary tract infection, tubo ovarian abscess, ovarian torsion, ectopic pregnancy, hemorrhagic ovarian cyst or corpus luteum remnants [figures 11 and 12]. Cecum and terminal ileum with thickened walls (arrows) and adjacent peritoneal fluid (f) confirm crohn's disease . An appendicolith (lateral right arrow) is seen at the base of the inflamed appendix, (shown in figure 9). Mri is an alternative method to ct for pregnant patients, offering high soft tissue contrast without ionizing radiation . The imaging protocol for mri evaluation of acute appendicitis in the pregnant population includes t1- and t2- weighted images, and has been extensively described in the literature . The normal appendix is seen as a tubular structure exiting from the cecum, greater than 7 mm in diameter, filled with air or contrast media [figure 13]. A 37- year - old - pregnant patient with lower abdominal pain and fever . A normal sized appendix with air into the lumen is seen in the right lower quadrant in this axial t1 weighted with fat suppression image . A previously performed us was negative for appendicitis, but the normal appendix could not be demonstrated . Acute appendicitis is seen as an enlarged appendix, greater than 7 mm in diameter, and void of air or contrast media . Signs of peri - appendicular inflammation, seen as band - like areas of high signal intensity on t2-weighted images, single - shot fast spin - echo images or fat saturation images, or the presence of an appendicolith, visualized as an intraluminal low signal intensity focus, confirm the diagnosis, especially in the borderline widened appendix [figure 14]. [1620] according to the american college of radiology guidelines for safe mri practices, contrast agents should not be routinely administered to pregnant patients . A 28-year - old - female of 26 weeks pregnancy, presenting with lower abdominal pain and fever . (a) coronal t2 weighted image showing an enlarged (10 mm in diameter) fluid filled appendix surrounded by a band - like area of high signal intensity (arrow) compatible with appendicitis with periappendicular inflammation . The inflamed appendix is seen as a tubular fluid filled structure (arrow) in the right lower quadrant, behind the pregnant uterus . On surgery, mri is also useful in identifying alternative sources of right lower quadrant pain in patients suspected of acute appendicitis [figure 15]. A 13- year - old - male with a history of crohn's disease, presenting with right lower abdominal pain . Axial t2 weighted image after diluted oral contrast media administration, showing the thickened - wall terminal ileum (long arrows) compatible with crohn's disease . In two previous review studies, us and ct performance for the diagnosis of acute appendicitis were compared . The respective mean specificities for ct and graded compression us were 90% and 83% . In a head - to - head comparison, ct offered a better test performance than graded compression us in diagnosing appendicitis . However, taking into consideration the drawbacks of radiation exposure in young, female and slender patients, graded compression us is recommended as the primary diagnostic test in these patients . Based on 26 studies in children and 31 studies in adults from 1988 to 2004, doria et al found the pooled sensitivity and specificity for us in the diagnosis of appendicitis in children to be 88% and 94% respectively, and for ct studies, 94% and 95% respectively . The pooled sensitivity and specificity for us studies in the diagnosis of appendicitis in adults were 83% and 93% respectively, and for ct studies, 94% and 94% respectively . However, the drawbacks of radiation exposure associated with ct should be considered, especially in children . Methodological shortcomings in both of these studies, however, could influence reported diagnostic test accuracy . In our present review, we selected studies performed from february 2006 to march 2011 comparing graded compression us and ct performance, using surgery or clinical follow - up as the standard reference . Us, ct, and mri performance for the diagnosis of acute appendicitis poortman et al developed an imaging diagnostic pathway using us as the primary test . Of a total population of 151 adult patients suspected of appendicitis, 79 patients diagnosed by us as positive for appendicitis were directed to appendectomy and 72 negative or inconclusive patients diagnosed by us proceeded to mdct . An alternative diagnosis was made in 12 patients and no source of abdominal pain was found in 39 patients . In this study, the sensitivity and specificity for ct were 100%, and for us, 77% and 86% respectively . Although less accurate than ct, us can be used as a primary diagnostic modality avoiding the disadvantages of ct . Gaitini et al conducted a retrospective study to evaluate the diagnostic accuracy of color doppler us and contrast - enhanced mdct in 420 adult patients referred from the emergency room with clinical suspicion of appendicitis . All the patients underwent graded compression us and color doppler of the right lower quadrant . Ct was performed in 132 patients due to inconclusive sonographic findings or a discrepancy between clinical and sonographic diagnoses . Sonography and ct correctly diagnosed acute appendicitis in 66 of 75 patients and in 38 of 39 patients, respectively, and correctly ruled out acute appendicitis in 312 of 326 and in 92 of 92 patients . Sonography was inconclusive in 17 of 418 cases and ct, in one of 132 cases . The sensitivity and specificity of us were 74% and 97% respectively, with a 93% negative predictive value, while ct had a sensitivity and specificity of 100% and 99% respectively, with a 100% negative predictive value . The positive predictive value and accuracy for sonography were 88% and 92% and for ct, 97% and 99% . Due to its high negative predictive value (93%), us should be used as the first imaging test in adult patients for the diagnosis of appendicitis and triage of acute abdominal pain, reserving ct as a complementary study for selected cases . Cuschieri et al, of the surgical care and outcomes assessment program (scoap) collaborative group evaluated the relationship between negative appendectomies (na) and negative ct / us over a two year period (20062007). There was a significant increase in the use of ct / us and decrease in na over the study period (p <.0001). Variation between hospitals was linked closely to ct / us accuracy, suggesting that ct / us accuracy should be considered a measure of quality in the care of patients with presumed appendicitis . Us is widely used for the diagnosis of appendicitis in pediatric patients due to concerns regarding risks from exposure to ionizing radiation . In a recent paper, wan et al analyzed cost - effectiveness of us versus ct in young children based on a decision analytic model using costs, utilities and probabilities . The most cost - effective method was to start with a us study and follow negative us studies with a ct examination . This strategy is in concordance with the previously published study of garcia pena et al . Pregnant patients present a special population, and clinical and laboratory findings lack specificity for the diagnosis of appendicitis in pregnancy . An unnecessary laparotomy increases the risk of pre - term contractions, while delay in the treatment of appendicitis leading to perforation increases fetal mortality to 637% . Pedrosa et al investigated the role of mri in 148 pregnant patients suspected of appendicitis in lowering the negative laparotomy rate (nlr) and the perforation rate (pr). An oral contrast media was administrated to facilitate identification of the appendix in mri examinations . Us had a low sensitivity (36%) but an excellent specificity (99%). The sensitivity and specificity of mri were 100% and 99% respectively . Among the patients with a negative diagnosis for appendicitis, the normal appendix could be visualized on us in less than 2% (2 of 126) of cases and on mr in 87% (116 of 134) of cases (p <.0001). The use of mr imaging yields favorable combinations of nlr and pr compared with values previously reported in the literature . Comparison of methods for imaging appendicitis the main advantage of us over ct is the lack of ionizing radiation, which is most important in the pediatric and young adult populations, among which appendicitis occurs more frequently, and who are most vulnerable to radiation's detrimental effects. [2731] in the pediatric population, us also obviates the need for sedation or general anesthesia . Us may be considered as an extension of the physical examination: patients can point to the region of tenderness and graded compression us may elicit a rebound with an increased sensation of pain . Contrast media ingestion, with a consequent delay in surgery, or contrast injection, carrying risks of allergic reaction and nephrotoxicity, are not required for us examinations . Us may not only diagnose an abscess or phlegmon in perforated appendicitis but can also guide percutaneous drainage . Several alternative conditions, especially in the female pelvis, may be diagnosed on us examination. [24] the main disadvantage of us is its operator dependence . Us has a lower sensitivity compared to ct for the diagnosis of appendicitis. [67222433] however, with a high prevalence of the disease and a strict protocol, a sensitivity and specificity of 98.5% and 98.2% respectively was achieved for the us diagnosis of appendicitis in children . The inability of us to properly scan a gas distended bowel or an obese patient, and lack of demonstration of a normal appendix, especially when retrocecal or deeply situated in the pelvis, may lead to indeterminate or false negative exams . In pregnant patients, the main advantage of ct for the diagnosis of appendicitis is its high sensitivity and specificity [table 1]. [3234] marked advancements in ct technology over the last decade have led to excellent image quality . The main disadvantage of ct is the radiation exposure, especially important in the radiosensitive pediatric and young adult appendicitis - suspected population . Allergic reaction and nephrotoxicity risks of iodinated contrast media injection, delay in the diagnosis and therapy due to time invested in ingestion of contrast media, higher cost and lower availability, particularly in small or peripheral centers, are further disadvantages of ct . Operator dependence related to ct protocols and radiologist skills for correct performance and interpretation are also limitations of ct exams . The main advantage of mri in pregnant patients is in avoiding radiation exposure to the fetus . Mri is less operator - dependent than us, lacks exposure to intravenous iodinated contrast media, and may afford alternative diagnoses, such as ovarian torsion or renal obstruction. [203739] mri disadvantages reside in a longer examination time, limitations related to claustrophobia and metal devices, low availability, and higher cost . Guidelines for the diagnosis of appendicitis are needed, related to choosing which is the optimal patient - tailored imaging modality to begin with, and an algorithm for further imaging procedures, if required . According to the present review, imaging of a patient of any age suspected of appendicitis should start with a graded compression us examination . A positive, good - quality us when there is a strong clinical suspicion is enough to proceed to surgery . A negative us exam for appendicitis, where the normal appendix is clearly visualized, or alternatively, when the appendix is not seen but the clinical suspicion for appendicitis is low, may end the investigation, leading to patient discharge or follow - up furthermore, if an alternative diagnosis for the clinical presentation is reached, treatment of the alternative condition will be followed . A negative or indeterminate us exam in a non - pregnant adult with a high clinical suspicion of appendicitis must be followed by ct . In pregnant patients, a negative us with a high suspicion for appendicitis should lead to an mri examination. [3840] [figure 16]. In the pediatric patient, a second us examination, especially when the first was performed by a less experienced professional, may render definitive results, while avoiding ct radiation risks . A positive us is enough to proceed to surgery or percutaneous drainage of a peri - appendicular abscess a negative us showing the normal appendix may end the investigation and lead to patient discharge . An indeterminate us, whether neither the normal nor the inflamed appendix is seen may lead to follow up or a repeated us if the clinical suspicion is low . This approach seems to offer excellent accuracy, with reported sensitivities of 94% to 99% and specificities of 94% to 95% . Imaging is increasingly important in the diagnosis and management of appendicitis, avoiding unnecessary interventions and delay in treatment that may lead to perforation . Us findings must be considered in conjunction with the clinical evaluation . If these guidelines are followed, the number of ct examinations performed will be considerably reduced . Clinical guidelines defining the use of each particular imaging modality allow uniformity, a targeted approach for different patient populations, and cost - effectiveness of medical services.
Role of n - acetylcystein (nac) in adults with non - acetaminophen induced liver failure was described in few studies in literature . Studies were particularly relevant to countries where, liver transplantation facilities are limited or unavailable . Found a significantly improved transplantation free survival at 3 weeks and at 1 year with the use of nac in non - acetaminophen related liver failure, the benefit being confined to those with early hepatic encephalopathy . Dengue infection is prevalent in southeast asia, and according to the epidemiological unit, ministry of health, sri lanka, during the last 4 months of the year 2012, 11148 suspected dengue cases and 46 deaths have been reported . The elevation of transaminases is usually less than five - fold greater than upper limit of normal . However, levels more than five - fold were reported 36.8% and 74.4% of patients with classical dengue and dengue hemorrhagic fever (dhf) respectively . Fulminant hepatitis tends to occur more often in dhf or dengue shock syndrome compared to classic dengue infection and case fatality rate of 50% being reported . Although, nac has shown benefit in non - acetaminophen related liver failure, it was not well studied in dengue associated severe hepatitis . A previously healthy 54-year - old mother of three admitted with 3 day history of fever, headache and body ache . Physical examination on investigation on the day of admission revealed platelets 84,000/cumm and haematocrit (hct) 37% . On the 2 day, she was transferred to intensive care unit (icu) as her platelets dropped to 41,000 per cu mm and hct increased to 47% . Liver transaminases showed mild to moderate rise with ((ast) aspartate transferase) 302 u / l and ((alt)alanine transferase) 262 u / l and patient was given total dose of 10 g of acetaminophen over 3 days at the time . She had stable hemodynamics apart from heart rate of 121 beats / min, but she developed right side moderate pleural effusion, icterus, mild ascites and right hypochondrial pain . In the icu, she deteriorated further, with a decline of glasgow coma scale (gcs) to 11, but no focal neurological signs . Urgent computed tomography brain was done and it neither showed intracranial hemorrhage nor evidence of increased intracranial pressure . Her liver functions continued to deteriorate and liver enzymes reached peak value of ast 16261 u / l and alt 4545 u / l, (pt / inr) prothrombin time / international normalized ratio 1.7 and total bilirubin 5.9 mg / dl on 4 day of admission (7 day of illness). Intravenous nac was started at 100 mg / kg / day as an infusion and continued for 5 days with liver failure regime . Marked improvement in liver enzyme was noted and sgot and sgpt levels dropped by more than half by 48 h of treatment . On the 9 day of admission, liver function revealed ast 300 u / l, alt 223 u / l and pt / inr 1.2, and her conscious level improved to gcs of 15 . During the course of illness, she had mild gum bleeding and few ecchymotic patches with lowest platelet count of 18,000 per cu mm . Her serology was positive for dengue antibodies but negative for hepatitis a and b. hepatitis e serology was not done due to unavailability . Co - infection of malaria was not excluded as she was from neither endemic area nor her symptomatology typical of malaria including fever pattern . Possibility of leptospirosis cannot be excluded in this case as serology was not done . However, she did not have any exposure and her renal functions were never abnormal . During her follow - up visit at 2 weeks after discharge, she had normal liver profile and did not have any evidence of chronic liver disease . Both virus itself and dysregulated immune response to virus are being described as possible mechanisms of liver damage in literature . Although, severe hepatitis associated with dengue fever is a rare occurrence, it carries significant mortality and morbidity . Nac, mostly used in acetaminophen poisoning, acts through its antidote effect of repletion of hepatocellular glutathione stores . Nac scavenges free radicals, improves antioxidant defense and acts as a vasodilator to improve oxygen delivery and consumption . These properties of nac have been postulated to improve outcome in patient with dengue associated acute liver dysfunction . Concluded that benefit is seen when nac is used early stage of liver failure rather than late stage . A retrospective analysis on nac in dengue associated liver failure by kumarasena et al . Showed that 5 patients who survived out of 8 were in early (coma grade 1, 11) liver failure stage at the time when nac was started . This case report also supports the view that intravenous administration of nac is safe and benefits patients, if started in early stage of liver failure . This patient was treated with intravenous nac 100 mg / kg / day infusion for 5 days compared to 150 mg / kg bolus over 15 min followed by 12.5 mg / kg / h for 4 h and then 6.25 mg / kg / h for 72 h was given by kumarasena et al . In their retrospective analysis . Reported a pediatric patient with dengue associated liver failure successfully treated with nac and they have given intravenous nac 100 mg / kg / day for 6 days . Large randomized trials should be carried out to establish its efficacy along with appropriate dosage, timing, and duration of treatment.
Gastric cancer is the fifth most common cancer in the world with about 952, 000 cases reported worldwide in 2012; and the third leading cause of cancer mortality.1 there is a wide geographical variation being highest in the far east (japan and china) and lowest in africa.23456 the age - standardised incidence range from 3.3 in west african men to 35.4 in east asian men; and from 2.6 in west african women to 13.8 in east asian women.1 in africa gastric carcinoma has a relatively higher incidence in nigeria and south africa than in francophone west africa, kenya and egypt.3 in nigeria the prevalence rate is reported to be between 1.64% and 4.1% being highest in the south - west region and lowest in the north - east.237 the relative ratio frequency of gastric carcinoma in the university college hospital (uch) ibadan has been declining over the past decades, from 3.6% in the 1980s to 2.73% in the 1990s as found by ogunbiyi.8 the most recent work on gastric carcinoma in the uch, ibadan by oluwasola and ogunbiyi in 2000 described the clinicopathological features of gastric carcinoma cases seen at the pathology department between 1980 and 1997.9 the aim of this study is to update the present data on the clinicopathological features of gastric carcinoma in uch, ibadan . All the gastric carcinoma cases diagnosed at the pathology department, uch, ibadan between january 2000 and september 2011 were included in this study . Other non - carcinomatous neoplasms of the stomach and all the cases of gastric carcinoma whose blocks and/or slides could not be retrieved were excluded from the study . The demographic and clinical data of the gastric carcinoma patients diagnosed in the pathology department, uch, ibadan, in the above period were retrieved from the cancer registry, pathology department records and the medical records of the uch and analysed for patients age, gender, clinical presentation, histological diagnoses including tumour types (using the lauren classification), stage, and presence of vascular permeation and/or lymph node status . Slides of cases which could not be retrieved were prepared from the archival (formalin - fixed, paraffin - embedded) samples of same patients and reviewed to confirm the histological diagnoses, histological types, stage and lymph node status . All statistical analyses were performed with the statistical package for social sciences (spss) version 19 . Student's chi square test was employed to define association between variables . However, when the expected count of any variable was less than 5, the fisher's exact test was applied . The student's t - independent test was used to compare the mean age for gender and different histologic type of gastric carcinoma . A total of 117 cases of gastric carcinoma were diagnosed at the uch, ibadan, between january, 2000 and september, 2011, accounting for 1.38% of the 8467 cases malignancies from all body systems it also accounted 18.4% of the 636 cases of gastrointestinal malignancies diagnosed within the same period . Of these 117 cases, 74 (63.2%) were males and 43 (36.8%) were females, giving a male to female ratio of 1.72:1 . About 63.5% of cases occurred between 41 and 70 years and the modal age - group of occurrence was 51 - 60 years [figure 1 and table 1]. When the patients ages were stratified into the social age - groups young (0 - 44 years), middle - age (45 - 64 years) and the elderly (65 years and above), the middle - age and the elderly accounted for 77.4% of the cases, while the young accounted for 22.6% . Bar chart showing the age distribution of gastric carcinoma patients diagnosed at the university college hospital between the years 2000 and 2011 age and sex distribution of gastric carcinoma patients diagnosed at the university college hospital between the years 2000 and 2011 the mean, median and modal ages for all cases were 53.36 15.31 years, 54.00 years and 50 years respectively . The mean and median ages for the male patients were 54.0 15.30 and 53.0 years respectively, while those for the female patients were 52.24 15.45 and 55 years respectively . There was no statistically significant difference between the mean ages of the male and female patients (p = 0.601, 95% confidence interval (ci) = 4.32 - 7.43). The modal age - group for the males was 61 - 70 years group while that for the females was 51 - 60 years [figure 2 and table 1]. The modal ages of the male and female groups are 50 years and 55 years respectively . Bar chart showing the age distribution of male and female gastric carcinoma patients diagnosed at the university college hospital between the years 2000 and 2011 the commonest anatomical site for gastric carcinoma was the pylorus / antrum accounting for 80% (59/75 cases) of all anatomic sites, followed by the body and the fundus / cardia which accounted for 5.3% (4/75 cases) each [table 2]. Anatomic site distribution of gastric carcinoma diagnosed at the uch, ibadan between 2000 and 2011 in 2.7% of cases (2/75) the tumour involved more than one site, the pylorus/ antrum and either the fundus / cardia or the body . In 6.7% of cases (5/75) the anatomical site was not known [table 2]. There was no association between the anatomical site of tumour and patients age (p = 0.923), nor between the anatomical site of tumour and the patients gender (p = 0.177). The commonest macroscopic morphology of gastric carcinoma was exophytic (62.3%, 33/53), followed by ulcero - infiltrative (22.6%, 12/53) and flat / diffusely infiltrating (15.1%, 8/53) [table 3]. No association was found between gross morphology of the tumour and patient's gender (p = 0.351), age (p = 0.805), or anatomic site of tumour (p = 0.262). Frequency of the gross morphological types of gastric carcinoma diagnosed at the uch, ibadan between 2000 and 2011 the histological type of gastric carcinoma according to the lauren classification was most commonly the intestinal type (47.0%, 55/117 cases). The diffuse type accounted for 35.1% (41/117) of cases, while the mixed / indeterminate type accounted for 17.9% (21/117) of cases [table 4 and figure 3]. The mean age for the intestinal histotype of gastric tumour was 51.36 15.24 years, 51.94 16.61 years for the diffuse and 53.67 16.17 years for the mixed histotype . Frequency of the histological types (lauren classification) of gastric carcinoma diagnosed at the uch, ibadan between 2000 and 2011 photomicrograph of intestinal (a and b), mixed (c and d) and diffuse (e and f) types of gastric carcinoma (haematoxylin and eosin). There was a significant association between the histological type of the tumour and the gross morphology of tumour (p = 0.002). Intestinal and mixed / indeterminate tumours were more likely to be exophytic or ulcero - infiltrative than tumours with diffuse histological morphology . Conversely, tumours with diffuse histological morphology had flat / diffusely infiltrating gross morphology than either exophytic or ulcero - infiltrative appearances . There was no significant difference in the mean ages of patients with the different histological types of gastric carcinoma [intestinal versus mixed (p = 0.748), diffuse versus intestinal (p = 0.91) and diffuse versus mixed (p = 0.828)]. However, the commonest histological type of gastric cancer in the female patients was diffuse (16/43) as against intestinal (15/43) and mixed / indeterminate (12/43) in the male patients . However, when the tumours were grouped into well - differentiated (mostly intestinal) and less differentiated grades (mostly diffuse and mixed), a significant association was found between grade and gender (p = 0.045) [table 5]. Tumours from the female cohorts were likely to be less differentiated than tumours from their male counterparts . Frequency of tumour grades of gastric carcinoma diagnosed at the uch, ibadan between 2000 and 2011 of the 117 cases only in 69 cases could the extent of the disease be assessed from clinical records, request cards and histology reports of tumour . The disease was classified as either advanced or localised on the bases of depth of tumour invasion on histology, presence of lymph node and/or other metastases . On the basis of this classification, about 92.8% (64/69) of patients presented with advance disease . The disease was only localised in 7.2% (5/69) of cases in which the tumour was histologically limited to the mucosa or the muscularis propria . There was no significant association between the depth of invasion of the tumour and the other clinicopathological features of the tumours . The relative ratio frequency of 1.38% of gastric carcinoma found in this study is significantly lower than the figures of 2.73% and 3.6% (for the periods 1980 - 1988 and 1989 - 1996, respectively) obtained previously from the uch by ogunbiyi in 2000.8 this reduction in frequency further supports the earlier assertion that there is a steady decline in the relative frequency of gastric carcinoma diagnosed in the department of pathology, uch, ibadan.38 this decline may be due to an increase in pathological diagnosis of tumours from other sites rather than an absolute decrease in the incidence of gastric carcinoma . This relative frequency also falls outside the national relative frequency range of 1.64% and 4.1% found in other studies across the country over three decades, although it is nearly comparable to the 1.64% reported by abdulkareem et al ., from lagos in 2010.2347 a rate of 18.4% for gastric carcinoma relative to other gastrointestinal tumours is somewhat higher than the 12% that was reported by abdulkareem et al ., in lagos in 2009.4 in keeping with the fact that gastric carcinoma is predominantly a disease of middle age and the elderly, 77.4% of our cohort were 45-years - old and above . The overall mean age of patients found in this study is similar to what had previously been reported in ibadan and elsewhere . Furthermore, when the patients were stratified into gender the modal age for male patients was 61 - 70 while that for the female was 51 - 60 . This shows a tendency for the female gastric cancer patients to be younger than their male counterparts, although the difference in the median ages between the genders was not statistically significant . The modal age - group found in this study is similar to what was reported in 2010 from three different centres (lagos, maiduguri and ile - ife).2710 it however, contrasts with the peak age incidences of the seventh to ninth decades obtained in studies from japan, china, korea, australia, the united states and the united kingdom.5111213141516 the male predominance of 1.72:1 found in this study concurs withthe results of several other studies nationwide and worldwide.23579101112131415171819 this preponderance of pyloric / antral tumours was similarly reported by oluwasola in ibadan in 1998 accounting for 83.2% of gastric carcinomas in that study.18 pyloric / antral predominance was also reported from ife, maiduguri, and in iran and the united states.6710172021 a significant association (p = 0.002) was found between the macroscopic growth pattern and the histological morphology of the tumour . This association has not been previously reported in local studies, probably because it had not been sought for in these other studies, and deserves to be considered in future studies of gastric cancer from this environment.237101822 the intestinal type of gastric carcinoma remains the commonest histological variant but in contrast to the rate of 56 - 88.7% found in previous studies, it occurs at a lower frequency of 47.0% in the present study . This difference represents a rise in the rate of the diffuse type of gastric carcinoma (35.1%) since the rate for the mixed / indeterminate histological type has remained generally constant (17 - 18.4%) over the past decade in ibadan . This rise in the rate of the diffuse type of gastric carcinoma has similarly been reported in the united states over the past two decades and has been attributed to the relative fall in the intestinal type of gastric carcinoma globally.2223 the fall in the intestinal type of gastric carcinoma may be due to the widespread use of anti - h pylori treatment regimen for dyspeptic patients with chronic gastritis and peptic ulcer as this regimen has been shown to reverse precancerous lesions like chronic atrophic gastritis and intestinal metaplasia, which are associated with h pylori infection.24 there was also a significant association between tumour grade and the gross morphology of tumour . The flat or diffusely infiltrating tumours tended to be less differentiated than exophytic / ulcero - infiltrative tumours, while well - differentiated tumours are more likely to be exophytic / ulcero - infiltrative than flat . The better - differentiated gland - forming tumours typically form exophytic or ulcerated tumours, whereas, the poorly differentiated tumours composed mainly of discohesive cells, are diffusely infiltrating.22232425 contrary to the report by abdulkareem et al ., from lagos in 2010, and zheng et al ., from japan in 2007 where the diffuse histologic type of gastric carcinoma was significantly associated with the female gender, no association was found in this study between patients gender and the histological type of gastric carcinoma (p = 0.056).226 however, when the different histological types were re - grouped into well - differentiated (mostly intestinal type) and less differentiated (mostly diffuse and mixed) grades, the female gender was found to associate significantly with the less differentiated tumours (p = 0.045). Perhaps, the use of a larger sample size may have been able to clarify true relationship between gender and the lauren's histological type of the tumour in our centre . Similar to reports from several centres in nigeria as well as from western countries and australia.182227 this study found that 92.8% of cases presented in advanced stage that confers poor prognosis to these tumours.23710 although in recent times the prognosis of gastric cancer in nigerian patients has improved significantly form the 5 year survival of 3% reported by arigbagbu et al ., in 1988 to about 14% (and up to 28.1% in those who had surgery with curative intent), this retrospective study does not report the follow - up characteristics of patients.282930 this is due to a high rate of loss of patients to follow - up as well as poor follow - up record keeping at the medical records department . Under these circumstances the appropriate approach to obtain survival data for any study would have to be a prospective approach . The clinicopathological characteristics of gastric carcinoma observed in this study are similar to what has been previously described except in the following regards: the lower relative ratio frequency of gastric carcinoma found in this study confirms the notion that there has been a steady, gradual decline in the relative rate of gastric carcinoma over the past three decades in ibadan although they still make up a substantial proportion of all gastrointestinal malignancies in our centre . A rise in the diffuse histological type of gastric carcinoma, which has been reported in the united states over the past two decades,
The study was performed on age - matched nondiabetic c57bl/6j (n = 22) and diabetic ins2 (akita) c57bl/6j (n = 22) mice from the jackson laboratory (bar harbor, me, usa) at 12 weeks (nondiabetic: n = 12, diabetic: n = 11) and 24 weeks (nondiabetic: n = 10, diabetic: n = 11) of age . The mice were treated in compliance with the arvo statement for the use of animals in ophthalmic and vision research . Prior to imaging, mice were anesthetized with intraperitoneal injections of ketamine (100 mg / kg) and xylazine (5 mg / kg) with additional injections given to maintain anesthesia as necessary . Nonfasting blood glucose levels in blood from a tail puncture were measured with a commercially available blood glucometer (freestyle lite; abbott, alameda, ca, usa). Mice were then placed in an animal holder and their pupils were dilated with 2.5% phenylephrine and 1% tropicamide . A glass cover slip with 1% hydroxypropyl methylcellulose was applied to the cornea to minimize its refractive power and prevent dehydration . For retinal vascular oxygen tension (po2) imaging, an oxygen - sensitive molecular probe, pd - porphine (frontier scientific, logan, ut, usa), was dissolved (12 mg / ml) in bovine serum albumin solution (60 mg / ml) and administered through the femoral arterial catheter (20 mg / kg). For retinal blood velocity imaging, 2-m polystyrene fluorescent microspheres (invitrogen, grand island, ny, usa) were injected through the catheter . Typically, two to three injections of the microspheres were given, and the volume of each injection was approximately 0.4 ml (10 microspheres / ml). For retinal vascular caliber measurement, fluorescein angiography (fa) was performed by the intravascular injection of 10% fluorescein sodium (5 mg / kg, ak - fluor; akorn, decatur, il, usa). Briefly, a laser line was projected onto the retina after intravenous injection of the pd - porphine probe . Due to the angle between the excitation laser beam and imaging path, optical section phosphorescence images were acquired in which the retinal vessels were depth - resolved from the underlying choroid . Po2 was measured in individual major retinal arteries (po2aind) and retinal veins (po2vind) at locations within 3 optic disc diameters from than edge of the optic nerve head, as shown in figure 1a . An average arterial (po2a) and venous (po2v) po2 value was calculated from the individual artery and vein measurements in each animal, respectively . Examples of images used to evaluate oxygen delivery, metabolism, and oxygen extraction fraction in a 12-week - old diabetic mouse . (a) cross - sectional retinal vascular po2 maps (rectangles) overlaid on a fluorescein angiogram . (b) fluorescein angiogram on which the boundaries of retinal vessels (red lines) have been detected and outlined within a circumpapillary region (green circles). (c) four successive image frames acquired at 105 hz superimposed on a red free image that display locations of a single fluorescent microsphere over time, which were used to determine blood velocity . Our previously described prototype blood flow imaging system was used for fluorescent microsphere imaging to assess retinal venous blood velocity and for performing fa to measure retinal arterial and venous vessel diameters . A slit - lamp biomicroscope with the standard light illumination (carl zeiss, oberkochen, germany) was equipped with a 488-nm diode laser (melles griot, carlsbad, ca, usa) and an emission filter (560 60 nm; spectrotech, inc ., saugus, ma, usa) for fluorescent microsphere imaging . Image sequences of the intravascular motion of the microspheres were captured at 105 hz using an electron multiplier charge coupled device camera (quantem; photometrics, tucson, az, usa). The camera sensor was binned to maximize the frame rate, allowing the motion of the microspheres to be resolved in time . Multiple image sequences, each 5 seconds in duration, were recorded over several minutes immediately following the injection of the microspheres . After microsphere imaging, fa retinal images were captured using the slit - lamp white light illumination with a narrow band optical filter (480 5 nm; edmund optics, barrington, nj, usa) and the above emission filter . Fluorescein angiography retinal images were obtained using the full resolution of the camera (512 512 pixels) to maximize the spatial resolution for vessel diameter measurements . Diameters of all individual major retinal arteries (daind) and veins (dvind) were measured from the fa images over a fixed vessel length (100 m), spanning approximately 150 to 250 m from the center of the optic disk, as shown in figure 1b . Daind and dvind were determined by the average full width at half maximum of seven intensity profiles perpendicular to the blood vessel axis . A mean arterial (da) and venous diameter (dv) was calculated from all daind and dvind values in each mouse, respectively . As shown in figure 1c, blood velocity in all individual veins was measured by manually tracking displacements of the microspheres over time, following our previously reported method . Typically, 20 to 30 microsphere velocity measurements were obtained in each individual vein and averaged to derive a velocity measurement per vessel (vind). A mean velocity (v) in each mouse v was measured in veins because they are less affected by pulsation and have larger diameters as compared with arteries . Blood flow in each major vein was calculated based on dvind and vind measurements: (vind /4). These blood flow measurements were summed over all veins to determine the total venous blood flow in the retinal circulation (f) for that animal . Because the retinal circulation is an end - artery system, f was equivalent to the total retinal blood flow . Measurements of f were obtained within 15 minutes after po2 imaging . The oxygen content of blood in each retinal artery (o2aind) and vein (o2vind) was calculated as the sum of oxygen bound to hemoglobin and dissolved in blood: o2ind = so2 c hgb + po2ind k, where so2 is the oxygen saturation (%), c is the oxygen - carrying capacity of hemoglobin (1.39 ml o2/g), hgb is the hemoglobin concentration, and k is the oxygen solubility in blood (0.003 ml o2/dlmm hg). So2 was calculated from the hemoglobin oxygen dissociation curve in mice by using the measured po2ind and an assumed ph value of 7.4 . A constant hgb value of 13.8 mg / dl, derived by averaging the hgb concentration of blood samples from three separate nondiabetic mice, was used for o2ind calculations in all mice . In each animal, a mean arterial (o2a) and venous (o2v) oxygen content were determined from o2ind measurements and the arteriovenous oxygen content difference was calculated as: o2a - v = o2a o2v . Do2, defined as the rate that oxygen becomes available to the inner retinal tissue supplied by the retinal circulation, was calculated as the product of f and o2a . Mo2, defined as the rate that oxygen is extracted from the retinal circulation and metabolized by the inner retinal tissue, was calculated as the product of f and o2a - v . It varies between 0 and 1, and it can be calculated as o2a - v / o2a . Twelve continuous outcome variables (po2a, po2v, o2a, o2v, o2a - v, da, dv, v, f, do2, mo2, and oef) were evaluated to assess the relationship of each with age and the presence of diabetes . Regression diagnostics including cook's distance were performed on do2, mo2, and oef to identify data points that were outliers, had leverage, or were influential . Two outliers were identified, which were removed from further analyses, both from the 24-week diabetic group . One mouse was an outlier due to abnormally high do2 and mo2 values, and another mouse was identified as an outlier due to an abnormally high mo2 value . The effects of diabetes (absence or presence) and age (12 or 24 weeks) on body weight, blood glucose, and the above outcome variables were determined using 2-way anova . In 1 of the 12-week and 4 of the 24-week diabetic mice, blood glucose exceeded the maximum level (600 mg / dl) that could be measured by the glucometer . To compare values in nondiabetic with diabetic mice we assigned the value of 600 mg / dl to any measurement that exceeded the maximal level . When a significant interaction was found, simple main effects were determined by the independent samples t - test . Because the weights of the diabetic mice were lower than those of the nondiabetic mice, we also performed two - way analysis of covariance with weight as the covariate on all outcome variables . However, the statistical results did not change, except a marginally significant reduction in o2a - v present in the diabetic mice . The study was performed on age - matched nondiabetic c57bl/6j (n = 22) and diabetic ins2 (akita) c57bl/6j (n = 22) mice from the jackson laboratory (bar harbor, me, usa) at 12 weeks (nondiabetic: n = 12, diabetic: n = 11) and 24 weeks (nondiabetic: n = 10, diabetic: n = 11) of age . The mice were treated in compliance with the arvo statement for the use of animals in ophthalmic and vision research . Prior to imaging, mice were anesthetized with intraperitoneal injections of ketamine (100 mg / kg) and xylazine (5 mg / kg) with additional injections given to maintain anesthesia as necessary . Nonfasting blood glucose levels in blood from a tail puncture were measured with a commercially available blood glucometer (freestyle lite; abbott, alameda, ca, usa). Mice were then placed in an animal holder and their pupils were dilated with 2.5% phenylephrine and 1% tropicamide . A glass cover slip with 1% hydroxypropyl methylcellulose was applied to the cornea to minimize its refractive power and prevent dehydration . For retinal vascular oxygen tension (po2) imaging, an oxygen - sensitive molecular probe, pd - porphine (frontier scientific, logan, ut, usa), was dissolved (12 mg / ml) in bovine serum albumin solution (60 mg / ml) and administered through the femoral arterial catheter (20 mg / kg). For retinal blood velocity imaging, 2-m polystyrene fluorescent microspheres (invitrogen, grand island, ny, usa) were injected through the catheter . Typically, two to three injections of the microspheres were given, and the volume of each injection was approximately 0.4 ml (10 microspheres / ml). For retinal vascular caliber measurement, fluorescein angiography (fa) was performed by the intravascular injection of 10% fluorescein sodium (5 mg / kg, ak - fluor; akorn, decatur, il, usa). Briefly, a laser line was projected onto the retina after intravenous injection of the pd - porphine probe . Due to the angle between the excitation laser beam and imaging path, optical section phosphorescence images were acquired in which the retinal vessels were depth - resolved from the underlying choroid . Po2 was measured in individual major retinal arteries (po2aind) and retinal veins (po2vind) at locations within 3 optic disc diameters from than edge of the optic nerve head, as shown in figure 1a . An average arterial (po2a) and venous (po2v) po2 value was calculated from the individual artery and vein measurements in each animal, respectively . Examples of images used to evaluate oxygen delivery, metabolism, and oxygen extraction fraction in a 12-week - old diabetic mouse . (a) cross - sectional retinal vascular po2 maps (rectangles) overlaid on a fluorescein angiogram . (b) fluorescein angiogram on which the boundaries of retinal vessels (red lines) have been detected and outlined within a circumpapillary region (green circles). (c) four successive image frames acquired at 105 hz superimposed on a red free image that display locations of a single fluorescent microsphere over time, which were used to determine blood velocity . Our previously described prototype blood flow imaging system was used for fluorescent microsphere imaging to assess retinal venous blood velocity and for performing fa to measure retinal arterial and venous vessel diameters . A slit - lamp biomicroscope with the standard light illumination (carl zeiss, oberkochen, germany) was equipped with a 488-nm diode laser (melles griot, carlsbad, ca, usa) and an emission filter (560 60 nm; spectrotech, inc ., image sequences of the intravascular motion of the microspheres were captured at 105 hz using an electron multiplier charge coupled device camera (quantem; photometrics, tucson, az, usa). The camera sensor was binned to maximize the frame rate, allowing the motion of the microspheres to be resolved in time . Multiple image sequences, each 5 seconds in duration, were recorded over several minutes immediately following the injection of the microspheres . After microsphere imaging, fa retinal images were captured using the slit - lamp white light illumination with a narrow band optical filter (480 5 nm; edmund optics, barrington, nj, usa) and the above emission filter . Fluorescein angiography retinal images were obtained using the full resolution of the camera (512 512 pixels) to maximize the spatial resolution for vessel diameter measurements . Diameters of all individual major retinal arteries (daind) and veins (dvind) were measured from the fa images over a fixed vessel length (100 m), spanning approximately 150 to 250 m from the center of the optic disk, as shown in figure 1b . Daind and dvind were determined by the average full width at half maximum of seven intensity profiles perpendicular to the blood vessel axis . A mean arterial (da) and venous diameter (dv) was calculated from all daind and dvind values in each mouse, respectively . As shown in figure 1c, blood velocity in all individual veins was measured by manually tracking displacements of the microspheres over time, following our previously reported method . Typically, 20 to 30 microsphere velocity measurements were obtained in each individual vein and averaged to derive a velocity measurement per vessel (vind). A mean velocity (v) in each mouse v was measured in veins because they are less affected by pulsation and have larger diameters as compared with arteries . Blood flow in each major vein was calculated based on dvind and vind measurements: (vind /4). These blood flow measurements were summed over all veins to determine the total venous blood flow in the retinal circulation (f) for that animal . Because the retinal circulation is an end - artery system, f was equivalent to the total retinal blood flow . The oxygen content of blood in each retinal artery (o2aind) and vein (o2vind) was calculated as the sum of oxygen bound to hemoglobin and dissolved in blood: o2ind = so2 c hgb + po2ind k, where so2 is the oxygen saturation (%), c is the oxygen - carrying capacity of hemoglobin (1.39 ml o2/g), hgb is the hemoglobin concentration, and k is the oxygen solubility in blood (0.003 ml o2/dlmm hg). So2 was calculated from the hemoglobin oxygen dissociation curve in mice by using the measured po2ind and an assumed ph value of 7.4 . A constant hgb value of 13.8 mg / dl, derived by averaging the hgb concentration of blood samples from three separate nondiabetic mice, was used for o2ind calculations in all mice . In each animal, a mean arterial (o2a) and venous (o2v) oxygen content were determined from o2ind measurements and the arteriovenous oxygen content difference was calculated as: o2a - v = o2a o2v . Do2, defined as the rate that oxygen becomes available to the inner retinal tissue supplied by the retinal circulation, was calculated as the product of f and o2a . Mo2, defined as the rate that oxygen is extracted from the retinal circulation and metabolized by the inner retinal tissue, was calculated as the product of f and o2a - v . It varies between 0 and 1, and it can be calculated as o2a - v / o2a . Twelve continuous outcome variables (po2a, po2v, o2a, o2v, o2a - v, da, dv, v, f, do2, mo2, and oef) were evaluated to assess the relationship of each with age and the presence of diabetes . Regression diagnostics including cook's distance were performed on do2, mo2, and oef to identify data points that were outliers, had leverage, or were influential . Two outliers were identified, which were removed from further analyses, both from the 24-week diabetic group . One mouse was an outlier due to abnormally high do2 and mo2 values, and another mouse was identified as an outlier due to an abnormally high mo2 value . The effects of diabetes (absence or presence) and age (12 or 24 weeks) on body weight, blood glucose, and the above outcome variables were determined using 2-way anova . In 1 of the 12-week and 4 of the 24-week diabetic mice, blood glucose exceeded the maximum level (600 mg / dl) that could be measured by the glucometer . To compare values in nondiabetic with diabetic mice we assigned the value of 600 mg / dl to any measurement that exceeded the maximal level . When a significant interaction was found, simple main effects were determined by the independent samples t - test . Because the weights of the diabetic mice were lower than those of the nondiabetic mice, we also performed two - way analysis of covariance with weight as the covariate on all outcome variables . However, the statistical results did not change, except a marginally significant reduction in o2a - v present in the diabetic mice . The body weights of the nondiabetic mice were 28 3 and 32 3 g (mean sd) at 12 and 24 weeks of age, respectively . The body weights of the diabetic mice were 24 2 g at both 12 and 24 weeks of age . There was a significant interaction effect between the presence of diabetes and age (p = 0.029). The simple main effect of diabetes (presence or absence) on weight was significant at both 12 and 24 weeks of age (p 0.001). The simple main effect of age (12 or 24 weeks) on weight was significant in nondiabetic mice (p = 0.008), but it was not significant in diabetic mice (p = 1). In nondiabetic mice, blood glucose measurements obtained on the day of imaging were 151 19 and 139 19 mg / dl at 12 and 24 weeks of age, respectively . Mean blood glucose measurements (assigning the value of 600 mg / dl to measurements above the glucometer maximum) in diabetic mice on the day of imaging were 444 and 505 mg / dl at 12 and 24 weeks of age, respectively . There was a significant difference in blood glucose levels between diabetic and nondiabetic mice in both age groups (p <0.001). Mean values of retinal vascular po2 and o2 content in nondiabetic and diabetic mice at 12 and 24 weeks of age are summarized in table 1 . There was a significant main effect of diabetes on po2v and o2v, such that diabetic mice had a 15% reduction in po2v and a 30% reduction in o2v as compared with nondiabetic mice (p 0.01). There were no significant interactions between diabetes and age or significant main effects of age on po2v or o2v . There were no significant interactions between diabetes and age or main effects of diabetes or age on po2a, o2a, or o2a - v . Retinal arterial (po2a) and venous (po2v) oxygen tension, and arterial (o2a) and venous (o2v) oxygen content, and arteriovenous oxygen content difference (o2a - v) of nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) as expected, dv was larger than da in both nondiabetic and diabetic mice (p <0.001). Mean values of da, dv, v, and f in nondiabetic and diabetic mice in both age groups are summarized in table 2 . There was a significant interaction effect between diabetes and age on f (p = 0.01), and their simple main effects are presented in table 3 . There was no significant simple main effect of diabetes on f at 12 weeks, but at 24 weeks, f was 36% higher in the nondiabetic than in the diabetic mice (p = 0.003). F increased by 37% between 12 and 24 weeks in the nondiabetic mice (p = 0.01), but did not change significantly over that time interval in the diabetic mice . No significant effect of age, diabetes, or their interaction was found on da, dv, or v. retinal arterial diameter (da), venous diameter (dv), venous velocity (v), and total retinal blood flow (f) of nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) simple main effects of total retinal blood flow (f) and inner retinal oxygen delivery (do2) in nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) mean values of do2, mo2, and oef in nondiabetic and diabetic mice at 12 and 24 weeks of age are summarized in table 4 . There was a significant interaction effect between diabetes and age on do2, and their simple main effects are presented in table 3 . No effect of diabetes on do2 was found at 12 weeks, but at 24 weeks do2 was 43% higher in the nondiabetic mice (p <0.001). Also, although do2 did not change significantly between 12 and 24 weeks within the diabetic mice, it increased by 32% in the nondiabetic mice (p = 0.03). There was no significant interaction between diabetes and age or main effect of diabetes or age on mo2 . Inner retinal oxygen delivery (do2), oxygen metabolism (mo2), and oxygen extraction fraction (oef) in nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) values of oef in individual mice are displayed in figure 2, illustrating that the mean oef was higher in the diabetic mice than in the nondiabetic mice at both ages . There was a significant main effect of diabetes on oef such that oef in diabetic mice exceeded that in nondiabetic mice by 17% (p = 0.01). There was no significant interaction between diabetes and age or a main effect of age on oef (p 0.28). Oxygen extraction fraction (oef) in nondiabetic (gray circles) and diabetic (white circles) mice at 12 and 24 weeks of age . The body weights of the nondiabetic mice were 28 3 and 32 3 g (mean sd) at 12 and 24 weeks of age, respectively . The body weights of the diabetic mice were 24 2 g at both 12 and 24 weeks of age . There was a significant interaction effect between the presence of diabetes and age (p = 0.029). The simple main effect of diabetes (presence or absence) on weight was significant at both 12 and 24 weeks of age (p 0.001). The simple main effect of age (12 or 24 weeks) on weight was significant in nondiabetic mice (p = 0.008), but it was not significant in diabetic mice (p = 1). In nondiabetic mice, blood glucose measurements obtained on the day of imaging were 151 19 and 139 19 mg / dl at 12 and 24 weeks of age, respectively . Mean blood glucose measurements (assigning the value of 600 mg / dl to measurements above the glucometer maximum) in diabetic mice on the day of imaging were 444 and 505 mg / dl at 12 and 24 weeks of age, respectively . There was a significant difference in blood glucose levels between diabetic and nondiabetic mice in both age groups (p <0.001). Mean values of retinal vascular po2 and o2 content in nondiabetic and diabetic mice at 12 and 24 weeks of age are summarized in table 1 . There was a significant main effect of diabetes on po2v and o2v, such that diabetic mice had a 15% reduction in po2v and a 30% reduction in o2v as compared with nondiabetic mice (p 0.01). There were no significant interactions between diabetes and age or significant main effects of age on po2v or o2v . There were no significant interactions between diabetes and age or main effects of diabetes or age on po2a, o2a, or o2a - v . Retinal arterial (po2a) and venous (po2v) oxygen tension, and arterial (o2a) and venous (o2v) oxygen content, and arteriovenous oxygen content difference (o2a - v) of nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) as expected, dv was larger than da in both nondiabetic and diabetic mice (p <0.001). Mean values of da, dv, v, and f in nondiabetic and diabetic mice in both age groups are summarized in table 2 . There was a significant interaction effect between diabetes and age on f (p = 0.01), and their simple main effects are presented in table 3 . There was no significant simple main effect of diabetes on f at 12 weeks, but at 24 weeks, f was 36% higher in the nondiabetic than in the diabetic mice (p = 0.003). F increased by 37% between 12 and 24 weeks in the nondiabetic mice (p = 0.01), but did not change significantly over that time interval in the diabetic mice . No significant effect of age, diabetes, or their interaction was found on da, dv, or v. retinal arterial diameter (da), venous diameter (dv), venous velocity (v), and total retinal blood flow (f) of nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) simple main effects of total retinal blood flow (f) and inner retinal oxygen delivery (do2) in nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) mean values of do2, mo2, and oef in nondiabetic and diabetic mice at 12 and 24 weeks of age are summarized in table 4 . There was a significant interaction effect between diabetes and age on do2, and their simple main effects are presented in table 3 . No effect of diabetes on do2 was found at 12 weeks, but at 24 weeks do2 was 43% higher in the nondiabetic mice (p <0.001). Also, although do2 did not change significantly between 12 and 24 weeks within the diabetic mice, it increased by 32% in the nondiabetic mice (p = 0.03). There was no significant interaction between diabetes and age or main effect of diabetes or age on mo2 . Inner retinal oxygen delivery (do2), oxygen metabolism (mo2), and oxygen extraction fraction (oef) in nondiabetic and diabetic (akita) mice at 12 and 24 weeks of age (mean sd) values of oef in individual mice are displayed in figure 2, illustrating that the mean oef was higher in the diabetic mice than in the nondiabetic mice at both ages . There was a significant main effect of diabetes on oef such that oef in diabetic mice exceeded that in nondiabetic mice by 17% (p = 0.01). There was no significant interaction between diabetes and age or a main effect of age on oef (p 0.28). Oxygen extraction fraction (oef) in nondiabetic (gray circles) and diabetic (white circles) mice at 12 and 24 weeks of age . The present study revealed an increase in oef in diabetic akita mice at both 12 and 24 weeks of age, due to reductions in po2v and o2v . Furthermore, there was a lack of normal increase in do2 with age in diabetic mice, driven by failure of f to increase with age in the diabetic group . These changes in oef and do2 were in the context of no discernible abnormalities in mo2 in the diabetic mice . There are two major ways for the retina to maintain adequate tissue oxygenation and mo2 . First, do2 can be increased by adjusting f. at 24 weeks of age, do2 (and f) was significantly lower in the diabetic group than in the nondiabetic group because the normal increase in do2 (and f) from 12 to 24 weeks did not occur . Do2, which is based on both f and o2a, has not been measured previously in diabetic mice, but there are several reports of f. studies by both wright et al . And muir and colleagues found decreases in f of 40% and 28%, respectively, in akita mice after approximately 6 months of diabetes . These decreases are very similar to the 37% decrease in f at 24 weeks in the current study . They are also similar to other reported reductions in f values in stz - diabetic mice . Had these investigators measured o2a, it is likely that they would have found decreases in do2 similar to the decrease of 43% in the current study . These defective do2 and f responses in diabetes likely correspond to the well - known impaired autoregulation seen in humans with diabetes . The second way for the retina to maintain adequate tissue oxygenation and mo2 is by extracting a greater fraction of the oxygen supplied by the blood, that is, by increasing oef . Because mo2 did not differ between nondiabetic and diabetic mice, it was not limited by the availability of oxygen . However, in the diabetic mice, the retina had to increase oef to 0.63 at 24 weeks as opposed to 0.54 in the nondiabetic mice in order to maintain mo2 . This means that much of the oxygen supply reserve was already spent solely by being diabetic . We note that oef increased similarly from 0.55 to 0.63 in a previous study in rats in which the inspired gas was reduced from 21% to 10% oxygen, and mo2 could not be maintained . It appears that the retina of the akita diabetic mouse exists in a state of vulnerability to superimposed metabolic stress due to its limited reserve of oxygen supply . However, it is now possible to calculate oef from data acquired in two previous studies on stz - diabetic rats from our laboratory . In one study, oef at 4 weeks of diabetes was 0.47, whereas it was 0.53 in the nondiabetic rats . In another study, the oef values were 0.46 and 0.51 in the diabetic rats at 4 and 6 weeks of diabetes, respectively, and 0.55 in the nondiabetic rats . Showed a number of biochemical abnormalities were more prominent in stz - diabetic rats than in stz - diabetic mice, but they did not measure po2 . Another possible explanation for the difference may be the longer duration of diabetes in the mice, because cataract prevented us from making measurements in diabetic rats with more than 6 weeks of diabetes . In humans oef tends not to increase in diabetes (blair np, unpublished data, 2016), so there may be differences in the pathogenesis of dr between humans, rats, and mice . We reported mo2 in diabetic rodents for the first time in rats with stz diabetes, and no abnormality was found, as in the current study . And sutherland and collegues found total retinal oxygen consumption to be decreased in diabetic rabbits in vitro, but most of the inner retina is avascular in this species . Increased total retinal oxygen consumption in excised alloxan - diabetic rat retinas was reported by de roetth . It is expected that mo2 will be maintained unless the inner retinal tissue po2 is reduced enough for hypoxic energy failure to supervene and threaten cell survival . Using oxygen microelectrodes, lau and linsenmeier found no decrease in inner retinal po2 in rats with stz - induced diabetes for 4 to 12 weeks . Furthermore, the inner retinal po2 actually was elevated relative to the choroidal po2 at 12 weeks of diabetes . These findings are consistent with the finding of no difference between mo2 in mice with and without diabetes in the current study . On the other hand, linsenmeier et al . Found foci of hypoxia with oxygen microelectrodes in cats with diabetes of 6 years duration . Evaluation of retinal tissue hypoxia using pimonidazole has yielded conflicting results . Reduced retinal po2 stimulates hypoxia - inducible factors, but assessments of these factors in diabetic rodents have not yielded consistent abnormalities . Unfortunately, these methods do not permit rigorous estimation of mo2 in the inner retina . Taken together, these results suggest that inner retinal hypoxia is not a major abnormality in these rodent models of diabetes . Hypoxia could be a factor at longer durations of diabetes or if there are localized areas of hypoxia . The extent to which the findings of the current study resemble the conditions in human dr remains to be elucidated . The safest extrapolation of our results to the clinic would be that mo2 may not be limited by hypoxia in the absence of significant nonproliferative or proliferative dr . Akita diabetic mice are known to have reduced body weight and this appears, at least in part, to be related to leptin . Similarly, stz - diabetic rats have reduced body weight and humans who become diabetic prior to puberty may have retarded growth . We do not know if differences in body weight alone are a cause for higher sensitivity of akita mice toward metabolic stress and subsequent longitudinal alterations in retinal oxygen delivery . However, including body weight as a covariate did not change the results of the statistical analysis, thus differences in body weight did not substantially influence the conclusions . The limitations of this work include image clarity, but we excluded eyes in which the images could not be evaluated with confidence . The number of mice in the four groups was relatively small so that we may not have been able to identify certain differences as statistically significant . Our measurement of mo2 assumes that the volumes of tissue supplied by the retinal vessels and choroid are the same in the nondiabetic and diabetic mice . The retinal po2 profiles obtained from microelectrodes in normal and stz - diabetic rats suggest that this assumption is reasonable . In conclusion, our finding of normal inner retinal oxygen metabolism in diabetic akita mice indicates that elevation of the oxygen extraction fraction adequately compensates for reduced oxygen delivery and prevents oxidative metabolism from being limited by hypoxia . However, these retinas may have inadequate capacity to compensate for common superimposed stresses so that pathologic changes may develop.
We present a case of a cardiac transplant candidate with an ejection fraction of less than 15% who underwent a laparoscopic cholecystectomy . This is the first case in which intraoperative hemodynamic measurements were recorded in a patient with cardiopulmonary disease this severe . The patient underwent the procedure while measurements were made at crucial intervals (baseline, with incremental insufflation, reverse trendelenberg, at desufflation) using a pulmonary artery catheter, transesophageal echocardiography with fractional area measurements, radial arterial line, as well as standard monitoring . There was a rise in mean arterial and systemic vascular resistance with insufflation to 10 mm hg, which was further exaggerated by reverse trendelenberg . We conclude that if a patient with congestive heart failure is medically optimized, and intra - abdominal pressures and surgical times are minimized, laparoscopic cholecystectomy may be performed with minimal risk to the patient . Increasingly, laparoscopic procedures are being utilized to decrease postoperative pain and length of hospital stay . The one group in which laparoscopy may have adverse effects, that outweigh the potential benefits, is the patient with severe cardiopulmonary disease . There has not been a case reported of a laparoscopic procedure with hemodynamic measurements on a patient with an ejection fraction of less than 20% . We present a case of a patient with end - stage cardiac disease with an ejection fraction estimated at less than 15% who underwent laparoscopic cholecystectomy prior to becoming eligible for cardiac transplant . The patient had asymptomatic cholelithiasis and was to undergo a laparoscopic vs. open cholecystectomy prior to becoming eligible for cardiac transplant . An echocardiogram two weeks prior to admission revealed a normal sinus rhythm, global hypokinesis with an ejection fraction of less than 15%; valvular abnormalities included mild mitral and tricuspid regurgitation . A right and left cardiac catheterization one week prior to this admission confirmed the above, and revealed pulmonary hypertension with pulmonary artery pressures of 72/35 . Pertinent medications included metropolol 6.25 mg po bid, digoxin 0.125 mg po q d, enalapril 20 mg po bid, furosemide 40 mg po q d, and coumadin 4 mg po q d. physical exam was without evidence of congestive heart failure . Upon arrival in the operating room, the patient was connected to standard monitoring (i.e., 5-lead ekg, pulse oximetry, arterial blood pressure, capnography, and body temperature). Prior to induction, a radial arterial line and oximetric pulmonary artery catheter were placed through the right internal jugular vein . Induction was accomplished with etomidate 0.3 mg / kg, fentanyl 10 meg / kg, and succhinylcholine 1.0 mg / kg . After entubation, a hewlett - packard biplanar transesophageal echocardiography probe (model #21362a) at 5.0 mhz frequency was positioned to obtain a transgastric short axis view of the midpapillary level of the left ventricle . Anesthesia was maintained with isoflurane at 0.35 - 0.45%, nitrous oxide 50% in oxygen 50% . Muscle relaxation was accomplished with vecuronium maintaining 1 of 4 twitches on a train - of - four . Ventilation was controlled with the ph ranging between 7.35 - 7.40 and pco2 40 - 48 mm hg . Blood loss was minimal, and the patient received a total of 10ml / kg of lactated ringer's intravenously . Measurements were taken pre - induction, immediately post - induction, five minutes after pneumoperitoneal insufflation to 5 mm hg, five minutes after insufflation to 10 mm hg, five minutes after 10 degrees of reverse trendelenburg position, and five minutes after return to the supine position and desufflation . Parameters measured included heart rate, blood pressure, pulmonary artery pressures, cardiac output, pulmonary artery occlusion pressure, and central venous pressure . With each change in table position, pressure transducer height was adjusted to maintain a constant phlebostatic axis with respect to the right atrium . Videotaped recordings of the intraoperative echocardiogram were used to evaluate for wall motion abnormalities and to calculate left ventricular fractional area change postoperatively . Analysis was performed via manual planimetry by a skilled observer (dck) blinded to intraoperative events . End - diastolic (eda) and end - systolic areas (esa) were planimetered on two consecutive cardiac cycles and averaged using the leading edge of the left ventricular endocardial border . Eda was determined by the greatest cross - sectional area obtained at the peak of the electrocardiogram r wave . Percent ejection fraction area (% efa) was determined from the formula% efa = (eda - esa / eda) 100 . Laparoscopic cholecystectomy is becoming an increasingly popular alternative to open cholecystectomy . The laparoscopic procedure combines the benefit of completely removing the gallbladder with the advantages of a shorter hospital stay, less pain, and less chance of postoperative ileus compared to the open cholecystectomy . These advantages should provide theoretical benefits for patients with heart disease requiring cholecystectomy; however, the use of peritoneal insufflation, co2 as the insufflating gas, and reverse trendelenburg position necessary for a laparoscopic cholecystectomy have been shown to cause significant alterations in hemodynamic parameters . Some of these changes might discourage the use of laparoscopic cholecystectomy in high - risk cardiac patients, especially those with very low ejection fractions . The three variables that effect the patient's hemodynamic parameters are insufflation, the use of co2 as the insufflant, and patient position . The extent of the cardiovascular changes associated with creation of pneumoperitoneum depends on the intra - abdominal pressure (iap) attained . With our patient, the iap was limited to 10 mm hg . Because of her thin body habitus, this limitation was not difficult to accomplish, yet still provided adequate visualization for surgery . In healthy patients undergoing laparoscopic cholecystectomy, the filling pressures and mean arterial pressure (map) may increase, but cardiac output (co) typically remains unchanged . In patients with severe heart disease, however, changes in hemodynamic parameters have varied between investigators . Utilizing iaps of 12 mm hg, have described hemodynamic changes similar to our patient . However, safran et al ., using 15 mm hg iaps, noted that several high - risk cardiac patients experienced decreases in co and svo2 . They felt that these patients were relatively volume depleted preinsufflation, then worsened with a further decrease in preload when insufflation was applied . Similarly describe a significant fall in cardiac index (ci) and stroke volume (sv) despite preoperative optimization of preload and cardiac output using fluid boluses . Neither safran nor iwase reported any intraoperative cardiac complications or any increase in postoperative morbidity or mortality . Portera, on the other hand, reported two cases of postoperative congestive heart failure . He observed a progressive decrease in ci, sv, and left ventricular stroke work following desufflation in these patients; but, he could not identify any obvious feature preoperatively, or at the time of insufflation, that might predict which patients might be at risk . The hemodynamic course of our patient was uneventful (figure 1). At 10 mm hg, our patient had changes similar to a healthy patient, documented by an increase in map and pulmonary artery wedge pressure (pawp); end diastolic volume (estimated as end - diastolic area - eda) and co remained unchanged . Systemic vascular resistance (svr) also increased with insufflation . With our patient's complicated preoperative course, consisting of two admissions within four weeks for chf, one would have expected her to be relatively volume depleted and as such respond with an increase in bp and hr . Prior to desufflation, the most unusual finding was the decrease in echocardiographic fractional area change (fac) to 12%; this finding mirrors (and is most probably explained by) a peak increase in svr . Following desufflation, all hemodynamic parameters corrected in an appropriate fashion: increased stroke volume secondary to improved preload, decreased svr (reduced afterload) with subsequent increase in cardiac output, and a decrease in heart rate . Pulmonary artery wedge pressure (pawp), fractional area change (fac) by transesophageal echo ., systemic vascular resistance (svr), mean arterial pressure (map), cardiac output (co), heart rate (hr) recorded at 5 minutes after an intra - abdominal pressure of 5 mm hg, 10 mm hg, assuming 10 degrees reverse trendelenberg (rt), and on return to supine and desufflation of the abdomen . Hypercarbia over 50 mm hg in healthy patients causes myocardial depression and is a direct vasodilator; as a result, there is a decrease in co, svr, and ph . Hypercarbia at levels less than 50 mm hg has been shown to produce minimal hemodynamic effects . In patients with ischemic heart disease, paco2 levels of 55 - 65 mm hg resulted in an increase in systolic bp, hr, and co. there was also a two to threefold increase in plasma catecholamine concentrations which suggests hypercarbia stimulates the sympathetic nervous system in this group of patients . We had no difficulty maintaining a paco2 below 50 mm hg in our patient; her lean body habitus was a contributing factor in our ability to maintain adequate ventilation . It is unclear whether her upward trend in hr, map, and svr (suggestive of an increase in sympathetic stimulation) was related to the mild hypercarbia present, or to a drop in preload (see edv, figure 1). Theoretically, the change to reverse trendelenburg should cause cardiovascular compromise in the form of decreased venous return (vr) and co. this decline may be offset by a decrease in afterload leading to an insignificant change in co in otherwise healthy patients . The study done by safran looking at laparoscopic cholecystectomy in patients with severe cardiac disease utilized an iap of 15 mm hg and maintained normal end - tidal co2 (etco2). The reverse trendelenburg position was used in most of the 15 asa physical class iii and iv patients evaluated . Unfortunately, hemodynamic parameters were not measured after the establishment of pneumoperitoneum independently of assuming reverse trendelenburg . A significant increase in map and svr accompanied by a decrease in co was found . These changes were thought to be due to aggressive treatment of congestive heart failure preoperatively, thus leading to a volume depleted patient with little intravascular volume reserve . In our patient, the only significant finding on assuming 10 degrees of reverse trendelenburg position was an increase in the svo2 from 83% to 90% . This elevation in svo2 came in conjunction with a decline in etco2 from 38 mm hg to 30 mm hg, adding further credence to the improvement in pulmonary function as the probable mechanism . We evaluated transesophageal echocardiography (tee) as an aid to monitoring this patient during laparoscopic cholecystectomy . Following the establishment of pneumoperitoneum and assumption of the reverse trendelenburg position, the fac via tee was assessed as an indirect measure of ejection fraction (ef). No significant changes or trends could be identified utilizing this technique, perhaps due to subtle changes in wall thickening and short axis diameters in a patient with a low ef . Were also unable to find benefit of tee use as an intraoperative monitor in laparoscopic patients with cardiac disease . We conclude from this case that laparoscopic cholecystectomy can be performed safely in patients with severe cardiac disease and ejection fractions less than 20% . Under these circumstances it is imperative that appropriate monitoring be utilized, and iap, reverse trendelenburg and surgical time minimized.
In japan, people aged 65 years and older comprise> 21% of the population, and the nation is becoming a super - aged society more rapidly than any other country . In 2007, the japanese orthopaedic association (joa) proposed the concept of locomotive syndrome, which refers to those elderly individuals who require nursing - care services because of problems with the locomotor organs or because they are at risk for conditions for which they may need such services in the future.1 locomotive syndrome is caused mainly by age - related locomotor organ diseases such as osteoporosis and osteoarthritis (oa) or age - related locomotor dysfunctions such as decline in walking speed and loss of muscle strength.2,3 oa is the most common joint disease, which causes pain in elderly patients . The knee is most frequently affected by oa because it is a weight - bearing joint, and knee oa impairs locomotor functions such as walking speed.4 according to the research on osteoarthritis against disability (road) study,> 25 million japanese people aged 40 years and older have been estimated to have radiographically determined kellgren lawrence (k l) grade ii or higher joint - space narrowing.5,6 previous studies reported that walking speed was associated with survival in older adults and that muscle weakness is the highest risk factor for falling, which can result in being bedridden.7,8 therefore, preventing these locomotor dysfunctions is considered essential to maintain quality of life . We previously demonstrated that an orally administered supplement containing glucosamine hydrochloride, chondroitin sulfate, and quercetin glycosides (gcq) was effective for relieving knee pain.9,10 supplements containing glucosamine and chondroitin sulfate have been widely used for the management of knee pain in oa.1113 nonsteroidal anti - inflammatory drugs (nsaids) relieve knee pain associated with oa and improve locomotor functions in patients with knee oa.14 these facts led us to speculate that glucosamine - containing supplements, as well as nsaids, could contribute to improved locomotor functions such as knee - extensor strength and walking speed . Quercetin, one of the flavonoids, which is widely distributed in plants and fruits, has been reported to suppress the atrophy of skeletal muscle in vivo.15 imidazole peptides are distributed mainly in the muscles of animals and have been reported to attenuate muscle fatigue in humans.16 furthermore, vitamin d is a nutrient that supports bone metabolism, and several studies have demonstrated its benefit in maintaining musculoskeletal functions and reducing the risk of falls in the elderly.17,18 in the present study, we investigated the effects of 16 weeks of treatment with a supplement containing glucosamine hydrochloride, chondroitin sulfate, type ii collagen peptides, quercetin glycosides, imidazole peptides, and vitamin d (gcqid) on locomotor functions in subjects with knee pain . A randomized, double - blind, placebo - controlled, parallel - group comparative study was designed to assess the efficacy and safety of gcqid supplementation in japanese women and men aged 4074 years . Inclusion criteria were the presence of knee pain, confirmed using the walking subscale of the joa criteria (25 points for either the left or right knee joint);19 visual analog scale (vas) score for knee pain (20 mm using the first (i) item of the japanese knee osteoarthritis measure [jkom]);20 and k l grades 0ii.5 all participants received an explanation about the study from the medical investigators, and written informed consent was obtained prior to enrollment in the study . Exclusion criteria were the following: faster walking speed (1.6 m / s); presence of hyperuricemia, diabetes, cardiovascular disease, hepatic disease, renal disease, or heart disease; treatment with exercise, diet, or medicine that may have an effect on the weight; presence of rheumatic arthritis that may cause joint pain; surgical treatment of knee joint(s) undergone or its necessity; needs to undergo pharmacological articular treatments during the study period; intra - articular hyaluronic acid within 2 weeks or corticosteroids within 3 months before inclusion; a history of osseous or articular diseases other than oa within the past 3 months; routine use of health food or medicine that may have an effect on the evaluation of the study; daily use of a cane; performing daily or occasional vigorous exercise; treatment with warfarin; bronchial asthma, respiratory disease, or potential for developing allergy to the test supplement; pregnant women; nursing mothers or women of child - bearing potential; and presence of any medical condition judged by the medical investigator . One hundred participants were enrolled in the study, which was performed from february 2012 to september 2012 at a clinical services center in japan . All subjects, who had never taken the same or similar type of supplement before, were recruited in and around osaka, kyoto, japan, through a volunteer bank managed by huma r&d co, inc . The study protocol was approved by the institutional ethics committee of east one medical clinic (tokyo, japan) and was conducted in accordance with the principles of the amended declaration of helsinki and ethical guidelines for epidemiological research (issued by the japanese government in 2008). The gcqid contained 1,200 mg of glucosamine hydrochloride, 300 mg of shark cartilage extract (60 mg as chondroitin sulfate, 45 mg as type ii collagen peptides), 90 mg of quercetin glycosides, 100 mg of fish meat extract (10 mg as imidazole peptides [anserine and carnosine]), and 5 g (200 iu) of vitamin d in eight tablets . Placebo just contained base material such as dextrin, and it did not contain components such as glucosamine hydrochloride, chondroitin sulfate, type ii collagen peptides, quercetin glycosides, imidazole peptides, and vitamin d in eight tablets . Subjects were randomly distributed to the two treatment groups in terms of normal walking speed, age, sex, average steps walked in a week, body mass index, and the aggregate scores for the joa criteria and sequentially assigned to receive either gcqid (gcqid group) or placebo (placebo group). The randomization codes for enrolled subjects were held by an appointed person who was not involved in the study . The gcqid and placebo tablets were manufactured by suntory wellness ltd specifically for the purpose of the present study to ensure that the placebo tablet was indistinguishable from the gcqid tablet in appearance, taste, and packaging . Allocation was preassigned on the basis of randomization numbers and was concealed from the subjects, the investigators, and the researchers who recruited and assessed participating subjects . All subjects were instructed to take eight tablets once a day and to record in their study diary whether they took the tablets or not . The efficacy of gcqid supplementation was assessed on the basis of measures of knee - joint functions and locomotor functions . Data on knee - joint functions were collected at baseline and at weeks 4, 8, 12, and 16 . Serum 25-hydroxy vitamin d (25-ohd) levels were measured at baseline and week 16 as an assessment of efficacy . Exclusion criteria based on efficacy assessment were as follows: taking <80% of the test supplement, performing actions that affected the reliability of the efficacy assessment (subjects who had too great a difference [0.25 m / s] in measured walking speed between the screening period and baseline), and noncompliance with the clinical protocol . Knee - joint functions were measured as score for jkom item i (vas score for jkom knee pain), total score for jkom items ii v (jkom total score),20 and vas score for pain on walking . Vas score was measured on a scale from 0 to 100, where 0 indicated no pain and 100 indicated the worst pain ever experienced . Subjects self - reported these measures on a website available only to the study participants . Jkom total score is a self - administered, disease - specific measure, consisting of 25 items that cover four different categories: ii: pain and stiffness in knees, iii: conditions in daily life, iv: general activities, and v: health conditions . An overall result was assessed by summing the scores from the 25 items, with results ranging from 0 (no complaint) to 100 (most severe condition possible). Locomotor functions were measured as walking speed and knee - extensor strength . For the measurement of walking speed, the time it took to walk the intermediate 6 m was measured, and the average of two times was calculated as a subject s walking speed . We measured the knee - extensor strength (torque) using a procedure previously described.21 for the measurement of knee - extensor strength, subjects performed isometric knee extensions on a custom dynamometer mounted force transducer (lu-100kse; kyowa electronic instruments, tokyo, japan). During the contraction of muscle, both knee joint and hip angles were flexed at 90 (180 was fully extended). For the maximal voluntary contractions of subjects, knee extension force exerted by knee extensor muscles was gradually increased from baseline to maximum in 23 seconds and then sustained at maximum for 2 seconds . Subjects carried out three trials with each leg, and the highest of the three was used . The sum of the values for both legs per unit body weight represented knee - extensor strength . The number of steps walked weekly, performed in leisurely fashion, and associated with activities of daily living was monitored using a pedometer . A stratified analysis of subjects with mild - to - severe knee pain (vas score for jkom knee pain 20 at baseline) was also performed . Using the oa criteria from the road study,6 subgroups with or without radiographic oa (k l grade ii or i, respectively) were then analyzed further . The safety of gcqid supplementation was assessed on the basis of the incidence and severity of treatment - related adverse events reported throughout the 16-week intervention period . Furthermore, enrolled subjects were requested to visit a clinic to undergo laboratory tests (hematology, blood biochemistry, and urinalysis) and physical examinations at baseline and week 16 as part of the safety assessment . Blood samples were obtained with the subjects in a fasting state, and urine samples from the morning s second void were collected . Baseline data were compared between the groups using the unpaired t - test for quantitative variables and -test for qualitative variables . Two - way repeated - measures analysis of variance was used for analyzing the differences in the effect of intervention, and post hoc analysis was conducted . In case of no significant group time interaction, comparisons between groups at each time point were performed using the mann whitney u - test for jkom total score and the unpaired t - test for physical and laboratory test variables, vas score for jkom knee pain, vas score for pain on walking, normal walking speed, and knee - extensor strength . For reference, changes in measurements during the intervention were compared with baseline using a paired t - test for physical and laboratory test variables; the steel test for jkom total score; and dunnett s test for vas score for jkom knee pain, vas score for pain on walking, normal walking speed, and knee - extensor strength . Effect sizes (d values for parametric variables and r values for nonparametric variables) were calculated in the measures in which there were significant differences between the groups . All statistical analyses were carried out using ibm spss statistics for windows, version 21.0 (ibm corporation, armonk, ny, usa) and ekuseru - toukei 2010 for windows (social survey research information co, ltd, tokyo, japan). A randomized, double - blind, placebo - controlled, parallel - group comparative study was designed to assess the efficacy and safety of gcqid supplementation in japanese women and men aged 4074 years . Inclusion criteria were the presence of knee pain, confirmed using the walking subscale of the joa criteria (25 points for either the left or right knee joint);19 visual analog scale (vas) score for knee pain (20 mm using the first (i) item of the japanese knee osteoarthritis measure [jkom]);20 and k l grades 0ii.5 all participants received an explanation about the study from the medical investigators, and written informed consent was obtained prior to enrollment in the study . Exclusion criteria were the following: faster walking speed (1.6 m / s); presence of hyperuricemia, diabetes, cardiovascular disease, hepatic disease, renal disease, or heart disease; treatment with exercise, diet, or medicine that may have an effect on the weight; presence of rheumatic arthritis that may cause joint pain; surgical treatment of knee joint(s) undergone or its necessity; needs to undergo pharmacological articular treatments during the study period; intra - articular hyaluronic acid within 2 weeks or corticosteroids within 3 months before inclusion; a history of osseous or articular diseases other than oa within the past 3 months; routine use of health food or medicine that may have an effect on the evaluation of the study; daily use of a cane; performing daily or occasional vigorous exercise; treatment with warfarin; bronchial asthma, respiratory disease, or potential for developing allergy to the test supplement; pregnant women; nursing mothers or women of child - bearing potential; and presence of any medical condition judged by the medical investigator . One hundred participants were enrolled in the study, which was performed from february 2012 to september 2012 at a clinical services center in japan . All subjects, who had never taken the same or similar type of supplement before, were recruited in and around osaka, kyoto, japan, through a volunteer bank managed by huma r&d co, inc . The study protocol was approved by the institutional ethics committee of east one medical clinic (tokyo, japan) and was conducted in accordance with the principles of the amended declaration of helsinki and ethical guidelines for epidemiological research (issued by the japanese government in 2008). The gcqid contained 1,200 mg of glucosamine hydrochloride, 300 mg of shark cartilage extract (60 mg as chondroitin sulfate, 45 mg as type ii collagen peptides), 90 mg of quercetin glycosides, 100 mg of fish meat extract (10 mg as imidazole peptides [anserine and carnosine]), and 5 g (200 iu) of vitamin d in eight tablets . Placebo just contained base material such as dextrin, and it did not contain components such as glucosamine hydrochloride, chondroitin sulfate, type ii collagen peptides, quercetin glycosides, imidazole peptides, and vitamin d in eight tablets . Subjects were randomly distributed to the two treatment groups in terms of normal walking speed, age, sex, average steps walked in a week, body mass index, and the aggregate scores for the joa criteria and sequentially assigned to receive either gcqid (gcqid group) or placebo (placebo group). The randomization codes for enrolled subjects were held by an appointed person who was not involved in the study . The gcqid and placebo tablets were manufactured by suntory wellness ltd specifically for the purpose of the present study to ensure that the placebo tablet was indistinguishable from the gcqid tablet in appearance, taste, and packaging . Allocation was preassigned on the basis of randomization numbers and was concealed from the subjects, the investigators, and the researchers who recruited and assessed participating subjects . All subjects were instructed to take eight tablets once a day and to record in their study diary whether they took the tablets or not . The efficacy of gcqid supplementation was assessed on the basis of measures of knee - joint functions and locomotor functions . Data on knee - joint functions were collected at baseline and at weeks 4, 8, 12, and 16 . Serum 25-hydroxy vitamin d (25-ohd) levels were measured at baseline and week 16 as an assessment of efficacy . Exclusion criteria based on efficacy assessment were as follows: taking <80% of the test supplement, performing actions that affected the reliability of the efficacy assessment (subjects who had too great a difference [0.25 m / s] in measured walking speed between the screening period and baseline), and noncompliance with the clinical protocol . Knee - joint functions were measured as score for jkom item i (vas score for jkom knee pain), total score for jkom items ii v (jkom total score),20 and vas score for pain on walking . Vas score was measured on a scale from 0 to 100, where 0 indicated no pain and 100 indicated the worst pain ever experienced . Subjects self - reported these measures on a website available only to the study participants . Jkom total score is a self - administered, disease - specific measure, consisting of 25 items that cover four different categories: ii: pain and stiffness in knees, iii: conditions in daily life, iv: general activities, and v: health conditions . An overall result was assessed by summing the scores from the 25 items, with results ranging from 0 (no complaint) to 100 (most severe condition possible). Locomotor functions were measured as walking speed and knee - extensor strength . For the measurement of walking speed, the time it took to walk the intermediate 6 m was measured, and the average of two times was calculated as a subject s walking speed . We measured the knee - extensor strength (torque) using a procedure previously described.21 for the measurement of knee - extensor strength, subjects performed isometric knee extensions on a custom dynamometer mounted force transducer (lu-100kse; kyowa electronic instruments, tokyo, japan). During the contraction of muscle, both knee joint and hip angles were flexed at 90 (180 was fully extended). For the maximal voluntary contractions of subjects, knee extension force exerted by knee extensor muscles was gradually increased from baseline to maximum in 23 seconds and then sustained at maximum for 2 seconds . Subjects carried out three trials with each leg, and the highest of the three was used . The sum of the values for both legs per unit body weight represented knee - extensor strength . The number of steps walked weekly, performed in leisurely fashion, and associated with activities of daily living was monitored using a pedometer . A stratified analysis of subjects with mild - to - severe knee pain (vas score for jkom knee pain 20 at baseline) was also performed . Using the oa criteria from the road study,6 subgroups with or without radiographic oa (k l grade ii or i, respectively) the safety of gcqid supplementation was assessed on the basis of the incidence and severity of treatment - related adverse events reported throughout the 16-week intervention period . Furthermore, enrolled subjects were requested to visit a clinic to undergo laboratory tests (hematology, blood biochemistry, and urinalysis) and physical examinations at baseline and week 16 as part of the safety assessment . Blood samples were obtained with the subjects in a fasting state, and urine samples from the morning s second void were collected . Baseline data were compared between the groups using the unpaired t - test for quantitative variables and -test for qualitative variables . Two - way repeated - measures analysis of variance was used for analyzing the differences in the effect of intervention, and post hoc analysis was conducted . In case of no significant group time interaction, comparisons between groups at each time point were performed using the mann whitney u - test for jkom total score and the unpaired t - test for physical and laboratory test variables, vas score for jkom knee pain, vas score for pain on walking, normal walking speed, and knee - extensor strength . For reference, changes in measurements during the intervention were compared with baseline using a paired t - test for physical and laboratory test variables; the steel test for jkom total score; and dunnett s test for vas score for jkom knee pain, vas score for pain on walking, normal walking speed, and knee - extensor strength . Effect sizes (d values for parametric variables and r values for nonparametric variables) were calculated in the measures in which there were significant differences between the groups . All statistical analyses were carried out using ibm spss statistics for windows, version 21.0 (ibm corporation, armonk, ny, usa) and ekuseru - toukei 2010 for windows (social survey research information co, ltd, tokyo, japan). Three subjects dropped out of the study because of adverse events (gcqid: 1, placebo: 1) or personal reasons (gcqid: 1), and 15 subjects were excluded from efficacy assessment because of efficacy - assessment exclusion criteria: taking <80% of the test supplement (gcqid: 1); performing actions that affected the reliability of the efficacy assessment (too great a difference [0.25 m / s] in measured walking speed between the screening period and baseline) (gcqid: 1, placebo: 2); and noncompliance with the clinical protocol (gcqid: 5, placebo: 6). Thus, 41 subjects in the gcqid group and 41 in the placebo group were deemed eligible for efficacy assessment . Table 2 shows the changes in knee - joint functions and locomotor functions during the 16-week intervention period . There was no significant difference between the groups in all measures for efficacy assessment except for serum 25-ohd levels . Serum 25-ohd levels were significantly greater in the gcqid group than in the placebo group at week 16 (31.90.8 ng / ml vs 28.51.1 ng / ml, p<0.05, d=0.49). A stratified analysis of subjects with mild - to - severe knee pain (vas score for jkom knee pain 20 at baseline) was performed . No significant difference was observed between the groups for any characteristic (table 3). There was no significant group time interaction in all measures for efficacy assessment except for serum 25-ohd levels, and knee - extensor strength was significantly greater in the gcqid group than in the placebo group at week 8 (p<0.05, d=0.65; table 4). The change in jkom total score at week 8 (p<0.05, r=0.37) and the change in normal walking speed at week 16 (p<0.05, d=0.62) were significantly greater in the gcqid group than in the placebo group (figure 1). There was a significant group time interaction in serum 25-ohd levels, and the change in serum 25-ohd levels at week 16 was significantly greater in the gcqid group than in the placebo group (11.91.8 ng / ml vs 6.91.3 ng / ml, p<0.05, d=0.80). L grade in subjects with mild - to - severe knee pain at baseline was also performed . There was no significant group time interaction, and there was no significant difference between the groups in jkom total score and normal walking speed both in subjects with k l grades ii, the change in jkom total score at week 8 was significantly greater in the gcqid group than in the placebo group (20.312.5 points vs 0.51.5 points, p<0.05, r=0.74). The change in normal walking speed tended to be greater in the gcqid group than in the placebo group at week 16, but the difference was not significant (p=0.08). L grade #i, the change in normal walking speed tended to be greater in the gcqid group than in the placebo group at week 16 (p=0.08). L grade i (14 in the gcqid group and 12 in the placebo group) showed that there was no significant group time interaction in jkom total score and normal walking speed, and the change in jkom total score was significantly greater in the gcqid group than in the placebo group at week 8 (6.21.5 points vs 1.80.9 points, p<0.05, r=0.42). Normal walking speed in the gcqid group was significantly greater than in the placebo group at week 16 (1.360.05 m / s vs 1.210.02 m / s, p<0.05, d=0.68). Significant changes in several blood biochemical and hematological variables were observed in both groups during the 16-week intervention (table s1), but the values were judged by the investigators to have remained within the normal range and to be medically unrelated to the treatment . There were also no abnormal changes in physical parameters and urinalysis, including proteinuria, glucosuria, and hematuria (data not shown). Some subjects in both groups reported experiencing one or more adverse events during the intervention . However, there was no between - group difference in frequency or pattern of events (table s2). Moreover, no adverse effect of treatment was identified when these results were analyzed on an individual - subject basis . The present study was conducted to evaluate effects of a glucosamine - containing supplement (gcqid) on locomotor functions in subjects with knee pain . The efficacy assessment revealed that gcqid supplementation improved jkom total score, normal walking speed, and knee - extensor strength in subjects with mild - to - severe knee pain at baseline better than the placebo (table 4 and figure 1). L grade was performed to investigate the efficacy of gcqid supplementation with or without radiographic oa . We found that gcqid supplementation only tended to improve normal walking speed more than placebo in subjects with k l grade ii or i, and it significantly improved both jkom total score and normal walking speed more than placebo in subjects with k l grade i. these results suggest that gcqid supplementation can be effective at improving knee - joint functions and locomotor functions in subjects with mild - to - severe knee pain, especially those with k the ability of gcqid supplementation to relieve knee pain may be explained mainly by the anti - inflammatory and chondroprotective activities of glucosamine hydrochloride,22,23 chondroitin sulfate,24 and quercetin,25,26 as described in a previous study on gcq supplementation.10 pain signals suppress muscle performance, and knee oa impairs locomotor functions such as walking speed.4 najm et al14 showed that nsaids improved knee - joint functions and locomotor functions at the same time in patients with knee . Similarly, the improvement in knee - joint functions observed with gcqid supplementation in the present study may partially contribute to improved locomotor functions in subjects with knee pain . Mukai et al15 confirmed that quercetin can prevent atrophy caused by muscle disuse by attenuating the expression of ubiquitin ligases, and horii et al27 revealed that a type of imidazole peptide increased muscle blood flow via changes in muscle sympathetic nerve activity, suggesting that quercetin glycosides and imidazole peptides in gcqid can directly affect muscle functions . Lower serum 25-ohd levels are associated with impaired locomotor functions.28 the 16-week intervention with gcqid supplementation elevated serum 25-ohd levels compared with the placebo group in subjects with mild - to - severe knee pain, suggesting that the elevated serum 25-ohd levels in the gcqid group may partially contribute to improved locomotor functions, although there was a significant group time interaction . In japan, serum 25-ohd levels are lowest at the end of winter and highest at the end of summer,29 and the present study was conducted from winter to summer . Therefore, the time effect in elevated serum 25-ohd levels can be partly explained by seasonal changes, which may also partially contribute to improved locomotor functions . In the present study, an increase in muscle strength with gcqid supplementation might be expected to lower the risk of falling because muscle weakness is the highest risk factor for falling.6 it has been reported that walking speed is associated with survival in older adults5 and that the ingestion of glucosamine or chondroitin is associated with decreased total mortality.30 therefore, gcqid supplementation may have a beneficial effect on survival rate . Further research is needed to elucidate the effect of supplements such as gcqid, which improve locomotor functions, on survival rate . In the safety assessment, no abnormalities in any laboratory tests were observed and no treatment - related adverse events were experienced during the intervention period, which suggests that gcqid is a safe supplement . First, the study was conducted over a relatively short period and the sample size was relatively small . Therefore, a long - term study with a large sample size may be needed to clarify the effect of gcqid supplementation on locomotor functions . Second, because subjects daily dietary intake, which might have affected knee - joint functions or locomotor functions, was not monitored, it must be acknowledged that daily diet might have partially affected the efficacy observed in the present study . Third, because the gcqid had several components, further studies should be conducted to clarify the role of each component of the gcqid on locomotor functions . In conclusion, our findings suggest that gcqid supplementation was safe and effective in improving knee - joint functions and locomotor functions in subjects with knee pain . Changes in laboratory values between baseline and week 16 notes: all values are expressed as mean standard error for gcqid group (n=48) and placebo group (n=49). Of the 100 subjects eligible for safety assessment, three (two in the gcqid group and one in the placebo group) dropped out of the study and did not undergo laboratory examinations at week 16 . Values below the detectable limit for crp and insulin are calculated as 0.05 mg / dl and 2.0 u / ml, respectively . P<0.01 compared with baseline . Abbreviations: alb, albumin; alp, alkaline phosphatase; alt, alanine aminotransferase; ast, aspartate aminotransferase; bun, blood urea nitrogen; cl, chloride; cpk, creatinine phosphokinase; cr, creatinine; crp, c - reactive protein; gcqid, glucosamine hydrochloride, chondroitin sulfate, quercetin glycosides, imidazole peptides, and vitamin d; -gtp, gamma - glutamyl transpeptidase; hb, hemoglobin; hba1c, hemoglobin a1c; hct, hematocrit; hdl - c, high - density lipoprotein cholesterol; k, potassium; ldh, lactate dehydrogenase; ldl - c, low - density lipoprotein cholesterol; na, sodium; plt, platelet; rbc, red blood cell; t - bil, total bilirubin; tc, total cholesterol; tp, total protein; ua, uric acid; wbc, white blood cell . Incidence of adverse eventsa incidence of adverse events (%) = 100 number of subjects with adverse events / number of subjects eligible for safety assessment . Abbreviation: gcqid, glucosamine hydrochloride, chondroitin sulfate, quercetin glycosides, imidazole peptides, and vitamin d.
Many studies have reported that patients with renal cell carcinoma (rcc) may have underlying risk factors for chronic kidney disease (ckd) [1 - 4]. Barlow et al . Suggested that patients undergoing renal surgery have a high rate of new - onset renal insufficiency . Understanding the possibility of renal function change in rcc patients before making any treatment decisions is important because it has been proven that ckd leads to anemia, renal osteodystrophy, uremic malnutrition, hyperlipidemia, and cardiovascular disease . For many years, radical nephrectomy has been considered the treatment of choice for localized renal cortical tumors . However, over the past decade, advances in imaging have allowed for the detection of small, asymptomatic renal tumors, which has altered the management of renal tumors less than 4 cm in size to focus on preventing postoperative renal insufficiency . Many studies have reported the oncological efficacy and functional advantages of nephron - sparing surgery [1,6 - 8], and it has been a preferred treatment for small renal tumors . However, in cases with large, endophytic renal tumors, radical nephrectomy is still the gold standard unless the patient has only one kidney or bilateral renal tumors . Many recent studies have found that nephron - saving surgery applied to tumors greater than 4 cm provides equivalent oncologic efficacy and superior renal functional outcomes compared with radical nephrectomy . This has allowed for flexibility in choosing the appropriate surgical procedure for patients, especially those who have a higher probability of postoperative renal insufficiency . Therefore, it is important to consider predictive preoperative factors for renal insufficiency before renal surgery and to predict postoperative renal function in patients with rcc . Recently, some studies have reported that the parenchymal volume, which is measured by use of computed tomography (ct), strongly correlates with differential renal function on nuclear renal scanning for normal or chronically obstructed kidneys . A significant correlation was identified between preoperative kidney volume and the glomerular filtration rate (gfr) as well as between postoperative kidney volume and gfr for the unaffected kidney after unilateral nephrectomy . We investigated the predictive preoperative factors for renal insufficiency following radical nephrectomy by focusing on the association between the measured parenchymal volume of the unaffected and the affected kidneys before radical nephrectomy and the postoperative estimated gfr (egfr) in rcc patients . Two hundred thirty - six patients who underwent radical nephrectomy for rcc between january 2001 and december 2005 at a single center were included in our study . Patient characteristics, including age, gender, and body mass index (bmi), were measured at hospital admission . Contrast - enhanced ct or magnetic resonance imaging (mri) was performed and serum creatinine levels were measured before and 5 years after surgery . The gfr was estimated by using the modification of diet in renal disease (mdrd) formula: mdrd egfr=186(serum creatinine) age (0.742 if female) all individuals with an mdrd egfr of less than 60 ml / min/1.73 m at 5 years after radical nephrectomy were classified as having renal insufficiency . We excluded those patients whose renal parenchymal volumes were not measurable by use of a perioperative imaging modality (ct or mri) or whose egfr was less than 60 ml / min/1.73 m before surgery . After patients with the aforementioned conditions were excluded, 89 patients were included in our study . We categorized patients into 2 groups on the basis of the egfr 5 years after radical nephrectomy: egfr <60 ml / min/1.73 m (group a) and egfr 60 ml / min/1.73 m (group b). Kidney volume before radical nephrectomy was measured by using ct or mri (ct: ge 64 channel vct, ge healthcare, waukesha, wi, usa; mri: archieva 3.0 t tx, philips, best, the netherlands) by using the standard clinical abdominopelvic imaging protocol for ct and the kidney imaging protocol for mri in our institution . Venous scans of entire abdomens for ct scan were performed with a 70-s delay after starting the 2-ml / kg intravenous injection of iodinated contrast agent through an antecubital vein . In mri, t1- and t2-weighted scans of the kidney were performed with a 180-s delay after starting the 0.1-ml / kg intravenous injection of gadolinium contrast agent through an antecubital vein . All axial images were checked by one urologist who was blinded to patient characteristics in order to distinguish normal - functioning tissue excluding tumor tissue or nonenhanced areas from the axial side of the delayed ct or mri images with a slice thickness of 5 mm (fig . 1). The process of distinguishing the tissue depended only on the gross appearance of the images . The two - dimensional extent of normal - functioning tissue from each image was measured by hand, and the kidney volume was calculated by integrating that extent of normal - functioning tissue . Preoperative factors such as age, gender, history of diabetes, history of hypertension, body mass index, preoperative egfr, serum uric acid, urine albumin, normal renal parenchymal volume, tumor size, and the ratio of the normal parenchymal volume of the removed kidney to that of the remaining kidney were compared between groups a and b. for the comparison, the mann - whitney u test was used for continuous variables and the chi - square test was used for categorical variables . Predictive factors for renal insufficiency 5 years after radical nephrectomy were analyzed by using univariate and multivariate logistic binary regression . Because the egfr 5 years after radical nephrectomy can be influenced by the preoperative egfr, we reanalyzed the preoperative predictive factors for the downgrading of ckd stage on the basis of the egfr (table 1), which may be a more objective measure of kidney function change . For that purpose, 89 patients were categorized into 2 groups: downgrade in ckd stage (group c) and no change in ckd stage (group d). A receiver operating characteristic (roc) curve was plotted to determine the cutoff value of the parameters found to be significant for predicting an egfr greater than 60 ml / min/1.73 m at 5 years after radical nephrectomy . All calculated p - values were two - sided, and values less than 0.05 were considered statistically significant . All data analyses were performed with ibm spss ver . 19.0 (ibm co., armonk, ny, usa). Two hundred thirty - six patients who underwent radical nephrectomy for rcc between january 2001 and december 2005 at a single center were included in our study . Patient characteristics, including age, gender, and body mass index (bmi), were measured at hospital admission . Contrast - enhanced ct or magnetic resonance imaging (mri) was performed and serum creatinine levels were measured before and 5 years after surgery . The gfr was estimated by using the modification of diet in renal disease (mdrd) formula: mdrd egfr=186(serum creatinine) age (0.742 if female) all individuals with an mdrd egfr of less than 60 ml / min/1.73 m at 5 years after radical nephrectomy were classified as having renal insufficiency . We excluded those patients whose renal parenchymal volumes were not measurable by use of a perioperative imaging modality (ct or mri) or whose egfr was less than 60 ml / min/1.73 m before surgery . After patients with the aforementioned conditions were excluded, 89 patients were included in our study . We categorized patients into 2 groups on the basis of the egfr 5 years after radical nephrectomy: egfr <60 ml / min/1.73 m (group a) and egfr 60 ml / min/1.73 m (group b). Kidney volume before radical nephrectomy was measured by using ct or mri (ct: ge 64 channel vct, ge healthcare, waukesha, wi, usa; mri: archieva 3.0 t tx, philips, best, the netherlands) by using the standard clinical abdominopelvic imaging protocol for ct and the kidney imaging protocol for mri in our institution . Venous scans of entire abdomens for ct scan were performed with a 70-s delay after starting the 2-ml / kg intravenous injection of iodinated contrast agent through an antecubital vein . In mri, t1- and t2-weighted scans of the kidney were performed with a 180-s delay after starting the 0.1-ml / kg intravenous injection of gadolinium contrast agent through an antecubital vein . All axial images were checked by one urologist who was blinded to patient characteristics in order to distinguish normal - functioning tissue excluding tumor tissue or nonenhanced areas from the axial side of the delayed ct or mri images with a slice thickness of 5 mm (fig . The process of distinguishing the tissue depended only on the gross appearance of the images . The two - dimensional extent of normal - functioning tissue from each image was measured by hand, and the kidney volume was calculated by integrating that extent of normal - functioning tissue . Preoperative factors such as age, gender, history of diabetes, history of hypertension, body mass index, preoperative egfr, serum uric acid, urine albumin, normal renal parenchymal volume, tumor size, and the ratio of the normal parenchymal volume of the removed kidney to that of the remaining kidney were compared between groups a and b. for the comparison, the mann - whitney u test was used for continuous variables and the chi - square test was used for categorical variables . Predictive factors for renal insufficiency 5 years after radical nephrectomy were analyzed by using univariate and multivariate logistic binary regression . Because the egfr 5 years after radical nephrectomy can be influenced by the preoperative egfr, we reanalyzed the preoperative predictive factors for the downgrading of ckd stage on the basis of the egfr (table 1), which may be a more objective measure of kidney function change . For that purpose, 89 patients were categorized into 2 groups: downgrade in ckd stage (group c) and no change in ckd stage (group d). A receiver operating characteristic (roc) curve was plotted to determine the cutoff value of the parameters found to be significant for predicting an egfr greater than 60 ml / min/1.73 m at 5 years after radical nephrectomy . All calculated p - values were two - sided, and values less than 0.05 were considered statistically significant . All data analyses were performed with ibm spss ver . 19.0 (ibm co., armonk, ny, usa). The clinical characteristics of the 89 patients included in the present study are shown in table 2 . On the basis of the egfr 5 years after radical nephrectomy, 43 patients with an egfr of less than 60 ml / min/1.73 m (group a) and 46 patients with an egfr of 60 ml / min/1.73 m or higher (group b) were compared . Of these patients, the median age of the patients was 61 years (range, 53 to 68 years) in group a and 48 years (range, 42 to 57 years) in group b. the median parenchymal volume of the remnant kidney was 156.28 ml in group a and 181.82 ml in group b. median age (61.0 years vs. 48.0 years, p<0.001), preoperative egfr (73.97 vs. 83.95, p<0.001), serum uric acid level (5.5 vs. 4.3, p=0.011), and parenchymal volume of the remnant kidney (156.28 vs. 181.92, p=0.003) were significantly different between groups a and b. table 3 shows the results of the univariate and multivariate binary regression analysis to identify the predictive factors for renal sufficiency (an egfr less than 60 ml / min/1.73 m) 5 years after radical nephrectomy . With control for bmi, urine albumin, and serum uric acid, the parenchymal volume of the remnant kidney (odds ratio [or], 9.185; p=0.001), a history of diabetes (or, 20.129; p=0.035), and preoperative egfr (or, 0.929; p=0.011) showed an association with renal insufficiency . Because egfr 5 years after radical nephrectomy was found to correlate with the parenchymal volume of the remnant kidney, an roc curve was plotted by using data from group a and group b (fig . The area under the curve was 0.678 (95% ci, 1.677 to 10.189). The optimal cutoff value of the parenchymal volume of the remnant kidney was 170 ml with a sensitivity of 58.7% and a specificity of 74.4% for predicting an egfr of less than 60 ml / min/1.73 m. the clinical characteristic of the 89 patients categorized by change in ckd stage are shown in table 4 . Sixty - one patients showed a downgrade in ckd stage (group c) and 28 patients showed no change in ckd stage (group d). The median parenchymal volume of the remnant kidney was 159.33 ml for group c and 185.91 ml for group d. no parameters were significantly different between groups c and d. table 5 provides the results of the univariate and multivariate binary regression analysis to identify the predictive factors for the downgrading of ckd category 5 years after radical nephrectomy . Only the parenchymal volume of the remnant kidney was statistically significant for the downgrading of ckd category in the multivariate linear regression analysis (or, 3.164; p=0.021). It has been noted that a decrease in the egfr is associated with an increased risk of cardiovascular events and many other complications . Many studies have found that patients with rcc have a high rate of renal insufficiency and that renal surgery, the treatment of choice for renal tumors, can negatively affect postoperative renal function [1 - 4]. Nephron - sparing surgery is an option that can reduce the chance of postoperative renal insufficiency if it is technically feasible, especially in patients who have more risk factors for postoperative renal insufficiency . Recently, as experience with partial nephrectomy has accumulated, nephron - saving surgery has been performed even for large (> 4 cm), endophytic tumors . Many studies have reported satisfactory results, which suggests that it is possible to perform nephron - saving surgery for more patients . However, nephron - saving surgery is still technically more difficult than radical nephrectomy for large (> 4 cm), endophytic renal tumors and can result in more postoperative complications, a longer operation time, and greater estimated blood loss . Determining the preoperative risk factors and predicting renal insufficiency after radical nephrectomy are important for patients with large, endophytic tumors and could allow nephron - saving surgery to be performed in patients with a higher risk of renal insufficiency, even those at a higher risk of complications after partial nephrectomy . Several studies have been conducted to identify the predictors of ckd or renal insufficiency after radical nephrectomy . Older age, hypertension, smoking, low preoperative renal function, and diabetes have all been identified as potential risk factors for renal dysfunction . Recently, functional kidney volume has been identified as an additional predictor for postoperative renal function deterioration . Studies have reported a correlation between the percentage functional volume preservation after renal surgery and the percentage decrease in the gfr . Simmons et al . Reported that preoperative nephron volume and percentage functional volume preservation are the primary determinants of long - term functional outcomes . Regarding donor nephrectomy, jeon et al . Reported that preoperative kidney volume measurement could be used to predict delayed kidney function recovery . These findings suggest that rcc patients with insufficient normal renal parenchymal volume could have a higher rate of renal insufficiency after radical nephrectomy and that surgeons should consider partial nephrectomy in these cases . In order to use the functional volume of either the affected or the normal kidney as a predictor of renal insufficiency after the surgery, proper measurement of the parenchymal volume of the kidney is important . Because immediate adaptation, such as hypertrophy and hyperfiltration, occurs in the normal kidney after unilateral nephrectomy, several methods have been used by different groups to identify hypertrophy by use of imaging technologies in donors and patients with renal diseases . Used image - directed color doppler ultrasonography and identified a mean hypertrophy of approximately 20% . However, the ellipsoid method is less accurate and is affected both by interobserver and intraobserver variability . Evaluated renal hypertrophy in 42 patients (mean age of 61.5 years) after they underwent unilateral nephrectomy by using a dimercaptosuccinic acid renal scan . Recently, ct and mri have been used to accurately assess functional kidney volume, because these techniques can exclude nonfunctional tissues such as the great renal vessels, perirenal fat, central sinus fat, and renal mass, which allowed for accurate functional kidney parenchymal assessment in our study . Recent studies have shown that tumor size is a significant risk factor for new - onset renal insufficiency in patients treated with radical nephrectomy . In a study by ohno et al ., the incidence of renal function deterioration (greater than 30% gfr decrease) was higher in patients with tumors sized 7 cm or less than in those with tumors greater than 7 cm (74.7% vs 32.8%, p<0.001). In our study, however, no significant association was identified for tumor size in the univariate analysis (or, 0.913; p=0.278). We focused on the influence of the absolute and relative renal parenchymal volume of the remaining kidney on postoperative egfr . A ratio (parenchymal volume of remnant kidney / total parenchymal volume) of more than 0.5 and less than 0.5 had no significant difference in the univariate analysis (or, 1.231; p=0.629). Nonetheless, when the absolute renal parenchymal volume of the remaining kidney is larger, especially when the measured volume was greater than 170 ml, the chance of postoperative renal insufficiency (or, 9.185; p=0.001) or ckd downgrading (or, 3.164; p=0.021) was significantly lower . We suggest that patients with an unaffected kidney that is already hypertrophied and hyperfiltrated have a lower chance of postoperative renal insufficiency . They reported that a group with an increase in kidney volume of less than 15% at 1 week after surgery as compared to the presurgical volume showed a 4.1-fold percentage increase in their risk of experiencing a 10% reduction in their postsurgical gfr at 6 months compared to the group who experienced an increase in kidney volume of greater than or equal to 15% . When undergoing renal surgery, patients should be informed of the renal functional outcome in addition to the oncological outcome so that they may make an informed preoperative treatment decision . We believe that our predictive model for new - onset renal insufficiency is useful for preoperative patient counseling . However, our study had several limitations . First, the patients analyzed in this study represent a retrospective, single - institution experience . Furthermore, the lack of randomization introduces a significant selection bias in the observed functional outcomes of patients treated with partial nephrectomy versus radical nephrectomy . Second, in order to differentiate the nonenhanced area of the kidney from the ct and mri scans, we depended on one urologist's opinion about the gross appearance of the images . Third, there is no standard method currently available for measuring kidney volume by ct or mri scan . Last, prospective data are needed to validate our predictive model for renal insufficiency and to analyze postoperative renal function longitudinally . The results of this study suggest that preoperative egfr, history of diabetes, and the radiologic volume of the remaining kidney parenchyma could be useful factors to predict postoperative renal function . Patients with a remaining kidney parenchymal volume of less than 170 ml should be considered candidates for nephron - sparing surgery if technically feasible, because this group has a higher chance of downgrading of ckd category 5 years after radical nephrectomy.
A 10-week - old boy presented with a history of rapidly progressive swelling over the left upper alveolus of a few weeks duration . The past medical and obstetric history was unremarkable and there was no history of trauma . Physical examination revealed a healthy infant with a reddish - bluish, firm, fixed and non - tender orofacial swelling involving the left upper vestibule and anterior hard palate, crossing the midline towards the right side (fig . Figure 1orofacial swelling involving the left upper vestibule, alveolar margin and anterior hard palate . Computed tomography (ct) and magnetic resonance (mr) scans were done to evaluate the lesion . A ct examination of the face was done on a multi - detector computed tomography (mdct) scanner (emotion duo; siemens medical systems, erlangen, germany). Contrast - enhanced scans were obtained after manual intravenous injection of 20 ml of nonionic iomeprol (iomeron-300, bracco, ferentino, italy) containing 300 mg / ml iodine . The ct scan revealed a bilobular, expansile bone lesion with homogenous soft - tissue density content, involving the anterior maxilla and adjacent hard palate, and displacing the developing dentition (fig . Figure 2axial contrast - enhanced ct scan images in (a) soft - tissue window and (b) bone window settings show an expansile, bilobed, well - circumscribed, solid lesion with epicenter in the anterior maxillary alveolar ridge . Axial contrast - enhanced ct scan images in (a) soft - tissue window and (b) bone window settings show an expansile, bilobed, well - circumscribed, solid lesion with epicenter in the anterior maxillary alveolar ridge . An mr scan of the face was done on a 1.5-tesla scanner (avento; siemens medical systems, erlangen, germany). (t2w) turbo spin echo sequences were obtained in multiple planes with 3-mm - slice thickness . Contrast - enhanced t1-weighted turbo spin echo scans were obtained after intravenous injection of 0.1 ml / kg of gadobenate dimeglumine (multihance, bracco, milan, italy). The mri scan revealed a bilobular bone lesion with lobulated margins and internal soft - tissue content involving the anterior maxillary alveolar margin and adjacent hard palate . The lesion showed hyperintense signals on t1w images and mildly hyperintense signals on t2w images (fig . Figure 3mr axial (a) t1-weighted, turbo spin echo, 630/17, (b) t2-weighted, turbo spin echo 4400/72, (c) gadolinium - enhanced t1-weighted turbo spin echo images . Images demonstrate a well - defined bilobed, solid, homogenous mass centered at the anterior maxillary ridge . The mass is hyperintense to muscles and tongue on both t1w (a) and t2w (b) images, contrary to the expected findings of melanin pigment . Mr axial (a) t1-weighted, turbo spin echo, 630/17, (b) t2-weighted, turbo spin echo 4400/72, (c) gadolinium - enhanced t1-weighted turbo spin echo images . Images demonstrate a well - defined bilobed, solid, homogenous mass centered at the anterior maxillary ridge . The mass is hyperintense to muscles and tongue on both t1w (a) and t2w (b) images, contrary to the expected findings of melanin pigment . A core needle biopsy revealed small round cells with peripherally arranged larger pigment - containing cells in alveolar distribution lying in fibrous stroma (fig . A full panel of immunohistochemistry markers were run on the biopsy tissue, which was strongly reactive for cytokeratin (ck), hmb45 and neuron - specific enolase (nse) and negative for s-100, cd99 and leukocyte common antigen (lca), confirming the diagnosis of melanotic neuroectodermal tumor of infancy (mnti) (fig . Figure 4histopathological image (magnification 300; hematoxylin and eosin stain) of the excised mass showing a nest of small cells (small arrow) and larger melanin - containing cells (large arrow) in a fibrous stroma (open arrow). Figure 5immunohistochemical assay images showing marked uptake of (a) ck, (b) hmb45 and (c) nse . Histopathological image (magnification 300; hematoxylin and eosin stain) of the excised mass showing a nest of small cells (small arrow) and larger melanin - containing cells (large arrow) in a fibrous stroma (open arrow). Immunohistochemical assay images showing marked uptake of (a) ck, (b) hmb45 and (c) nse . The excised specimen was a bluish - black oval mass consistent with a pigment - containing tumor (fig . Figure 6image of the extirpated specimen showing an oval mass with bluish - black pigmented areas . Image of the extirpated specimen showing an oval mass with bluish - black pigmented areas . Mnti was first described by krompecker in 1918; he called it congenital melanocarcinoma . Since then, there has been uncertainty about its cellular origin . The origin presumed earlier was from odontogenic or retinal rests, and this led to a plethora of names such as retinal anlage tumor, pigmented ameloblastoma, melanotic adamantinoma, retinal choristoma, melanotic epithelial odontoma, melanotic progonoma, pigmented teratoma, atypical melanoblastoma, pigmented epulis and retinoblastic teratoma . However, based on immunohistochemical, ultrastructural, and electron microscope studies and on occasional high urinary excretion of vanillylmandelic acid (vma), it is now agreed that the tumor originates from the neural crest . Therefore, the tumor is most commonly referred to as a melanotic (melanocytic) neuroectodermal tumor of infancy, a term first coined by borello and gorlin in 1966 . . It may be locally aggressive in 1536% of cases; 37% of tumors have an overtly malignant behavior with distant metastases . Mtni usually (95%) occurs in children less then 1 year of age peaking between 2 and 6 months, although occasionally it has been described in adults . The tumor usually occurs in the head and neck region because of its origin from the neuroectoderm . Anterior maxilla (premaxilla) is the most common site of origin, occurring in almost 71% of cases . Rarely, it can arise in the skull, mandible, brain, meninges, transverse sinus, retina, mediastinum, bones, soft tissue, uterus, and epididymis . In the skull, the anterior fontanelle is the most common site; the epididymis is the most common extracranial site . The classic clinical presentation is a sessile, firm swelling involving the upper anterior alveolar ridge and anterior hard palate . The presence of melanin gives it a bluish hue, often mimicking a vascular malformation . It may result in facial asymmetry, dentition displacement and difficulty in feeding . On gross examination, two groups of cells are noted with larger pigment - containing melanocyte - like cells arranged in an alveolar pattern between the fibrovascular septae and smaller non - pigmented neuroblast - like cells dispersed in the center of nests . Histopathologic examination is usually unreliable to differentiate benign from malignant changes as cellular atypia and sparse mitosis may be seen even in benign lesions . Immunohistochemistry is characteristic, being strongly reactive to cytokeratin, hmb45, synaptophysin, nse, epithelial membrane antigen, glial fibrillary acidic protein and leu-7, but negative for s-100 protein . Electron microscopy shows ultrastructural findings typical of neural, epithelial and melanocytic cells, such as the presence of intracellular melanosomes in various stages of development, numerous intercellular junctions, neurofibrils and desmosomes . Pathological differential diagnosis would include other small round cell tumors such as neuroblastoma, ewing sarcoma, rhabdomyosarcoma, lymphoma, primitive neuroectodermal tumor, etc . The plain radiograph of mnti shows a well - circumscribed, radiolucent, expansile lesion involving the anterior maxilla . The mass appears as an expansile bone lesion centered at the anterior maxilla, with a thin continuous bone shell and internal soft - tissue density content that enhances on intravenous contrast . Bone expansion, erosion, hyperostosis and spiculations can all be seen in the same lesion . Mr imaging findings should follow the paramagnetic effect of its melanin content, that is, the characteristic hyperintense signals on t1w images and hypo- to iso - intense signals on t2w images . The enhanced t1 relaxation (shortened) of melanin is probably due to chelation of paramagnetic metal ions by melanin . The enhanced (shortened) t2 relaxation is explained by melanin - induced heterogeneity in the magnetic susceptibility of the surrounding environment, resulting in rapid t2 decay . However, this typical mr signal pattern of melanin is only seen in very densely pigmented tumors, and when present can often clinch the diagnosis in an appropriate clinical setting . More often than not and as in our case, the mr signals are unlike a melanin signal, because of the variable proportion of melanin in the predominantly non - melanin content . Multiple foci with variable signals representative of melanin, bone and soft tissue can be seen within the same tumor . Melanin signals may be mimicked by a paramagnetic effect of methemoglobin or a highly cellular tumor . The differential diagnosis of mnti includes: (1) developmental cysts (nasopalatine cyst, globulomaxillary cyst); (2) odontogenic lesions (odontoma, ameloblastoma, ameloblastic fibroma, odontogenic myxoma, adenooameloblastoma, odontogenic keratocyst); (3) non - odontogenic non - neoplastic lesions (central giant cell granuloma, fibrous dysplasia and arteriovenous malformation); and (4) non - odontogenic neoplastic lesions (rhabdomyosarcoma, burkitt lymphoma, langerhans cell histiocytosis, ewing sarcoma). However, this long list can be reduced to just a few based on the clinical and radiological features . Management is usually by wide surgical excision with at least 5 mm of healthy margins . In the absence of free margins, the recurrence rate is increased by five times, most of which occur within 4 months and local radiotherapy with combination chemotherapy is then recommended . Delayed recurrences can be seen which makes close post - operative follow - up essential in all cases . In summary, mnti, although rare, has a distinctive clinicopathological and imaging features and it should be considered in the differential diagnosis of all patients presenting with head and neck masses particularly if present in the maxillary region.
All strains were derived from haploid cells by4741 (mating type a, euroscarf). Cooperator strain has an intact suc2 gene, defective his3 gene (his31), and yfp expressed constitutively by the adh1 promoter (inserted using plasmid prs401 containing met15). The mutant cheater strain lacks the suc2 gene (euroscarf y02321, yil162w::kanmx4), has an intact his3 gene, and has tdtomato expressed constitutively by the pgk1 promoter (inserted using plasmid prs301 containing his3). 2 and 5a were done using a strain containing yfp driven by the suc2 promoter (inserted using plasmid prs306 containing ura3). Co - culture experiments were performed in 5ml batch culture at 30c using synthetic media (minus histidine) supplemented with 5% sucrose and variable concentrations of glucose and histidine . The 20% sucrose stock solution was filter - sterilized and stored with 1 mm tris ph 8.0 to prevent acid - catalyzed autohydrolysis . Serial dilutions were performed daily (23 hours of growth) such that the starting optical density was 0.0025, corresponding to ~150,000 cells . Equilibrium data in figures 1c and 3b, c were recorded after five days of competition between the two strains . All strains were derived from haploid cells by4741 (mating type a, euroscarf). Cooperator strain has an intact suc2 gene, defective his3 gene (his31), and yfp expressed constitutively by the adh1 promoter (inserted using plasmid prs401 containing met15). The mutant cheater strain lacks the suc2 gene (euroscarf y02321, yil162w::kanmx4), has an intact his3 gene, and has tdtomato expressed constitutively by the pgk1 promoter (inserted using plasmid prs301 containing his3). 2 and 5a were done using a strain containing yfp driven by the suc2 promoter (inserted using plasmid prs306 containing ura3). Co - culture experiments were performed in 5ml batch culture at 30c using synthetic media (minus histidine) supplemented with 5% sucrose and variable concentrations of glucose and histidine . The 20% sucrose stock solution was filter - sterilized and stored with 1 mm tris ph 8.0 to prevent acid - catalyzed autohydrolysis . Serial dilutions were performed daily (23 hours of growth) such that the starting optical density was 0.0025, corresponding to ~150,000 cells . Equilibrium data in figures 1c and 3b, c were recorded after five days of competition between the two strains.
-adrenoceptor ligands comprise one of the most commonly used classes of drugs in clinical practice . -agonists have been used since the 1940s for their beneficial effects in respiratory disease and -antagonists (-blockers) since the 1960s for the control of cardiovascular disease, and both classes remain among the most commonly prescribed drugs today . The major therapeutic aim of -blockade is antagonism of endogenous catecholamines at the cardiac 1-adrenoceptors to reduce myocardial demand and workload . This reduces mortality following myocardial infarction, and therefore -blockers are used for both symptomatic control and life prolongation in ischemic heart disease and acute coronary syndrome and are increasingly being used earlier in the disease process . -blockers also reduce mortality in patients with heart failure and are used in the management of arrhythmias, hypertension, portal hypertension, benign essential tremor, thyrotoxicosis, anxiety, migraine, and glaucoma . The major therapeutic aim of -agonist administration is to mimic adrenaline and stimulate the 2-adrenoceptors in bronchial smooth muscle, thus increasing airway caliber and relieving the breathlessness and wheeze of bronchospasm . -agonists are used in the emergency setting for short - term rescue of bronchospasm in asthma and copd (e.g., salbutamol inhalers and nebulizers) as well as in the daily prevention of bronchospasm, particularly using longer acting molecules (labas, e.g., salmeterol, formoterol, indacaterol, and vilanterol). As -ligands have been used in clinical practice for around for 70 years, and a great many different agonist and antagonist molecules have been generated, it would be expected that good tool agonist and antagonist compounds for each of the human -adrenoceptor subtypes are available . However, it is surprising that there are actually very few truly selective molecules, especially for the 1-adrenoceptor . Drug discovery efforts driven by the clinical need have yielded several highly selective 2-adrenoceptor agonists for the treatment of asthma and copd (e.g., salmeterol, formoterol, indacaterol, and vilanterol) and one highly selective 2-antagonist (ici 118551) that has become a very useful tool in pharmacological studies of -adrenoceptors . For the 1-adrenoceptor, despite the fact that more 1-selective -blockers would be preferable to minimize the 2-mediated respiratory side effect of bronchospasm in the clinical setting, the selectivity of the clinically available -blockers for the human 1-adrenoceptor over the human 2-adrenoceptor is poor . For this reason, -blockers remain absolutely contraindicated in patients with asthma and relatively contraindicated in people with copd . Our desire to address this significant unmet clinical need initially led us to assess reported ligands that displayed very high 1-adrenoceptor versus 2-adrenoceptor selectivity . From the literature, one of the most highly 1-selective antagonists described to date was 1 (lk 204 - 545). Although this ligand provided an ideal structural template to investigate further, there was no published synthetic route and, in reality, very little pharmacological data available for this compound . Furthermore, it was not commercially available and therefore not readily accessible for pharmacological and other studies . As we also observed, there were also very few 1-selective -agonists, with xamoterol and ici 89406 being the most selective 1-partial agonists reported to date . Here, we report a synthesis and the in vitro pharmacological characterization of 1 and several novel, highly 1-selective -adrenoceptor ligands, most of which display significant agonist activity . Whereas the synthesis of 1 is not described in the literature, a synthetic route to the related compound, 1-(2-cyano-4-(2-cyclopropylmethoxyethoxy)phenoxy)-3-(2-(3-phenylureido)ethylamino)-2-propanol, is outlined as an example of this class of compound in a patent . The introduction of the cyano group employed an undesirable cyanation step, alongside several protection and deprotection steps, complicating the synthesis and compromising overall yield . Conscious that we would require appreciable quantities of this ligand to benchmark any newer ligands against, we embarked upon a new and more expeditious route to its synthesis (scheme 1). Reagents and conditions: (a) et3n, pivaloyl chloride, dmf, 0 c rt, 72%; (b) (i) nah, dmf; (ii) p - methoxybenzyl bromide, 0 c rt, 33%; (c) 37% nh3 (aq), i2, thf, 98%; (d) sodium tert - butoxide, meoh, 34%; (e) allyloxyethanol, diad, pph3, dcm, 100%; (f) et2zn, ch2i2, toluene 0 c rt, 25% 8 and 29% 9; (g) can, h2o, mecn; (h) et3n, rac - epichlorohydrin, 80 c, 100%; (i) 4-(benzyloxy)phenylisocyanate, dcm, 94%; (j) (i) concd hcl; (ii) 2 m naoh (aq), neutralization, 73%; (k) 13, propan-2-ol, reflux, 15%; (l) allyloxyethanol, diad, ph3p, dcm, rt, 71%; (m) et2zn, ch2i2, toluene, 0 c rt, 97%; (n) h2, 10% pd / c, etoh, 100%; (o) (i) 2 m naoh(aq); (ii) rac - epichlorohydrin, 60 c, 62%; (p) 13, propan-2-ol, reflux, 21% . The new synthesis of 1 relied upon an alternative synthesis of epoxide 10, with subsequent aminolysis of 10 using amine 13 . The availability of methods to easily convert an aryl aldehyde to the corresponding nitrile allowed 2,5-dihydroxybenzaldehyde (2) to be selected as an appropriate starting material in the synthesis of epoxide 10 . A procedure reported by chen et al . Facilitated selective monoprotection of 2 using pivaloyl chloride to generate pivaloate ester 3 in good yield . The remaining hydroxy group then required protection orthogonal with respect to the pivaloate ester; hence, the p - methoxybenzyl (pmb) group was selected for this purpose . The ability to cleave a pmb ether under oxidative conditions, using reagents such as ceric(iv) ammonium nitrate (can), was anticipated, because this would allow selective removal of the pmb group in the presence of the nitrile later in the synthesis . Conversion of aldehyde 4 to the desired nitrile was achieved cleanly by oxidative amination in nearly quantitative yield to give 5 . Alkoxide - mediated removal of the pivaloate ester then revealed phenol 6, which was subsequently submitted to mitsunobu coupling with 2-(allyloxy)ethanol to generate ether 7 . The desired pmb - protected product 8 was isolated and subjected to can - mediated oxidative pmb group cleavage conditions . Unfortunately, this did not yield the desired phenol 8, possibly due to instability of the molecule to can, for example, through dealkylation of the aralkyl ether . Re - examination of crude reaction products from the cyclopropanation step revealed that apart from the expected pmb - protected product 9, the oxidative conditions of the simmons smith procedure had caused partial removal of the pmb group to afford phenol 8 directly, although in modest yield . Consequently, phenol 8 was recovered, albeit as a side product and in relatively low yield . Alkylation of phenol 8 with rac - epichlorohydrin in the presence of tea afforded epoxide 10 in quantitative yield, with no further purification required . In this preliminary study, although it was well understood that the s - form was the more pharmacologically active of the two aryloxypropanolamine enantiomers, we elected to synthesize racemic derivatives throughout, as a more expeditious and cost - effective route to explore early sar, with the intention that favorable pharmacology would prompt synthesis of individual stereoisomers if warranted . Construction of aminophenol 13 required initial addition of 1,2-ethanediamine (11) to 4-(benzyloxy)phenyl isocyanate . Dropwise introduction of a solution of the isocyanate into a solution containing an excess of 11 allowed selective mono - addition to occur, as the product urea 12 was found to precipitate on formation, allowing facile isolation and purification by filtration . Conversely, the poor solubility of 12 in a variety of appropriate solvents meant that attempted hydrogenolysis progressed slowly . Acidolytic o - benzyl deprotection was therefore chosen as an alternative to give aminophenol 13 in good yield . Finally, aminolysis of epoxide 10 with 13 under neutral conditions, by reflux in propan-2-ol, afforded 1 after purification by flash column chromatography . Table 1 and pharmacology discussion) indicated a slightly lower 1/2-selectivity than previously reported . In addition to this, we noted with interest that previous studies indicated that the presence of substituents of the phenyl ring ortho to the oxypropanolamine moiety did not improve 1/2-selectivity but led to either increased or decreased affinity at both receptors simultaneously . Because our aim was to devise 1-selective ligands, we were therefore encouraged to explore whether removal of the cyano group was a worthwhile modification because, if successful, it would allow a more expeditious route into a range of new analogues . We therefore embarked upon the more efficient synthesis of the decyanated analogue of 1 (19), which had not previously been described, to assess the impact of the nitrile group on 1/2-receptor pharmacology (scheme 1). Mitsunobu coupling of 2-(allyloxy)ethanol with commercially available 4-(benzyloxy)phenol (14) furnished the desired phenylether 15, whose terminal allyl ether function was efficiently converted to the corresponding cyclopropylmethyl ether 16 using simmons subsequent benzyl ether hydrogenolysis of 16 over pd(0) on charcoal, using standard conditions, afforded phenol 17 in quantitative yield . Alkylation of 17 was achieved in moderate yield by initial deprotonation in aqueous 2 m naoh solution, followed by heating in excess rac - epichlorohydrin to afford oxirane 18 . Finally, oxirane 18 was subjected to aminolysis with 13 under basic conditions by refluxing in propan-2-ol . The desired aryloxypropanolamine 19 was obtained as the hydroformate salt, after plc and subsequent preparative rp - hplc purification . Pharmacological and in vivo analysis of our early lead ligand 19 gave mixed results . Whereas its selectivity was somewhat diminished compared to that of 1 (cf . Pharmacology results), both compounds possessed significant partial agonism, rendering them unsuitable for future therapeutic development as a selective 1-adrenoceptor antagonist . Therefore, to explore the sars of this class of molecule further and to ascertain if selectivity could be enhanced while decreasing partial agonism, the more facile synthesis of 19 now allowed access to new analogues in a more expeditious manner . Initially we tried to ascertain the effect of the alkoxyalkylether and the phenylurea termini on affinity, selectivity, and partial agonism . To explore the influence of the alkoxyalkoxy terminus on pharmacology, and on the basis of established literature precedent, we synthesized oxiranes 28a c in moderate yield (scheme 2), essentially in the same manner as for 10 . Although 2-ethoxyethanol (25) was commercially available, the analogous 2-(cyclopentyloxy)ethanol (21) and 2-(4-fluorophenethyloxy)ethanol (24) starting materials required independent synthesis (scheme 2). Reagents and conditions: (a) nabh4, zrcl4, thf 05 c, 81%; (b) (i) nah, dmf 60 c; (ii) chloroacetic acid, 60 c, 50%; (c) lialh4, thf, 0 c, 67%; (d) ph3p, 14, di - tert - butyl azodicarboxylate or diethyl azodicarboxylate, dcm, 3585%; (e) h2, 10% pd / c, etoh, 68100%; (f) (i) nah, dmf, 0 c rt; (ii) rac - epichlorohydrin, 7184%; (g) (i) 2 m naoh(aq); (ii) rac - epichlorohydrin, 60 c, 84%; (h) 13, propan-2-ol, reflux, 2129% . 2-(cyclopentyloxy)ethanol (21) was obtained in good yield from ketal 20, according to the nabh4/zrcl4 reductive cleavage conditions reported by chary et al . In the case of 2-(4-fluorophenethyloxy)ethanol (24), this entailed initial alkylation of 4-fluorophenethyl alcohol (22) with chloroacetic acid to give alkoxyacetic acid 23 . Subsequent reduction was achieved with lialh4, to give alcohol 24 in acceptable yield . The alcohols 21, 24, and 25 were subjected to mitsunobu coupling with 4-(benzyloxyphenol) (14) in a similar manner as previously described, to give o - benzyl ethers 26a c . Subsequent alkylation of phenol 27c was carried out using the previously described method to give oxirane 28c . In the case of phenols 27a, b, nah was used as a base in anhydrous dmf, with subsequent alkylation proceeding at room temperature to furnish oxiranes 28a, b . The target aryloxypropanolamines 29a c were obtained in the free base form, from oxiranes 28a c through aminolysis with 13 as previously detailed . Encouraged by the pharmacological data of the novel compounds possessing the 2-(cyclopentyloxy)ethoxyphenyl terminus (cf . Table 4 and pharmacology discussion), we kept this group constant to investigate the pharmacological effects of modifying the phenylurea substituent at the opposite pole of the molecule . This required synthesis of a small set of phenyl - substituted 1-(2-aminoethyl)-3-phenylureas (scheme 3). Reagents and conditions: (a) o - tolylisocyanate (30), dcm, 0 c rt, 64%; (b) di - tert - butyl dicarboxylate, dcm, 87%; (c) substituted phenylisocyanate, dcm, 0 c rt, 46 95%; (d) meoh / concd hcl, 80100%; (e) phthalic anhydride, 150 c, 94%; (f) (i) diphenylphosphoryl azide, tea, toluene; (ii) reflux; (iii) 2-aminophenol or 3-aminophenol, 7576%; (g) hydrazine monohydrate, etoh, reflux; (ii) acidic workup, 5160%; (h) 33a r, 10 m naoh(aq), propan-2-ol, reflux, 731%; (i) 33t u, et3n, propan-2-ol, reflux, 2125% . In accordance with the synthesis of 12, attempted condensation of o - tolylisocyanate (30) with an excess of ethylenediamine (11) afforded urea 33b in only modest yield, alongside the undesirable bis - urea side product . To avoid this complication with the remaining analogues, tert - butyl-2-aminoethylcarbamate (31) selective mono - boc protection of ethylenediamine (11) was accomplished in excellent yield using a literature method to give amine 31 (scheme 3). The stoichiometric condensation of 31 with corresponding substituted phenylisocyanates permitted preparation of the boc - protected intermediates 32a and 32c r in moderate to good yield, with the pure products being easily isolated by filtration following precipitation with hexanes . Boc - deprotection of these intermediates with concentrated hcl in methanol proceeded smoothly and in excellent yields, affording the desired amines 33a and 33c r as their respective hydrochloride salts . The remaining 1-(2-aminoethyl)-3-(hydroxyphenyl)urea analogues (33t u) required an alternative synthetic strategy, due to the lack of commercially available phenylisocyanate starting materials . However, the availability of 2-amino and 3-aminophenol allowed urea synthesis via condensation with the appropriate alkylisocyanates (scheme 3). Using -alanine (34) as a starting material, the conversion of the primary amine to the phthalimide eliminated possible side reactions during isocyanate formation; monoacylated amine protecting groups still have the propensity to attack the isocyanate functionality . Phthalimide protection of 34 was effected through a solvent - free method, by heating directly with phthalic anhydride to give acid 35, which was converted to the corresponding acyl azide with the aid of diphenylphosphoryl azide in the presence of tea; careful reflux of this intermediate promoted curtius rearrangement to the isocyanate . The isocyanate solution was divided into two equal portions, prior to addition of either 2- or 3-aminophenol, producing protected intermediates 36a and 36b . The desired amines 33 t and 33u were once again isolated as the hydrochloride salts after standard hydrazinolytic cleavage of the phthalimide group and subsequent acidic workup . Finally, aminolysis of oxirane 28a with amines 33a u and subsequent plc purification afforded the target aryloxypropanolamine compounds 37a u (scheme 3). We initially evaluated and compared the affinity of 1 with that of the decyanated analogue 19 at the human 1-, 2-, and 3-adrenoceptors, each stably expressed in chinese hamster ovary cells . The kd values for -adrenoceptor binding of h - cgp 12177 in these cell lines have previously been established and are 0.42, 0.17, and 109.2 nm for the 1-, 2-, and 3-adrenoceptors, respectively.1 completely inhibited specific binding to the human 1-adrenoceptor to yield a log kd of 8.04 0.03, n = 9, and 5.29 0.04, n = 11, for the human 2-adrenoceptor, thus giving a selectivity for the 1-adrenoceptor of 562-fold (figure 1; table 1). Likewise, 19 was also shown to inhibit specific binding to the human 1-adrenoceptor to yield a log kd of 7.49 0.03 . However, its affinity for the human 2-adrenoceptor was so low that measurement of complete inhibition of specific h - cgp 12177 binding was not possible (figure 1d), so that affinity (kd values) could not be calculated by this method . The affinity of both ligands for the human 3-adrenoceptor was also too low to establish reliable kd values from this method . Inhibition of h - cgp 12177 binding to whole cells by (a, b) 1 and (c, d) 19 in (a, c) cho 1 cells and (b, d) cho 2 cells . Bars represent total h - cgp 12177 binding, and nonspecific binding was determined in the presence of 10 m propranolol . The concentration of h - cgp 12177 present in each case was 0.73 nm . These single experiments are representative of (a) 9 and (b d) 11 separate experiments . Log kd values were obtained from h - cgp 12177 whole cell binding studies in cho cells stably expressing the human 1 or 2-adrenoceptors . Incomplete inhibition of specific h - cgp12177 binding meant that generation of an affinity value (log kd value) by this method was not possible . Because quantifiable antagonist affinity was difficult to determine for 19 at the human 2- and 3-adrenoceptors, an alternative assay was employed to establish accurate values via measurement from the shift of an agonist response (figure 2; table 2). H - camp accumulation in response to cimaterol in (a) cho 1 cells, (b) cho 2 cells, and (c) cho 3 cells in the absence and presence of 19 . Bars show basal h - camp accumulation, that in response to 10 m isoprenaline, and that in response to 30 nm, 100 nm, 300 nm, 1 m, 3 m, and 10 m 19 alone . Data points are the mean sem of triplicate values from single experiments that are representative of seven separate experiments in each case . Cimaterol was chosen as the agonist as it is a chemically stable nonselective agonist and thus could be used across all three human -adrenoceptors and because cimaterol agonist responses are also readily inhibited by classical -blockers, suggesting that its primary action is agonism of the catecholamine conformation of the human 1-adrenoceptor . Cimaterol stimulated responses were 96.9 1.1% (log ec50 = 8.13 0.03, n = 9), 98.9 1.7% (log ec50 = 8.78 0.06, n = 9), and 89.6 1.9% (log ec50 = 6.62 0.06, n = 9) of the isoprenaline maximum response at the human 1-, 2-, and 3-adrenoceptors, respectively (figure 2). These agonist responses were antagonized by both 1 and 19 (table 2). As both ligands examined demonstrated clear partial agonist stimulatory effects (e.g., figure 2), the data were analyzed by the partial agonist method of stephenson . Log kd values were obtained from h - camp accumulation assays following inhibition of the cimaterol agonist response in cho cells stably expressing the human 1-, 2-, or 3-adrenoceptors . Given the significant degree of agonist activity seen above, full concentration response relationships were investigated for the ligands . 1 caused an agonist response at the human 1-adrenoceptor that was 37.1 2.2% of the maximum response to isoprenaline (n = 5). Agonist responses were also seen at the human 2- and 3-adrenoceptors; however, the top of the concentration response curve was not reached in each case . At the maximum concentration of 10 m, 1 stimulated responses that were 15.2 0.7% (2) and 8.6 0.4% (3) of the isoprenaline maximum at each receptor . Log ec50 values and% isoprenaline maximal responses were obtained from h - camp accumulation for cells expressing the human 1-adrenoceptor . For the human 2- and 3-adrenoceptors, the top of the agonist concentration response curve was not obtained even with the maximum concentration of ligand . In these instances, the percentage of isoprenaline response is given for the response at 10 m of ligand or, in the case of xamoterol, 100 m . To assess the potential clinical effect of this degree of selectivity and partial agonism, these parameters were investigated in a freely moving conscious rat model . Previous studies with this model have shown that the heart rate response to isoprenaline is solely a 1-mediated response and that the hindquarters vascular conductance is solely a 2-mediated response . We employed the hydrochloride salt of 19 (2 mg / kg iv bolus, 1 mg / kg / h iv infusion) and observed that it significantly inhibited the 1-mediated heart rate responses to isoprenaline (at 40 and 120 ng / kg / min, 3090 min following administration) while having no effect on the 2-mediated hindquarters response (n = 4 rats), consistent with this compound s 1-adrenoceptor selectivity (figure 3). In addition, the ligand caused an increase in basal heart rate (from 424 9 to 465 3 beats / min, an increase that corresponds to 44% of the response to 120 ng / kg / min isoprenaline, n = 4 animals), in keeping with the partial agonist actions observed in the in vitro cell studies with 19 . Absolute values for heart rate (a) and hindquarters conductance (b) in conscious freely moving rats (n = 4 per group) before and at the end of 3 min infusions of isoprenaline (12, 40, and 120 ng / kg / min). Isoprenaline responses were measured before and at intervals after saline or 19 (hcl salt) administration . 19 (hcl salt) was given as a 2 mg / kg bolus followed by 1 mg / kg / h infusion for 90 min, and isoprenaline responses were measured during the infusion (3090 min) and after the 19 (hcl salt) infusion was turned off (45 and 2425 h). Isoprenaline responses measured in saline - treated animals at 3090 min, 45 h, and 2425 h were highly reproducible, but for the sake of clarity only the 3090 min time point is shown here . To investigate whether the structural extremities of our lead compounds could be modified to improve affinity and selectivity while attenuating partial agonism, we employed an iterative screening cascade approach . Initially we used a h - cgp 12177 whole cell binding assay to ascertain antagonist affinity measurements (table 4). Log kd values were obtained from h - cgp 12177 whole cell binding studies in cho cells stably expressing the human 1- or 2-adrenoceptors . Incomplete inhibition of specific h - cgp12177 binding meant that generation of an affinity value (log kd value) by this method was not possible . Informed from previously established sars, we explored three variants of the p - alkoxyalkoxy side chain present in 1 and 19, namely a para - positioned 2-(cyclopentyloxy)ethoxy- (29a), a 2-(4-fluorophenethyloxy)ethoxy- (29b), and an 2-(ethyloxy)ethoxy- (29c) moiety . Because the 2-(cyclopentyloxy)ethoxy side chain displayed the best combination of an increase in both binding affinity and 1/2-selectivity when compared to 19, we retained this moiety to undertake a sar evaluation of the phenylurea portion of the molecule (table 5). The unsubstituted phenylurea (37a) displayed a loss in affinity at both the 1- and 2-adrenoceptors yet maintained good 1-selectivity . Insertion onto the phenyl ring of individual electron - withdrawing or electron - donating groups at the ortho-, meta-, or para - positions displayed a set of measurable trends . In general, the ligand binding affinity remained within an order of magnitude for the respective adrenoceptor subtypes (1 log kd between 6.94 and 8.17 m; 2 log kd between 5.52 and 6.54 m) with all ligands showing selectivity for the 1-subtype . In most cases (37b, 37e, 37k, 37n, 37q, and 37 t) an ortho - positioned substituent resulted in the weakest 1-adrenoceptor binding affinity within each three - ligand subset alongside the smallest 1/2-selectivity . The only exception to this observation was that of the o - fluoro derivative (37h), which, while displaying the second highest affinity of the three ligands (37h j) for the 1-adrenoceptor, did possess the greatest, albeit modest, 1/2-selectivity (76-fold). Log kd values were obtained from h - cgp 12177 whole cell binding studies in cho cells stably expressing the human 1- or 2-adrenoceptors . The optimal ligands in terms of both affinity and selectivity were substituted in the meta- or para - positions with either a chlorine atom (37l and 37 m) or a hydroxyl group (29a and 37u) or, as mentioned above, were unsubstituted (37a). To cross - validate these results and attempt to determine 3-adrenoceptor affinity, we repeated the alternative assay for determining affinity whereby the shift of a cimaterol - mediated dose response curve is measured, focusing upon the best ligands from the phenylurea sar study and re - evaluating the original alkoxyalkoxy compounds (table 6). Log kd values were obtained from h - camp accumulation assays following inhibition of the cimaterol agonist response in cho cells stably expressing the human 1-, 2-, or 3-adrenoceptors . Interestingly, this assay gave values for ligand affinity (and selectivity) slightly higher than that of the binding assay, for example, with 29a 1-selectivity 478-fold (over 2) in the binding assay but 513-fold in the camp assay and with 37l 331-fold in the binding assay and 537-fold in the camp assay, similar to what was observed with previous ligands (tables 1 and 2). This method therefore provides a similar pattern of 1- versus 2-selectivity, allowing comparison between different chemical analogues . Even using this method, the affinity at the human 3-adrenoceptor once again could not be assessed, because the maximum concentration of test compounds did not cause a sufficient shift of the cimaterol concentration response curve (e.g., tables 2 and 6; figure 2). Although the affinity and selectivity profiles for many of the ligands were extremely encouraging, we were also aware that the original lead compounds (19 and 1) also displayed significant partial agonism at the -adrenoceptors . We therefore investigated the full concentration response relationships for the best ligands (table 7). Log ec50 values and% isoprenaline maximal responses were obtained from h - camp accumulation for cells expressing the human 1-adrenoceptor . For the human 2- and 3-adrenoceptors, the top of the agonist concentration response curve was not obtained even with the maximum concentration of ligand . In these instances, the percentage of isoprenaline response is given for the response at 10 m of ligand . Whereas the affinities of the second- and third - generation ligands were comparable to those of 1 and 19, there was a modest drop in efficacy at the 1-adrenoceptor, suggesting a marginal attenuation of partial agonism with these compounds . Interestingly, the one exception to this observation was 29c, which contained the linear 2-(ethyloxy)ethoxy side chain . In this example, the affinity at the 1-adrenoceptor was an order of magnitude less than that of 1 and the 2-(4-fluorophenethyloxy)ethoxy derivative (29b), yet this compound displayed a similar efficacy profile . As the 1-adrenoceptor is now accepted to exist in at least two agonist conformational states, it was important to assess at which state of the 1-adrenoceptor these novel -adrenoceptor ligands were exerting their partial agonist actions . Cgp 20712a (neutral high - affinity 1-adrenoceptor antagonist, capable of inhibiting both conformational states of the 1-adrenoceptor but with different affinities) measured in the presence of cimaterol yielded a high affinity value of log kd = 9.48 (supporting information table 1) compatible with the agonist activity occurring via the primary catecholamine site of the receptor . Cgp 12177 (log ec50 = 7.91 0.04, 58.2 2.6% isoprenaline maximum, n = 5) was antagonized by cgp 20712a to yield a much lower affinity (log kd = 7.22 0.03, supporting information table 1), demonstrating that a far higher concentration of cgp 20712a is required to block the cgp 12177 agonist response . The agonist activity of cgp 12177 was therefore occurring via the secondary site of the 1-adrenocptor (supporting information figure 1). The agonist response of our compounds was therefore inhibited by cgp 20712a to determine the site of their agonist actions . The affinity values for cgp 20712a were all high (log kd values between 9.51 and 9.09, supporting information table 1). Suggesting that, like the partial agonist xamoterol and ici 89406 (and unlike cgp 12177), our new partial agonists were indeed achieving their agonist actions via the catecholamine conformation of the receptor . The human 3-adrenoceptor, similar to the human 1-adrenoceptor, has also been demonstrated to exist in two conformational states . However, given the low affinity and efficacy of our novel compounds for the human 3-adrenoceptor, it was not possible to determine at which receptor conformational state the 3-agonist responses were occurring . Despite 70 years of experience and the availability of many adrenoceptor ligands, there remain very few truly selective ligands for the 1-adrenoceptor . To date, cgp 20712a is a widely known and truly selective 1-antagonist, whereas ici 118551 is a 2-selective antagonist . However, although several very highly 2-selective agonists exist (e.g., salmeterol and formoterol), there are no selective 1-agonist compounds noradrenaline, dobutamine, xamoterol, and ici 89406 all having relatively poor selectivity . Here we have characterized the pharmacological properties of 1 and some novel derivatives and thus present the most selective 1-partial agonists reported to date . 1 was confirmed as being a truly selective 1-adrenoceptor ligand with a 1 over 2 selectivity of 562-fold as measured by whole - cell binding . The first analogue of 1 made was 19, a decyanated version of the molecule, and the pharmacological effect of this maneuver was to reduce affinity at both human 1- and 2-adrenoceptors . It was clear from the antagonist studies that 1 and its derivative 19 had substantial agonist activity at the human -adrenoceptors . Given this observation, we investigated the potential clinical impact of this degree of partial agonism in a fully conscious rat model . Infusion of the hydrochloride salt of 19 resulted in a substantial increase in the basal (1-mediated) heart rate, in keeping with its 1-partial agonist effects, followed by inhibition of the 1-heart rate response to the isoprenaline (compare figure 2a in cho 1 cells and figure 3a in conscious rats). No effect on basal hindquarters vascular conductance or on the 2-mediated hindquarters response to isoprenaline was observed, consistent with high 1-selectivity, thereby demonstrating the 1-selective partial agonist effects in vivo . This agonist effect is significant because -ligands with agonist properties (e.g., xamoterol) have been shown to be less beneficial and, at times, even detrimental if used long - term in cardiovascular disease . Thus, although 19 maintained its 1-selectivity in vivo, its partial agonist properties were too great for it to be pursued as a potential clinical compound . Given 19 had reduced affinity and selectivity compared to 1, we designed a series of analogues of the molecule looking for evidence of structure activity relationships related to affinity, selectivity, and partial agonism . Substitution of the alkoxyalkoxy side chain afforded ligands with increased 1-adrenoceptor affinity (29a and 29b). Because all molecules remained partial agonists at the human 1-adrenoceptor (table 7), we therefore examined the phenylurea portion of the molecule in an attempt to attenuate this pharmacology . Generally, regardless of the substituent, substitution in the meta- and para - positions of the phenyl ring afforded higher 1-affinity (and therefore higher 1-selectivity) than substitutions at the ortho - position . Once again, however, these ligands retained their 1-selective partial agonist activity, and the agonist actions were demonstrated to occur via the catecholamine conformation . In summary, 1 and the new family of ligands that we have generated are 1-selective partial agonists . Compared with currently published ligands, for example, xamoterol and ici 89406, 1 and some of our novel analogues are the most 1-selective agonists to date and will prove to be useful pharmacological tools; however, the observed clinically deleterious effects of significant partial agonism in -blockers preclude any of these ligands from further clinical development . However, this preliminary body of work clearly demonstrates that it is possible to generate more 1-selective molecules than those currently available and in clinical use . Molecules of higher selectivity, but devoid of partial agonist effects, should prove to be a very powerful treatment for people with concomitant cardiovascular and respiratory disease and is the focus of ongoing work within our laboratories . Chemicals and solvents were purchased from standard suppliers and used without further purification . Merck kieselgel 60, 230400 mesh, for flash column chromatography (fcc), was supplied by merck kgaa (darmstadt, germany), and deuterated solvents were purchased from goss international limited (england) and sigma - aldrich co. ltd . Reactions were monitored by thin layer chromatography on commercially available precoated aluminum - backed plates (merck kieselgel 60 f254). A solution of ninhydrin (in ethanol) was used to visualize primary and secondary amines . All organic extracts collected after aqueous workup procedures were dried over anhydrous mgso4 or na2so4 before gravity filtration and evaporation to dryness . Organic solvents were evaporated in vacuo at 40 c (water bath temperature). Purification using preparative layer chromatography (plc) was carried out using fluka silica gel 60 pf254 containing gypsum (200 mm 200 mm 1 mm). Melting points (mp) were recorded on a reichert 7905 apparatus or perkin - elmer pyris 1 differential scanning calorimeter and were uncorrected . Fourier transform infrared (ft - ir) spectra were recorded as thin films or kbr disks in the range of 4000500 cm using an avatar 360 nicolet ft - ir spectrophotometer . Hig- resolution mass spectra (hrms)time - of - flight electrospray (tof es) were recorded on a waters 2795 separation module / micromass lct platform . H nmr spectra were recorded on a bruker - av 400 at 400.13 mhz . Chemical shifts () are recorded in parts per million (ppm) with reference to the chemical shift of the deuterated solvent / an internal tetramethylsilane (tms) standard . Coupling constants (j) and carbon fluorine coupling constants (jcf) are recorded in hertz and the significant multiplicities described by singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), multiplet (m), doublet of doublets (dd), and doublet of triplets (dt). Spectra were assigned using appropriate cosy, distortionless enhanced polarization transfer (dept), hsqc, and hmbc sequences . Analytical reverse - phase high - performance liquid chromatography (rp - hplc) was performed on a waters millenium 995 lc using both system 1 and system 2 described below and was used to confirm that all final products were 95% pure . Phenomenex onyx monolithic reverse phase c18 column (100 4.6 mm), a flow rate of 3.00 ml / min and uv detection at 287 nm . Solvent a, 0.1% formic acid (fa) in water; solvent b, 0.1% fa in mecn . Waters symmetry reverse phase c18 column (75 4.6 mm), a flow rate of 1.00 ml / min, and uv detection at 287 nm . Linear gradient 595% solvent b over 20 min . Solvent a, 0.1% fa in water; solvent b, 0.1% fa in meoh . Preparative hplc was performed using a phenomenex onyx monolithic reverse phase c18 column (100 10 mm), a flow rate of 14.10 ml / min, and uv detection at 287 nm . Solvent a, 0.1% fa in water; solvent b, 0.1% fa in mecn . A solution of 4-(benzyloxy) phenylisocyanate (3.739 g, 16.61 mmol) in anhydrous dcm (30 ml) was dripped into a flask containing vigorously stirred 1,2-ethanediamine (11) (6 ml, 89.80 mmol, 5.4 equiv) under nitrogen . Instant precipitation of a white solid was noted, and the reaction was allowed to stir for a further 3 h after addition of isocyanate solution was complete . After removal of all volatiles under reduced pressure, the crude solid was washed with et2o, before drying to give 4.472 g (94%) of white solid: mp 147149 c; ft - ir h, str), 2932, 2864 (alkyl c h, str), 1642 (urea c = o, str), 1604 (1 h, bend), 1111 (c o, str), 830 (aryl c h, bend, para - disubstituted ring), 741, 697 (aryl c h, bend, phenyl ring); h nmr (dmso - d6) 8.48 (s, 1h, nhar), 7.317.44 (m, 5h, aromatic benzyl ch), 7.28, 6.88 (d, j = 9.0 hz, 2 2h, para - disubstituted ring), 6.24 (t, j = 5.2 hz, 1h, nhconhar), 5.02 (s, 2h, phch2o), 4.27 (br s, 2h, nh2), 3.103.17 (m, 2h, ch2nh), 2.67 (t, j = 6 hz, ch2nh2); c nmr (dmso - d6) 155.72 (c = o), 152.88, 137.37, 133.97 (4 c), 128.37, 127.71, 127.63 (benzyl ch), 119.29, 114.87 (aryl ch), 69.37 (benzyl ch2), 40.87 (ch2nh2), 40.38 (chnh); m / z hrms (tof es) c16h20n3o2 [mh] calcd 286.1550; found 286.1547 . 1-(2-aminoethyl)-3-(4-(benzyloxy)phenyl) urea (12) (113 mg, 0.40 mmol) was stirred overnight in a solution of concd hcl (10 ml). The mixture was concentrated under reduced pressure and the residue redissolved in water (10 ml) before neutralization with aqueous 0.5 m naoh . After reconcentration, the residue was dissolved in the minimum amount of meoh and filtered (gravity), before purification by plc (eluent 37% aqueous nh3/meoh / dcm 2:25:73). This gave 56 mg (73%) of a brown semisolid: ft - ir 3339 (1 h, str), 3118 (br, o h, str), 1643 (urea c = o, str), 1615 (1 amine n h, bend), 1570 (aryl, str), 836 (aryl c h, bend, para - disubstituted ring); h nmr (meod - d4) 7.17 (d, j = 8.7 hz, 2h, aryl 3-h and 5-h), 6.73 (d, j = 8.7 hz, 2h, aryl 2-h and 6-h), 3.45 (t, j = 5.6 hz, 2h, ch2nh(c = o)nh), 3.05 (t, j = 6.0 hz, 2h, ch2nh2); c nmr (meod - d4) 159.38 (4 aryl 4-c), 154.70 (c = o), 132.02 (4 aryl 1-c), 123.61, 116.32 (aryl ch), 41.73 (ch2nh2), 38.79 (ch2nh(c = o)nh); m / z hrms (tof es) c9h14n3o2 [mh] calcd 196.1081; found 196.1081 . 2-(cyclopentyloxy)ethanol (21) (3.751 g, 28.81 mmol), triphenylphosphine (9.448 g, 36.02 mmol, 1.25 equiv), and 4-(benzyloxy)phenol (14) (5.769 g, 28.81 mmol, 1 equiv) were dissolved in dcm (70 ml). Di - tert - butyl azodicarboxylate (8.294 g, 36.02 mmol, 1.25 equiv) in dcm (20 ml) was added dropwise to the reaction mixture, which was allowed to stir overnight . After removal of approximately half of the solvent from the reaction mixture under reduced pressure, the resulting slurry was diluted with hexanes (100 ml) and washed with aqueous 1 m hcl (2 50 ml), aqueous 1 m naoh (2 50 ml), water (2 50 ml), and brine (1 50 ml). The organic layer was concentrated and redissolved in dcm (30 ml). The flask was left in the freezer for 1 h before filtration of the precipitate and washing with hexanes and et2o . After concentration of the filtrate, purification was achieved via fcc (eluent et2o / hexanes 10:90) to give 6.75 g (75%) of a clear colorless oil: ft - ir 2870, 2954 (alkyl c h, str), 1507 (aryl, str), 1109 (c o c, str), 824 (aryl c h, bend, para - disubstituted ring), 738, 696 (aryl c h bend, phenyl ring); h nmr 7.34 7.48 (m, 5h, aromatic benzyl ch), 6.91, 6.96 (d, j = 9.2 hz, 2 2h, aryl - dioxy ring), 5.04 (s, 2h, phch2o), 4.09 (t, j = 4.9 hz, 2h, ch2oarobn), 4.024.06 (m, 1h, pe ch), 3.76 (t, j = 5.3 hz, 2h, ch2ch2oarobn), 1.731.84 (m, 6h, pe ch2), 1.561.63 (m, 2h, pe ch2); c nmr 153.02, 153.26 (4 c, aryl - dioxy ring), 137.29 (4 benzyl c), 127.44, 127.83, 128.50 (benzyl ch), 115.62, 115.68 (ch aryl - dioxy ring), 81.89 (pe ch), 70.55 (benzyl ch2), 68.16 (ch2oarobn), 67.28 (ch2ch2oarobn), 32.27 (2-c and 5-c pe ring), 23.55 (3-c and 4-c pe ring); m / z hrms (tof es) c20h25o3 [mh] calcd 313.1798; found 313.1766 . 1-(2-(cyclopentyloxy)ethoxy)-4-(benzyloxy)benzene (26a) (6.326 g, 20.25 mmol) was hydrogenated according to the general procedure for o - benzyl deprotection to give the title compound in quantitative yield as a clear colorless oil: ft - ir 3381 (br, o h, str), 2960, 2871 (alkyl c h, str), 1510 (aryl, str), 1104 (c o c, str), 827 (aryl c h, bend, para - disubstituted ring); h nmr 7.60 (br s, 1h, oh), 6.69, 6.73 (d, j = 9.2 hz, 2 2h, para - disubstituted phenol), 3.963.99 (m, 3h, ch, ch2oar), 3.70 (t, j = 5.0 hz, 2h, peoch2), 1.621.78 (m, 6h, pe ch2), 1.451.53 (m, 2h, pe ch2); c nmr 150.05, 152.01 (4 c), 115.53, 115.88 (ch phenolic ring), 82.05 (pe ch), 67.82 (ch2oar), 67.11 (ch2ch2oar), 32.87 (2-c and 5-c pe ring), 23.20 (3-c and 4-c pe ring); m / z hrms (tof es) c13h17o3 [m h] calcd 221.1183; found 221.1191 . Nah 60% suspension in mineral oil (863 mg, equivalent to 518 mg of nah, 21.58 mmol, 1.1 equiv) was suspended in anhydrous dmf (20 ml) with stirring under a nitrogen atmosphere . After 5 min, 4-(2-(cyclopentyloxy)ethoxy)phenol (27a) (4.360 g, 19.61 mmol) in anhydrous dmf (20 ml) was added dropwise, with the vessel cooled over an ice bath . This mixture was then allowed to stir at rt for 20 min before addition of rac - epichlorohydrin (15.34 ml, 196.10 mmol, 10 equiv). After removal of all volatiles under reduced pressure, the crude residue was partitioned between water (30 ml) and et2o (30 ml) and the aqueous layer washed again with et2o (3 30 ml). The combined organic extracts were concentrated before purification through a silica plug (initial wash with hexanes, followed by etoh / dcm 5:95) to give 4.558 g (84%) of a clear yellow oil: ft - ir 3052 (epoxide c h, str, weak), 2961, 2870 (alkyl c h, str), 1508 (aryl, str), 1110 (c o c, str), 828 (aryl c h, bend, para - disubstituted ring); h nmr 6.80 (s, 4h, aryl c h), 4.11 (dd, j = 11.1/3.1 hz, 1h, aroch2ch), 3.99 (t, j = 4.9 hz, 2h, ch2oar), 3.923.96 (m, 1h, pe ch), 3.82 (dd, j = 11.1/5.7, 1h, aroch2ch), 3.67 (t, j = 5.3 hz, 2h, peoch2), 3.253.29 (m, 1h, epoxide ch), 2.82 (d, j = 4.9/4.9 hz, 1h, epoxide ch2), 2.67 (dd, j = 5.0/2.7 hz, 1h, epoxide ch2), 1.58 1.77 (m, 6h, pe ch2), 1.41 1.56 (m, 2h, pe ch2); c nmr 152.65, 153.36 (4 c), 115.46, 115.53 (aryl ch), 69.35 (aroch2ch), 68.08 (ch2oar), 67.18 (ch2ch2oar), 50.13 (epoxide ch), 44.49 (epoxide ch2), 32.17 (2-c and 5-c pe ring), 23.45 (3-c and 4-c pe ring); m / z hrms (tof es) c16h22nao4 [mna] calcd 301.1410; found 301.1414 . 2-((4-(2-(cyclopentyloxy)ethoxy)phenoxy)methyl)oxirane (28a) was opened with 1-(2-aminoethyl)-3-(4-hydroxyphenyl)urea (13) as described in the general procedure for aminolysis of oxiranes (cf . Supporting information). Yield: 29%, white amorphous solid; mp 135138 c; ft - ir 3311 (br, o h, str), 2929, 2869 (alkyl c h, str), 1636 (urea c = o, str), 1509, 1568 (aryl, str), 1111 (c o c, str), 820 (aryl c h, bend, para - disubstituted ring); h nmr (dmso - d6) 8.91 (br s, 1h, phenol), 8.20 (s, 1h, nh(c = o)nhar), 7.13 (d, j = 8.8 hz, 2h, aryl c h ortho to urea), 6.84 (s, 4h, aryl - dioxy ring), 6.62 (d, j = 8.8 hz, 2h, aryl c h ortho to phenol), 6.02 (t, j = 5.4 hz, 1h, nh(c = o)nhar), 4.96 (br s, 1h, nh), 3.793.97 (m, 6h, ch2oar, pe ch, ch(oh), aroch2), 3.61 (t, j = 4.8 hz, 2h, peoch2), 3.123.16 (m, 2h, nhch2ch2), 2.56 2.71 (m, 4h, ch(oh)ch2nh, nhch2ch2), 1.541.69 (m, 6h, pe ch2), 1.421.51 (m, 2h, pe ch2); c nmr (dmso - d6) 152.74, 152.49, 151.85, 132.14 (aryl 4 c), 155.65 (c = o), 119.72 (aryl c h ortho to urea), 115.33 (ch aryl - dioxy ring), 115.04 (aryl c h ortho to phenol), 80.84 (pe ch), 71.23 (aroch2), 68.08 (ch(oh)), 67.71 (ch2oar), 66.72 (peoch2), 52.20 (ch(oh)ch2nh), 49.38 (nhch2ch2), 39.15 (nhch2ch2), 31.78 (2-c and 5-c pe ring), 23.12 (3-c and 4-c pe ring); m / z hrms (tof es) c25h36n3o6 [mh] calcd 474.2599; found 474.2578; rp - hplc rt 3.17 (system 1), 10.30 (system 3). Fetal calf serum was from paa laboratories (teddington, middlesex, uk). White - sided view plates were supplied by thermo fisher scientific (basingstoke, uk). Microscint 20 scintillation fluid and ultima gold xr scintillation fluid were from perkinelmer . Radioligands (h - cgp 12177, h - adenine, and c - camp) were from amersham international (buckinghamshire, uk). Ici 89406, cimaterol, and cgp 20712a were from tocris life sciences (avonmouth, uk). All other reagents were from sigma chemicals (poole, dorset, uk). Cho - k1 stably expressing either the human 1 (1146fmol / mg protein), the human 2 (466fmol / mg protein), or the human 3-adrenoceptor (790fmol / mg protein) was used throughout this study . Cells were grown in dulbecco s modified eagle s medium nutrient mix f12 (dmem / f12) containing 10% fetal calf serum and 2 mm l - glutamine in a 37 c humidified 5% co2/95% air atmosphere . Cells were grown to confluence in white - sided, flat - bottom 96-well view plates . Briefly, the medium was removed from each well, and 100 l of serum - free medium containing the competing ligand at twice the final required concentration was added to each well . A fixed concentration of 100 l h - cgp 12177 was then immediately added to each well (1:2 dilution in well), and the cells were incubated for 2 h at 37 c, 5% co2 . The cells were washed twice by the addition and removal of 200 l of 4 c phosphate - buffered saline per well . One hundred microliters of microscint 20 was added to each well, a white sticky base was applied to the bottom of the plate, and a sealant top was applied to the top of the plate . The plates were left at room temperature overnight in the dark and then counted on a topcount at 21 c for 2 min / well . The cells were prelabeled with h - adenine by incubation for 2 h with 2 ci / ml the h - adenine was removed, and each well washed by the addition and removal of 1 ml of serum - free medium . One milliliter of serum - free medium containing 1 mm ibmx was added to each well, and the cells were incubated for 15 min . When used, antagonists were added at a final concentration at this stage and thus had 15 min of incubation . Agonist (in 10 l of serum - free medium) was added to each well, and the plates were incubated for 5 h. the reaction was terminated by the addition of 50 l of concentrated hcl per well . The plates were then frozen and thawed, and h - camp was separated from other h - nucleotides by sequential dowex and alumina column chromatography, as previously described . As measurements of partial agonism depend on many factors, including particularly the receptor expression level in the cells being examined, all camp experiments were performed in cells at the same passage throughout this entire study . In addition, whenever possible, experiments were performed with all ligands being investigated in parallel studies on the same day . Thus, every ligand was examined on the same split of cells on the same day, with each separate n number being performed on different days . All data points on each binding curve were performed in triplicate, and each 96-well plate also contained three to six determinations of total and nonspecific binding . A one - site sigmoidal binding curve (eq 1) was then fitted to the data using graphpad prism 2.01, and the ic50 was then determined as the concentration required to inhibit 50% of the specific binding.1 in eq 1, a is the concentration of the competing ligand, ic50 is the concentration at which half of the specific binding of h - cgp 12177 has been inhibited, and ns is the nonspecific binding . From the ic50 value and the known concentration of radioligand [h - cgp 12177], a kd (concentration at which half the receptors are bound by the competing ligand) value was calculated using eq 2:2 agonist responses were best described by a one - site sigmoidal concentration response curve (eq 3)3where emax is the maximum response, [a] is the agonist concentration, and ec50 is the concentration of agonist that produces 50% of the maximal response as several ligands have too low affinity at the human 2 and 3 receptors to assess accurately in the whole cell binding assay, the affinity (log kd value) of these antagonists was calculated in functional assays from the shift of the agonist concentration responses in the presence of a fixed concentration of antagonist using eq 44where dr (dose ratio) is the ratio of the agonist concentration required to stimulate an identical response in the presence and absence of a fixed concentration of antagonist [b]. Several of the compounds, however, displaced clear partial agonist activities; that is, they antagonized the more efficacious agonist cimaterol while stimulating an agonist response of their own . When clear partial agonism was seen, the affinity (log kd) was calculated according to the method of stephenson using eq 5:5 in eq 5 [p] is the concentration of the partial agonist, [a1] is the concentration of the agonist at the point where the partial agonist alone causes the same response, [a2] is the concentration of agonist causing a given response above that achieved by the partial agonist, and [a3] is the concentration of the agonist in the presence the partial agonist causing the same stimulation as [a2]. Isoprenaline (10 m) was included in all experiments, and therefore all maximal responses are expressed as a percentage of this maximum . Dawley rats (charles river, margate, kent, uk), weighing 300350 g, were housed in groups in a temperature - controlled (2123 c) environment with a 12 h light dark cycle (lights on at 6:00 a.m.) and free access to food (teklad global 18% protein rodent diet, bicester, oxon, u.k .) And water for at least 7 days after arrival from the supplier before any surgical intervention . Surgery was performed in two stages under general anesthesia (fentanyl and medetomidine, 300 g / kg of each ip, supplemented as required), with reversal of anesthesia and postoperative analgesia provided by atipamezole (1 mg / kg sc) and buprenorphine (0.02 mg / kg sc). At the first surgical stage, a miniature pulsed doppler flow probe was sutured around the distal abdominal aorta to monitor hindquarters hemodynamics . The wires from the probe were taped and sutured at the nape of the neck, and the animals were returned to the holding room . At the second surgical stage, which took place at least 10 days after the surgery for probe implantation, and following a satisfactory inspection from the named veterinary surgeon, catheters were implanted in the distal abdominal aorta via the caudal artery (for arterial blood pressure (bp) monitoring and the derivation of heart rate (hr)) and in the right jugular vein (for drug administration). Three separate intravenous catheters were placed in the jugular vein to enable concurrent administration of different substances . At this stage, the wires from the probe were soldered into a miniature plug (microtech inc ., boothwyn, pa, usa), which was mounted onto a custom - designed harness worn by the rat . The catheters emerged from the same point as the probe wires and were fed through a protective spring secured to the harness and attached to a counterbalanced pivot system . The arterial catheter was connected to a fluid - filled swivel for overnight infusion of heparinized (15 units / ml) saline to maintain patency . Experiments began 24 h after surgery for catheter implantation, with animals fully conscious and unrestrained in home cages, with free access to food and water . All procedures were carried out with approval of the university of nottingham local ethical review committee, under home office project and personal license authority . Cardiovascular variables were recorded using a customized, computer - based system (instrument development engineering evaluation (ideeq), maastrich instruments bv, the netherlands) connected to a transducer amplifier (gould, usa; model 13 - 4615 - 50) and a doppler flowmeter (crystal biotech (holliston, usa) vf-1 mainframe (pulse repetition frequency = 125 khz) fitted with high - velocity (hvpd-20) modules). Raw data were sampled by ideeq every 2 ms, averaged, and stored to disc every cardiac cycle . Hindquarters vascular conductance (hvc) changes were calculated from the changes in bp and doppler shift . In all experiments, atropine methyl nitrate (1 mg / kg / h; 0.4 ml / h) was infused continuously to remove any parasympathetic influence on the control of heart rate . Starting 2 h after the onset of the atropine infusion, rats were given 3 min infusions (0.15 ml / min) of isoprenaline (12, 40, and 120 ng / kg / min) in ascending order separated by at least 20 min . At least 45 min after the last infusion of isoprenaline, saline or 19 was given as an iv bolus (0.1 ml) maintained by continuous infusion (0.4 ml / h), and the isoprenaline infusions were repeated, starting 30 min thereafter . Responses to isoprenaline were measured as the difference between steady - state values immediately before the isoprenaline infusion and during the third minute of infusion . Changes in baseline were measured as the difference between the control values for the last dose of isoprenaline before, and the first dose of isoprenaline after, saline or 19 administration . Data were analyzed by t test or anova with bonferroni correction as appropriate; p <0.05 was taken as significant (graphpad prism version 5.02).
Chronic hepatitis c virus (hcv) infection is an important cause of chronic liver disease worldwide and a major indication for liver transplantation . Despite the exciting and promising discovery of direct - acting antiviral agents, pegylated interferon alpha (peg - ifn) and ribavirin numerous extrahepatic features are associated with chronic hcv infection, of which essential mixed (type ii) cryoglobulinaemia is well recognised . Mixed cryoglobulinaemia is a systemic vasculitis affecting small and medium - sized arteries and veins . It is characterized by the deposition of immune complexes containing rheumatoid factor, igg, hcv rna and complement on endothelial surfaces, eliciting vascular inflammation through poorly understood mechanisms . These signs and symptoms tend to occur in the setting of detectable circulating hcv rna levels and normally respond to therapy with peg - ifn and ribavirin . Exacerbations of vasculitic symptoms have been reported during interferon therapy, but they have almost always occurred in those with pre - existing vasculitis or cryoglobulinaemia [4, 5]. Occurrence of de novo cryoglobulinaemic vasculitis after successful viral suppression late in the course of interferon therapy is unusual, especially with use of the pegylated form . We report a case of severe de novo cryoglobulinaemic mononeuritis multiplex (mnm) that occurred during week 30 of antiviral treatment for chronic hcv infection . Cryoglobulins had been undetectable in the pre - treatment period and at the time of presentation the patient was hcv rna - negative . A 47-year - old caucasian man was referred by his primary care physician to the hepatitis clinic at our institution following the finding of abnormal liver function tests and elevated serum ferritin . Risk factors for liver disease included a brief period of injection drug use 20 years previously . His alcohol intake was within the national recommended limits (20 units / week) and there was no prior history of neurological, psychiatric or rheumatological illness . On examination screening blood tests showed positive serology for hcv, genotype 1a, with a viral load of 1.68 10 iu / ml (pcr, abbott). An exhaustive screen for other causes of chronic liver disease (including hfe gene mutation analysis) was negative and abdominal ultrasonography was unremarkable . Percutaneous liver biopsy revealed incomplete cirrhosis (ishak fibrosis stage 5), with a necroinflammatory score of 6/18, there being no histological evidence of iron overload . In view of the advanced hepatic fibrosis, antiviral treatment this included potential side effects such as flu - like and neuropsychiatric symptoms, haematological side effects and the small risk of hepatic decompensation . Pre - treatment bloods tests were as follows: haemoglobin 15.3 g / dl (13.518), white blood cells 5.5 10/l (4.011.0), platelets 203 10/l (150450), bilirubin 9 mol / l (021), alanine aminotransferase (alt) 226 iu / l (041), alkaline phosphatase 68 iu / l (40129), albumin 44 g / l (3448), inr 1.0 (0.81.2). Rheumatoid factor was elevated at 88 iu / ml (014) and c4 complement low at 0.03 whilst anti - nuclear antibody (ana) had been positive on initial referral (1:160 titre), this was negative on repeat testing and cryoglobulins were undetectable . In accordance with current guidelines, antiviral treatment with peg - ifn 2a (pegasys, roche) 180 g weekly and weight - based ribavirin (copegus, roche) 1,000 mg early virological response (evr; undetectable hcv rna at week 12 of treatment) was achieved and in the initial stages antiviral treatment was well tolerated, except for mild lethargy . The patient presented to hospital during week 30 of antiviral therapy with progressive painful bilateral foot drop of 2 weeks duration, associated with left ankle monoarthritis and weakness and sensory loss in the distribution of the left ulnar nerve . A week later the right ulnar nerve had become involved although less severely . A clinical diagnosis of mnm was made, corroborated by compatible neurophysiology studies suggesting an asymmetrical mixed sensory and motor peripheral neuropathy . Cerebrospinal fluid analysis and magnetic resonance imaging of the spine showed no abnormality . When repeated, cryoglobulin levels were found to be significantly elevated (8%) and analysis demonstrated a mixed (type ii) picture . Ana was again positive (1:160 titre) with an elevated rheumatoid factor (201 iu / ml vs. 88 iu / ml at initiation of antiviral therapy) and low c4 (0.03 g / l, unchanged from pre - treatment levels) with normal c3 levels . Peg - ifn and ribavirin were discontinued immediately and oral prednisolone 40 mg daily was commenced . Despite immunosuppression the patient's clinical state worsened over the following week with the development of cutaneous vasculitic stigmata and progressive bilateral median and common peroneal nerve palsies . In view of the progressive disease and persistence of cryoglobulinaemia, plasma exchange was performed (5 sessions over 1 week) and cylophosphamide initiated (150 mg / day). Cyclophosphamide was well tolerated and continued for 2 months before changing to azathioprine 75 mg twice daily . Prednisolone was increased to 80 mg daily and subsequently weaned progressively, aiming to taper completely over 12 months . A slow but steady partial neurological recovery was observed in the outpatient clinic over the following 4 months, with resolution of median and common peroneal nerve palsies and improvement in the other affected areas . Given that the severe mnm occurred during the course of antiviral therapy, we deemed it appropriate to discontinue peg - ifn and ribavirin . This was a difficult decision in view of the advanced hepatic fibrosis and encouraging initial response to treatment with achievement of evr . However, since mnm was the more immediately life - threatening condition with progressive weakness affecting all four limbs, discontinuation of antiviral therapy was justified . Not unsurprisingly, the patient developed virological rebound in hcv rna (5.6 10 iu / ml) approximately 1 month after the withdrawal of antiviral therapy and initiation of immunosuppression . The time course of alt, hcv rna, rheumatoid factor and cryoglobulin levels in relation to antiviral treatment, the development of mnm and immunosuppression are shown in fig . 1 . Unfortunately, 3 months after the onset of neurological disease the patient's mood worsened with the development of biological features of depression . Antidepressants were commenced but his depression worsened, and regrettably the patient died following a drug overdose 10 months after the onset of mnm . Post - mortem examination indicated that the cause of death was unrelated to liver disease or cryoglobulinaemia . We have described a patient with hcv - related cirrhosis who developed de novo cryoglobulinaemic mnm late in the course of antiviral therapy with peg - ifn and ribavirin . At the time of neurological presentation he had received 30 weeks of antiviral treatment with negative hcv rna since week 12 of therapy . There was no prior history of vasculitis and pre - treatment testing for cryglobulins had been negative . Our case is unique as occurrence of de novo cryoglobulinaemic vasculitis after successful viral suppression late in the course of interferon therapy is most unusual . Exacerbations of vasculitic symptoms have in fact been reported during interferon therapy, but almost always occur in those with pre - existing vasculitis or cryoglobulinaemia [4, 5]. Mnm is a well - documented complication of chronic hcv infection, normally associated with hcv viraemia, and typically responds to antiviral treatment . In our case the temporal association (both in terms of onset and recovery) between neurological symptoms, cryoglobulinaemia and the use of antiviral treatment raises the intriguing possibility that mnm could have been induced by antiviral therapy . However, it must be emphasised that at present this presumed association between peg - ifn 2a and mnm remains speculative . The various immunomodulatory effects of interferon that may have contributed to the development of mnm include upregulation of mhc class i and downregulation of mhc class ii expression, increased cytolytic activity of natural killer cells, increased dendritic cell activation and antigen uptake and presentation from apoptotic cells and finally increased production of autoantibodies [7, 8]. The association between hcv and vasculitis is well established, related usually to the development of cryoglobulinaemia or, less commonly, a non - cryoglobulinaemic polyarteritis nodosa - type disease . Estimates of mixed cryoglobulinaemia prevalence in patients with hcv infection vary widely from 10 to 70%, perhaps a reflection of population selection and lead time biases . High viral titres and the presence of type ii cryoglobulinaemia, as present in this case, increase the likelihood of clinically apparent vasculitic disease . When present pre - treatment these diseases usually abate and improve with the initiation of peg - ifn and ribavirin and hence antivirals can be used for treatment of symptomatic cryoglobulinaemia associated with hcv infection . However interferon therapy has also been associated with the development / exacerbation of various underlying autoimmune diseases including hcv - associated vasculitis, systemic lupus erythematosus and rheumatoid arthritis [11, 12]. There are also reports of the development and/or exacerbation of cryoglobulinaemic disease upon commencement of pegylated interferon therapy [4, 5], though these have almost always occurred in those with pre - existing vasculitis . The time span between the initiation of interferon therapy and the development or exacerbation of vasculitis is usually short, typically within the first few doses . De novo mnm during pegylated interferon therapy for hcv is more unusual, with only two prior published cases [13, 15]. In the case the patient achieved a rapid virological response (undetectable hcv rna by week 4 of treatment) and first presented with mnm 6 weeks into antiviral therapy . Describe a case of mnm occurring later in the course of peg - ifn treatment, with the onset of neurological symptoms at week 40 in a patient with negative hcv rna since week 24 . Similar to our case report, these two patients were also men and at least one also had advanced hepatic fibrosis . However in both cases it is unclear whether there was pre - existing cryoglobulinaemia before the initiation of antiviral therapy . In contrast, cryoglobulins were undetectable in our patient prior to therapy, though pre - treatment work - up did reveal a positive ana (titre 1:160), elevated rheumatoid factor and suppressed c4 level . The significance of these findings in the absence of detectable cryoglobulins or clinical evidence of vasculitis is uncertain . Conceivably they could be representative of low - level cryoglobulinaemia below the threshold for detection, resulting in an increased propensity to develop a nascent autoimmune process . When performed, nerve biopsy in patients with mixed cryoglobulinaemic mnm typically shows a perivascular lymphocytic infiltrate . However, as indicated above, it is possible for the direct neuropathic effects of both hcv and peg - ifn to result in an ischaemic rather than vasculitic neuropathy . Our patient did not undergo nerve biopsy and it may be argued that histology is required in order to discriminate between direct ifn-induced and mixed cryoglobulinaemic neuropathies . In the case we describe there was a significant deterioration in symptoms following withdrawal of peg - ifn and disease only stabilised and subsequently ameliorated once specific therapy for cryoglobulinaemic disease had been initiated . Additionally, the clinical picture was that of mnm rather than progressive peripheral neuropathy usually associated with ifn . We would therefore maintain that the clinical course in this case strongly favours mixed cryoglobulinaemic vasculitis over ifn-induced neuropathy . In summary we report a patient with hcv - related cirrhosis who developed de novo mnm associated with cryoglobulinaemia during week 30 of antiviral therapy, having achieved evr . While interferon therapy is the treatment of choice for hcv - related mnm, exacerbations have been reported during its use . Usually this has been reported in those with pre - existing cryoglobulinaemia and in the setting of detectable hcv viraemia . The de novo development of mnm during hcv therapy with undetectable hcv rna is most unusual . This raises the possibility that mnm could have developed as an adverse immunomodulatory effect of interferon therapy . However it must be re - emphasized that at present this association remains speculative . Nonetheless, such patients with advanced hepatic fibrosis present a challenging dilemma as the inevitable virological rebound after discontinuation of antiviral therapy can have grave implications . Clinical trials exploring interferon - free hcv treatment regimens are ongoing and these could ease the management of such patients in the future.
Trigeminal neuralgia (tn) is a debilitating pain syndrome that is characterized by agonizing, paroxysmal, and lancinating pain . The recommended first - line treatment for tn is medical therapy, but this often fails to provide pain relief . Thus, second - line treatment modalities are given to patients whose symptoms are intractable or who cannot tolerate medication . These treatment modalities include invasive procedures such as microvascular decompression (mvd), and ablative procedures such as gamma knife radiosurgery (gkrs), percutaneous balloon microcompression, radiofrequency rhizotomy, and glycerol rhizolysis . Recently, gkrs has been used as a treatment modality in several centers for patients with concurrent medical illness who are poor candidates for mvd or who refuse more invasive surgery17,20). Other treatment modalities provide short - term pain relief at a rate of 80 - 90%; gkrs also produces short - term alleviation of pain at similar rates, usually within 6 - 12 months after the operation2,3,7,9,11,15,18,23,29 - 31). Pain relief after mvd and radiofrequency rhizotomy for long - term periods has been well reported . In previous studies, patients who underwent mvd and radiofrequency reported favorable long - term pain relief of for more than 5 years1,5,10,25,28,32). However, long - term outcomes of gkrs for tn have been published in only a few reports12,22,29). Thus, we conducted the present study to analyze the long - term outcomes in a series of patients with tn who underwent single gkrs treatment and were followed up for a minimum of 60 months . Between march 1995 and march 2009, 40 patients with typical tn underwent gkrs at our clinic . Indications for gkrs included medically intractable pain,> 65 years of age, refusal of invasive procedures, or failure of previous invasive procedures . Among them, we retrospectively reviewed the medical records of patients with tn undergoing gkrs treatment that had a minimum follow - up of 60 months . Patient demographic characteristics, clinical presentation, treatment history, and the radiosurgical modality were reviewed in 22 patients whose median follow - up was 92.2 months (range: 60 - 144 months). All patients were treated with leksell gamma knife (eleckta instruments, stockholm, sweden) under local anesthesia . As a planning tool, the kula system was used until 2001, and the leksell gamma plan system (eleckta ab, stockholm, sweden) has been used since 2001 . All patients were treated using a single isocenter through a 4-mm collimator helmet targeting the root entry zone of the trigeminal nerve (fig . The mean maximal dose to the target was 77.1 gy (range, 65.2 - 83.6 gy). Long - term outcomes were established by face to face interview with a written questionnaire after the treatment in the outpatient clinic . The questionnaire was devised based on the barrow neurological institute (bni) pain scale24) but modified at our institution to better incorporate the use of adjuvant medications . Facial pain outcomes were categorized into one of the following classes: grade i, no pain, no medication; grade ii, no pain with medication; grade iii,> 50% improved pain with medication; grade iv, 50% improved pain with medication; and grade v, no pain relief . Patients with grade i - ii on the pain scale (equivalent to bni grades i and iiia) were considered to have a good treatment outcome . Patients with grade iv - v on the pain scale (equivalent to bni grades iv and v) were considered to have a poor treatment outcome (table 1). Statistical verification was determined using spss statistic software, version 13.0 (spss, inc . The mean age was 61.5 years (range 25 - 84 years), and the mean symptom duration from onset to treatment was 46.8 months (range 1 - 84 months). The most common distribution of pain involved the second and third divisions of the trigeminal nerve, observed in a total of 40.9% of patients (9 out of 22 patients). Thirteen patients had right - sided pain, and 9 had left - sided pain . During the last follow - up period, 6 patients (27.3%) had good treatment outcomes (represented by grades i and ii), and 7 patients (31.8%) had poor treatment outcomes (represented by grades iv and v) during a median follow - up period of 92.2 months (range 60 - 144 months). Of the 6 patients with good outcomes, 4 patients (18.2%) experienced complete pain relief without medication, and 2 (9.1%) had complete pain relief with medications . Of the 7 patients with poor outcomes, 5 patients (22.7%) experienced 50% improved pain with medication, and 2 (9.1%) had no pain relief (table 3). Subgroup analysis of excellent, good, and poor outcomes found 2 statistically significant factors related to achieving and maintaining complete pain relief: symptomatic periods before treatment (p=0.035) and previous surgery (p=0.008). However, sex, age, pain distribution, and laterality of pain did not correlate with facial pain improvement (table 4). Out of a total of 22 patients, 16 patients had been treated with medication only before gkrs (primary group), and 6 had gone through mvd prior to gkrs (secondary group). During the final follow - up period, patients with good outcomes were 37.5% and 0% in the primary and secondary group, respectively; patients with poor outcomes, on the other hand, were 12.5% and 83.3% in the primary and secondary group, respectively (table 5). Of the 22 patients, only one patient (4.54%) experienced facial numbness after gkrs, with grade iii pain on the pain scale . None of the patients had neuropathic pain, dry eye, keratitis, or jaw weakness after gkrs . Tn patients in whom medical therapy fails are recommended to undergo several treatment modalities, including mvd, radiofrequency rhizotomy, percutaneous balloon microcompression and gkrs . In general, the most effective surgical treatment is considered to be mvd in healthy patients because it has a demonstrated effective initial pain - free rate and good long - term outcomes . Barker et al.1) reported that the pain - free rate was maintained at 70% of 1185 patients for a median of 6.2 years after mvd . Severe complications that developed include 2 deaths (0.2%) and 16 patients (1%) with ipsilateral hearing loss . Tronnier et al.27) reported that 65% of patients had excellent outcomes at 10 years, and 63% at 20 years after mvd . In this study, broggi et al.4) reported that 53 - 94% of mvd patients were pain - free after a long - term follow - up . They also reported that the mortality of mvd was 0.3% and that cranial nerve dysfunction rates ranged from 0.1 - 3% . Additionally, radiofrequency rhizotomy also demonstrated an effective long - term outcome for tn patients . Taha et al.26) found that 75% were pain - free at 10 years, and 23% experienced facial dysesthesia . In previous studies, patients who underwent mvd reported long - term pain relief at rates of 62 - 89%, and patients who underwent radiofrequency reported long - term pain relief at rates of 20 - 82% for> 5 years after achieving a short - term pain relief rate of> 90%1,4,5,10,25 - 28,32). However, severe complications have occurred in tn patients who underwent mvd or radiofrequency rhizotomy at rates of approximately 2 - 19%1,4,5,10,25 - 28,32). In previous reports with long - term follow - up, gkrs for tn patients has long term outcomes slightly inferior to mvd and radiofrequency rhizotomy, but with lower complication rates . Urgosik et al.29) reported that 80% of 107 patients had good outcomes (patients with 60% <residual pain) at a median of 5 years . Little et al.12) reported that 83% of 136 patients had good outcomes (bni pain score of class i, ii, or iii) at a median of 6.3 years . Riesenburger et al.22) reported that 58.5% achieved good outcomes (bni pain score of class i, ii, or iii) at a follow - up period of 48 months . These reports have demonstrated that gkrs is a safe technique for the management of tn, as trigeminal sensory disturbance, the main complication of radiosurgery for tn, only developed in approximately 10% of the patients . In addition, gkrs patients did not develop any of the severe complications which patients that had undergone mvd or radiofrequency rhizotomy experienced14). However, most of the other reports on gkrs and tn combined the longer - term follow - up patients with more recently treated patients, thus making it more difficult to identify long - term results . In the present study, we strictly evaluated long - term outcomes at a follow - up period of a minimum of 5 years . Thus, our data demonstrate that patients experience lasting pain relief with few complications after gkrs, although patients with good outcomes are fewer than in the previous long - term follow - up studies of outcomes after gkrs . This seems to be due to the fact that only patients with no pain regardless of medication were included in the " good outcome " patient group in our study, whereas published reports on long - term follow - up studies on gkrs included patients with medication - controlled pain in the " good outcome " group . Because pain negatively impacts upon the quality of life of chronic tn patients, we think that the goal of treatment in tn should be complete pain relief . Therefore, we believe it is better to grade patient outcome by the reporting of pain - free outcomes that clearly separates effective pain relief from continued pain . In the current study, patients with pain that had decreased by 50% or more with the use of medication were classified separately as patients with fair outcomes . As a result, our data demonstrated that patients with good and fair outcomes comprise 72.7% of all treated patients, which is a similar outcome to that in previous reports . Therefore, from the results of this 5-year long - term follow - up study, we suggest that the proportion of patients with good outcomes, consisting purely of pain - free patients with or without medication, after gkrs was 27.3% . Published reports have used radiation dose protocols ranging from 70 - 100 gy7,8,21,31). In general, higher doses of radiation are related to better outcomes, but complications, including facial sensory loss and paresthesia, increase at doses greater than 90 gy19,21). Generally, maximal radiation doses between 70 - 90 gy are used to treat tn . In previous long - term follow - up studies, the mean maximal radiation dose was approximately 80 gy, and complications included facial numbness, which affected approximately 10% of the treated patients12,22,29). In the present study, the mean maximal dose was 77.7 gy, which is lower than that utilized in other recent studies; this is partly due to the long follow - up period which started from 1995, and partly because the dose was slightly lowered to avoid the development of lasting complications . In our study, only one patient (4.54%) experienced facial dysesthesia . Thus, we believe that lower radiation doses are associated with lower complication rates . The presence of previous surgery is regarded as a predictive factor significantly associated with a poor short - term outcome6,13,16,20). However, long - term pain relief factors have not been well studied until recently . Little et al.12) recently reported that at long term follow - up, 75% of patients who were treated with primary gkrs achieved long - term pain relief at 7 years, but only 10% of patients in whom previous surgery had achieved a similar outcome . Similarly, our study found that good outcomes in primary gkrs group (37.5%) was better than in secondary gkrs group (0%), respectively . Also, the presence of previous surgery was observed to be a statistically significant factor related to achieving and maintaining complete pain relief according to our subgroup analysis of good and poor outcomes . Based upon these findings, we have come to believe that there is significant correlation between previous surgery and poor long - term outcomes . First, although the pain intensity scale is validated tools for the quantification of pain, they are subjective outcome measures because they are dependent on personal interpretations and variation . Second, the small sample size and retrospective study design limit the power of our outcome observations . Gkrs performed at lower radiation doses not only decreased complication rates, but also lessened the development of more severe complications in the long run . However, the long - term results of gkrs for tn are not as satisfactory as those of mvd and other conventional modalities . Although gkrs achieves relatively good outcomes of initial pain relief, our results suggest a steady rate of late failure, particularly among patients who performed secondary gkrs following prior surgery . Gkrs is a safe, effective and minimally invasive treatment modality for only limited group of patients who are not suited to more invasive treatment.
Acute exacerbations of copd are characterized by a change in cough, sputum production, and respiratory symptoms from baseline that cause the patient to seek medical attention and involve increasing the dose and/or frequency of existing bronchodilator therapy . Systemic corticosteroids are also recommended in patients with more advanced copd that may require hospitalization . Exacerbations may be mild, necessitating only outpatient therapy such as antibiotics and/or systemic steroids but often may be severe enough to require hospitalization and admission to the intensive care unit . Therefore, prevention of exacerbations is one of the most important goals of chronic obstructive pulmonary disease (copd) management (global initiative for chronic obstructive lung disease 2005) because of the impact on patients daily activities and health - related quality of life (hrqol), with these effects lasting for several weeks or months (haughney and gruffydd - jones 2004). Frequent exacerbations also lead to a poorer recovery in terms of improvement in health status (haughney and gruffydd - jones 2004). Furthermore, the frequency of acute exacerbation has a significant impact on the accelerated decline of lung function (donaldson et al 2002) and also on healthcare costs (haughney and gruffydd - jones 2004). Consequently, strategies for preventing copd exacerbations may have an important effect on the natural course, cost of management, and mortality in copd (martinez and anzueto 2005). In recent years, several large - scale clinical trials in copd have analyzed the exacerbation - preventing aspects of different treatments (niewoehner 2004; solr 2005). These include inhaled bronchodilators, inhaled corticosteroids (icss), and a limited number of antimicrobial strategies . However, most antimicrobial strategies have been used therapeutically in the form of antibiotic treatment during an established exacerbation, apart from vaccinations against influenza and pneumococci, which can protect from these infections which are particularly dangerous for copd patients (cazzola et al 2005). Long - acting 2-agonists (labas) have both bronchodilator and non - bronchodilator properties, whereas icss are potent anti - inflammatory agents (cazzola and dahl 2004). It is, therefore, obvious that labas and icss treat different aspects of copd (cazzola and dahl 2004). Labas are central in the symptomatic control of copd (american thoracic society / european respiratory society 2005), whereas conflicting results have been reported with icss (barnes 2000; calverley 2000). This may reflect the fact that the inflammation in copd is poorly suppressed by corticosteroids, with small reduction in inflammatory cells, cytokines, or proteases in induced sputum even with oral corticosteroids (barnes 2004), although there are some subgroups of patients (eg, those with eosinophilia) who respond very well (chanez et al 1997). Nonetheless, findings from several clinical studies have shown that treatment with icss improve expiratory airflow and reduce symptoms and the rate of exacerbations associated with copd (alsaeedi et al 2002), and, moreover, reduce all - cause mortality in copd (sin et al 2005). Icss in relatively high doses (eg, budesonide 800 g / day or fluticasone propionate 1 mg / day) reduce exacerbations by 20%30% and improve the health status of copd patients by a similar amount compared with placebo (man and sin 2005). In particular, a meta - analysis of six trials, which evaluated the long - term effects (6 months) of icss in stable copd and provided information about total exacerbations, demonstrated that icss reduce exacerbation rates in copd by nearly one third relative to placebo (relative risk [rr], 0.70; 95% ci, 0.580.84) (alsaeedi et al 2002). In the isolde (inhaled steroid in obstructive lung disease in europe) study, the major benefit of therapy was seen in those whose lung function was less than 50% of predicted; however, the number of patients having at least one exacerbation per year was reduced with icss irrespective of baseline lung function (jones et al 2003). A convincing demonstration of the effectiveness of these agents in copd patients has come from the cope study (an investigation of copd in the department of pulmonology, enschede, the netherlands) (van der valk et al 2002). This study showed that the discontinuation of therapy with icss was associated with a more rapid onset and a higher risk of recurrence of exacerbations, and with a significant deterioration in aspects of hrqol, in the majority of patients . However, 40% of subjects in the cope study experienced no untoward effect from the withdrawal of icss . This finding indicates that there is an urgent need to identify which subgroups of copd patients respond well to prolonged icss therapy . In any case, a recent article that has reviewed the diverse approaches to data analysis used in the randomized trials of icss in the treatment of copd and their meta - analysis, has concluded that the reports that icss reduce copd exacerbation rates are the result of improper statistical analysis techniques (suissa 2006). The only two studies that used the correct statistical approach found insignificant effects with these drugs . It is not a surprise, therefore, that the evaluation of a cohort of newly treated copd patients formed from the administrative databases of saskatchewan health documented that icss do not seem to be beneficial in preventing the risk of a first exacerbation of copd (de melo et al 2004). Interestingly, a recent paper has documented that immortal time bias cannot account for the risk reduction associated with icss exposure in observational studies (kiri et al 2005). This finding is strengthened by the observed relationship between increased regularity of icss prescriptions and reductions in event rates . Although labas have been shown to reduce exacerbation rates of copd, the magnitude of the effect appears modest at best . Published trials reveal that salmeterol is the only laba with a significant effect on the rate of exacerbations . In a 1-year study comparing salmeterol with placebo, salmeterol significantly reduced the mean number of exacerbations per patient by 20% and the mean number of exacerbations requiring oral corticosteroids per patient by 29% (calverley et al 2003a). Recently, it has been documented that the addition of salmeterol to existing treatment in patients with a history of copd exacerbations and poor reversibility of airflow obstruction reduces exacerbations in patients who comply with treatment (stockley et al 2006). On the contrary, formoterol, in twice daily doses, failed to show such an effect in two other trials (calverley et al 2003b; szafranski et al 2003), although a subsequent study has documented that it can influence exacerbation surrogates, for example bad days salmeterol has been shown in several model systems to reduce inflammatory cell activation, to reduce formation of edema and to enhance edema clearance, and to mitigate epithelial damage induced by bacteria (rennard 2004). Whether these effects have any clinical importance remains to be determined, but they may account for its ability to reduce frequency of exacerbations of copd . Formoterol may have similar non - bronchodilator effects; however, studies are needed to document these effects . Combining two therapies that possess different modes of action could be expected to have a greater benefit in the management of copd . There is an exciting possibility that the potential benefit in combining icss and labas might be due to a synergistic interaction . However, the basic molecular mechanism of such an interaction has still to be fully identified . Recent in vitro and in vivo evidence suggest a mechanistic interaction at the molecular level between icss and labas . Corticosteroids have been shown to up - regulate the 2-adrenoceptor in the human airways, which in turn may provide more receptors for 2-agonists to activate (mak et al 1995; baraniuk et al 1997). At the same time, labas may potentiate the molecular mechanism of corticosteroid actions by increasing the nuclear translocation of glucocorticoid receptors (grs) and thus causing an additive or sometimes synergistic suppression of inflammatory mediator release (eickelberg et al 1999; korn et al 2001; pang and knox 2000, 2001). Similar interactions between icss and labas in vivo are a likely explanation for the effects seen in the study of barnes and coworkers (2006) documenting that the combination of inhaled salmeterol and fluticasone propionate significantly reduced the absolute numbers of biopsy (cd45) leukocytes, cd8 cells, and cd4 cells together with decreases in cells expressing genes for the proinflammatory mediators ifn- and tnf-. In the complementary airway compartment sampled by induced sputum, combination treatment significantly reduced sputum differential neutrophils and total eosinophils . Furthermore, in vitro studies suggest that the addition of a laba to an ics may counter some of the potential negative effects of the corticosteroid (tse et al 2003) or alternatively, some of those elicited by laba (seeto et al 2003). In addition to the complementary interaction of labas and icss at the molecular level, the delivery of these medications together in a single device may also help simplify treatment regimens and improve patient adherence (cazzola and dahl 2004). It is widely recognized that adherence to treatment of chronic obstructive airways diseases declines as the regime becomes more complicated, either by increasing the number of medications and/or the number of daily doses (coutts et al 1992; chapman et al 2000). As a result, improved compliance would be expected to occur through the use of a single combination inhaler simply because of the reduction in the number of medications and actuations required with such a regime . In particular, it would be expected that the use of combination therapy should improve compliance with ics therapy as its use is linked with laba therapy, which patients are more likely to take because of the greater symptomatic benefit . Because of the above effects, it is not unexpected that long - term use of icss with labas may be associated with a reduction in copd exacerbations (cazzola and dahl 2004). However, the capacity of both icss and labas to reduce the total number of bacteria adhering to the respiratory mucosa in a concentration - dependent manner without altering the bacterial tropism for mucosa, and to preserve ciliated cells, is important in this context (dowling et al 1999). Icss and labas, when administered together at low concentrations, exhibited a synergistic effect with respect to the preservation of ciliated cells, showing a trend toward reduced damage and a significant preservation of the number of ciliated cells compared with either agent alone at the same concentrations . This result may have clinical significance as it is thought that ciliated cells are the most sensitive to damage by bacterial infection (tsang et al 1994). It is well - known that airway colonization and chronic infection contribute to progressive pulmonary damage in copd patients via the action of proinflammatory substances in what is known as the vicious circle theory (wilson 1991). Recently, the synergistic effects of salmeterol and fluticasone in human rhinovirus - induced proinflammatory cytokine production have also been documented (edwards et al 2006). Rhinoviruses are implicated in many acute exacerbations of copd, perhaps by inducing proinflammatory cytokines (seemungal et al 2001). Combining two therapies that possess different modes of action could be expected to have a greater benefit in the management of copd . There is an exciting possibility that the potential benefit in combining icss and labas might be due to a synergistic interaction . However, the basic molecular mechanism of such an interaction has still to be fully identified . Recent in vitro and in vivo evidence suggest a mechanistic interaction at the molecular level between icss and labas . Corticosteroids have been shown to up - regulate the 2-adrenoceptor in the human airways, which in turn may provide more receptors for 2-agonists to activate (mak et al 1995; baraniuk et al 1997). At the same time, labas may potentiate the molecular mechanism of corticosteroid actions by increasing the nuclear translocation of glucocorticoid receptors (grs) and thus causing an additive or sometimes synergistic suppression of inflammatory mediator release (eickelberg et al 1999; korn et al 2001; pang and knox 2000, 2001). Similar interactions between icss and labas in vivo are a likely explanation for the effects seen in the study of barnes and coworkers (2006) documenting that the combination of inhaled salmeterol and fluticasone propionate significantly reduced the absolute numbers of biopsy (cd45) leukocytes, cd8 cells, and cd4 cells together with decreases in cells expressing genes for the proinflammatory mediators ifn- and tnf-. In the complementary airway compartment sampled by induced sputum, combination treatment significantly reduced sputum differential neutrophils and total eosinophils . Furthermore, in vitro studies suggest that the addition of a laba to an ics may counter some of the potential negative effects of the corticosteroid (tse et al 2003) or alternatively, some of those elicited by laba (seeto et al 2003). In addition to the complementary interaction of labas and icss at the molecular level, the delivery of these medications together in a single device may also help simplify treatment regimens and improve patient adherence (cazzola and dahl 2004). It is widely recognized that adherence to treatment of chronic obstructive airways diseases declines as the regime becomes more complicated, either by increasing the number of medications and/or the number of daily doses (coutts et al 1992; chapman et al 2000). As a result, improved compliance would be expected to occur through the use of a single combination inhaler simply because of the reduction in the number of medications and actuations required with such a regime . In particular, it would be expected that the use of combination therapy should improve compliance with ics therapy as its use is linked with laba therapy, which patients are more likely to take because of the greater symptomatic benefit . Because of the above effects, it is not unexpected that long - term use of icss with labas may be associated with a reduction in copd exacerbations (cazzola and dahl 2004). However, the capacity of both icss and labas to reduce the total number of bacteria adhering to the respiratory mucosa in a concentration - dependent manner without altering the bacterial tropism for mucosa, and to preserve ciliated cells, is important in this context (dowling et al 1999). Icss and labas, when administered together at low concentrations, exhibited a synergistic effect with respect to the preservation of ciliated cells, showing a trend toward reduced damage and a significant preservation of the number of ciliated cells compared with either agent alone at the same concentrations . This result may have clinical significance as it is thought that ciliated cells are the most sensitive to damage by bacterial infection (tsang et al 1994). It is well - known that airway colonization and chronic infection contribute to progressive pulmonary damage in copd patients via the action of proinflammatory substances in what is known as the vicious circle theory (wilson 1991). Recently, the synergistic effects of salmeterol and fluticasone in human rhinovirus - induced proinflammatory cytokine production have also been documented (edwards et al 2006). Rhinoviruses are implicated in many acute exacerbations of copd, perhaps by inducing proinflammatory cytokines (seemungal et al 2001). The efficacy of combination therapy with icss and labas delivered via a single inhaler to copd patients has repeatedly been documented (table 1). In particular, the impact on acute exacerbations of long - term treatments with combined therapy is shown in figures 1 and 2, and table 2 . It is very important to also mention that the definition of exacerbation and the patient population enrolled in these studies are not uniform (table 3), and these facts have to be taken in consideration when analyzing their results . In a multicenter, randomized, placebo - controlled study, hanania et al (2003) compared the efficacy and safety of fluticasone propionate (250 g) and salmeterol (50 g), when administered twice daily together in a single device for 6 months, with that of placebo and the individual agents alone in patients with copd . This study was unable to observe significant differences among treatment groups in the number of exacerbations or the time to first exacerbation . However, this study was of short duration and was designed and powered to evaluate the treatment effect on fev1, which was the primary measure of efficacy, rather than to evaluate the rates of exacerbations . In a study with a similar design but evaluating the higher dose formulation of this combination therapy, fluticasone propionate (500 g) with salmeterol (50 g) twice daily given for 6 months (mahler et al 2002), there were no statistically significant differences between treatment groups in time to exacerbation . This study also demonstrated no difference in the number of participants who exacerbated once (placebo: 8.8%; salmeterol: 5.6%; fluticasone: 10.1%; fluticase / salmeterol combination [fsc]: 8.5%), but no p values were presented . Also this study was of short duration and was designed and powered to evaluate the treatment effect on fev1, which was the primary measure of efficacy, rather than to evaluate the rates of exacerbations . The tristan study (calverley et al 2003a), a randomized controlled trial over 1 year of combination treatment with salmeterol (50 g) and fluticasone (500 g) administered together in a single device twice daily versus each of the components alone and versus placebo, enrolled patients with a baseline fev1 before bronchodilation that was 25%70% of that predicted . In this study, the rate of exacerbations requiring medical intervention, defined as a worsening of copd symptoms that required treatment with antibiotics or oral corticosteroids, or both, fell by 25% in the combination group and by 20% and 19% in the salmeterol and fluticasone groups, respectively, compared with placebo . In particular, there was a significant difference in the mean exacerbation rate per participant per year in favor of the combination therapy when compared with placebo (fsc: 0.97 vs placebo: 1.30, p<0.0001), but, interestingly, differences among the three active treatment arms were small and not statistically significant . The treatment effect was more pronounced in patients with severe disease (ie, a baseline fev1 <50% of predicted), who showed a 30% reduction with combination therapy compared with placebo, compared with a 10% reduction in patients who had a baseline fev1 greater than 50% of predicted . However, this beneficial effect was also seen for exacerbations treated with oral corticosteroids, which have conventionally been thought to be more severe episodes . The combination therapy was associated with an approximate 39% reduction in the number of this type of exacerbations compared with placebo (0.46 vs 0.76 exacerbations per year, p<0.0001) but, again, the differences between fsc and salmeterol or fluticasone were not significant . It is worth mentioning that the tristan was longer, but still based on fev1 . Hence, the ability to observe exacerbation differences may have been helped by study duration . In a study using a design similar to that of the tristan study but including patients with an fev1 below 50% predicted, szafranski et al (2003) compared the effects of two inhalations twice daily of either budesonide / formoterol combination (bfc) 160/4.5 g (delivered dose) in a single inhaler, budesonide 200 g (metered dose), formoterol 4.5 g, or placebo for 12 months . Data from this study demonstrate a significant reduction in the number of severe exacerbations by 24% in the bfc group vs placebo, 23% vs formoterol, and 11% vs budesonide . This reflects a mean reduction in the mean annual exacerbation rates of 0.42 (95% ci 0.140.70), 1.841.42 exacerbations per year when combination therapy was used . Compared with placebo, both bfc and budesonide significantly reduced the number of oral steroid courses used in association with exacerbations (31% and 29%, respectively, compared with 3% for formoterol vs placebo). In addition, bfc significantly reduced the number of oral steroid courses compared with formoterol (28%). Mild exacerbations were also significantly reduced on bfc when compared with placebo (62%) and budesonide (35%), but not with formoterol (15%). In another study investigating bfc, calverley et al (2003b) studied patients who initially received formoterol alone (9 g delivered dose twice daily) and a short course of oral prednisolone (30 mg once daily) for 2 weeks before randomization . The patients were subsequently randomized to twice daily inhaled bfc 320/9 g (delivered dose), budesonide 400 g (metered dose), formoterol 9 g, or placebo for 12 months, bfc prolonged time to first exacerbation requiring medical intervention, defined as a need for antibiotics and/or oral corticosteroids and/or hospitalization due to respiratory symptoms, compared with all other treatments . Hazard rate analysis showed that the risk of having an exacerbation while being treated with bfc was reduced by 22.7%, 29.5%, and 28.5% vs budesonide, formoterol, and placebo, respectively . The exacerbation rate with bfc was reduced compared with placebo (23.6%) and formoterol (25.5%) but not with budesonide alone (13.6%). Furthermore, bfc prolonged the time to first course of oral corticosteroids after randomization; risk reductions were 32.7% and 33.8% vs budesonide and formoterol, respectively, and 42.3% vs placebo . Bfc also reduced the rate of oral corticosteroid courses by 28.2%, 30.5%, and 44.7% vs budesonide, formoterol, and placebo, respectively; budesonide alone reduced the number of oral corticosteroid courses compared with placebo but formoterol did not . Data of three large studies (calverley et al 2003a, 2003b; szafranski et al 2003) that have examined the effects of a combination of laba and ics on the risk of developing exacerbation in copd have shown that the patients receiving combination therapy were less likely to report an exacerbation than those receiving the laba alone . Nevertheless, a recent cochrane review (nannini et al 2004) concluded that there were conflicting results when the different combination therapies were compared with the single components alone . In particular, the results of the studies showed that bfc and fsc were effective and reduced the frequency of exacerbations compared with dummy medication to a level of three quarters of the previous rates, with one exacerbation prevented every 24 years in the participants in the clinical trials . When the combination treatment was compared with one of the component drugs given as single treatments, bfc was better than its component laba at preventing the frequency of exacerbations, but fsc did not show a significant advantage over laba . Recently, two different reviews (halpin 2005; cazzola 2006), which have evaluated the applicability and clinical relevance of number needed to treat (nnt) analysis for determining the effectiveness of combination therapies against copd exacerbations, have calculated that for every 100 patients treated with bfc for 1 year, between 42 and 47 exacerbations requiring medical intervention would be prevented vs laba therapy alone, whereas for every 100 patients treated with fsc for 1 year, only 7 exacerbations would be prevented vs laba alone (table 2). It should be noted, however, that the nnt value relating to fsc should be interpreted with caution as there was no significant difference between fsc and salmeterol alone (p=0.345 for treatment ratios for combination vs salmeterol therapy). This means that the hypothetical 95% ci would include infinity and that an nnt of infinity should be more appropriately applied to the difference between the two treatments, which indicates no true net clinical benefit of fsc over salmeterol alone . In any case, in a recent study investigating the effect withdrawal of fluticasone from patients with severe copd (fev1 <50% predicted) using fsc (cosmic study) for 3 months, wouters et al (2005) demonstrated an acute and sustained decrease in fev1, increase in symptoms, and an increase in mild exacerbations in patients treated with salmeterol alone compared with those who continued treatment with fsc over the one year of the study . The conflicting findings between the results of the different studies may be accounted for by dissimilar study designs . In particular, definitions of exacerbation that have been used in the examined studies have been rather different (table 3). For example, in mahler s study (mahler et al 2002), exacerbation was defined by treatment (mild = increased use of salbutamol; moderate = use of either oral antibiotics and/or corticosteroids; severe = hospitalization). In the szafranski s study (szafranski et al 2003), the definition of mild exacerbation was a day with 4 inhalations of reliever medication above the mean run - in use, whereas that of severe exacerbation was a requirement for oral steroids and/or antibiotics and/or hospitalization due to respiratory symptoms . In the tristan study (calverley et al 2003a), exacerbations were defined a priori as a worsening of copd symptoms that required treatment with antibiotics or oral corticosteroids, or both . Pauwels et al (2004) correctly highlighted that considerable heterogeneity in the aetiology and manifestation of copd exacerbations makes identification and quantification of defining symptoms extremely difficult . However, with the emergence of a number of drugs designed or developed specifically to treat copd and associated exacerbations, the absence of a uniform definition is a significant issue . In fact, the choice of definition can significantly affect study outcomes, with varying criteria likely to result in different levels of demonstrated treatment success . Furthermore, emerging data confirm that exacerbation is a term not easily understood by patients, who prefer to use simpler words such as crisis it is also interesting to stress that there was some difference in the patient populations enrolled in the studies described above . Patients enrolled in the tristan study (calverley et al 2003a) had to have suffered from at least one episode of acute copd symptom exacerbation per year in the previous 3 years, and at least one exacerbation in the year immediately before trial entry that required treatment with oral corticosteroids, antibiotics, or both . In the szafranski study (szafranski et al 2003), patients had to have suffered from at least one severe (use of oral corticosteroids and/or antibiotics and/or hospitalization due to respiratory symptoms) copd exacerbation within 212 months before the first clinic visit . Moreover, the mean pre - trial fev1 was 45% predicted in the tristan study (calverley et al 2003a) and 36% predicted in the szafranski s study (szafranski et al 2003). Moreover, the mean total exacerbation rate per patient per year under placebo was 1.30 in the tristan study (calverley et al 2003a) and 1.9 in the szafranski study (szafranski et al 2003). This differences indicate that the szafranski s study (szafranski et al 2003) probably enrolled patients with more severe disease or, at least, with a higher possibility of suffering from acute exacerbation . In fact, also in the calverley study (calverley et al 2003b), the mean pre - trial fev1 was 36% predicted and the mean total exacerbation rate per patient per year under placebo was 1.80 . As correctly highlighted by calverley et al (2003b), the more severe disease in the patients enrolled in bfc studies is the likely explanation of the greater number of episodes that they observed compared with other studies, a difference that increased the power of the study to detect an effect of treatment . It is also important to highlight that the authors of the three studies did not report the number of exacerbations in the year before the enrolment in the studies . This is a real lack of information because, as recently documented by donaldson et al (2003), annual exacerbation frequency remains constant over time . As rabe (2003) stated in his editorial comment on the calverley paper (calverley et al 2003b), the data of the combination therapy trials in copd seem to indicate that patients experiencing more severe exacerbations benefit the most from combination therapy, whereas, if the definition of a (mild) exacerbation for clinical trials also includes aggravation of symptoms, such as in the paper by szafranski et al (2003), the relative effect of a long - acting bronchodilator might be more pronounced . This clearly highlights the importance of definitions of exacerbations for clinical trials and calls for studies comparing the effect of maximal bronchodilation with, for example, the combination of labas with long - acting anticholinergics (lamas) in copd with mild and severe exacerbations as an outcome . In effect, some studies are documenting the clinical value of combining a laba with a lama in copd (cazzola et al 2004; van noord et al 2005, 2006), although the information of the impact of such a type of combination on exacerbations is still lacking . Borrowing jones opinion (jones 2004), we believe that there are a number of possible mechanisms to account for the apparent additive effect of inhaled combination therapy with icss and labas in preventing exacerbations and to explain the discrepancies observed between the studies . It is likely that: (1) the individual drugs were not at the top of their dose response curve for this outcome; (2) they have different, and additive, mechanisms of action, which can also be linked to the specific compound; or (3) the different agents were preventing different types of exacerbation . Jones (2004) has highlighted that an operational definition based upon a worsening of symptoms and the treatment required (moderate exacerbations are those that can be treated with oral corticosteroids and/or antibiotics, severe exacerbations are those requiring hospital admission) does not identify the type of exacerbation . This emphasizes again the importance for the need of a uniform definition of copd exacerbation for clinical trials in order to reach more solid conclusions . Whatever the case may be, it must be emphasized that a treatment regimen with administration of an ics and laba in a single inhaler prolongs the time to a first copd exacerbation, compared with treatment with the single components in patients with an fev1 <50% predicted (calverley et al 2003b). Intriguingly, this finding has been observed with bfc (calverley et al 2003b), but not with fsc (calverley et al 2003a). The prolonged time to first exacerbation may delay the deterioration of the disease and help maintain health status and patient well - being . It is an important outcome because it indicates that following aggressive treatment of an acute exacerbation with an oral corticosteroid, therapy with an ics / laba combination may better control the disease than treatment with bronchodilator alone . In particular, as highlighted by calverley (2004), combining treatments is certainly something to be considered in patients who have reported exacerbations on a regular basis but have not yet received ics or laba therapy in combination, although there are concerns about the increased risk of side - effects and cost of using laba / ics therapy in copd . Data of three large studies (calverley et al 2003a, 2003b; szafranski et al 2003) that have examined the effects of a combination of laba and ics on the risk of developing exacerbation in copd have shown that the patients receiving combination therapy were less likely to report an exacerbation than those receiving the laba alone . Nevertheless, a recent cochrane review (nannini et al 2004) concluded that there were conflicting results when the different combination therapies were compared with the single components alone . In particular, the results of the studies showed that bfc and fsc were effective and reduced the frequency of exacerbations compared with dummy medication to a level of three quarters of the previous rates, with one exacerbation prevented every 24 years in the participants in the clinical trials . When the combination treatment was compared with one of the component drugs given as single treatments, bfc was better than its component laba at preventing the frequency of exacerbations, but recently, two different reviews (halpin 2005; cazzola 2006), which have evaluated the applicability and clinical relevance of number needed to treat (nnt) analysis for determining the effectiveness of combination therapies against copd exacerbations, have calculated that for every 100 patients treated with bfc for 1 year, between 42 and 47 exacerbations requiring medical intervention would be prevented vs laba therapy alone, whereas for every 100 patients treated with fsc for 1 year, only 7 exacerbations would be prevented vs laba alone (table 2). It should be noted, however, that the nnt value relating to fsc should be interpreted with caution as there was no significant difference between fsc and salmeterol alone (p=0.345 for treatment ratios for combination vs salmeterol therapy). This means that the hypothetical 95% ci would include infinity and that an nnt of infinity should be more appropriately applied to the difference between the two treatments, which indicates no true net clinical benefit of fsc over salmeterol alone . In any case, in a recent study investigating the effect withdrawal of fluticasone from patients with severe copd (fev1 <50% predicted) using fsc (cosmic study) for 3 months, wouters et al (2005) demonstrated an acute and sustained decrease in fev1, increase in symptoms, and an increase in mild exacerbations in patients treated with salmeterol alone compared with those who continued treatment with fsc over the one year of the study . The conflicting findings between the results of the different studies may be accounted for by dissimilar study designs . In particular, definitions of exacerbation that have been used in the examined studies have been rather different (table 3). For example, in mahler s study (mahler et al 2002), exacerbation was defined by treatment (mild = increased use of salbutamol; moderate = use of either oral antibiotics and/or corticosteroids; severe = hospitalization). In the szafranski s study (szafranski et al 2003), the definition of mild exacerbation was a day with 4 inhalations of reliever medication above the mean run - in use, whereas that of severe exacerbation was a requirement for oral steroids and/or antibiotics and/or hospitalization due to respiratory symptoms . In the tristan study (calverley et al 2003a), exacerbations were defined a priori as a worsening of copd symptoms that required treatment with antibiotics or oral corticosteroids, or both . Pauwels et al (2004) correctly highlighted that considerable heterogeneity in the aetiology and manifestation of copd exacerbations makes identification and quantification of defining symptoms extremely difficult . However, with the emergence of a number of drugs designed or developed specifically to treat copd and associated exacerbations, the absence of a uniform definition is a significant issue . In fact, the choice of definition can significantly affect study outcomes, with varying criteria likely to result in different levels of demonstrated treatment success . Furthermore, emerging data confirm that exacerbation is a term not easily understood by patients, who prefer to use simpler words such as crisis it is also interesting to stress that there was some difference in the patient populations enrolled in the studies described above . Patients enrolled in the tristan study (calverley et al 2003a) had to have suffered from at least one episode of acute copd symptom exacerbation per year in the previous 3 years, and at least one exacerbation in the year immediately before trial entry that required treatment with oral corticosteroids, antibiotics, or both . In the szafranski study (szafranski et al 2003), patients had to have suffered from at least one severe (use of oral corticosteroids and/or antibiotics and/or hospitalization due to respiratory symptoms) copd exacerbation within 212 months before the first clinic visit . Moreover, the mean pre - trial fev1 was 45% predicted in the tristan study (calverley et al 2003a) and 36% predicted in the szafranski s study (szafranski et al 2003). Moreover, the mean total exacerbation rate per patient per year under placebo was 1.30 in the tristan study (calverley et al 2003a) and 1.9 in the szafranski study (szafranski et al 2003). This differences indicate that the szafranski s study (szafranski et al 2003) probably enrolled patients with more severe disease or, at least, with a higher possibility of suffering from acute exacerbation . In fact, also in the calverley study (calverley et al 2003b), the mean pre - trial fev1 was 36% predicted and the mean total exacerbation rate per patient per year under placebo was 1.80 . As correctly highlighted by calverley et al (2003b), the more severe disease in the patients enrolled in bfc studies is the likely explanation of the greater number of episodes that they observed compared with other studies, a difference that increased the power of the study to detect an effect of treatment . It is also important to highlight that the authors of the three studies did not report the number of exacerbations in the year before the enrolment in the studies . This is a real lack of information because, as recently documented by donaldson et al (2003), annual exacerbation frequency remains constant over time . As rabe (2003) stated in his editorial comment on the calverley paper (calverley et al 2003b), the data of the combination therapy trials in copd seem to indicate that patients experiencing more severe exacerbations benefit the most from combination therapy, whereas, if the definition of a (mild) exacerbation for clinical trials also includes aggravation of symptoms, such as in the paper by szafranski et al (2003), the relative effect of a long - acting bronchodilator might be more pronounced . This clearly highlights the importance of definitions of exacerbations for clinical trials and calls for studies comparing the effect of maximal bronchodilation with, for example, the combination of labas with long - acting anticholinergics (lamas) in copd with mild and severe exacerbations as an outcome . In effect, some studies are documenting the clinical value of combining a laba with a lama in copd (cazzola et al 2004; van noord et al 2005, 2006), although the information of the impact of such a type of combination on exacerbations is still lacking . Borrowing jones opinion (jones 2004), we believe that there are a number of possible mechanisms to account for the apparent additive effect of inhaled combination therapy with icss and labas in preventing exacerbations and to explain the discrepancies observed between the studies . It is likely that: (1) the individual drugs were not at the top of their dose response curve for this outcome; (2) they have different, and additive, mechanisms of action, which can also be linked to the specific compound; or (3) the different agents were preventing different types of exacerbation . Jones (2004) has highlighted that an operational definition based upon a worsening of symptoms and the treatment required (moderate exacerbations are those that can be treated with oral corticosteroids and/or antibiotics, severe exacerbations are those requiring hospital admission) does not identify the type of exacerbation . This emphasizes again the importance for the need of a uniform definition of copd exacerbation for clinical trials in order to reach more solid conclusions . Whatever the case may be, it must be emphasized that a treatment regimen with administration of an ics and laba in a single inhaler prolongs the time to a first copd exacerbation, compared with treatment with the single components in patients with an fev1 <50% predicted (calverley et al 2003b). Intriguingly, this finding has been observed with bfc (calverley et al 2003b), but not with fsc (calverley et al 2003a). The prolonged time to first exacerbation may delay the deterioration of the disease and help maintain health status and patient well - being . It is an important outcome because it indicates that following aggressive treatment of an acute exacerbation with an oral corticosteroid, therapy with an ics / laba combination may better control the disease than treatment with bronchodilator alone . In particular, as highlighted by calverley (2004), combining treatments is certainly something to be considered in patients who have reported exacerbations on a regular basis but have not yet received ics or laba therapy in combination, although there are concerns about the increased risk of side - effects and cost of using laba / ics therapy in copd.
Antibiotics are among the most frequently used pharmaceuticals in both the inpatient and outpatient setting . Cephalosporins are grouped in b - lactam class of antibiotics, along with penicillins and carbapenems . While these antimicrobial agents are generally well tolerated, these drugs are not without their associated side effects, and neurotoxic effects are perhaps much less recognized . Neurotoxicity of cephalosporins has been reported with first generation cephalosporins such as cefazolin, second generation such as cefuroxime, third generation such as ceftazidime and fourth generation such as cefepime and can range from slurred speech, tremor, seizures and ncse.1 the threshold of neurotoxicity is decreased in settings of reduced creatinine clearance, impaired renal function, pre - existing cns conditions, and with use of third and fourth generation cephalosporines.2 ncse is a conditions in which electrographic seizure activity is prolonged and results in non - convulsive clinical symptoms, usually lasting> 30 min.3 cephalosporines, particularly cefepime, have been associated with ncse, especially in adult patients with impaired renal function . We present a case of 71-year - old women who developed ncse after use of cefepime after recovering from acute renal failure . A 71-year - old woman with a history of diabetes mellitus and liver cirrhosis associated with hepatitis b viral infection was hospitalized for right lower lobe lobectomy after diagnosis of lung cancer . Although she had successful lobectomy, she underwent several post - operative complication including operation site bleeding, acute renal failure, acute respiratory distress syndrome, and atypical pneumonia . Her renal failure was prerenal type after massive operation site bleeding, and continuous renal replacement therapy (crrt) were started for renal replacement treatment . Post operation (op) day 9, she experienced confusion, and disorientation and started on quetiapine with diagnoses of delirium . Post op day 13, she had antibiotic resistance for atypical pneumonia, which had to switch from imipenem and vancomycin to cefepime . Although her serum creatinine level was much improved from 2.7 mg / dl to 1.87 mg / dl, after 5 days of renal replacement therapy, she was started on cefepime renal dose of 2 g every 12h . Plasma concentration of cefepime was not measured because the analytical technique was not available in our hospital . After 5th day of cefepime use, the neurologic examination revealed change in mental status, and minimal response to noxious pain stimulation . The brain magnetic resonance imaging (mri) including diffusion and gradient echo images revealed no acute lesions which can explain sudden mental status change . Serum blood cell counts, glucose, ammonia and electrolytes were within the normal range . Renal profile showed blood urea nitrogen (bun) of 27 mg / dl (normal range: 725 mg / dl), creatinine of 1.33 mg / dl, creatinine clearance (ccr) of 25.35 ml / min/1.73 m . The electroencephalograph (eeg) revealed continuous 23 hz generalized sharp and wave (fig . Ncse was diagnosed and administered lorazepam 4 mg intravenous injection with continuous eeg monitoring were started . She was than further treated with levetiracetam 500 mg and valproic acid 900 mg intravenously . She stay on maintenance doses of valproic acid (900 mg / day) and phenytoin (300 mg / day), clonazepam (1.5 mg / day). 2). As we discontinued cefepime, considering as a causative agent for ncse, clinical symptoms improved 3 days after discontinuation of cefepime . Alteration of mental status in patients with post - operative complications and medical illnesses may have potential causes of toxic metabolic alterations, infections, hypoxia, cerebral stroke or ncse, etc . Cefepime as a causative agent was derived by similar eeg finding of cefepime induced ncse of previous reports.4,5 and temporal relationship of consciousness change, with onset of clinical symptom in 5 days after the administration of cefepime, and improvement in 3 days after the withdrawal . The typical time period for encephalopathy induced by cephalosporin use is a latency of 1 to 10 days following start of medication, and resolution in 2 to 7 days following discontinuation.6 the clinical symptoms other than consciousness changes have been reported including myoclonic seizure, abnormal behavior, mutism, ataxia, asterixia, hallucinations, tremor, clonus, and hyperreflexia . The brief myoclonic seizure preceding ncse, as in our patient, has been seen in 9 of 25 case reports.4 cefepime is a fourth - generation cephalosporin which is bactericidal for a broad spectrum of organisms, including pseudomonas aeruginosa.7 cefepime is predominantly cleared by renal excretion and the half - life of cefepime in adults with normal renal function is approximately 2 hours.8 the neurotoxic effects of cephalosporins can range from slurred speech, tremor, confusion, aphasia, agitation, coma, myoclonus, seizure, to ncse.5 the ncse, in particular has been reported in patients with renal impairment and with use of third and fourth generation agents such as ceftazidime, ceftriaxone, and cefepime.9,10 the mechanism of cefepime - induced ncse appears to be r - aminobutyric acid (gaba)-a receptor antagonism, reducing the gaba mediated inhibitory response, therefore generating a pro - epileptogenic activity.11 since the first description in 1945, several reports of penicillin - induced neurotoxicity involving adults have been reported . Mostly those with acute or chronic renal failure . Pathogenesis of neurotoxicity in patients with renal impairment appears to be mediated by rise in serum concentrations, increased permeability of the blood - barrier as well as buildup of toxic organic acids within the cerebrospinal fluid.12 increased circulating unbound antibiotic also contributes to the vulnerability of patients to cns toxicity, especially in renal impairment.13 with much emphasis on renal function to neurotoxic effects of cefepime, ncse have been reported mostly in patients with acute or chronic renal failure with only one case report with normal renal failure.4 however, even in this case of normal renal function, subtle renal impairment was seen with below normal range of creatinine clearance of 44.47 ml / kg / min.14 our case is an example of ncse due to neurotoxicity of cephalosporin at therapeutic dose . And it is different from previous cases in the aspect that renal function was recovered in our patient and her creatinine level was within normal limit . In previous cases, thus, our case suggest more strongly that cefepime induced neurotoxicity may occur in patients with renal dose treatment and with recent history of acute renal failure . In conclusion, awareness of the potential neurotoxic clinical manifestations of various antibiotics and high degree of vigilance in critically ill patients is essential in identifying potentially serious though reversible complications of antibiotic therapy particularly with the advent of newer antimicrobial agents.
Gastric cancer (gc) is the fourth most common cancer and the second leading cause of cancer death in the world [1, 2]. The principal risk factors in development of gc are helicobacter pylori infection, atrophic gastritis, intestinal metaplasia, dysplasia, male gender, cigarette smoking, partial gastrectomy, menetrier's disease, and genetic factors . Global incidence of primary tumour locations and the histological types are constantly changing: in united states and in western europe the incidence of esophagogastric junction (barrett's type) and gastric cardia adenocarcinoma is increasing while there has been a reduction of incidence of distal gc since the 1970s, especially in western countries . Although gc mortality has been reduced, it remains a disease with poor prognosis and high mortality, second only to lung tumour . The prognosis of gc depends on stage and location: proximal gastric tumours (i.e., cardia tumor) have poorer prognosis compared to those in the pyloric antrum and when the disease is confined to the stomach mucosa, 5-year survival is near to 95%, while the reported 5-year survival rate for advanced gc varies from 10 to 20% . Metastatic dissemination in gc may occur through the hematic torrent or by dissemination to the peritoneal cavity; this last condition is called peritoneal carcinomatosis (pc), and it is considered a stage iv of gc . Only 40% of gc deaths have hepatic metastases, while in 5360% disease evolves through pc . Pc is considered the end stage of primary peritoneal malignant disorders (such as peritoneal mesothelioma) and a common manifestation of digestive - tract and gynaecological advanced cancers (such as appendicle tumour, ovarian cancer, colorectal cancer, or gastric cancer). It is generally associated with a poor prognosis; patients with pc of gastric origin have an extremely bad prognosis with a median survival estimate at 13 months [3, 14]. Data from the literature show that 15% of patients present pc ab initio and 35% of patients die of intraperitoneal recurrence for pc confined exclusively into the peritoneum . Systemic chemotherapy improves median survival in metastatic gastric cancer to 710 months, but in patients with pc from gc the same improvement has not been reported . Currently, at the intraoperative abdominal examination, peritoneal seeding is found in 1020% of patients scheduled for potentially curative resection and in 40% of those at stage ii - iii [15, 18, 19]; 2050% of patients treated with radical surgery will develop postoperatory peritoneal recurrence, and intraperitoneal spread of tumour cells is observed in 54% of patients who died of recurrence after surgery in advanced gc . During the last 30 years multimodal therapeutic approaches on pc improved, resulting in a modified role of surgery: not a simple debulking operation anymore but a complete tumour cytoreduction with no macroscopic residual disease . Sugarbaker investigated the synergism of the effects of hyperthermia and intraperitoneal anticancer chemotherapy against tumour cells; he found the existence of pc originating by low grade malignancy tumours without invasion capacity (like pseudomyxoma peritonei) that can be treated with cytoreductive surgery (crs) and hyperthermic intraperitoneal chemotherapy (hipec). In 1995 sugarbaker definitively codified, in terms of rationale and surgical technique, the procedure of peritonectomy . Following these innovative studies, peritoneal dissemination of free cancer cells happens through exfoliation and leads to direct invasion of the serosa . Tumour cells can also diffuse passing through the stomata: big communicating orifices present in the peritoneal surface, between peritoneal cavity and lymphatic vessels . Cells distribution into peritoneal cavity is also conditioned by physical factors: tumour primary site, effects of gravity, presence or presence of fluids (ascites, mucus, etc . ), and intrinsic biological aggressiveness [2325]. Some studies showed that there are tumours with a distinct capability to give peritoneal metastases, without giving distant metastases . For preoperative diagnosis of pc, useful imaging techniques are ultrasound (us), computerized tomography (ct), magnetic resonance imaging (mri), and 18f - fluorodeoxyglucose positron emission tomography - ct (fdg pet - ct), but all these imaging techniques have major limitations in diagnosing pc because of the low - volume density of peritoneal nodules . Ct and mri are important mainly in evaluating unresectable disease and cancer staging [26, 27]. Pet - ct seem to be a good option, but are expensive and have drawbacks for lesions smaller than 5 mm in diameter . Concerning pc from gc, yang et al . Report an accuracy of pet - ct of 87%, with a sensitivity and specificity of 72.7% and 93.6%, respectively, with a sensitivity better than ct, while for primary gc and lymph node metastases the accuracy for pet - ct is 54% . Ct is not accurate (817% of sensitivity) particularly for malignant granulations less than 5 mm in diameter and for small bowel nodulations . Due to the low accuracy given by the imaging, the main diagnosing methods currently used to evaluate peritoneal surface are diagnostic laparoscopy or laparotomy and peritoneal cytological examination that show a greater accuracy in diagnosing pc . Diagnostic laparoscopy, with or without peritoneal washing for malignant cells, has only a iii b degree of recommendation; it is used to exclude metastatic disease in tumours that are considered potentially resectable . A standardized technique minimizes the risk of tumour contamination of the trocars insertion sites; this method, compared with laparotomy, is also free from risks related to the complications of diagnostic laparotomy and allows staging before and after crs + hipec and during the follow - up . Moreover, exploratory laparoscopy could be used in order to evaluate the efficacy of neoadjuvant chemotherapy . The three main different scoring systems for intraperitoneal cancer dissemination that have been published until today are as follows.japanese rules of gc [14, 29] is a classification into five categories that only considers the presence of cancerous implants and/or of malignant cells in the peritoneal washing fluid, without considering the size of malignant nodules.gilly staging system for pc [14, 19], also called the lyon score, is based on the size and distribution of malignant granulations (localized or diffused). It demonstrates that the use of complete (r0-r1) or incomplete (r2) cytoreduction, in order to assess the entirety of surgical clearance of cancer, is successful . R0 and r1 can be grouped together as the outcome of these two groups is very similar . This system was also revealed to be an important prognostic indicator, as the median survival of patients with stage i or ii is significantly higher than those with stage iii or iv, and they can be candidates for crs and hipec . However, this system does not clearly indicate the potential resectability of pc .jacquet and sugarbaker peritoneal cancer index (pci) is based on quantitative distribution and size of peritoneal nodules . The abdomen cavity is divided into 13 regions and the lesion size is scored in each region . After a meticulous intraoperative inspection, the extent of the disease can be summed as a numerical score (0 to 39). The pci has a prognostic value, allowing for an estimate of the probability of complete cytoreduction; it is the only method that shows in detail the nodules localization . Japanese rules of gc [14, 29] is a classification into five categories that only considers the presence of cancerous implants and/or of malignant cells in the peritoneal washing fluid, without considering the size of malignant nodules . Gilly staging system for pc [14, 19], also called the lyon score, is based on the size and distribution of malignant granulations (localized or diffused). It demonstrates that the use of complete (r0-r1) or incomplete (r2) cytoreduction, in order to assess the entirety of surgical clearance of cancer, is successful . R0 and r1 can be grouped together as the outcome of these two groups is very similar . This system was also revealed to be an important prognostic indicator, as the median survival of patients with stage i or ii is significantly higher than those with stage iii or iv, and they can be candidates for crs and hipec . Jacquet and sugarbaker peritoneal cancer index (pci) is based on quantitative distribution and size of peritoneal nodules . The abdomen cavity is divided into 13 regions and the lesion size is scored in each region . After a meticulous intraoperative inspection, the extent of the disease can be summed as a numerical score (0 to 39). The pci has a prognostic value, allowing for an estimate of the probability of complete cytoreduction; it is the only method that shows in detail the nodules localization . The completeness of cytoreduction (ccr) could be evaluated using the sugarbaker and the lyon scores combined, as they indicate a direct relation between ccr, prognosis, and survival . Pharmacokinetic and peritoneal permeability studies demonstrate a higher intraperitoneal concentration of drugs with chemotherapy administered intraperitoneally than with systemic administration [23, 24]. The peritoneal plasma barrier maintains a positive gradient of chemotherapy in the peritoneum, increasing the local effects of the drugs and reducing the systemic toxicity . Moreover, when chemotherapy treatment is associated with hyperthermia, the locoregional effects are considerably extended, with an increased penetration up to 36 mm into malignant nodules and an increased antimitotic effect . Several studies confirm that hyperthermia (42 - 43c) enhances the effects of antitumoral drugs, especially of oxaliplatin, mitomycin c, doxorubicin, cisplatin, paclitaxel, and irinotecan, also increasing the chemosensibility of neoplastic cells . The intraabdominal temperature, however, should not exceed the temperature of 43c, in order to avoid the risk of bowel perforation . According to surgical - oncologic principles, the treatment of non - metastatic gc consists of resection with total gastrectomy and d1 and/or d2 lymphadenectomy . Different trials proposed multiple chemotherapy protocols using drugs like epirubicine, cisplatin and 5-fluorouracil (ecf), or epirubicin, cisplatin and capecitabina (ecx), that do not need a central venous access device . S-1, a new drug recently introduced in japan, combined with cisplatinum, paclitaxel, docetaxel or irinotecan has become the standard treatment for pc from gc . Correct radiological, clinical, and cytological stadiation is essential requirement for a better prognosis after hipec in gc . It is necessary to distinguish pc in early or advanced gc from pc as a recurrence of already operated gc . In fact, in the former case it is easier to succeed with a complete cytoreduction (ccr-0, r0), while in the latter, previous surgical treatment and adhesion development decrease the possibility to achieve a complete cytoreduction . Common contraindications for hipec are age> 70 yrs, important comorbidities, clinical aggravation with systemic chemotherapy, malnutrition, extra abdominal metastases, liver metastases when unresectable, and massive retroperitoneal bulk disease or lymph node involvement . Other minor exclusion criteria are body mass index (bmi)> 40, history of pelvic irradiation, carcinomatosis extended at the ct or clinically significant, more than 4 surgical procedures, occlusion, and no drop markers with neoadjuvant chemotherapy . Different studies presented contrasting data about survival rates; however, they all agreed with the necessity of a complete (tables 1 and 2) cytoreduction to improve survival . Show that in patients with a cc (completeness of cytoreduction) score of 0 or 1 overall median survival was 15 months (versus 7.9 months in patients with cc 2 score), with an overall mortality rate of 4.8% . Gc randomized into 3 groups (hipec and surgery, intraperitoneal normothermic chemotherapy plus surgery, and surgery alone) show that in the first group the survival rate was significantly higher (61% versus 43% and 42% of the other two groups), particularly in patients with serosal infiltration and lymph node positive metastases . Ii - iii gc patients, and by kim and bae in patients affected by stage iii and iv gc treated with hipec + crs versus surgery alone . Kim and bae analysed 103 patients with gc stage iii - iv: 51 underwent surgical resection alone and 52 received surgery plus hipec . It was higher but not statistically significant in the hipec + crs group (32.7% versus 27.1% control group). The difference, considering exclusively the survival rate of the 65 patients with stage iii gc, was statistically significant (58.6% versus 44.4% control group). In a retrospective multicentric french study undertaken between february 1989 and august 2007, glehen et al . Evaluated 159 patients that underwent cytoreductive surgery and perioperative intraperitoneal chemotherapy (hipec and epic or both) and showed 1-, 3-, and 5-year survival rates of 43%, 18%, and 13%, respectively, that increase up to 61%, 30%, and 23%, respectively, in patients with a complete cytoreduction . Thanks to multivariate analysis, the authors reported the completeness of cytoreduction as being the principal independent prognostic factor . In order to correctly execute cytoreduction, the study showed that if cytoreductive surgery does not allow a sufficient downstaging, particularly in hipec, the survival rates are poor (median survival of 68 months). Three recent meta - analysis [1113] of rcts, assessing patients with gc (with or without pc), demonstrates the survival benefit offered by hipec . Already in 2007, yan et al . Conducted 13 studies on 1648 patients and demonstrated the positive effects in terms of overall survival rates, when adding hipec or hipec with epic to surgery . Jin - yu et al . Analyzed 15 rcts and demonstrated that hipec and niic should be recommended in treating patients with gc, as they significantly improve the overall survival rates . This meta - analysis demonstrated that adding postoperative intraperitoneal chemotherapy (pic) to hipec has no additional effect on overall survival rates but it improves costs and toxicity . This study also showed that intraperitoneal chemotherapy (ipc) has no effect on prevention of lymph metastasis, but could decrease by 73% the rate of hepatic metastasis . Authors demonstrated that ipc does not increase perioperative mortality and postoperative anastomotic leaks, ileus, or bowel perforation rates, but it increases the risk of marrow depression, intra - abdominal abscess, and fever . In the last decade an interesting new drug, called catumaxomab, has been developed in germany . The catumaxomab is a rat - mouse hybrid monoclonal antibody that is now used for patients with malignant ascites in phase ii / iii randomized trial . Two studies [32, 33] demonstrate that this drug seems to improve progression - free survival in patients with gc (median 71 versus 44 days; p = 0.03) and that it seems to improve the survival in gastrointestinal epcam+ tumours (epcam: antiepithelial cell adhesion molecule) in intraperitoneal use . Crs and hipec are associated with significant morbidity and mortality, also in high volume centers, and reported rates are included between 0.95.8% and 1252%, respectively . The main postoperative complications after crs and hipec are intra - abdominal abscess, gastric or small intestinal perforation, postoperative ileus, anastomotic leakage, postoperative bleeding, fistula, sepsis, respiratory distress, hematologic toxicity, and urinary disturbance [6, 19]. In the same group of patients, the main causes of death include anastomotic leakage, sepsis, postoperative bleeding, intestinal fistula, and disseminated intravascular coagulation (dic). Recently, yonemura et al . [9, 14] proposed a multimodal strategy that associates neoadjuvant intraperitoneal and systemic chemotherapy (nips), crs + hipec and epic . The rationale of this method is to reduce tumour burden before surgery with nips, a bidirectional chemotherapy that attacks pc from both sides of peritoneum (from the peritoneal cavity and from subperitoneal blood vessels), together with crs and hipec, in order to reduce macroscopic and microscopic pc . The aim of epic is then to eradicate residual intraperitoneal cancer cells before the development of adhesions . The nips technique is characterized by a first phase in which patients are treated with 60 mg / m of oral s-1 for 21 days, followed by one week of rest . A port system has been previously placed into the abdominal cavity under local anesthesia, with the tip in the douglas pouch . On the 1st, 8th, and 15th postoperative days, 30 mg / m of taxotere and 30 mg / m of cisplatinum with 500 ml of saline are infused into the peritoneal cavity . Complications after nips have been reported in 4 out of 79 patients (1 with grade 4 of bone marrow toxicity, 3 with a renal dysfunction). In 3 patients, infections around the periportal space, that led to the port remotion, this study shows a washing cytology negativization in 41 out of 79 patients (63%)., in a retrospective multicentre study, recommend the routine use of neoadjuvant chemotherapy to improve surgical results and to exclude patients who do not respond to the therapy form hipec treatment . The aforementioned procedures should be exclusively performed in highly experienced centres because of the special surgical expertise needed to achieve high rates of complete cytoreduction [14, 19]. Patient selection is very crucial and should be carried out by a multidisciplinary group of specialists (anaesthetists, surgeons, clinicians, and oncologists) in order to achieve better results and to reduce the high costs related to these procedures and relevant complications . An interdisciplinary approach should be developed further: the association of nips, crs, hipec, and epic could increase the rate of cc-0 procedures and consequently survival rates, particularly for pci 6 [5, 11, 21, 32]. Neoadjuvant chemotherapy, routinely recommended for management of gc without pc, may improve survival also in pc from gc [10, 3437] and adjuvant chemotherapy could prevent recurrence from gc . Finally, the study of molecular and serum tumour markers could provide valuable prognostic information and would allow for a better selection of subsequent treatment combinations [14, 38].
Genome reduction in bacteria is usually associated with a genome - wide shift toward increased at content (moran 2002; bentley and parkhill 2004; mccutcheon et al . 2009). This pattern is especially pronounced in bacteria that live exclusively in the cytoplasm of host cells; for example, the two most extremely at - biased bacterial genomes yet reported are from the insect nutritional endosymbionts candidatus zinderia insecticola (13.5% gc) (mccutcheon and moran 2010) and candidatus carsonella ruddii (16.5% gc) (nakabachi et al . 2006). First, endosymbionts tend to lose genes involved in dna repair and recombination during genome reduction (dale et al . Second, endosymbionts have small effective population sizes and decreased rates of recombination, which reduces the efficacy of selection and allows more slightly deleterious mutations to be fixed by random genetic drift (moran 1996; woolfit and bromham 2003). Combined with what seems to be an at mutational bias in bacteria lacking dna repair enzymes (lind and andersson 2008), these forces are thought to shift the gc at equilibrium toward at in endosymbiont genomes . Until recently, empirical data from complete bacterial genomes universally supported this hypothesis . Remarkably, the only known exceptions to this trend are from the two bacteria with the smallest reported genomes: candidatus hodgkinia cicadicola (hereby referred to as hodgkinia for simplicity, 144 kb, 58.4% gc; mccutcheon et al . 2009) and candidatus tremblaya princeps (tremblaya, 138 kb, 58.8% gc; mccutcheon and von dohlen 2011; lpez - madrigal et al . Hodgkinia is a member of the alphaproteobacteria, a group in which most free - living members have gc - rich genomes and most obligate intracellular members have reduced genomes that show the expected decrease in gc content (mccutcheon et al . These observations led to the hypothesis that the high gc content of hodgkinia resulted from the retention of a gc mutational bias that was present in its free - living alphaproteobacterial ancestor (mccutcheon et al . 2009). That the gc content at third positions of 4-fold degenerate codons (gc4) in hodgkinia is higher than the overall gc content in the genome (62.5% vs. 58.4%) seemed to support this hypothesis, as these positions are expected to be under little or no selection for protein - coding sequence and were therefore thought to more clearly reflect the mutational biases inherent in hodgkinia's replication machinery (mccutcheon et al . Two recent reports provide evidence that an at mutational bias exists in all bacteria (hershberg and petrov 2010; hildebrand et al . 2010). The authors of both papers conclude that selection for increased gc content, or a selection - like process such as biased gene conversion, is the most likely explanation for the diverging patterns of at - biased mutation and gc - biased substitution observed in most bacterial genomes (hershberg and petrov 2010; hildebrand et al . Both papers also single out hodgkinia as an outlier and a possible exception to this rule (hershberg and petrov 2010; hildebrand et al . 2010). To help clarify the roles of mutational biases and selection on the gc content of the hodgkinia genome, we sought to determine the direction of hodgkinia's mutational bias (if any) from existing population data generated during genome sequencing . The published hodgkinia genome was generated by combining samples from 10 wild - caught individuals of the cicada diceroprocta semicincta (mccutcheon et al . We reasoned that it might be possible to calculate mutational patterns from these population genomic data . We first reconfirmed that the pooled sample was from a single species of cicada by verifying a low level of sequence polymorphism in its mitochondrial cytochrome c oxidase i (coi) gene (about 0.6% of 815 sites were polymorphic, well within the 12% divergence levels typically seen in conspecific pairs of animal coi sequences; hebert et al . We then calculated the number of single nucleotide polymorphisms (snps) in the hodgkinia genome falling into all possible nucleotide change categories and found that the majority of the mutations (115 of 179 or 64%) were in the gc to at direction . (the tremblaya genome was generated from only three lab - reared insects, and no high - quality snps were observed in these data .) To unambiguously assign a mutational direction to these snps, we used a draft hodgkinia genome assembly from a closely related but undescribed cicada species (referred to here as the cryptic species) as an outgroup to verify the ancestral state of each position where a snp was identified (for a complete description of the methods, see supplementary materials online). The pairwise nucleotide divergence between partial mitochondrial coi sequences from diceroprocta semicincta and the cryptic species was 3.5% . Of the 179 snps initially identified, 12 were not covered by contigs from the cryptic species . These 12 were removed from the data set, resulting in 167 snps in which the direction of mutation could be confidently determined (fig . 1 and table 1; table s1, supplementary materials online). The expected equilibrium gc content (gceq) given the mutational patterns observed in the polarized data is 42%, significantly lower than the observed genomic value of 58% (table 1). Raw snp counts, mutation rates, and expected gc equilibrium values for synonymous (s), nonsynonymous (ns), and intergenic (ig) sites note.the numbers in parentheses are 95% confidence intervals; significant values are bolded . The values do not sum to 167 because five snps did not alter the gc content (i.e., a g to c mutation). The majority of snps in the hodgkinia genome are g to a or c to t transitions and collectively show a pronounced at mutational bias . Snps are shown as a percentage of the total number in each category (synonymous, nonsynonymous, and intergenic sites). To estimate the strength of selection acting on these snps, we calculated the ratio of nonsynonymous and synonymous polymorphisms per nonsynonymous and synonymous site (dn / ds) and found evidence for weak purifying selection (dn / ds = 0.37). This value is slightly lower but consistent with values reported previously for populations of clonal bacterial pathogens, which range from 0.45 to 0.64 (hershberg and petrov 2010). Differences in the magnitude of dn / ds need to be interpreted with caution in this situation, as this measure assumes that sequence polymorphisms are fixed substitutions between species and not intraspecific mutations segregating in a population (kryazhimskiy and plotkin 2008). Some snps in the pooled data set include those at high frequencies, and we assume that these snps have been segregating in the population for some time and may have been exposed to significant levels of purifying selection . To assess whether we could measure differences in 1) the levels of purifying selection and 2) the magnitude of the at mutational bias for snps partitioned into different frequency bins, we calculated dn / ds and gceq values for snps binned at 0.1 frequency intervals (fig . As 10 individuals were pooled for sequencing, an ideal experiment would reveal snps clustering at frequencies of 0.1, 0.2, 0.3, and so on up to 0.9 . We did not observe an increased number of snps near these expected frequencies, and attribute this nonideal behavior to numerous potential experimental and computational artifacts (for a full discussion, see the supplementary materials online). Nevertheless, these results confirm that the snps present in the population at lower frequencies have been exposed to less purifying selection (indicated by a higher dn / ds value) and are more strongly at biased than snps present at higher frequencies (fig ., the gceq content of the hodgkinia genome is calculated to be 37% using only snps called at a frequency of 0.1 or less, lower than the 42% calculated when all snps are included . The true hodgkinia gceq is therefore probably closer to 37%, or perhaps even lower . From these data, plotting gceq (gray line) and dn / ds (black) at different snp frequency cutoffs shows that snps present at lower frequencies (which are likely more recent mutations) have been subjected to less selection and are more at biased . Although our data clearly show an at mutational bias in hodgkinia, they do not directly implicate the force(s) responsible for the disparity between the observed patterns of mutation and substitution . Hershberg and petrov (2010) and hildebrand et al . (2010) suggest selection, or a selection - like process such as biased gene conversion, as the force driving the difference in bacteria . In bacteria, biased gene conversion involves horizontal gene transfer, recombination, and dna repair - based mechanisms (rocha and feil 2010). As hodgkinia encodes no gene homologs capable of these processes (mccutcheon et al . 2009), biased gene conversion seems unlikely to be responsible for hodgkinia's elevated gc content . Therefore, it appears that an unidentified selective force (or forces) is the most likely explanation for the gc compositional bias in the hodgkinia genome, although other explanations cannot be ruled out given the present data . For example, it is possible that the gc content in hodgkinia is mostly driven by mutational patterns, and that it recently underwent a shift from a gc to an at mutational bias . Were this true, we would have had to have measured the mutational pattern soon after the change from a gc to an at bias, but before this shift would have had the chance to alter the genome - wide nucleotide composition . Rather, given the results of hershberg, hildebrand, and coworkers, we favor the explanation that hodgkinia has, and has always had, an inherent at mutational bias (hershberg and petrov 2010; hildebrand et al . Our results seem to present a paradox in the way that the population genetics of endosymbionts are normally considered . The prevailing view that endosymbionts have less efficacious selection, resulting from reduced effective population sizes (moran 1996; andersson and kurland 1998; woolfit and bromham 2003; fares et al . 2004), fits well with some features of hodgkinia genome, in particular with its tiny size and overall rapid rate of sequence evolution . The disparity between the hodgkinia's at - biased mutational pattern and gc - biased genome does not fit easily into this framework because these results seem to require either an atypically large effective population size for hodgkinia or an unusually large selection coefficient for each individual at gc polymorphism in the population, or some combination of the two . It is possible that the population size of the host cicada is large and thus inflates the effective population size of hodgkinia; theoretical work has shown that host population size can have large effects on mutation accumulation in buchnera aphidicola in the context of its symbiosis with aphids (rispe and moran 2000). Why g or c nucleotides would be globally favored over a or t nucleotides is unclear and is an interesting area of future study . Hodgkinia is found as a symbiont throughout the cicada lineage (data not shown), and it will be of interest to examine the gc contents and mutational biases of hodgkinia across the diversity of cicadas . If gc - poor lineages of hodgkinia are found, then it may be possible to narrow the list of possible selective forces responsible for the elevated gc levels in hodgkinia from d. semicincta, by considering factors such as the environmental conditions and population structures of the insect hosts . The mutational results reported here would predict that a lineage of hodgkinia in which the selective restraints on elevated gc content were severely reduced or eliminated would have a genomic gc content as low as, or possibly lower than, 37% . Snps were identified using both roche 454 gsmapper and swap454 (brockman et al . 2008) software . Genome - wide dn / ds and gceq calculations were performed as described (hershberg and petrov 2010). The confidence intervals in table 1 were estimated using the rpois and quantile functions of r (2.13.0) by generating 1,000 value poisson distributions with means equal to the mutation counts; rate, equilibrium, and 95% confidence interval calculations were performed on these distributions . Full materials and methods, including a detailed description of how the snps were polarized, are included in the supplementary materials online . Supplementary materials, figure s1, and table s1 are available at genome biology and evolution online (http://www.gbe.oxfordjournals.org/).
Conventional inguinal hernia repair in children involves ligation of the hernial sac at the internal inguinal ring . Laparoscopic surgery has been applied in children, and the repair is based on the same principle . This study aimed to document the authors' experience with laparoscopic inguinal hernia repair in children . Patients were admitted in the evening 1 day before surgery and discharged within 24 hours following surgery . Two lateral ports of 3-mm were made through the right and left pararectal region to maintain a triangular orientation . In cases with small defects, laparoscopic ring closure (lrc) was done with 4 0 absorbable purse - string suture (figures 1, 2, and 3). In early cases and when difficulty was noted, saline was injected to separate the peritoneum from cord structures . The procedure was modified in 24% of children with a dilated internal ring (figure 4). Ligature of the hernial sac at the internal ring is inadequate in such cases . Here the sac was identified and dissected from the cord structures and then divided (figure 5). In patients with a large hernial sac, the landmarks identified included the iliopubic tract (ipt), arching of fascia transversalis, cord structures, and the peritoneal reflection (figure 6). The ipt was identified as a shiny white band running under the cord structures at the inferior border of the internal ring . The tranversus arch was identified as the arching of the transversalis fascia immediately above and lateral to the internal ring . The ipt was approximated to the transversus arch by using nonabsorbable 2 0 interrupted suture to narrow the internal ring (figure 7). All patients were evaluated after 5 days, 4 weeks, 6 months, 1 year, and then annually, when possible . Laparoscopic ring closure: continuation of the purse string on the inferior aspect of the internal ring . Laparoscopic ring closure: completion of the purse string suture and occlusion of the internal ring . Laparoscopic iliopubic tract repair: completion of the dissection and landmarks identified: (a) dilated internal ring, (b) iliopubic tract, (c) cord structures, (d) transversalis fascial arch . Ninety - three indirect inguinal hernial sacs were closed (28 right, 7 left, 58 bilateral) in 64 children (56 boys and 8 girls), ranging in age from 3 years to 13 years (median, 5.1 years). The mean operating time for lrc was 25 minutes (range, unilateral 21 to 35; bilateral 28 to 50). The contralateral processus vaginalis was patent in 20% of children . In 24% of children, of the 29 bilateral hernias, 20 underwent lrc, 7 underwent liptr, and 2 underwent lrc on one side and liptr on the other side . The median follow - up was 30 months (range, 2 to 84). The first recurrence was in a 3-year - old female child after 8 months, following a right lrc . The second one was in a 4-year - old boy after 5 months, following a left lrc . The essential step in the conventional method for inguinal hernia repair in children is simple ligation of the hernial sac without narrowing the open ring . From our point of view first and the most obvious is the confirmation of the hernia and looking for the contralateral side . First championed by ger, ring closure is essentially a high ligation of the indirect hernia sac, which is the preferred technique in pediatric patients . This technique re - establishes the usual anatomic relations of the medial aspect of the internal ring and cord structures, giving them a flush union with the transversus abdominis muscle and eliminating the lead point that allows the intestine to enter the inguinal canal . Ligature of the hernial sac at the internal ring alone is inadequate in patients with dilated internal ring (24% of children), as it does not take care of the component of the dilated ring . Reports exist in the literature of methods to tighten this dilated ring . In hernias exceeding 4 mm to 5 mm in diameter, the external hemicircumference of the neck was opened to bring the conjoined tendon closer to the crural arch with a nonresorbable suture without the use of a prosthesis . Ipt repair has been described as ringplasty, wherein the deep structures of the lateral iliopubic tract were approximated to the proximal arching musculotendinous fibers of the transversus abdominis muscle gazayerli described a laparoscopic preperitoneal ipt repair by approximating the transversus abdominis to the ipt, similar to the anterior preperitoneal approach described by nyhus and associates . We believe that in children with a dilated internal ring, a routine lrc would be inadequate . This is also observed in our 2 recurrences with one of them occurring within 5 months of lrc . The recurrence rate of inguinal hernias is slightly higher with laparoscopic herniorrhaphy than with the conventional technique . Following liptr, there have been no recurrences . In children with recurrences after lrc, the surgeon has an undisturbed anatomy for the groin incision; the risk of an injury to the vas deferens, subsequent testicular atrophy, and the risk of superior displacement of the testicle seem less likely . The problems we faced during liptr were trivial and related to the little scar tissue at the internal ring . This could be explained by some peritoneal fluid passing in between the knots placed during ring closure . Our series shows that both techniques are safe, reproducible, and technically easy for experienced laparoscopic surgeons . The long - term follow - up of liptr would help assess any technical modifications . Laparoscopic inguinal hernia repair in children can be offered, as it is safe, reproducible, and technically easy for experienced laparoscopic surgeons.
Pseudohypoparathyroidism (php) is a disorder of end - organ resistance primarily affecting the actions of parathyroid hormone (pth)1). It is characterized by hypocalcemia and hyperphosphatemia in association with increased secretion of pth due to decreased target tissue responsiveness to pth . Patients with php ia are not only resistant to pth, but also to other hormones that bind to receptors coupled to stimulatory g protein (gs alpha)2). The gnas gene is located on chromosome 20q13.11 and consists of 13 exons and 12 introns3). Php - ia is caused by heterozygous mutations affecting one of the 13 gnas exons encoding gs alpha4). We report here a 9-yr - old boy with php ia with a nonsense mutation of c.637 c> t in the gnas gene, which is novel . 9-year - old boy was admitted to hallym university medical center with painful subcutaneous soft tissue masses with calcifications on the right toes and posterior chest wall . He had a history of repeated convulsive episodes from the age of 2 months . Physical examination showed short stature (120 cm, 3rd percentile), round face (fig . 2) and subcutaneous ossifications on the 2nd toe tip and 1st web space of the right foot and the left lower posterior chest wall (fig . 3). His weight was 30 kg (50th percentile), thus having a normal body mass index (20.83 kg / m, 85th-90th percentile). He was born at full term by cesarean section with a birth weight of 3.3 kg . Family history revealed that his maternal grandmother had features suggestive of aho, such as short stature, round face, obesity and brachydactyly . His mother had normal appearance apart from short stature, but two of her siblings had short stature, round face and obesity . We could not make further investigation because the patient lost contact with his mother and maternal family after his parents got divorced . Laboratory tests revealed hypocalcemia (4.2 mg / dl; normal range, 8.8 - 10.8 mg / dl), hyperphosphatemia (8.9 mg / dl; normal range, 3.7 - 5.6 mg / dl) and elevated serum pth (146.5 pg / ml; normal range, 9 - 65 pg / ml). He also showed elevated plasma thyroid - stimulating hormone (6.978 iu / ml; normal range, 0.5 - 4.8 iu / ml), follicle stimulating hormone (5.6 iu / ml; normal range, 0.26 - 3.0 iu / ml), and luteinizing hormone (1.2 iu / ml; normal range, 0.02 - 0.3 iu / ml), which suggest multiple hormone resistance . Urinary phosphorous and cyclic adenosine monophosphate response after infusion of synthetic human pth (teriparatide acetate) was attenuated (ellsworth - howard test) (table 1). His intellegence scale (korean educational development institute - wechsler intelligence scale for children) indicated mild mental retardation (intelligence quotient, 67). He was diagnosed as having php ia based on physical and laboratory findings; hypocalcemia, hyperphosphatemia, pth resistance, presence of aho, multiple hormone resistance and mental retardation . He was started on phosphate - binding substance (aluminum hydroxide, 80 mg / kg / day), vitamin d (alfacalcidol, 1 mcg / day) and calcium carbonate (50 mg / kg / day). Three months later, his serum calcium was elevated from 4.2 to 6.0 mg / dl, and phosphorus was decreased from 8.9 to 8.6 mg / dl . The patient's clinical symptoms of pain, seizure and hyperactivity were reduced . To support the diagnosis of php ia, we performed dna analysis of the gnas gene . Genomic dna was extracted from peripheral blood leukocytes using the wizard genomic dna purification kit following the manufacturer's instructions (promega, madison, wi, usa). All coding exons and their flanking introns of the gnas gene were amplified using primer sets designed by the authors . Polymerase chain reaction was performed with a thermal cycler (model 9700, applied biosystems, foster city, ca, usa) as follows: 32 cycles of denaturation at 94 for 30 seconds, annealing at 60 for 30 seconds, and extension at 72 of 30 seconds . After treatment of amplicon (5 ul) with 10 u shrimp alkaline phosphatase and 2 u exonuclease i (usb co., cleveland, oh, usa), direct sequencing was performed with the bigdye terminator cycle sequencing ready reaction kit (applied biosystems) on the abi prism 31001 genetic analyzer (applied biosystems). Sequencing of the amplified gnas genomic dna fragments revealed a heterozygous nonsense mutation within exon 11 (c.637 c> t) (fig . 5). The c> t transversion results in an amino acid substitution from gln to stop codon at codon 213 (p.gln213). To our knowledge, this is a novel mutation in gnas . We also performed dna sequencing of the gnas gene of his father, but no mutation was detected . Php is a heterogeneous disease characterized by pth resistance and classified as types ia, ib, ic, and ii, according to its different pathogenesis and phenotype5). In contrast to the situation in hypoparathyroidism, in php the parathyroid glands are normal or hyperplastic and they can synthesize and secrete pth6). Heterozygous inactivating mutations within gs alpha - encoding gnas exons are found in patients with php - ia, who also show resistance to other hormones and a constellation of physical features called aho . Patients who exhibit aho features without evidence for hormone resistance, who are said to have pseudopseudohypoparathyroidism (pphp), also carry heterozygous inactivating gs alpha mutations1,7). There is some evidence to suggest that the gs alpha mutation is paternally transmitted in patients with pphp and maternally transmitted in patients with php - ia . The gene may be imprinted in a tissue - specific manner6,8,9). In our case, dna sequencing of the patient and his father revealed that only the patient had mutations . The presence of slight aho in the mother's side of the patient may suggest that the mutation came from his mother, but we were unable to confirm this because the dna of the patient's mother or maternal family was not available at the time of the study . Although we could not obtain molecular data from the maternal family of the patient, considering variation of phenotypes due to genomic imprinting, we were able to explain that the patient had the genotype and phenotype of php ia . In the maternal family of the patient, his mother had milder features suggestive of aho, whereas her mother and two siblings had characteristics that were more strongly suggestive of aho . We can suspect the possibility of a hypofunctioning gnas mutation like mosaicism, but it was impossible to validate . Mutational analysis of gnas gene is a useful method for identifying genetic abnormalities as well as making diagnosis and differentiation of various subtypes of php10). There are previous reports of korean patients with php ia and pphp who were confirmed by genetic analysis . Park et al.11) reported a nonsense mutation of c.94a> t (p.k32x) and a frame shift mutation of c.344_345inst (p.v117rfsx23) in patients with php ia, of which the former was novel . In a study of patients with php ia and pphp, jin et al.10) identified two novel mutations; c.569_570del mutation (p.tyr190cysfsx19) and a splicing mutation (c.659 + 1g> a). Gnas has been termed one of the most complex gene loci in the human genome3). Although many mutations have been identified in gnas, c.637 c> t nonsense mutation is deemed novel.
Sea ice seasons have shortened by at least 5 days / decade over most of the arctic across 1.9 million km ice seasons have shortened by at least 25 days / decade counter to most of the arctic ice seasons have lengthened in the bering sea arctic sea ice has received considerable attention in recent years because of the downward trend in both the areal extent [e.g., parkinson et al ., 1999; stroeve et al ., 2012] and the thickness [e.g., rothrock et al ., 1999; kwok and rothrock, 2009] of the ice cover . The decreasing ice cover coincides with significant arctic warming [e.g., jeffries et al ., 2013; ding et al ., 2014], and indeed the positive feedback between the ice and the temperature with warming leading to ice reductions that in turn lead to more warming because of the resulting decreased surface albedo is thought to be a principal reason why the arctic region has warmed more than the global average [e.g., screen and simmonds, 2010]. Passive - microwave satellite data have provided the primary data source for the quantification of the decreasing areal extent of the ice . These data have important advantages for sea ice monitoring . Among them: the microwave signature of ice is quite different from the signature of liquid water, allowing a clear identification of the ice edge and an estimation of ice concentration; the microwaves are coming from within the earth system, and thus the data can be collected during periods of darkness as well as sunlight; and at selected microwave frequencies, the microwave radiation can pass through clouds nearly unaffected . These advantages allow a monitoring of the ice cover under all lighting and most weather conditions . This has enabled a robust determination of the areal coverage of the arctic sea ice since the beginning of the multi - channel satellite passive - microwave record in late 1978 . Decreases in the areal extent of the arctic sea ice as determined from the multi - channel satellite record were pointed out in the late 1980s, although with due cautions in view of the brevity of the record and the minor nature of the decreases up to that point [parkinson and cavalieri, 1989]., 1995; parkinson et al ., 1999] and have accelerated in the 21st century [e.g., meier et al ., 2007; cavalieri and parkinson, 2012; stroeve et al ., 2012], although with continuing interannual variability [e.g., screen et al ., important as the quantification of the ice extent decreases are, they fail to tell the full story of what the satellite passive - microwave data can reveal about the changing arctic sea ice cover . They allow quite thorough temporal detail, even down to showing changes on a daily timescale, but much less spatial detail . In this paper, instead of looking at the extent changes, the focus is on changes in the length of the sea ice season, defined (for each pixel and each year) as the number of days with sea ice coverage . The season - length calculations cannot show the temporal detail of the ice - extent calculations, as they aggregate to yearly values, but they show much greater spatial detail, down to the pixel level . The length of the sea ice season was first defined and mapped from satellite data in the early 1990s . At that time, it was found that trends in the length of the sea ice season over the 19791986 period showed coherent patterns of shortening sea ice seasons in most of the eastern hemisphere of the arctic seasonal ice zone and in the greenland sea and lengthening sea ice seasons in most of the remainder of the seasonal ice zone, particularly in baffin bay, the labrador sea, hudson bay, the beaufort sea, and portions of the bering sea [parkinson, 1992]. (a location map appears in the supporting information, as figure s1 .) With an additional 27 years of data, the results in this paper show the area with lengthening sea ice seasons to be vastly reduced, as by now shortening sea ice seasons dominate almost the entire seasonal ice zone . In view of the high albedo of sea ice relative to liquid water, the shortened length of the sea ice season in much of the seasonal sea ice zone results in less reflection of solar radiation back to space and more absorption of solar radiation into the ocean, providing a forcing toward further warming the system . This forcing, well known as the ice - albedo feedback [e.g., kellogg, 1975; perovich et al . 2012], is so impactful that calculations with the global model of the nasa goddard institute for space studies (giss) show that, at least in that model, a full 37% of the global warming calculated for a doubling of atmospheric carbon dioxide (co2) is due to the inclusion of sea ice in the calculations [rind et al ., 1995]. The sea ice reductions also result in increased transfer of energy between the ocean and atmosphere, a decreased strength of the arctic atmospheric temperature inversion, and increased evaporation, with consequences to atmospheric moisture, atmospheric circulation, and precipitation [e.g., screen et al ., 2013; vihma, 2014]. Impacts of ice losses on the arctic ecosystem extend all the way through the polar food web, from helping to control algal production to impacting such large animals as walruses, seals, arctic foxes, and the iconic polar bears, whose primary hunting season shortens or lengthens in line with the sea ice season [post et al . Sea ice reductions affect species interactions, disease transmission, and a wealth of other aspects of the ecological system [post et al . Humans are affected by the ice reductions through the climate and ecosystem impacts and also through such economically relevant impacts as the anticipated opening of new trans - arctic shipping routes [smith and stephenson, 2013] and effects on proposed offshore oil and gas exploration in the arctic [galley et al ., the data used for this study are from satellite multi - channel passive - microwave instruments from the national aeronautics and space administration (nasa) and the united states department of defense (dod). The earliest of these instruments was nasa's scanning multichannel microwave radiometer (smmr), launched on the nimbus 7 satellite in october 1978 . Fortunately, by the end of life of the smmr instrument, the first of the dod special sensor microwave imager (ssmi) instruments had been launched on the defense meteorological satellite program's (dmsp's) f8 satellite, in june 1987 . Since june 1987, the dod has maintained at least one ssmi or its follow - on ssmi sounder (ssmis) in orbit at all times . The results in this paper are from the nimbus 7 smmr, the f8, f11, and f13 ssmis, and the f17 ssmis . The smmr, ssmi, and ssmis instruments are also the instruments used in many studies of the decreasing arctic sea ice extents [e.g., johannessen et al ., 1995; parkinson et al ., 1999; meier et al ., 2007; cavalieri and parkinson, 2012; stroeve et al ., 2012]. For those studies as for this one, the data have been mapped onto a rectangular grid overlaid on a north polar stereographic projection with grid squares (or pixels) representing earth areas of 25 25 km [nsidc, 1992], and the results are based on the fundamental sea ice variable derived from the satellite data, the sea ice concentration . The ice concentration data were generated at nasa goddard space flight center using the nasa team algorithm [gloersen et al . Rigorous intercalibration was done between the smmr and three ssmi sensors [cavalieri et al ., 1999] and between the ssmi and ssmis sensors [cavalieri et al ., the data are archived and available at the national snow and ice data center (nsidc) in boulder, colorado . The length of the sea ice season is calculated for each year at each pixel by counting the number of days the pixel had at least 15% sea ice concentration . The 15% cutoff is also commonly used in the ice extent studies [e.g., parkinson et al ., 1999; trends in the length of the sea ice season are then calculated for each pixel by obtaining the slope of the line of least squares fit through the data for the time period of interest . Trends are mapped in the results section for the first 10, 20, and 30 years of the record and for the full 35 years 19792013 . Due to the orbits of the satellites and the characteristics of the instruments, the data sets do not include the area in the immediate vicinity of the north pole . The smmr data extend northward to 84.6n, and the ssmi and ssmis data extend northward to 87.6n . Hence, results for the smmr years (19791987) and for the trends extend only to 84.6n, whereas results for individual years within the ssmi and ssmis time frame (1988 and beyond) extend to 87.6n . Examining maps of the length of the sea ice season for each of the 35 years 19792013, it is clear that many fundamentals have remained the same throughout the record . For instance, comparing the starting and ending years (figure 1), which are typical of the early and late portions of the record: the area of year - round (or at least 360 days) ice coverage continues to include much of the central arctic and a portion of the canadian archipelago; the overall outer edge of the ice area continues to reflect the strong influence of the gulf stream and north atlantic current, with year - round ice - free areas extending well into the polar region to the west and north of scandinavia; and for any longitude, the general (although not universal) tendency is for the season length to decrease southward (figure 1). There are also, however, some clear differences in the 2013 versus 1979 conditions, among those being the reduction in the area of year - round ice coverage and the shifting of substantial areas of the sea of okhotsk and the barents sea from being within the seasonal ice zone to being ice - free year round (figure 1b versus 1a). Length of the sea ice season for (a) 1979 and (b) 2013 . Mapping the trends in the length of the sea ice season for the first 10, 20, and 30 years of the record and for the full 35 years (figure 2) dramatically illustrates the transition in the arctic sea ice cover . Specifically: for the first 10 years, 19791988, the trends show substantial regions with marked shortening of the ice season and substantial regions of lengthening sea ice seasons (figure 2a); with an additional 10 years, the 19791998 trends have a noticeably larger area of shortening versus lengthening seasons (figure 2b); and with the 30 and 35 year records, the seasonal ice zone has shortening sea ice seasons almost everywhere, the main exceptions being in the bering sea and portions of the canadian archipelago (figures 2c and 2d). This complements well the aforementioned acceleration of the decrease in ice extents since the 1990s . Trends in the length of the sea ice season for the following periods: (a) 19791988, (b) 19791998, (c) 19792008, and (d) 19792013 . The statistical significance of the trends has increased markedly as the data set has lengthened . Trends for the first 10 years of the record are generally not statistically significant even at the 95% confidence level, with the exception of a sizeable portion of the sea of okhotsk and smaller portions of the northeastern greenland sea and the eastern barents sea (figures s2a and s3a for 95% and 99% confidence levels, respectively). However, the area of statistical significance is larger for the 20 year trends, 19791998 (figures s2b and s3b) and much larger for the 30 year (figures s2c and s3c) and 35 year (figures s2d and s3d) trends . The 30 year trends, 19792008, show statistical significance at the 95% level over most of the seasonal sea ice zone (figure s2c) and at the 99% level over a lesser but still substantial portion of it (figure s3c). The 35 year trends, 19792013, show statistical significance at the 95% level over almost the entire seasonal sea ice zone (figure s2d) and at the 99% level over most of it (figure s3d). Figure 3 presents histograms quantifying the area of shortening and lengthening sea ice seasons . Summing the relevant values in the histograms, for the 10 year 19791988 record, the area with ice seasons that lengthened by at least 5 days / decade is 5.7 10 km, and the area with ice seasons shortened by at least 5 days / decade is only 14% greater than that, at 6.5 10 km (table 1). Adding another 10 years, the 20 year record has a far greater difference, with values of 2.5 10 km and 9.1 10 km, respectively . The difference is even greater for the 30 year and 35 year records; and the contrast in areas of shortened versus lengthened sea ice seasons over the full 35 year record increases even further, percentage - wise, when considering some of the higher rates . For instance, for the 35 year record, 1.9 10 km had ice seasons shortened by at least 25 days / decade while only 78,000 km had ice seasons lengthened by at least 25 days / decade (table 1). Histograms of the area of the arctic region with trends in the length of the sea ice season 75 days / decade, between 75 and 65 days / decade, between 65 and 55 days / decade, and on up to between 35 and 45 days / decade, and 45 days / decade . Histograms are shown for the 10 year, 20 year, 30 year, and 35 year periods, corresponding to figures 2a, 2b, 2c, and 2d, respectively . (in constructing the histograms, only pixels with an average of at least 1 day of 15% sea ice coverage per year are included .) Areal extent of arctic sea ice coverage in different trend categories, presented for the 10 year, 20 year, 30 year, and 35 year trends in the length of the sea ice season the season - length trends are affected very little by the selection of 15% for the ice concentration cutoff used in the calculations . A higher ice concentration cutoff would require more ice to be present before including a given day in the ice season and hence would mean shorter ice season lengths overall in each year of the record . However, sensitivity studies on the ice concentration cutoff yield very little effect on the trends . This is illustrated in figure 4 with results for a 50% ice concentration cutoff . With a 50% cutoff, the season length throughout the seasonal sea ice zone is naturally shorter than with a 15% cutoff (figure 4a versus figure 1b, illustrating for the 2013 case), but when trends are calculated, results with a 50% cutoff are very similar to those with a 15% cutoff, retaining the basic pattern of the trends and confirming the primary conclusion of the dominance of shortening sea ice seasons (figure 4b versus figure 2d). (a) length of the 2013 sea ice season when considering a location to have ice only when the ice concentration is at least 50% (versus the 15% ice - concentration cutoff used in figures 1 and 2). (b) trends in the length of the sea ice season over the period 19792013 when using a 50% ice - concentration cutoff . This paper examines changes in the length of the sea ice season over the period 19792013, showing a large predominance of shortening versus lengthening ice seasons (figure 2). These results complement the more frequently discussed decreases in ice extent, showing much greater spatial detail although not nearly as much temporal detail as is shown in the ice - extent results . The season lengths show, on a pixel - by - pixel basis, at 25 25 km resolution, how much of the year has an ice cover (figure 1) and how that ice - season length has changed over time (figure 2). Defining the length of the ice season simply as the number of days of ice coverage avoids the considerable complication of establishing start and end dates of ice coverage . Throughout the seasonal ice zone, both the autumn onset of ice coverage and the spring / summer disappearance of ice can involve multiple appearances and disappearances, some quite transitory . Simply counting the days of ice coverage gives a pure accounting of the portion of the year with a reflective, insulating ice cover present and has resulted in trend maps that show a high degree of spatial coherence, with almost no speckling . Throughout the entire grid, adjacent pixels show very similar results (figure 2). Furthermore, the 35 year trends are statistically significant over almost the entire seasonal sea ice zone (figures s2 and s3). Several studies have examined the related issue of the length of the melt season and in doing so have tackled the problem of determining dates of melt onset and freezeup [smith, 1998; belchansky et al . In particular define the melt season as extending either from early melt onset to continuous freezeup (the outer melt season) or from continuous melt onset to early freezeup (the inner melt season). The mapped 19792013 trends in the length of the melt season [stroeve et al ., 2014] show considerably more speckling than appears in figure 2, but the overall pattern of trends corresponds well, with the melt season having lengthened over much of the arctic region except for the bering sea [stroeve et al ., 2014], in line with the opposite trends for the length of the sea ice season (figure 2). The shortened sea ice seasons throughout much of the arctic provide opportunities for shipping and increased air - sea exchanges, increase the region's absorption of solar radiation, force polar bears in some locales onto land for a longer portion of the year, and necessitate many additional adjustments within the polar ecosystem . Furthermore, the impacts of sea ice decreases are not confined to the polar regions, as illustrated by the aforementioned rind et al . Study in which 37% of the global warming simulated from a doubling of atmospheric co2 was due explicitly to the inclusion of sea ice in the calculations . The highly interconnected nature of the earth system ensures that major changes in one region will have at least some impact on adjacent regions . In fact, considerable evidence has been compiled to link changes in the arctic to consequent changes in weather conditions at lower latitudes [e.g., francis and vavrus, 2012; vihma, 2014], and conversely, changes in lower latitudes have been viewed as forcings on recent arctic warming [ding et al ., although there are different approaches, at times leading to different conclusions on the specifics [e.g., barnes, 2013; screen and simmonds, 2013], there is no controversy that the earth system is interconnected and that major changes in one region are likely to propagate beyond.
Malaria remains one of world s major infectious diseases and an impediment to economic development . One third of the world s population are at risk of infection, around 250 million people develop clinical infections annually, and at least half a million die each year; most are children aged under five years . The disease is caused by infection of circulating red blood cells by one of five species of the protozoan parasite, plasmodium; p. falciparum causes most morbidity and mortality . The majority of antiplasmodial drugs are now severely compromised due to acquired resistance by the parasite, and the development of efficacious vaccines remains a challenge . Novel approaches to treatment and a greater understanding of disease pathogenesis and the host response to infection are desperately needed . Outcome to malaria infection is determined largely by the increasing parasite mass and the response of the host to the infection . Importantly, less than 1% of infections progress to life - threatening stages, underscoring the efficacy of host protective mechanisms . The innate protective response limits parasite growth early in an infection in a non antigen - specific manner . It also allows time for the subsequent development of an adaptive response, which is capable of clearing the infection and protecting against clinically symptomatic malaria . It is also antigen - specific, and several years and many exposures are needed to build an effective immunity against the multitudinous array of parasite antigens in any given endemic region . Innate immune mechanisms are therefore crucial in all malarial infections to buffer against the early growth of blood - stage parasites . We recently reported that platelets are an important component of the host innate immune response against malaria infection . In addition to their well - defined role in hemostasis, platelets are increasingly implicated in immunological processes, including direct pathogen - killing functions (reviewed by yeaman and colleagues, ref . They express receptors that bind host immune response modulators (e.g., antibodies and cytokines) and toll - like receptors that bind microbial products . They also express the cd154 co - stimulatory molecule and influence the development of adaptive immune responses . Importantly, their location in the circulation makes them ideal sentinels against any nascent infection . Platelets respond to a variety of microbial cells by releasing immunomodulatory molecules and by directly killing microbial pathogens . Malaria infections are commonly accompanied by a thrombocytopenia or loss of platelets, the severity of which closely mirrors the increasing parasite mass . It is now clear from our study and others that platelets protect the host during erythrocytic infection . Mice with pre - existing platelet deficiencies are more susceptible to infection and exhibit higher loads of viable parasites . Treatment of normal mice with aspirin, a platelet activation and aggregation inhibitor, also reduces survival to infection . Pre - treatment of these platelets with inhibitors (including aspirin) blocks the parasite killing effect . Platelets bind preferentially to p. falciparum ie, mainly through interactions between the platelet - expressed scavenger receptor protein, cd36 and the p. falciparum erythrocyte membrane protein (pfemp1), produced by the parasite and trafficked to the erythrocyte surface . We believe that platelets are active early in infection to slow the initial growth of malaria parasites in the bloodstream, providing greater opportunity for other defense mechanisms to control the infection and ensure survival . More recent studies by our group and others have provided additional mechanistic and molecular insight to how platelets kill the intraerythrocytic parasite . Central to these findings is a platelet derived cxc - type chemokine called platelet factor 4 (pf4/cxcl4), which is released by activated platelets and kills the parasite . Approximately 25% of the protein released by platelets comprises of pf4, and concentrations surrounding activated platelets reach high micromolar ranges . The parasite - killing activity of platelets appears to be entirely due to pf4 . Neutralizing anti - pf4 antibodies completely block the activity of human platelet lysate and pf4-deficient platelets from mice fail to kill parasites . Love and colleagues elegantly demonstrated that upon entering the cell, the protein relocates to the parasite digestive vacuole (dv, site of hemoglobin digestion), resulting in specific lysis of the organelle and death of the parasite . Therefore pf4 is a unique example of a host - derived molecule with direct plasmocidal activity . Pf4 belongs to growing list of chemokine molecules called kinocidins, which have a remarkable capacity to function as both chemotactic and antimicrobial molecules . The different functions are localized to structurally distinct domains, and the latter at least is separable . Chemotactic activity resides in the n - terminal portion of the protein where the classical cxc chemokine motif exists . This domain interacts with chemokine receptors on various target cells, leading to receptor - mediated signaling events . The c - terminal domain, rich in positive charged amino acids, mediates the antimicrobial effects of pf4 and other kinocidins . Peptide scanning analyses have mapped the bactericidal and fungicidal activity of the rabbit pf4 ortholog, platelet microbiocidal protein 1 to the carboxyl terminal 15 residues, 5 of which are lysines . Notably, love and colleagues showed that a peptide encompassing the carboxyl terminal 12 residues of human pf4 (c12) had plasmocidal activity . This function was abrogated when three out of the four lysine residues were substituted to alanines . The charged, amphipathic nature of kinocidin c - terminal domains gives them propensity to interact with cell membranes . In bacteria, this has been variously shown to result in disruption of membrane potential, cell permeabilization and inhibition of protein and dna synthesis . Consistent with these former effects, pf4 and c12 treatment of p. falciparum results in lysis of the dv . However, exactly how and why the dv is specifically targeted by pf4 is unclear, especially given that the molecule must cross three separate membranes in order to reach the dv (red cell plasma membrane (pm), parasitophorous vacuole membrane and parasite pm). Pf4 binds to duffy, a red cell antigen receptor for chemokines (darc / fy) and our results show that platelet and pf4-mediated killing of intraerythrocytic parasites requires darc . The likely mechanism involves the binding of pf4 to darc on the infected cell surface, following by internalization of the pf4-darc complex . Binding occurs via a domain on darc known to bind a number of other chemokines . Also, other chemokines with a high affinity for darc out - compete the pf4 parasite binding and killing of parasites . The molecular details of how the complex may translocate into the parasite (as well as the existence of the complex within the parasite) remain to be demonstrated . But interestingly the uptake of host membrane proteins by intraerythrocytic plasmodium, including darc, has been documented . Uptake of darc appears to be an active process and requires the tubovesicular membrane (tvm) network that derives from the parasitophorous vacuole membrane and connects the parasite with the erythrocyte pm . The tvm develops within the first 24 h following merozoite invasion and host proteins accumulate in conjunction with parasite maturation . Uptake is also selective for certain host proteins, especially those that reside in cholesterol - rich detergent - resistant host membranes; more abundant cytoskeletal molecules such as glycophorin - a, band-3 and glut1 are excluded . Interestingly, establishment of the tvm and maximal accumulation of darc coincide with the parasite stage most sensitive to pf4 (trophozoites). The intracellular fate (and function) of these imported host proteins remains to be determined . In the case of darc, details of parasite intracellular localization are not known . As well as establishing if pf4-directed lysis of the dv requires darc, it will be of interest to determine if darc translocates to the parasite dv membrane, and thereby provide a plausible mechanism for the delivery of pf4 from the activated platelet to this organelle . The protective role played by platelets in human malarial infections has yet to be directly answered, but there are a number of lines of evidence that support the affirmative . Depletion of platelets (or inhibition of platelet function) in murine models of malaria where outcome is determined by bloodstream parasitemia (as opposed to the inflammatory response - based cerebral malaria syndrome) results in reduced survival . Although a similar platelet depletion study is difficult to perform in humans, we do know that thrombocytopenia, which is a common clinical accompaniment of all malarial infections, has been correlated with a poor outcome in falciparum malaria . Platelets have also been implicated as susceptibility factors in the development of cerebral malaria (cm), a complex collection of syndromes specific to p. falciparum infections and a major cause of death . Central to the pathophysiology of cm is the accumulation or sequestration of ie in the cerebral microvasculature, causing the obstruction of blood flow, leukocyte accumulation, localized intravascular inflammation, activation and damage of the endothelium and disruption of the blood - brain barrier . Platelets are often found at sites of ie sequestration in both human cm and mouse cm models where they are believed to mediate ie binding in the microvasculature, and release molecules that affect endothelial cell viability and promote leukocyte adhesion . The parasite - killing activity of platelets may also contribute to the pathophysiology of cm . Platelet - directed killing may moderate the local inflammatory responses to live parasites, or dead parasite toxins and exudate could contribute to the inflammatory and cell - damaging milieu . The effect of non - steroidal anti - inflammatory drugs, especially aspirin, may add an additional level of complexity in assigning the importance of platelets in human malaria . We showed previously that aspirin prevented platelet - mediated killing of cultured p. falciparum and reduced survival in p. chabaudi - infected mice . Aspirin is cheap and readily available, and its usage as an antipyretic remains widespread (although contraindications such as dengue fever and reyes syndrome have led to revisions on usage guidelines in many westernized countries). A recent cochrane review found that the available data are insufficient to conclude if aspirin (as well as other antipyretics) are beneficial or detrimental in malaria . Therefore studies to access the impact of aspirin on malaria are highly warranted . The polymorphism responsible for the duffy - negative blood group, a t to c substitution in a gata1 promoter binding site, prevents erythrocytic expression of the duffy gene . The c allele is very common in african populations; frequencies are close to 100% in most of central and western africa . Our findings on the requirement for darc in killing of parasites by platelets have led us to speculate that platelet - mediated protection may be inadequate in duffy - negative (darc deficient) individuals . Duffy - negativity provides protection against p. vivax infection by virtue of the requirement of the receptor for merozoite invasion (although the simplicity of this assumption has been recently challenged), and p. vivax is rarely seen in africa . However since erythrocytic expression of duffy is also necessary for the platelet - mediated killing of p. falciparum parasites, we might expect to see increased severity or a poorer outcome to falciparum malaria in duffy - negative populations . At a broad level, this appears to be the case . Using the best available global estimates, rates of infection and death due to p. falciparum are much higher in africa compared with other parts of world (asia and south america), where duffy - negativity is rare . But many other factors also contribute to this difference, such as the type of parasite and its level of virulence, vector control measures, rates of host immunity, access to healthcare and other socioeconomic factors . In africa as much as half of malaria - associated deaths are attributable to cm . The failure of platelets to kill sequestered parasites growing in duffy - negative red cells may counter intuitively exacerbate this complication by allowing viable parasites to remain within the cerebral vasculature . Importantly, studies are needed to test the relationship between duffy - antigen status and platelet protection where, ideally, p. falciparum disease severity, incidence and outcome would be compared within duffy - negative and positive individuals living in the same area and affected by the same parasite strains and other environmental factors . If a lack of platelet protection is indeed detrimental in duffy - negative individuals, it is challenging to reconcile evolutionarily why the duffy - negative polymorphism should remain common in africa . However the events that led to, and maintained, the duffy - negative allele in africans are likely to be complicated . It is widely held that the polymorphism arose through positive selection at a distant time in human evolutionary history to protect against, what was presumably then, a lethal form of p. vivax . Compared with p. vivax, the advent of p. falciparum infection in humans appears to have been a more recent event . Therefore fixation of the duffy - negative allele may have preceded p. falciparum in humans . There may also be other positive selective pressures for the polymorphism that we do not know about . The protective power of the platelet in malaria infection is only beginning to be appreciated . While convincing evidence of their importance in clinical infection is still needed, advances in understanding the molecular detail of parasite killing process have led to some very interesting findings . Useful novel antimalarial molecules based on the pf4 platelet effector molecule are an exciting prospect . The findings may also help to explain why p. falciparum is a particular problem in africa . We must continue to direct our efforts to control this scourge of humankind, especially in this part of the world.
Understanding the changes occurring within an aging immune system is essential if public health authorities are to be equipped to deal with an aging population . Specifically, knowledge of altered immune responses to infectious agents is required if rational clinical interventions are to be tailored to these aging individuals . Aging is a continuous and slow process that compromises the normal functioning of various organs and systems . As the population ages, there is growing interest in understanding host - parasite interaction and eventual prevention of chronic parasitic diseases, including cysticercosis, in elderly individuals . Cysticercosis is emerging as a serious public health problem in many poor countries in latin america, africa and asia . Although theoretically easy to control, and declared eradicable, cysticercosis remains neglected in most endemic countries . This parasitosis may be asymptomatic or it may cause a variety of clinical manifestations depending on the number, location, and stage of cysticercus lesions . Pleomorphic disease is a result of the presence of the parasite itself (cysticerci), of the inflammatory process that surrounds the larvae, and of residual fibrosis and calcification [46]. It is likely that the combination of several factors is responsible for such differences, one of which may be gender - related . The relevance of gender in host susceptibility has been explored in cysticercosis infection . In experimental murine taenia crassiceps cysticercosis, female mice were found to be more susceptible than males in different syngeneic and congenic strains of mice . The finding that gonadectomy equalized susceptibility between sexes, by reducing parasite loads in females and increasing it in males, first clearly indicated the relevance of sexual hormones . The cysticercus contains a large number of antigens that can elicit a host immune inflammatory reaction . The inflammatory cellular infiltrate, if present, may be discrete with lymphocytes and eosinophils in the initial stage, or it may be a more intense lymphocyte infiltrate with giant multinucleated foamy macrophages in the necrotic stage . Recent evidence suggests that immunosenescence associated to an immunological alteration caused by cysticercosis leads to a favorable condition for neoplasia development in elderly individuals attacked by the parasitosis . Moreover, it is likely that the patients continue to be infected with cysticercosis as they age . The aim of this study was to quantify the inflammatory infiltrate in the heart and in the encephalon of the elderly with cysticercosis . Our hypothesis is that it is possible to quantify the infiltrated inflammatory among male and female elderly patients, due to the fact that those individuals are undergoing immunosenescence . This research paper was approved by triangulo mineiro federal university research ethics committee under protocol no . 486 . As this research regards autopsy material, the only risk was the loss of confidentiality . However, as a precautionary measure, the cases were identified by letters and numbers . Also, consent for the autopsy was given in writing by the next of kin after the death of the patient . Then the document was filed in the general hospital and the general pathology discipline records . A retrospective transversal study of 3639 autopsies of adults collected at the general hospital of triangulo mineiro federal university, located in uberaba, mg, brazil, from 1970 to 2008, was carried out . Diagnosis of cysticercosis was made through histological demonstration or through direct visualization of the cysticercus, meeting the diagnostic criteria proposed by other authors, in 75 autopsies, 55 non - elderly, and 20 elderly patients . Information regarding age, gender, body weight, height, heart weight, brain weight, and the number, location, and evolutionary stage of the cysticercus was registered . In order to analyze the heart and brain inflammatory infiltration, we obtained 33 (13 elderly and 20 non - elderly) samples of heart and brain of patients with cysticercosis the 7 heart samples, amongst which 3 belonged to elderly patients and 4 belonged to non - elderly patients with cardiac cysticercosis, and 26 brains with neurocysticercosis, 10 of which belonged to elderly patients and 16 to non - elderly patients . The other organs were not found at the anatomical specimens' archives from the department of general pathology . The brain and heart samples affected by cysticercosis were fixed in formaldehyde 10% and subjected to routine histological processing so as to obtain 4 m thick sections, stained with hematoxylin and eosin (he), for general morphological analysis and for quantification of the inflammatory cell infiltrate . A video camera coupled to a standard light microscope and an interactive image analysis system (ks 300 carl zeiss) were used . We analyzed ten fields per quadrant; that is, 40 measurements were carried out in each slide . The he - stained slides were examined using a standard light microscope with a 20x objective and 800x magnification range . The digital image showed the field where the number of inflammatory cells was counted in absolute value . Quantification was carried out by the observer's identification of such cells and through the staining performed by point - counting method . The variables were tested in order to verify the type of distribution using the kolmogorov - smirnov test and variance analysis . Student's t - test (t) or mann - whitney (t) was used in the comparison of two groups, and anova (f) or kruskal - wallis (h) for comparison between three or more groups, followed by bonferroni or dunn test when necessary . Correlations between two variables were analyzed by pearson's or spearman correlation coefficient (r). Amongst the patients with cysticercosis, the average age of the non - elderly was 47.3 years, ranging from 23 to 58 years old, whereas the elderly had an average age of 66.7 years, ranging from 61 to 75 years old . Male and caucasian patients predominated in both groups, and analysis of nutritional status showed that the non - elderly had an average body mass index (bmi) of 21.2 4.4 kg / m and that the elderly with cysticercosis had an average bmi of 20.2 9.9 kg / m . Heart weight and brain weight of the non - elderly were found to be higher than those of the elderly with cysticercosis, and both elderly and non - elderly male patients had heart weight and brain weight higher than female patients (table 1). It was possible to ascertain the evolutionary stage of the parasite in 8 cysticerci of elderly individuals, among whom 4 (50%) were vesicular stage, 2 (25%) colloidal vesicular stage, 1 (12.5%) granular nodular stage and (12.5%) nodular calcified stage . Amongst the non - elderly, 4 (21.1%) cysticerci were in the first evolutionary stage, 5 (26.3%) colloidal vesicular stage, 4 (21.1%) granular nodular stage, and 6 (31.5%) nodular calcified stage . The elderly had an average of 3.1 2.5 cysticerci, whereas the non - elderly had 2.7 3.8 parasites . At all stages was observed some degree of inflammatory reaction around the cysticercus, its intensity decreased with the succession of evolutionary stages . The colloidal vesicular stage showed the highest inflammatory infiltrate, followed by the granular nodular stage in elderly and non - elderly groups (table 2). Analysis of the cardiac inflammatory infiltrate indicated that the non - elderly had significantly more inflammation than the elderly patients with cardiac cysticercosis (figures 1 and 2). In the non - elderly group, although men had more cardiac inflammation than women, this difference was not significant . Nonetheless, elderly females had significantly more inflammation than the elderly males (table 3). Encephalic inflammation was more acute amongst the non - elderly when compared to the elderly with neurocysticercosis (figure 1). In the elderly group, female patients had significantly more encephalic inflammation than male patients . When contrasting both genders of the non - elderly group, male patients had more inflammation, yet without any significant difference (table 3). A positive and not significant correlation between encephalic inflammation and cardiac inflammation was found (r = 0, 032; p = 0,247), as well as a negative correlation between age and encephalic inflammation (r = 0,518; p = 0,03) or cardiac inflammation (r = 0,385; p = 0,186) in male group . Population aging, which has increased since the last decades of the twentieth century, has changed the demographic and epidemiological profiles of countries such as brazil . The increase in chronic degenerative diseases, which have replaced infectious and parasitic diseases, has demanded that more emphasis be placed on the prevention and treatment of such diseases, which leads to the need to know about their pathological changes during the aging process . In the present study, heart and brain weights of non - elderly patients with cysticercosis were found to be higher than those of the elderly group with the parasitosis, without significant difference . Male patients had higher heart weight and brain weight, regardless of age . According to the literature, heart weight ranges from 347 g to 487 g in individuals over 60 years old, and brain weight ranges from 1105 g to 1264 g [1416]. In an experimental study, not only did adult male rats have higher heart weight, but they also had larger myocardiocytes compared with female rats, which might be related to a higher risk of cardiovascular disease in males . Studies involving patients without encephalopathy showed that the brain weight and the volume and density of the cell undergo a steady decrease with age, whereby male patients have higher brain weight than female patients . Analysis of the inflammatory infiltrate showed that the non - elderly had significantly more cardiac and encephalic inflammation than the elderly, and that such inflammatory infiltrate decreases with age and depends upon the evolutionary stage of the cysticercus . The inflammatory process caused by cysticerci in the cerebral parenchyma and in the myocardium comprises mononuclear and polymorphonuclear cells, mainly eosinophils, macrophages, and lymphocytes [19, 20]. During the aging process, changes in the expression of functionally important cell receptors, reduction in the population of polymorphonuclear cells, and reduction in the capability of producing antibodies are verified, and these factors may lead to immune dysfunction [21, 22]. Therefore, our data might be related to changes in the immune response, mainly in t cells, which were found in the elderly individuals . The vesicular stage was more prevalent among the elderly and nodular calcified stage among non - elderly, and colloidal vesicular stage showed higher inflammatory infiltrate in both groups . Researches show that a more intense inflammation with lymphocyte and macrophage infiltrate can be found around the cysticercus in colloidal vesicular stage [10, 24, 25]. With cysts degenerates, the inflammatory reaction tends to decrease in the granular nodular stage, denoting continuity in the host reaction against the parasite remnants without, however, having an association with the type or intensity of the inflammatory response [24, 26]. The duration of each of the progressive stages in the natural history of cysticercosis has not been established because there are considerable differences between individuals, particularly in relation to the intensity of the endogenous immune response . Whereas the parasite typically dies few years after infection stimulating a vigorous inflammatory response, probably the acquisition of the parasite occurred most recently in the elderly than in non - elderly patients, or the elderly, due to changes in the immune system with aging, preserve the cysticercus in the initial phase for a long time . There are also some indications that, in human neurocysticercosis caused by t. solium, women show a more intense inflammatory profile in the cerebrospinal fluid than men do and, likewise, are more prone to develop a severe and generalized encephalitic process . These observations are in accordance with previous studies in which gender has been associated with the intensity of the inflammatory response against the parasite, possibly promoted by the female sex - steroid levels [7, 9, 2832]. Therefore, our data showed that even during senescence, when a decrease in the levels of female sex steroids is noticed, women have a more intense immune response towards cysticercosis in comparison with men . Studies have showed that the presence of multiple parasites is more common in older individuals . Encephalic inflammation and cardiac inflammation were more commonly found in the analyzed material, and most of the individuals had cysticercosis in more than one location . It was found that multiple cysticerci lesions and multiple vesicular cysts were more frequently observed in the elderly without an increase in severity of the clinical symptoms . This observation could indicate that susceptibility to become infected increases with age, whereas susceptibility to follow a pathogenic course of the infection decreases . The reverse effect of age upon susceptibility to infection and to resistance against severe disease has been found in other parasite infections such as schistosomiasis [33, 34] and it suggests that susceptibility and pathogenicity involve distinct physiological pathways that are independently regulated . This study presents important findings on the influence of gender on cardiac and encephalic inflammation in the elderly with cysticercosis, although it has some limitations, such as small number of samples for analyses, particularly of the gender influence, loss of many biopsies and retrospective design . Future researches are needed to determine the mechanisms of the differences related to gender and immunosenescence associated to immunological alteration caused by cysticercosis . In this study, we showed that the non - elderly had significantly more cardiac and encephalic inflammation than the elderly, and that such inflammatory infiltrate decreases with age and depends upon the evolutionary stage of the cysticercus . Furthermore, there are differences, concerning gender, in the intensity of the inflammatory response due to cysticerci in the heart and brain parenchyma during senescence . Even during this period, women continue to have a more intense response to the parasitosis.
There is a large and developing market for biodegradable bone substitutes in many fields of surgery . Special attention has been paid to develop bone substitutes for the cranio - maxillofacial skeleton since bone defects requiring grafting are easily created in routine procedures such as cranioplasty and facial asymmetry corrections . Numerous surgical techniques have been developed to reconstruct intra - operatively created cranio - maxillofacial bony defects [1 - 3]. Although bone substitutes, such as tricalcium phosphate (tcp) and bioactive glass (bag), are already widely used in humans, there is little published histological and radiological data comparing their bony healing . In vivo animal experiments using the rabbit calvarial critical - sized defect (csd) model is a well - established and especially suitable for cranial bone defect model as the rapid healing process is similar to the common human patient group of pediatric patients with a postoperative cranioplasty condition . The rabbit csd model serves as a basis to evaluate bone substitute products and compare their healing characteristics to the current gold standard, autogenous bone . Inorganic synthetic bone substitutes have also been combined with autologous differentiated stem cells [6 - 8] or even with bone marrow or adipose derived stem cells without osteogenic induction [9 - 11] with the goal of speeding up the ossification process in larger bone defects . There is a growing interest to gain an understanding of the fate of implanted cells within a porous solid scaffold and how extracellular mineralization induced by the scaffold affects the cells [12 - 17]. The bag scaffolds are known to be non - cytotoxic, bacteriostatic and capable of supporting both cell attachment and proliferation in vivo . Early results indicate that the inclusion of bag promotes precipitation of calcium phosphate on the scaffold surfaces leading to earlier cell differentiation and matrix mineralization [18 - 19]. Micro - computed tomography (micro - ct) serves as a new and accurate tool to analyze bone healing and to evaluate the bone formation on biomaterials such as tcp and bag . The aim of this study was to evaluate bone healing in rabbit critical - sized bicortical calvarial defects comparing two different synthetic scaffold materials, solid bioactive glass and tricalcium phosphate granules versus solid or particulated autogenous bone . Since granular materials were used in the form of tricalcium phosphate and particulated autogenous bone chips no mechanical testing was planned in this study since granular scaffolds are known to be characteristically non - loadbearing materials . The following animal care and experimental protocol received ethical approval (decision eshl-2008 - 07701/ym-23) from the oulu university hospital ethical committee . The study was performed in accordance with the declaration of helsinki and its later amendments . A total of 15 white new zealand male rabbits, aged 6 months or older and weighing at least 3.5 kilograms were included in this study . The anaesthesia was induced with subcutaneous injection of 15 mg / kg ketamine (ketalar 50 mg / ml, pfizer oy, helsinki, finland) and 0.25 mg / kg medetomide (domitor vet 1 mg / ml, orion oyj, espoo, finland). The eyes were protected against drying by applying carbomer gel (viscotears 2 mg / g, alcon finland, vantaa, finland). An intravenous catheter was inserted to the lateral ear vein and a continuous infusion of sodium hydrochloride solution 0.9% (natriumklorid 0.9%, fresenius kabi ab, helsinki, finland) was given under the operation . Antibiotic prophylaxis with 60 mg / kg cefuroxime (zinacef 750 mg, glaxosmithkline oy, espoo, finland) was given intravenously before the operation as a single dose . The animals were protected against temperature loss with special covers and warming pads in standard animal laboratory manner . Under general anaesthesia, the fur on the planned operation area on the rabbit head was shaved and cleaned properly with povidone iodide (betadine 75 mg / ml, oy leiras finland ab, helsinki, finland) solution . A double cover and sterile instrumentation was used individually on each animal for the surgery according to the standard or protocol . For local anaesthesia, 2 ml of lidocaine (lidocaine c. adrenalin 2%, orion oyj, espoo, finland) was infiltrated in the skin around the planned incision line in the midline of the skull . From an approximately 5 cm long sagittal incision of the skin the periosteum was elevated and the rabbit was operated bilaterally bicortical full thickness circular critical - sized (15 mm in diameter) defects, producing a total of 30 defects . Ten defects were filled with autologous particulated calvarial bone combined with fibrin glue to fix the bone mass within the defects mimicking particulate bone harvesting during cranioplasty . Five defects were filled with an autologous calvarial bone block mimicking the standard cranioplasty maneuver . Five defects were filled with a porous solid circular 15 mm diameter resorbable commercially available three - dimensional porous bag fiber scaffolds (inion biorestore, tampere, finland). These solid scaffolds were characterized by a nominal composition of 11.1 - 12 wt% na2o; 15 - 17.1 wt% k2o; 2.8 - 3.3 wt% mgo; 12.7 - 15.2 wt% cao; 2.7 - 3.8 wt% p2o5; 1 - 1.4 wt% b2o3; 0 - 0.6 wt% tio2; and 48.5 - 52 wt% sio2 . The solid scaffolds were made of melt spun bioactive glass fibers of 75 m diameter which were sintered under defined conditions to produce a rigid scaffold with total porosity of 70% . Five defects were filled with 0.5 g of biphasic -tcp granules (straumann bone ceramic, straumann ag, basel, switzerland). The granules were 100% crystalline being composed of 60% hydroxyl apatite and 40% -tricalcium phosphate . Straumann bone ceramic granules are available in 2 sizes: 0.4 to 0.7 mm and 0.5 to 1 mm . In this study granule sizes ranging from 500 to 1000 after the operation the soft tissue and skin was sutured tightly to cover the operation areas with vicryl 3 - 0 (ethicon inc ., all animals received intensive supervision and care at the animal care facilities 24 h / day for the first three days following the surgery and for weeks after surgery three times a day . For postoperative buprenorphine (temgesic, rb pharmaceuticals ltd, slough, england, uk) and against opioid related intestinal motility problems 3 mg metoclopramide s.c . (primperan 5 mg / ml, sanofi - aventis oy, helsinki, finland,) three times a day for three postoperative days . Decrease in eating, drinking and moving or clear suffering from pain were determined to be the humane end points and the animals would have been terminated immediately if these signs were exhibited . Qualitative evaluation of the samples following harvest healing of the calvarial csds was allowed up to the 6 weeks post placement time point to evaluate the early stage healing and ossification process in defects . The animals were terminated by giving an overdose of pentobarbital (mebunat vet, orion oyj, espoo, finland) intravenously after sedation with a subcutaneous injection of 0.25 mg / kg medetomide (domitor vet 1 mg / ml, orion oyj, finland) and 15 mg / kg ketamine (ketalar 50 mg / ml, pfizer oy, helsinki, finland). Immediately after termination, the skulls of the animals were exposed and a parietal bone block including the defect area and its surrounding bone was taken as a specimen . One rabbit had traumatized its cranial wound resulting in an ectopic position of the implanted scaffold which led to a lack of contact with the surrounding bone resulting in disturbed healing . After sacrifice the calvarial bone blocks including the bilaterally created defects with filling materials were harvested for ex vivo micro - ct imaging (figure 1). The samples were scanned with an in vitro micro - ct device (skyscan 1272, bruker micro - ct) with scanning parameters of: 50 kv, 200 ma, 1200 projections, exposure 1400 ms / frame, average of 2 frames per projection, 0.5 mm aluminium filter and isotropic 8 m voxel side length . Reconstructions for x - ray projections were made with skyscan nrecon - software (v. 1.6.9, brker micro - ct, kontich, belgium). Volume of interest (voi, 250 x 350 x 250 pixels) was selected from the reconstructed cect image stacks . A = empty negative control defect showing a minimum of healing with uneven, centric ingrowth from the margins of the defect (arrows) and small bone islets in the center of the defect (arrowhead). B = defect filled with combination of particulated autogenous bone (yellow and orange granules inside of dotted circle) and fibrin glue (dark red areas pointed with arrowhead). The margins of the bone block have been partially resorbed (long arrow) and partial osseous continuity of the margin the bone block can be seen (inside of dotted circle). Ingrowth of new bone from the defect margin can be seen as well as very small ossifying spots in the middle of scaffold material (yellow / orange spots inside of dotted circle). E = tcp granules (blue spots pointed with arrowheads) filling a calvarial defect . Bone formation on the material surfaces can be seen (yellow and orange spots pointed with arrows). Radiological analysis a volume of interest was manually selected from the defect and fully mineralized bone was thresholded to calculate the ratio of between the deposited bone and defect volume . Analyses were conducted with ctan (v. 1.14.4.1, brker micro - ct, kontich, belgium). Mean percentages of bone formation with standard deviation (sd) were calculated from micro - ct analyses . The negative control group was compared separately with all filling materials using mann - whitney s u test . The negative control group revealed a noticeable variance in bone formation between individuals with micro - ct, 21.8 (23.7)%, presented in table 1 . The micro - ct analysis at the 6 weeks post implantation time point revealed new bone formation in all defects . Particulated bone with fibrin glue and solid bone block were superior to bag and tcp (p = 0.012, p = 0.025, p = 0.019 and p = 0.024, respectively; table 1). The micro - ct analysis also showed significantly more new bone formation with tcp granules than with bag scaffolds (p = 0.024, table 1). Micro - computed tomography results significance from the analysis of mann - whitney test, each filling material compared with negative control . Comparison of particulated bone graft + fibrin glue vs. bone block, bag and tcp, benjamini - hochberg procedure . Sd = standard deviation; bag = bioactive glass; tcp = tricalcium phosphate . In treating cranial deformities, often the cranial vault must be reshaped by either recontouring or by sectioning the cranial vault into pieces . When fragments are reassembled there may be palpable or visible defects between the bone pieces which may be unsightly when the scalp and pericranial tissues are redraped over the recontoured bone . Such unattractive bony defects can be filled with fragments of bone, particulated pieces of bone and bone dust collected during craniotomy . Some clinicians collect bone dust and particulated bone during cranioplasty and mix it together with fibrin glue to form a slurry - like bone paste . The fibrin glue in this paste helps fixate the bone pieces in the slurry and prevents their migrating from the wound . While the use of bone slurry is common practice in some craniofacial units, there is little evidence to show that it is beneficial . This study attempted to use an animal model to show that solid cortical bone grafts and bone slurry could have a role in managing cranial bony defects . This study also tried to illustrate the differences in bone defect healing with solid versus granular synthetic scaffolds . Advances in imaging have led to improved resolution and to the ability to reveal both newly calcifying and already calcified tissue in healing bone defects . Micro - ct proves to be a novel and accurate tool for quantitative analysis of bone formation (figure 1) whereas traditional histology better illustrates the cellular changes and histological properties of the healing area . The micro - ct results of this study illustrate some of the key differences in bone defect healing when the defect is left empty or when a solid or granular scaffold is used . Remarkably, the results showed a notable variance in new bone formation between individual rabbits with empty control defects . The percentages in micro - ct range from 1.9 to 57.6, which is due to individual variation in the healing of large bone defects . In some individual rabbits however, what is common in the negative control group is the pattern of healing . Since these are critical sized defects the healing is incomplete at the six week time point . In contra - distinction to this it must be realized that an empty void defect is dramatically different from a defect filled with a solid material . In the case of a solid scaffold or a bone block, this leaves little empty space available for new bone growth which is the parameter of interest measured in this experiment . In the case of a slowly resorbing solid bag scaffold the only space allowed for tissue ingrowth is either into the three - dimensional porous space within the scaffold or around the solid implant on the dural surface of the implant . Solid biodegradable materials obstruct the ingrowth of bone to the defect area by its shear physical presence, unless the scaffolds resorb or have accessible porosity to allow the ingrowth . Thus sufficient blood flow by ingrowth of fibrous tissue and blood vessel is essential for the degradation and eventual replacement of a solid biomaterial by autologous tissues . In the case of solid bone blocks, which are analogous to replacing a devascularized bone flap during cranioplasty, these grafts become incorporated by replacement resorption and little physical space exists for new bone growth at the six week post grafting time point . Granular materials are inherently different from solid configurations of the same material [25 - 28]. There is a far greater surface area available for cellularization with the granular configurations over solid structures . Moreover there is space between the granules to permit autologous tissue ingrowth and new bone formation which is evident at the six week time point for both particulate bone and -tcp granules . The pore size of the implanted tcp granular scaffold plays an important role in revascularization as showed earlier by other investigators . In general granular scaffolds are more quickly incorporated into the healing of a bony defect when compared to solid scaffolds . However, scaffolds in a granular format lack any load bearing capacity, while scaffolds in a solid form possess physical properties that allow them to be used to replace large cranial defects despite their slow resorption and replacement compared to granular scaffolds . The micro - ct images show the three - dimensional curved structure of the tcp granules which permit locking of the particles, preventing their migration out of the bony defects . The ingrowth of the fibrous tissue and new vessels between the granules enables the granules to remain locked with bone bridging which makes the structure of the graft construct even more stable . On the other hand mechanical stability and good vascularity seems to hasten the resorption of the bag scaffold more than seen with the granules . Both biomaterials used in this study, tcp and bag, seemed to induce islets of bone growth histologically underneath the implanted area . Whether this phenomenon is caused by local irritation or stimulation of the stem cells in dura lying between the brain and the implant material remains unknown and will require more detailed investigation in the future . A major limitation of this study is related to the absence of density differentiation between assayed grafts, since the material density of autologous bone, tricalcium phosphate, bioactive glass, can be similar to that of the newly formed bone, thus rendering data interpretation difficult . In a future in vivo study the authors will attempt to apply synchrotron based computed tomography imaging, where various artefacts can be avoided and superior resolution achieved even though the study protocol might require the additional use of bone deposition seeking labels such as strontium . This should allow for improved separation between newly formed bone and residual graft remains, thus greatly enhancing the relevance of the attained results . The variability within the negative control group also leads to the question if the used model truly reflects a critical size defect - since up to approximately 60% of regeneration was attained . Longer time points such as 12 weeks would help answer the question of how complete the healing of an unfilled defect would be in the longer - term . Larger sample sizes in the future may also help lessen the effect of such inter - subject variability . Other sources of variability may arise, for instance, from the defect location, the inclusion of cranial sutures, the presence of dural tears, from thermal damage to the wound by electrocautery or by heat generation during the drilling of bone . The findings of this study suggest that particulated autogenous bone with fibrin glue and solid autogenous bone blocks were superior in new bone formation to bioactive glass and tricalcium phosphate, while tricalcium phosphate granules were found to be superior to bioactive glass with more new bone formation in the rabbit critical - sized defect model . The authors wish to express their gratitude for the financial support provided to this project by the iti foundation, basel, switzerland (iti grant number 619 - 2009), the university of oulu evo and vtr grant fund, the stiftelsen alma och k.a . Snellman foundation, oulu, finland, academy of finland (grants no . 268378 and 273571), the sigrid juselius foundation and european research council under the european union s seventh framework programme (fp/2007 - 2013)/erc grant agreement no . 336267, for their generosity.
Privacy is an individual right, which covers situations related to the intimacy of each person, to respect for dignity, and to family and social relationships . When hospitalized, people find themselves in a situation of extreme fragility, in which they often need care that invades their intimacy . Being ill generates some feelings, such as incapacity, dependency, insecurity, and a feeling of loss of control over oneself . Patients face hospitalization as a depersonalization factor, since they recognize the difficulty in maintaining their identity, intimacy, and privacy . Observing the practice of care, a lack of concern regarding body exposure is noted in many situations, along with little preoccupation with the patient s modesty . This person, who should be perceived as the subject of care, becomes an object, losing his / her identity . Ethical and technical aspects should always be observed by the players involved in patient care . The situation of exposure of the patient s body at the time of medical care has been reported as a moment of potential breach of a patient s privacy . However, healthcare professionals should be attentive to one more action which can compromise the patient s dignity: the registration of images . The use of the cell phone with photographic camera by many healthcare professionals has facilitated the capture and reproduction of images of patients with a compromise of the level of consciousness . Particularly in the emergency scenario, filming raises specific ethical issues, since the patients are often vulnerable, many times without the capacity to consent, or they feel under pressure to give their consent . One international study analyzed the commercialization of images of medical care in the hospital settings . As a result, it was shown that many times filming violated the privacy of patients, which generally could be avoided with the adequate consent from the individual . We point out, however, that patients could feel obligated or compelled by the person who was in command of his / her care and well - being . In brazil, the legislation is very clear as to the right to images . According to the federal constitution of 1988, article 5, item x, intimacy, private life, honor, and the image of persons are inviolable, assuring the right to compensation for the material or moral damage resulting from its violation . Although the federal constitution and some professional boards prohibit making and/or using images of patients without their consent, within the hospital setting this is still a frequent practice on the part of some professionals . In times when technology is highly popularized, and when most professionals have cell phones with photographic cameras, the capture and reproduction of patient images are facilitated . It is believed that the evaluation of knowledge of the healthcare professionals as to the rights of images and privacy of the patient is important, since it generates a basis for conducting educational interventions on the theme . To evaluate the knowledge of healthcare professionals as to the capture and reproduction of patient images within a hospital environment . This is an observational and cross - section study, carried out at the teaching hospital of the universidade federal de so paulo, in the city of so paulo (sp), during the period from february 2013 to july 2013 . The study sample was made up of nurses, licensed practical nursing and nursing technicians, medical residents, and physical therapists that performed their activities in the hospital environment . Based on the estimate of a population proportion of 2,590 professionals, the formula for determination of sample size of 335 individuals was used, with a confidence interval of 95% and sample error of 5% . A subject characterization questionnaire was used with the purpose of investigating some sociodemographic aspects (sex, age, profession, time of professional experience, type of professional bond, and unit where they worked) and those related to capture and reproduction of images within the hospital setting . Data collection was carried out during the period of february to july, 2013 . Before the collection, the informed consent form was presented to the study participants, and upon their agreement to participate in the research, they were presented with the data collection instruments . The data were presented by means of descriptive statistics . To compose the variables of interest, the test was used, and when this was not appropriate, the likelihood ratio was used . The project was approved by the research ethics committee of the universidade federal de so paulo, under caae number 09250112.2.0000.5505 . Of the 360 healthcare professionals interviewed, there was a 72.8% predominance of females, 89.7% were aged 40 years, 31.4% were nurses, 36.7% reported a professional experience time of 1 to 3 years, 43.9% (n=158) were residents, and regarding the place of work in the hospital, 43.0% worked at inpatients units . Most participants (81.3%) declared that they had witnessed other healthcare professional making images of patients, 9.7% of which had seen this once, 23.3% from two to four times, 48.3% more than four times, 5.3% reported not remembering, and 13.3% were not witnesses . When asked if the professional had photographed or filmed patients over the previous year, 57.8% answered yes, and 71.1% declared they had not photographed or filmed anyone who was unconscious . Among the professionals who responded positively as to having captured images (n=147), most reported having requested verbal authorization (61.2%) and the minority requested written authorization (10.9%) (table 1). Table 1aspects of the authorization to make images (n=147)questionsauthorization to make images total n (%) yesnonot enough timedoes not remembern (%) n (%) n (%) n (%) 1 . Did you request verbal authorization from the patient or guardian to make these images?90 (61.2)34 (23.1)11 (7.5)12 (8.2)147 (100)2 . Did you request written authorization from the patient or guardian to make these images?16 (10.9)109 (74.2)14 (9.5)8 (5.4)147 (100)3 . Did you confirm the existence in the medical records of authorization from the patient or guardian for capturing images?10 (6.8)115 (78.2)9 (6.1)13 (8.8)147 (100) in questioning if they considered it important to request authorization from the patient to capture the images, 90% (n=324) responded yes, and 8.8% (n=32) responded yes, but that frequently there was not enough time to request it . When the participant was questioned as to if they thought making images of a person without their prior authorization had legal implications, 97.5% (n=351) responded yes . Of the 147 participants that affirmed having made images, 41.5% (n=61) used them for the presentation of clinical cases and studies, 12.2% (n=18) showed friends and relatives outside of work, and 0.7% (n=1) published them on social networks . The results as to knowledge of the professionals on capturing images demonstrated that most professionals knew of the prohibition of making images (97.5%) and of the need to preserve the individual s image (98.1%) (table 2). Table 2aspects as to the preservation of the image and legal implications (n=360)questionsknowledge of professionals total n (%) yesnoi do not known (%) n (%) n (%) 1 . Do you think that making images of a person without prior authorization has legal implications?351 (97.5)3 (0.8)6 (1.7)360 (100)2 . Do you think that the individual s image should be preserved?353 (98.1)4 (1.1)3 (0.8)360 (100)3 . In the brazilian federal constitution, civil code, and penal code do you know about any item that provides for the capture and use of the images of persons?126 (35.0)168 (46.7)66 (18.3)360 (100)4 . In the code of ethics of your profession, do you know about any legislation that provides for the capture and use of images of patients?167 (46.4)116 (32.2)77 (21.4)360 (100) in making the association of sociodemographic variables with taking images, the test was used, which showed that the male professionals made more images / films (p=0.0058), and in the age group 41 years, a significant number of professionals had not made images (p=0.000). In order to obtain the list of the professional category with the number of individuals who captured images, the test was also used, which demonstrated that in the category of nursing technicians / licensed practical nursing, there was a significantly greater number of individuals who did not photograph / film patients relative to the other professionals (p=0.0000). Additionally, the same statistical test revealed that professionals with an experience of more than 5 years photographed / filmed significantly less relative to the employees with less time of experience (p=0.0022). As to a bond, medical residents photographed / filmed significantly more relative to those with other bonds (p=0.0287) (table 3). Table 3association between sociodemographic characteristics and making patient s images (n=360)variablefrequency totalp valueonce2 - 4 times>4 timesi do not rememberi did not film and i did not photographsex * 0.0058male882935098female2736347158262 total35446310208360 age, years * 0.000040354361101743234101203437 total35446310208360 profession * 0.0000nurse121817165113nursing technician / licensed practical nursing936489111physician9163033795physical therapist571021741 total35446310208360 time of professional experience, years * 0.0022<1469342641 - 31426333561323 - 556612543>512615385121 total35446310208360 bond * 0.0287[employed under] clt131119476123resident142836476158specializing112149approved in public contest74615270 total35446310208360 * test . Clt: consolidated labor laws . As to knowledge of any item in the brazilian federal constitution, civil code, and penal code as to the capture of images of persons currently, technology has given great support to the work of healthcare professionals . Nevertheless, the fact that the majority of healthcare professionals has access to new technologies, such as cell phones with photographic cameras, has created a new ethical confrontation in healthcare organizations, since situations experienced by the patients are easily captured and reproduced, with rare obtaining of prior consent from the patient . Patients should have a chance to give or not give their consent for making of images . This should be documented in a way that fulfills the recommendations of the code of ethics and of the patient s data and confidentiality protection . Once the subject photographed authorizes the use of the image, it is important to observe that the consent should be interpreted restrictively, since acceptance to be photographed may not include its publication; in the same way, the agreement for publication does not include other uses . In this study, as well as in the similar study result the institute of medical illustrators published, in 2006, a manual on obtaining consent for images of patients, which emphasizes that obtaining consent is the responsibility of the professional . This term should have specified the purpose of the images, so that the patient may agree or not agree with its use . It is a common practice to offer three levels of authorization: for use only in the medical records; for use in teaching healthcare professionals and students; for publication and public domain . Good practices indicate that the consent for publication should only be obtained for one specific use; it is not a comprehensive release . If the publication is in a journal, book, electronic media, or the internet, the patient should receive the orientation that, once published, the consent cannot be withdrawn, especially for publication on the internet, since the images are in public domain . Some experts on civil law still defend that the right to image includes cases in which the image might be violated without there being any graphic reproduction of it, not restricting it only to the physical form of the subject . The image - picture and the image - attribute compose the identity of each individual, and its violation may cause moral damage to the person . They support that every and any lesion that affects the individual s being will have enough characteristics to be considered as moral damage . Despite moral damages and invasion of privacy, there are law researchers defending that public interest is a directive of extreme importance and therefore, if the divulging of an image is justified in this, the law of freedom of expression prevails . Others consider that public interest should be presumed in a democratic rule - of - law state, in order to make feasible the free circulation of ideas . Although this study verified that the participating professionals used verbal authorization, the consent process should be in written form . It is important to have documented proof to support a legal defense, when necessary . Written consent is the requirement of many journals before publication and edition of photographs with consent forms from the patients . It is important to point out that, in the institution where the study was developed, there is no policy for image capture, with the exception of the burn unit, where at the time of admission the patient or guardian is requested authorization for capturing images . The results of this study allow inferring there are gaps in knowledge of the healthcare professionals during the undergraduate course relative to the right to image, since 46.7% said they were not aware of any item in the brazilian federal constitution, civil code, and penal code as to this capture, as well as to the use of images of the individuals . The lack of information on the right of images by healthcare professionals can be directly related to the absence of formal orientation for the lecturers on this topic . According to a study that evaluated the orientation given by universities of the united states and the united kingdom to their lecturers on the topic of consent for images and publications, it was identified that of the nine participating european universities, only one issued specific orientation for the faculty as to how to show photographs of patients in their presentations . And of the three american participants, none gave orientation, although the legislation of the united states refers to confidentiality . This study revealed that the capture and reproduction of patient images within a hospital environment are carried out by a considerable part of the professionals investigated, despite most being aware of the prohibition of this practice and the need to preserve the individual s image . Nurse technicians and licensed pratical nursing, as well as professionals with longer time of experience perform the capture and reproduction of images with less frequency . Residents of the various fields of health made up the category that most captured and reproduced images and that showed the least amount of knowledge as to the items provided for in the brazilian federal constitution, civil code, and penal code . Avaliar o conhecimento dos profissionais da sade sobre a captao e a reproduo de imagens de pacientes em ambiente hospitalar . Estudo observacional e seccional, realizado com 360 profissionais de sade (equipe de enfermagem, fisioterapeutas e mdicos), que atuam em um hospital universitrio localizado no municpio de so paulo (sp). Foi aplicado um questionrio com informaes sociodemogrficas e relacionados captao e reproduo de imagens no ambiente hospitalar . Dos 360 entrevistados, 142 haviam captado imagens de pacientes no ltimo ano, e 312 afirmaram ter visto outro profissional fazendo imagens de pacientes . Dos participantes que captaram imagens, 61 disseram que as utilizaram para estudos e apresentao de casos clnicos, e 168 profissionais relataram no conhecer nenhuma legislao do cdigo penal brasileiro a respeito de captao e do uso de imagens . H uma lacuna na formao dos profissionais de sade em relao ao uso da imagem de pacientes, sendo necessrio incluir, na graduao, disciplinas que contemplem essa temtica, assim como a regulamentao por parte das instituies de sade . A privacidade um direito individual, que abrange situaes relacionadas intimidade de cada um, ao respeito dignidade, e aos relacionamentos familiares e sociais . As pessoas, quando hospitalizadas, encontram - se em uma situao de extrema fragilidade, na qual, muitas vezes, necessitam de cuidados que invadem sua intimidade . As condies de enfermidade geram sentimentos como incapacidade, dependncia, insegurana e sensao de perda do controle sobre si mesmo . Os pacientes encaram a hospitalizao como fator de despersonalizao, por reconhecerem a dificuldade para manter sua identidade, intimidade e privacidade . Observando a prtica assistencial, percebem - se, em muitas situaes, a falta desvelo com a exposio corporal e pouca preocupao com o pudor do paciente . Este, que deveria ser percebido como sujeito do cuidado, torna - se objeto, perdendo sua identidade . Aspectos ticos e tcnicos devem sempre ser observados pelos atores envolvidos na assistncia ao paciente . A situao de exposio do corpo do paciente no momento do cuidado tem sido relatada como um momento de potencial infrao da privacidade do paciente . Entretanto os profissionais da sade devem ficar atentos a mais uma ao que pode comprometer a dignidade do paciente, o registro de imagens . O uso do telefone celular com cmera fotogrfica por muitos profissionais da sade tem facilitado a captura e a reproduo de imagens dos pacientes no momento de seu atendimento, principalmente dos pacientes com comprometimento do nvel de conscincia . Particularmente no cenrio de emergncia, as filmagens trazem questes ticas especficas, pois os pacientes esto vulnerveis, muitas vezes sem a capacidade de consentir ou sentem - se sob presso para dar o consentimento . Como resultado, encontrou que, muitas vezes, a filmagem violava a privacidade dos pacientes e isso, geralmente, podia ser evitado com o consentimento adequado do indivduo . Ressalta - se, no entanto, que os pacientes podiam sentir - se obrigados ou compelidos por quem estava no comando de seu cuidado e bem - estar . No brasil, a legislao bem clara quanto o direito imagem . De acordo com a constituio federal de 1988, art . 5, inciso x, so inviolveis a intimidade, a vida privada, a honra e a imagem das pessoas, assegurado o direito a indenizao pelo dano material ou moral decorrente de sua violao . Embora a constituio federal e os conselhos de algumas profisses probam fazer e / ou usar imagens de pacientes sem a autorizao dos mesmos, no ambiente hospitalar pode - se observar que essa ainda uma prtica frequente por parte de alguns profissionais . Em tempos em que a tecnologia est altamente popularizada, e que a maioria dos profissionais tm celular com cmera fotogrfica, a captao e a reproduo de imagens do paciente so facilitadas . A avaliao do conhecimento dos profissionais de sade sobre os direitos de imagem e privacidade do paciente importante, uma vez que gera subsdios para a conduo de intervenes educativas sobre o tema . Avaliar o conhecimento dos profissionais da sade sobre a captao e a reproduo de imagens de pacientes em ambiente hospitalar . Trata - se de um estudo observacional e transversal, realizado no hospital de ensino da universidade federal de so paulo, na cidade de so paulo (sp), no perodo de fevereiro de 2013 a julho de 2013 . A amostra do estudo foi constituda por enfermeiros, auxiliares e tcnicos de enfermagem, mdicos residentes e fisioterapeutas, que desempenhavam suas atividades no ambiente hospitalar . Com base na estimativa da proporo populacional de 2.590 profissionais, foi utilizada a frmula para determinao do tamanho da amostra de 335 indivduos, com nvel de confiana de 95% e erro amostral de 5% . Foram includos 360 questionrios, por meio de amostra de convenincia . Foi utilizado um questionrio de caracterizao dos sujeitos com a finalidade de investigar aspectos sociodemogrficos (sexo, idade, profisso, tempo de experincia profissional, tipo de vnculo profissional e unidade de atuao) e aqueles relacionados captao e reproduo de imagens no ambiente hospitalar . Antes da realizao da coleta, foi apresentado o termo de consentimento livre e esclarecido aos participantes do estudo e, mediante sua concordncia em participar da pesquisa, foram apresentados os instrumentos de coleta de dados . Para comparar as variveis de interesse, foi utilizado o teste e, quando este no era apropriado, utilizou - se o teste da razo de verossimilhana . O projeto foi aprovado pelo comit de tica em pesquisa da universidade federal de so paulo sob nmero caae 09250112.2.0000.5505 . Dos 360 profissionais de sade entrevistados, houve predominncia de 72,8% do sexo feminino, 89,7% possuam idade 40 anos, 31,4% eram enfermeiros, 36,7% relatavam experincia profissional de 1 a 3 anos, 43,9% (n=158) eram residentes e, em relao ao local de atuao no hospital, 43,0% trabalhavam em unidade de internao . A maior parte dos participantes (81,3%) afirmou que presenciou algum outro profissional de sade fazendo imagens de pacientes, dos quais 9,7% haviam presenciado uma vez, 23,3% de duas a quatro vezes, 48,3% mais de quatro vezes, 5,3% relataram no lembrar e 13,3% no presenciaram . Quando perguntado se o profissional j havia fotografado ou filmado pacientes no ltimo ano, 57,8% afirmaram que sim, e 71,1% disseram no ter fotografado ou filmado algum inconsciente . Dentre os profissionais que responderam afirmativamente sobre ter captado imagens (n=147), a maioria relatou ter solicitado autorizao verbal (61,2%) e a minoria solicitou autorizao escrita (10,9%) (tabela 1). Tabela 1aspectos sobre a autorizao para realizao das imagens (n=147)questesautorizao para realizao de imagens total n (%) simnono houve tempo hbilno lembron (%) n (%) n (%) n (%) 1 . Voc pediu autorizao verbal ao paciente ou responsvel por ele para fazer essas imagens?90 (61,2)34 (23,1)11 (7,5)12 (8,2)147 (100)2 . Voc pediu autorizao escrita ao paciente ou responsvel por ele para fazer essas imagens?16 (10,9)109 (74,2)14 (9,5)8 (5,4)147 (100)3 . Voc confirmou a existncia no pronturio de autorizao do paciente ou responsvel para captao de imagens?10 (6,8)115 (78,2)9 (6,1)13 (8,8)147 (100) ao questionar se consideravam importante solicitar autorizao do paciente para captar as imagens, 90% (n=324) responderam que sim e 8,8% (n=32) responderam que sim, porm frequentemente no havia tempo hbil para solicit - la . Quando o participante era questionado se achava que fazer imagens de algum sem sua prvia autorizao tinha implicaes legais, 97,5% (n=351) responderam que sim . Dos 147 participantes que afirmaram ter feito imagens, 41,5% (n=61) usaram para apresentao de casos clnicos e estudos, 12,2% (n=18) mostraram para amigos e parentes fora do trabalho, e 0,7% (n=1) publicou em redes sociais . Os resultados sobre o conhecimento dos profissionais sobre a captao de imagens demonstrou que a maioria dos profissionais sabia da proibio da realizao de imagens (97,5%) e da necessidade de preservar a imagem do individuo (98,1%) (tabela 2). Tabela 2aspectos quanto preservao da imagem e implicaes legais (n=360)questesconhecimento dos profissionais total n (%) simnono sein (%) n (%) n (%) 1 . Voc acha que fazer imagens de algum sem sua prvia autorizao tem implicaes legais?351 (97,5)3 (0,8)6 (1,7)360 (100)2 . Voc acha que a imagem do indivduo deve ser preservada?353 (98,1)4 (1,1)3 (0,8)360 (100)3 . Voc conhece, na constituio federal, no cdigo civil e no cdigo penal brasileiro, algum dispositivo que disponha sobre a captao e o uso de imagem de pessoas?126 (35,0)168 (46,7)66 (18,3)360 (100)4 . Voc conhece, no cdigo de tica da sua profisso, alguma legislao que disponha sobre a captao e o uso de imagem de pacientes?167 (46,4)116 (32,2)77 (21,4)360 (100) ao realizar a associao de variveis sociodemogrficas com a realizao de imagens, foi utilizado o teste, que evidenciou que os profissionais do sexo masculino realizaram mais imagens / filmagens (p=0,0058), assim como na faixa etria 41 anos se observou um nmero significativo de profissionais que no realizaram imagens (p=0,000). Para obter a relao da categoria profissional com o nmero de indivduos que captaram imagens, tambm foi utilizado o teste, que demonstrou que, na categoria de tcnicos / auxiliares de enfermagem, havia um nmero significativamente maior de indivduos que no fotografaram / filmaram os pacientes em relao as demais profisses (p=0,0000). Alm disso, o mesmo teste estatstico revelou que profissionais com tempo de experincia maior que 5 anos fotografaram / filmaram significativamente menos em relao aos funcionrios com menor tempo de experincia (p=0,0022). No que se referia ao vnculo, residentes fotografaram / filmaram significativamente mais em relao aos profissionais com outros vnculos (p=0,0287) (tabela 3). Tabela 3associao entre as caractersticas sociodemogrficas e a realizao de imagens de pacientes (n=360)varivelfrequncia totalvalor de p1 vez2 - 4 vezes>4 vezesno lembrono filmei e no fotografeisexo masculino882935098 * 0,0058feminino2736347158262 total35446310208360 idade, anos 4035436110174323 * 0,00004101203437 total35446310208360 profisso enfermeiro121817165113 * 0,0000tcnico / auxiliar de enfermagem936489111mdico9163033795fisioterapeuta571021741 total35446310208360 tempo de experincia profissional, anos <146934264 * 0,00221 - 31426333561323 - 556612543>512615385121 total35446310208360 vnculo clt131119476123 * 0,0287residente142836476158especializando112149concursado74615270 total35446310208360*teste . Clt: consolidao das leis do trabalho . No que se refere ao conhecimento de algum dispositivo existente na constituio federal, no cdigo civil e no cdigo penal brasileiro sobre a captura de imagens de pessoas, os residentes foram os que relataram menor conhecimento (p=0,0465). Atualmente, a tecnologia tem dado um grande suporte para atuao dos profissionais de sade . No entanto, o fato de a maioria dos profissionais de sade ter acesso a novas tecnologias, como celulares com cmeras fotogrficas, criou um novo confronto tico nas instituies de sade, uma vez que situaes vivenciadas pelos pacientes so facilmente capturadas e reproduzidas, com rara obteno de consentimento prvio do paciente . Os pacientes devem ter a chance de conceder ou no o consentimento para realizao de imagens . Isso deve ser documentado de forma que se cumpram as recomendaes do cdigo de tica e de proteo de dados e confidencialidade do paciente . Uma vez que o sujeito fotografado autoriza a utilizao da imagem, importante observar que o consentimento deve ser interpretado restritivamente, uma vez que o aceite em permitir a fotografia pode no incluir a publicao da mesma; tampouco a concordncia em publicao no inclui outros usos . Neste estudo, bem como no resultado de estudo semelhante, foi identificado que a principal finalidade das imagens foi o ensino . O institute of medical illustrators publicou, em 2006, um manual sobre a obteno de consentimento de imagens dos pacientes, no qual est frisado que a obteno do consentimento de responsabilidade do profissional . Esse termo deve ter especificada a finalidade das imagens, para que o paciente possa ou no concordar com seu uso . Prtica comum oferecer trs nveis de autorizao: para uso nos pronturios apenas; para uso no ensino de profissionais de sade e estudantes; para a publicao e domnio pblico . A boa prtica indica que o consentimento para a publicao s deve ser obtido para um uso especfico, no sendo uma liberao abrangente . Caso a publicao seja em um jornal, livro, mdia eletrnica ou internet, o paciente deve receber a orientao de que, uma vez publicado, o consentimento no pode ser retirado, visto que as imagens passam a ser de domnio pblico, principalmente para a publicao na internet . Alguns estudiosos de direito civil defendem ainda que o direito imagem inclui casos em que a imagem possa ser violada sem haver reproduo grfica da mesma, no restringindo - se apenas forma fsica do sujeito . A imagem - retrato e a imagem - atributo compem a identidade de cada indivduo, e sua violao pode acarretar danos morais pessoa: toda e qualquer leso que atinja o ser do indivduo ter caractersticas suficientes para considerar - se como dano moral . Apesar dos danos morais e da invaso de privacidade, h pesquisadores de direito que defendem que o interesse pblico diretriz de extrema importncia e, portanto, se a divulgao de uma imagem encontra nele sua justificativa, prevalece liberdade de expresso . Outros consideram que o interesse pblico deve ser presumido em um estado democrtico de direito, a fim de viabilizar a livre circulao de ideias . Embora este estudo tenha verificado que os profissionais participantes usaram autorizao verbal, o processo de consentimento deve se dar de forma escrita . . O consentimento escrito exigncia de muitas revistas antes da publicao e da edio de fotografia com formulrios de consentimento dos pacientes . Vale ressaltar que, na instituio em que foi desenvolvido o estudo, no h politica para a captao de imagens, com exceo da unidade de tratamento de queimados, onde solicitada ao paciente ou responsvel, no momento da internao, autorizao para captao de imagens . Os resultados deste estudo permitem inferir que h lacuna de conhecimento dos profissionais da sade durante o curso de graduao em relao ao direito imagem, pois 46,7% disseram desconhecer algum dispositivo na constituio federal, no cdigo civil e no cdigo penal brasileiro sobre a captao, bem como sobre o uso de imagens dos indivduos . A falta de informao sobre direito de imagem por parte dos profissionais de sade pode estar diretamente relacionada ausncia de orientao formal para aos professores sobre esse assunto . Segundo estudo que avaliou as orientaes dadas por universidades dos estados unidos e do reino unido a seus professores, sobre o termo de consentimento para imagens e publicaes, foi identificado que, das nove universidades europeias participantes, apenas uma emitiu orientaes especficas para docentes sobre como mostrar fotografias dos pacientes em suas apresentaes e, das trs participantes americanas, nenhuma dava orientaes, embora a legislao dos estados unidos refira - se confidencialidade . Este estudo revelou que a captao e a reproduo de imagens de pacientes em ambiente hospitalar so realizadas por parte considervel dos profissionais investigados, apesar de a maioria ter conhecimento sobre a proibio dessa prtica e da necessidade de preservar a imagem do indivduo . Auxiliares e tcnicos de enfermagem e profissionais com maior tempo de experincia realizaram a captao e a reproduo de imagens com menos frequncia . Residentes das diversas reas da sade consistiram na categoria que mais captou e reproduziu imagens e que apresentou menos conhecimento sobre os dispositivos existentes na constituio federal, no cdigo civil e no cdigo penal brasileiro.
Traumatic loss of thumb leads to significant loss of function as the thumb contributes to about 40% of the hand functions . When the loss is bilateral, it is more devastating . Replantation is indicated in all cases; however, may not be possible in cases with unsuitably amputated thumb . Toe to thumb transfer is an excellent option in these patients and helps restore both function and esthetics. [14] we have performed a bilateral second toe to thumb transfer in a patient who underwent bilateral traumatic thumb amputation . A 25 year old male presented to our department with the traumatic amputation of both the thumbs distal to the metacarpophalangeal (mcp) joint, and at the proximal 1/3 of the proximal phalanx while working on punch press machine [figure 1]. The amputated parts were not replantable . After discussing the pros and cons of the different modalities of reconstruction, within 12 hours of the injury, the left second toe was transferred to the right thumb . A week later, the right second toe was transferred to left thumb . Preoperative view of amputated bilateral thumbs surgery was performed under the combination of spinal, epidural and brachial blocks, and was supplemented with intravenous general anaesthesia . V shaped incision was taken at the base of the second toe and extended proximally . The slip of extensor digitorum longus (edl) and the deep peroneal and dorsal digital nerves, were dissected proximally . The plantar dissection was started by taking a v shaped incision extending proximally up to the instep area . Plantar digital arteries and nerves were identified and proximal intraneural dissection was done . The flexor hallucis longus (fhl) tendon was dissected proximally for adequate length . Vascularity to the toe was confirmed after the release of tourniquet . At this stage, the dorsal branch of the radial artery, its venae comitantes, tributary of cephalic vein, the dorsal digital nerve and the proximal end of the extensor pollicis longus (epl) were explored and tagged . Through volar incision, flexor pollicis longus (fpl) the toe was separated by ligating the pedicle and transferred to recipient site [figure 2 and 3]. After removing the articular surface of donor toe proximal phalanx, flexor and extensor tendons were repaired [flexor digitorum longus (fdl) to fpl, extensor digitorum longus (edl) to epl]. Bilateral plantar digital nerves were coapted to volar digital nerves of the thumb, and the dorsal digital nerve was coapted to the dorsal digital nerve of thumb . The medial arch vein and venae comitantes of donor toe were anastomosed with a tributary of cephalic vein and venae comitantes of the radial artery, respectively . Residual raw areas on the radial and ulnar aspect of the reconstructed thumb were grafted [figure 4]. Plantar view of harvested second toe dorsal view of harvested second toe intraoperative view of second toe transferred to thumb . Also seen the grafted the area to avoid wound closure with undue tension the second metatarsal was shortened . The gap between the first and third toe was reduced by repair of intermetatarsal ligament and transverse k wire . A similar procedure was carried out for the reconstruction of the left thumb using the right second toe . In the right foot, fdma was plantar dominant . Rehabilitation: after 3weeks, the k wires were removed, and the patient was supervised daily . Physiotherapy in the form of active flexion and extension was started and continued for 2 months . Patient had a recovery of protective sensation at 8 months, and was advised to resume his normal duties . Functional assessment at follow up at the end of 1, 2 & 3 years is shown in table 1, and in figures 57 . The patient is back to the same occupation, & has no disability or restricted flexion in terminal joint on the left sside; hence no further surgery has been carried out . Functional assessment of the patient in the reported case study with bilateral second toe to thumb transfer, at follow up at the end of 1, 2 3 years good function achieved after toe transfer excellent strength achieved after toe transfer(right thumb) excellent strength achieved after toe transfer (left thumb) surgery was performed under the combination of spinal, epidural and brachial blocks, and was supplemented with intravenous general anaesthesia . V shaped incision was taken at the base of the second toe and extended proximally . Superficial dorsal veins were dissected . The first dorsal metacarpal artery (fdma) was seen dorsal to the dorsal interosseous muscle, and dissected proximally . The slip of extensor digitorum longus (edl) and the deep peroneal and dorsal digital nerves, were dissected proximally . The dorsal branch of the radial artery, its venae comitantes, tributary of cephalic vein, the dorsal digital nerve and the proximal end of the extensor pollicis longus (epl) were explored and tagged . Through volar incision, flexor pollicis longus (fpl) proximal end and digital nerves the toe was separated by ligating the pedicle and transferred to recipient site [figure 2 and 3]. After removing the articular surface of donor toe proximal phalanx, flexor and extensor tendons were repaired [flexor digitorum longus (fdl) to fpl, extensor digitorum longus (edl) to epl]. Bilateral plantar digital nerves were coapted to volar digital nerves of the thumb, and the dorsal digital nerve was coapted to the dorsal digital nerve of thumb . The medial arch vein and venae comitantes of donor toe were anastomosed with a tributary of cephalic vein and venae comitantes of the radial artery, respectively . Residual raw areas on the radial and ulnar aspect of the reconstructed thumb were grafted [figure 4]. Plantar view of harvested second toe dorsal view of harvested second toe intraoperative view of second toe transferred to thumb . The gap between the first and third toe was reduced by repair of intermetatarsal ligament and transverse k wire . A similar procedure was carried out for the reconstruction of the left thumb using the right second toe . In the right foot, fdma was plantar dominant . Rehabilitation: after 3weeks, the k wires were removed, and the patient was supervised daily . Physiotherapy in the form of active flexion and extension was started and continued for 2 months . Patient had a recovery of protective sensation at 8 months, and was advised to resume his normal duties . Functional assessment at follow up at the end of 1, 2 & 3 years is shown in table 1, and in figures 57 . The patient is back to the same occupation, & has no disability or restricted flexion in terminal joint on the left sside; hence no further surgery has been carried out . Functional assessment of the patient in the reported case study with bilateral second toe to thumb transfer, at follow up at the end of 1, 2 3 years good function achieved after toe transfer excellent strength achieved after toe transfer(right thumb) excellent strength achieved after toe transfer (left thumb) reconstruction of the thumb requires careful evaluation and discussion of the patient's occupational and social profile, and the treatment options available . Microvascular toe transfer has become an invaluable tool to restore hand function in such patients . The advantage of second toe transfer is minimal donor site morbidity, excellent appearance and good function, making it the preferred digit for use for thumb transfer . However, it is important to consider this in the indian population as many here people walk barefoot or wear chappels . In our case, after discussing all the reconstructive options, a decision to perform a bilateral second toe transfer was taken . The surgery is of long duration, and also places significant physiological demands on the patient, and hence was performed in two stages . This was done to ensure the definitive survival of the transferred toe, and also as we do not expect any vascular event beyond one week . During the second surgery as well, the pedicle dissection has to done carefully, as the course of the fdma may be different here and not necessarily similar to the first surgery . Also, precautions need to be taken to maintain the proper tensioning of the tendons, and from an esthetic point of view, the same length of the reconstructed thumbs needs to be maintained on both the sides . We had a long term follow up (defined as> 3 years) for the patient, as by this time the neural recovery is almost complete, and most of the adaptation to the newly reconstructed thumb has occurred . In our case, thus to conclude, bilateral second toe to thumb microvascular transfer restores excellent hand function with minimal morbidity in the foot [figure 8]. Primary toe transfer, not only decreases the convalescent period and the medical costs, but also ensures early return to work . Besides, it gives bilaterally symmetrical and aesthetically pleasing thumbs [figure 9]. Minimal donor side morbidity which goes unnoticed excellent strength achieved after toe transfer
There is a lot of epidemiological evidence to suggest that vitamin d has a role in glucose homeostasis, especially in maintaining normal glucose homeostasis and protecting against type 2 diabetes [1, 2]. However, controlled supplementation trials with vitamin d or its analogues have not been able to produce similar results . In a recent systematic review and meta - analysis of 15 trials lasting from two months to 7 years two trials in subjects with impaired glucose tolerance (igt) did find a positive effect on glucose homeostasis with vitamin d3 supplementation [47], whereas three trials in subjects with igt and one in subjects with normal glucose tolerance found no effects [810]. Insulin resistance, metabolic syndrome, and type 2 diabetes are associated with a systemic, chronic inflammation, where, for example, circulating levels of proinflammatory cytokines and c - reactive protein are elevated [11, 12]. Although vitamin d has immunomodulatory and anti - inflammatory properties, especially based on experimental studies, bodies of evidence from human studies in the prediabetic state assessing the role of moderate to large dose supplementation of vitamin d in inflammation and glucose metabolism are still relatively few . As more evidence is clearly needed, we decided to study the placebo - controlled effect of 5-month 40 g / d (1,600 iu) or 80 g / d (3,200 iu) oral vitamin d3 supplementation on glucose homeostasis in an eastern finnish population with either impaired fasting glucose (ifg) or igt during winter time when serum vitamin d3 concentrations drop without supplementation . Furthermore, we assessed the anti - inflammatory effect of the supplementation by measurements of serum high - sensitive c - reactive protein (hscrp), plasma soluble tumor necrosis factor receptor type ii (pstnfrii), plasma interleukin-6 (pil-6), plasma interleukin-1 receptor antagonist (pil-1ra), and plasma interleukin-1 beta (pil-1) concentrations . The glucose metabolism effects of vitamin d supplementation in prediabetes (vitdmet) study was a 5-month randomized, double - blind, placebo - controlled supplementation trial that was conducted in winter 2011/2012 in eastern finland . The primary aim was to assess the effects of vitamin d3 supplementation on glucose homeostasis in subjects with ifg or igt . For inclusion the subjects needed to be 60 years of age with evidence of disturbed glucose homeostasis, that is, either ifg (fasting plasma glucose concentration 5.6 to 6.9 mmol / l) or igt (oral glucose tolerance test (ogtt) 120 min plasma glucose concentration 7.8 to 11.1 mmol / l), but not type 2 diabetes (fasting plasma glucose concentration 7.0 mmol / l or ogtt 120 min plasma glucose concentration 11.1 mmol / l), and to be overweight (bmi> 25), but not severely obese (bmi <35). Furthermore, subjects needed to have their serum 25(oh)d3 concentration <75 nmol / l . Exclusion criteria were any disease that could be affected by vitamin d, such as sarcoidosis, or a condition that could affect vitamin d metabolism, such as kidney disease . The adult voluntary subjects were recruited by advertisements in regional newspapers and after a prescreening on the phone for inclusion and exclusion criteria (self - reported elevated blood glucose, age, height, weight, and diseases) were invited for the first screening at the study clinic (visit 1). A written informed consent was collected, hba1c was measured with a point - of - care device, the subjects were interviewed, and their self - administered questionnaires were checked . If the subject's hba1c was 5.57.0%, fasting venous blood samples were drawn and analyzed for plasma glucose and serum 25(oh)d3 concentrations . Those fulfilling the inclusion criteria were scheduled for the ogtt (visit 2), after which those fulfilling all criteria were randomized to receive either placebo, 40 g / d, or 80 g / d vitamin d3 daily . Each dose comprised a combination of placebo and 20 g tablets, two in the morning and two in the evening . Randomization was carried out by a statistician, in the order of entry after visit 2 . Study nurse distributed the containers according to the statistician - generated list and neither the nurse nor a participant knew the allocation group . All tablets and containers were physically identical, the allocation arm code hidden in the serial number on the container label . The average time between visit 2 and the beginning of the supplementation was approximately one week . During the 5-month supplementation period between october 2011 and april 2012, the subjects visited the study site three times: 1 and 3 months after the start of the supplementation and at the end of the study (figure 1). We aimed to recruit 102 participants, 34 into each of the three supplementation groups, but due to lower than expected public interest to participate and generally higher than anticipated serum 25(oh)d3 concentrations in the base population, at closing of the recruitment window we had 73 randomized subjects in the three supplementation arms (63 men and 10 women). According to power computations, to reach 80% power (i.e., = 0.20) at = 0.05, with a difference of 1 sd to 0.75 sd in the effect measure between the groups, there needed to be 16 to 28 subjects per group . 1 sd equals an effect size of 10% to 50% in most of the outcome parameters . The research ethics committee of the northern savo hospital district has approved the study protocol . Serum 25(oh)d3 concentration was measured from venous blood samples by a high performance liquid chromatography with coulometric electrode array (hplc - cead), as described . The interassay cv% of the method is <8.0% and the intra - assay cv% is <7.3% . Plasma glucose concentration was assayed by the photometric hexokinase method (konelab 20xt, thermo fischer scientific, vantaa, finland) and plasma insulin concentration by the chemiluminescence assay (diasorin liaison, diasorin, dietzenbach, germany). Plasma glucose and plasma insulin concentrations were measured at three time points: at start (i.e., fasting), at 30 min, and at 120 min . Homeostatic modeling assessment (homa2) indices were computed according to the nonlinear function presented by wallace and coworkers . The insulinogenic index (igi) was computed as follows: igi = (30 min insulin 0 min insulin)/(30 min glucose 0 min glucose) in the ogtt, as described by phillips and coworkers, and the insulin sensitivity index (isi) was computed as follows: isi = 10000/sqr[(fasting glucose fasting insulin) (mean glucose mean insulin)], as described by matsuda and defronzo . Hba1c was measured with a point - of - care device from a capillary blood sample as instructed by the manufacturer (dca vantage, siemens healthcare diagnostics, deerfield, illinois, usa). Serum liver enzymes gamma - glutamyl transpeptidase (sggt) and alanine aminotransferase (salat) were determined with enzymatic photometric ifcc methods, kidney function marker serum creatinine (screa) with an enzymatic photometric test, and serum total calcium (sca) with colorimetric arsenazo iii test at site with a clinical chemistry analyzer (konelab 20xt, thermo fisher scientific, vantaa, finland). Serum parathyroid hormone (spth) and serum thyroid stimulating hormone (stsh) were assayed by chemiluminescence methods (diasorin liaison, diasorin, dietzenbach, germany). Total blood count (tbc) and inflammatory cytokines hscrp, pstnfrii, pil-6, pil-1ra, and pil-1 were measured in eastern finland laboratory centre, kuopio, finland . Plasma il-6, il-1ra, soluble tnfrii, and il-1 were measured with elisa from r&d systems (minneapolis, mn, usa). Body weight was measured to 0.1 kg with subjects wearing light underwear, with a recently calibrated vetek ti-1200 scale (vetek, sweden), and height in a frankfurt position to 0.5 cm by a wall - mounted device (kawe, germany). Body mass index (bmi) was computed as weight / height squared (kg / m). Blood pressure was measured after a 5 min rest from the upper arm at a supine position, using a recently calibrated semiautomatic device (omron intellisense m7, omron healthcare, netherlands). Systolic and diastolic blood pressure values were computed as the mean of the last two of three measurements . Participant compliance in taking supplements as instructed was assessed by counting the study supplements not consumed at the last study visit . From the statistical analyses we excluded two subjects who had an increase in their bmi to> 35 kg / m between the randomization and the start of the supplementation (visit 3), two subjects who reported at the end of the study that they had started to use a high - dose (50 g / day and 125 g / day) vitamin d3 supplement in addition to the study supplements during the study period, and one subject due to low compliance . The subject with the low compliance in the 80 g / day group had used only 17% of the study tablets . After the exclusions, the number of subjects in the analyses with the baseline data was 68 (table 1) and in the analyses with the change variables 66 (table 2), after the two dropouts (figure 1) were excluded . Glucose homeostasis was assessed by fasting glucose, insulin, and hba1c measurements and ogtt glucose and insulin measurements . The derived variables to be tested were the change in the chosen variables over the supplementation period . These were as follows: serum hba1c (concentration and%), fasting, 30 min and 120 min glucose and insulin concentrations, homa2 insulin resistance (homa2-ir), insulin sensitivity (homa2-is) and beta cell function (homa2-b%), igi, and isi . Inflammatory effects were assessed by analyzing the change in the hscrp, pstnfrii, pil-6, pil-1ra, and pil-1 concentrations over the supplementation period . Physiological and safety indicators were changes in the 25(oh)d3, spth, sca, screa, sggt, salat, and stsh . As a large proportion of the variables did not follow the normal distribution, we used the kruskal - wallis test (pk - w) to test the intergroup differences, mann - whitney u test (pm - wu) to test the pairwise intergroup differences (placebo versus 40 g / d and placebo versus 80 g / d), and the jonckheere - terpstra test (pj - t) for trend across the groups . Related - samples wilcoxon signed rank test (pwsr) was used to study the intragroup change over time . A two - sided p <0.05 was considered statistically significant for the kruskal - wallis test, a 1-sided p <0.05 for the jonckheere - terpstra test, and a two - sided p <0.025 for the mann - whitney u test (i.e., with the bonferroni correction). All statistical analyses were computed with the spss statistical package for windows, version 21.0 (armonk, ny: ibm corp . ). The supplementation effect analyses were conducted per protocol; as for the dropouts there were no data available at the end of the study to compute the change variables . Despite the moderately small sample size, the randomization yielded reasonably identical groups with regard to vitamin d and glucose metabolism - related parameters . Characteristics of the 68 study subjects at baseline are presented in table 1 . The median age of the subjects at entry was 65.7 (interquartile range 62.7 to 69.7) years, and the median serum 25(oh)d3 concentration was 57.2 (interquartile range 47.2 to 67.2) the arithmetic mean 25(oh)d3 concentration was 57.0 nmol / l with a standard deviation of 11.0 nmol / l . Of the 68 subjects that entered the supplementation period, 66 completed the study (figure 1). There was a marked dose - dependent increase in the average serum 25(oh)d3 concentration in the three study groups, from 55.3 to 59.0 nmol / l, from 57.7 to 85.4 nmol / l, and from 58.1 to 103.1 nmol / l, in the placebo, 40 g / d, and 80 g / d groups, respectively (mean ranks for change 17.1, 35.4, and 47.8, resp ., pk - w <0.001; see table 2). At baseline, one of the subjects in the 80 g / day group had serum 25(oh)d3> 75 nmol / l . At the end of the study, 3/21 (14%) subjects in the placebo group, 16/24 (67%) subjects in the 40 g / d group, and 18/21 (86%) subjects in the 80 g / d group had serum 25(oh)d3> 75 concomitant with the increase in the serum 25(oh)d3 concentrations, there was a dose - dependent decrease in the serum pth concentrations (table 2). Changes in the tested glucose homeostasis parameters over the supplementation period are given in table 2 . The only statistically significant effect between the groups was an increase in the 120 min plasma glucose concentration, that is, opposite to expected (pk - w = 0.039, pj - t = 0.021), although only the pairwise group difference for the placebo versus 40 g / d was statistically significant after bonferroni correction (p = 0.022), and a decreasing trend in the hba1c concentration (pj - t = 0.024) and in the 30 min insulin concentration (pj - t = 0.030). Borderline statistically significant trend was observed in the igi (pj - t = 0.063). In the tested inflammation markers, shown in table 2, the only borderline statistically significant finding was a decreasing trend in the plasma il-1ra concentration (pj - t = 0.070). There were detectable concentrations of pil-1 only for four subjects at entry and for two subjects at the end of the study and therefore the p values cannot be considered reliable . No statistically significant changes were observed in sca (table 2) or in the parameters of liver or kidney function or in the tbc (data not shown). No statistically significant differences between groups were observed in changes in waist circumference or bmi, in blood pressure, or in circulating lipids (data not shown). We repeated the analyses by including also those with bmi> 35 at the start of the supplementation period, those who started their own high - dose vitamin d supplementation during the trial, or the one with low compliance (n = 71). The results were generally similar to those with the exclusions, except for the p for trend for il1-ra, which was statistically significant (pj - t = 0.027). Our study showed that oral supplementation with daily 40 g or 80 g doses of vitamin d3 for five months markedly increases the circulating concentrations of 25(oh)d3, even over the winter months that usually are associated with a decrease in the 25(oh)d3 in this population . We also showed that the 80 g dose is more potent than the 40 g dose in increasing serum 25(oh)d3 and that a considerably large proportion of the subjects in both supplementation groups reached serum 25(oh)d3 concentrations above 75 nmol / l . Furthermore, we showed that the supplementation also resulted in a decrease in serum pth concentration but did not induce a rise in serum total calcium concentration . Vitamin d3 supplementation, even at 80 g per day, was also well tolerated and safe . The primary outcome of the study, body glucose homeostasis, remained for the most part practically unchanged, thus speaking against the role of vitamin d3 as an important regulator of glucose homeostasis in an ageing finnish population with prediabetes . Among the 13 glucose homeostasis indices analyzed, the 120 min plasma glucose was the only index that was statistically significant also in the pairwise analyses . We believe that other changes observed (30 min insulin, hba1c) may be related to random fluctuation of glucose metabolism in individuals with impaired glucose metabolism . Among the interventional studies on glucose homeostasis indicators in populations with prediabetes, an open - label study by nazarian et al . Showed an improvement in insulin sensitivity in 11 ifg subjects after a 4-week 10,000 iu / d (250 g / d) vitamin d3 intervention . In an earlier post, there was a lower rise in fasting plasma glucose concentration and lower increase in homa - ir in 3 years in the group taking 700 iu (17.5 g) vitamin d3 plus 500 mg calcium citrate daily as compared with subjects taking placebo . Glucose homeostasis was also improved in the study by mitri et al ., where 16-week vitamin d3 supplementation of 2,000 iu / d (50 g / d) improved -cell function and attenuated the rise in hba1c . In an open - label study by dutta et al . In subjects with igt or ifg, vitamin d3 supplementation of 60,000 iu (1500 g) once per week for 8 weeks and then monthly along with 1250 mg of calcium carbonate / d resulted in lower fasting blood glucose and improvement in insulin resistance during an average follow - up of 28 months, when compared with subjects taking only calcium carbonate . In a recent placebo - controlled study by gagnon et al, vitamin d3 upplementation for six months with a dose aimed at increasing the serum 25(oh)d3 to 75 nmol / l (20006000 iu / d, 50150 g / d) improved insulin sensitivity in subjects with prediabetes, but not in subjects without prediabetes . In contrast, in a study by harris et al . In 89 overweight or obese african american subjects, 12-week supplementation with 4,000 iu / d (100 g / d) of vitamin d3, compared with placebo, led to reduced insulin sensitivity and increased insulin secretion . In the 12-month supplementation trial by davidson et al . In 117 latino and african american subjects in california with hypovitaminosis d3 (i.e., serum 25(oh)d3 concentration <30 ng / ml (75 nmol / l)), the average weekly dose of 88,865 iu (2,222 g, or about 317 g / day) of vitamin d3 had no effect on insulin secretion, insulin sensitivity, or development of type 2 diabetes, as compared with placebo . Our results support these null findings on glucose homeostasis in finns, a caucasian population at northern latitudes, even though we have earlier observed vitamin d3 sufficiency to associate with the lower risk of type 2 diabetes in an observational study of the same source population . The observed increase in the 120 min plasma glucose is somewhat unexpected, but one explanation may simply be a chance finding due to a largish number of tests that were carried out . The increase was larger in the 40 g group than in the 80 g group, which supports that the finding occurred by chance as we believe is the case with other variables we monitored, that is, 30 min insulin and hba1c . Taken together, the evidence from the observational studies supports the association between low vitamin d status and impaired glucose metabolism, but the results from the experimental studies have not found improvements in glucose metabolism with vitamin d supplementation . A recent mendelian randomisation study that looked into the association between body vitamin d status and glucose homeostasis and type 2 diabetes with four snps and four glycaemic traits showed an association in two snps near genes related to 25(oh)d synthesis with fasting insulin, but not in any of the other eleven tests conducted . However, if a limitation is to be pointed out, many of the trials have been conducted in subjects with healthy glucose homeostasis or in subjects with diabetes, and only few have been carried out in subjects with prediabetes, the subjects that would probably show any effect on glucose homeostasis most easily . Therefore, well - designed randomized trials with large sample sizes and in people with insufficient vitamin d status may be needed to elucidate the role of vitamin d supplementation in glucose homeostasis and in prevention of type 2 diabetes . We found supplemental vitamin d3 to exert some anti - inflammatory action, when a borderline statistically significant decrease in the il-1ra concentration was found . Il-1ra is regarded as one of the most sensitive markers of inflammation, as it is readily secreted by many cell types and plasma levels are high enough to be reliably measured . Elevated values of il-1ra have also been shown to predict the onset of type 2 diabetes [2224]. The results from previous studies of the anti - inflammatory effects of vitamin d supplementation have been inconsistent . Some studies have found vitamin d supplementation to improve the levels of certain inflammatory markers, such as il-6, il-10, crp, or tnf- [6, 10, 2527], whereas others have found no effects on any of the studied markers [19, 2830]. However, only a few studies have been conducted in subjects with prediabetes [6, 19, 31] and, to our knowledge, no study thus far has investigated the impact of vitamin d supplementation on il-1ra . Our study was conducted during october to april, the period of low uvb light exposure from the sun in finland, to allow as unbiased supplemental vitamin d effect as possible . Strengths of the study were the homogenous study population and use of two different, sufficiently large vitamin d3 doses, which enabled studying possible dose response . A limitation of the study was the unbalanced gender distribution, especially the low number of females . Another limitation is the lower than planned total number of study subjects, which was due to the need to close recruitment to allow a sufficiently long supplementation during the low uvb exposure period, and the higher than anticipated average 25(oh)d3 concentration of the study population at the start of the study . However, the fairly consistent results of our study do not suggest a marked change in observed effects even if some more subjects would have been included, or if intention - to - treat analysis had been carried out instead of per - protocol analysis, given that per - protocol tends to exaggerate the treatment effects, if anything . In conclusion, our study does not support the role of winter - time relatively high - dose vitamin d3 supplementation as a means to improve glucose homeostasis in a general ageing population with prediabetes but suggests a modest anti - inflammatory effect.
Multiple sclerosis (ms), received its name in 1955, is an inflammatory disorder of brain and spinal cord where in body's immune system incorrectly attacks its own central nervous system (cns), causing variable and unpredictable symptoms (some examples including slurred speech, blurred vision, loss of balance, poor coordination, tremors, numbness, extreme fatigue, problems with memory and concentration, paralysis, and blindness). Even though a lot of advances have been made in past few decades, exact causes of this disorder remains unknown(environmental factor combined with genetic predisposition). There is a high cost associated with this disorder and it reduces life expectancy as well as quality of life . Unfortunately, there is no cure for ms, and most of the current drugs available in the market help treat the symptoms . Ms is not the same in any two people who have it, since the underlying cause is demyelination (along with axon degeneration), which can target any brain area, resulting in a broad range of clinical symptoms (compston and coles, 2002; alastair compston et al . In addition, brain lesions can sometimes outnumber the clinical symptoms by as much as 10:1, and lesions in noncritical areas may not result in obvious functional deficits, even if brain function is in fact altered . There are four subtypes of the disease - relapsing - remitting ms, primary progressive ms, secondary progressive ms and progressive relapsing ms, which differ in relapse rate, clinical symptoms, trajectory, underlying causes and approaches to disease management . Combined all these aspects of the disease (various locations for demyelination, clinically silent timeline, and a highly variable disease course) make the approach to care and treatment complex (compston and coles, 2008; trapp and nave, 2008). Current behavioral and imaging methods used to assess disease severity are insufficient by themselves to allow confident prediction of disease progression . The addition of functional modeling (modeling that correlates the functional loss to disease progression) and objective functional measurements (imaging as well as other assessments combined with disease status) may greatly improve such prediction . Currently, magnetic resonance imaging (mri) along with clinical manifestation of the disease are used as clinical tools for diagnosing ms (filippi and grossman, 2002). The 2010 revision to the mcdonald diagnostic criteria (polman et al ., 2011) uses a combination of lesion and clinical attacks for the most confident diagnosis of ms . Lesions must be disseminated in time or space or have positive identification from cerebral spinal fluid (csf) measure (lublin et al ., 2014). Unfortunately, discrepancy still exists between clinical symptoms and structural measurements, because of the poor correlation between the presence of lesions and symptoms . The two common measures used for diagnosis and monitoring with mri are hyperintense lesions on dual - echo mr, which is a nonspecific measure of macroscopic tissue injury, and enhanced abnormalities on t1 lesion weighted images, which is a measure of lesions . However, neither of these measures do provide extent and severity of inflammation, cellular component, or resultant tissue damage information (filippi and grossman, 2002). Thus, there is a need for supplemental information from alternative techniques to have a more holistic approach . There are emerging techniques that hold promise to quantify the brain imaging data, and relate that with clinical outcomes . Some promising examples include magnetization transfer mri (quantitative and continuous measure of loss of myelin and reduction in axonal density), diffusion weighted mri (quantitative measure of size, shape, geometry, and orientation of tissues), proton magnetic resonance spectroscopy (mrs, provides measure of two major pathologic aspects of ms - the active inflammatory / demyelinating process and axonal injury), functional mri (provides functional information about brain activation during motor, sensitive, and cognitive tasks), high field strength imaging (ex 7 t mri, leads to improved signal - to - noise ratio, speed, and resolution in both mri and mrs) and transcranial magnetic stimulation (tms, highly sensitive technique to evaluate cortico - spinal conduction abnormalities in ms). Since t1 scan is transient, it should be only used in patients that have frequent mri, and should not be used for comparison between subjects . Instead, given the availability of multiple brain imaging modalities, the need still exists for creating a combined assessment tool . Quite a few studies have attempted to combine various measures (petzold et al ., 2006; daumer et al ., 2007, 2009; castro - borrero et al ., 2012; sormani, 2013; giffroy et al ., multiple sclerosis severity scale (msss) was developed to relate expanded disability status scale (edss) to the disease duration . It has been shown that msss correlates with axonal biomarkers but not with glial biomarkers (petzold et al ., 2006). This scale was tested on 195 ms patients to predict accrual of disability over time to see if current therapies have impact on disease severity over time . Pachner and steiner (2009) concluded that the current disease modifying drug therapies lack emphasis on disease severity . An online analytical processing tool was developed that can be used for prognosis of near term future course of an individual patient (daumer et al ., 2007). This tool is based on matching algorithm (using statistical analysis) and contains data of 1,059 patients . Outcome was to show the probable progression over time which can be useful for subjects, physicians, researchers and other professionals who counsel the patients . In an attempt to test the developing treatment methods, sormani (2013) explored the use of various biomarkers that can be used to predict the clinical response to interferon beta (ifn-) treatment . He concluded with the need for precise, meaningful measures of disease progression, integrated with clinical measures to help with personalized treatment for ms . A computational classifier was modeled that can be used for predicting short term course of ms (bejarano et al ., 2011). It combined clinical data, with mri and motor evoked potential (mep), and it was found that the model did good job on predicting short time scale disability . A description of the use of various potential biomarkers with their pros and cons was created . It ranges from markers for immunological activation, as well as markers for demyelination, axonal damage, oxidative stress, remyelination, glycoses, and details of specimen such as blood, urine, tears and csf (bielecki et al ., 2010). (2010) concluded with a need for unification and standardization of results of various measurements and techniques . There have been many advances in modeling and designing tools and predictive capabilities for ms . (2015) studied 1,959 patients from 19952013 with clinically isolated syndrome and found that demographic and topographic characteristics had a low effect on prognostic factors for ms, while the presence of oligoclonal band (oligoclonal bands are proteins called immunoglobulins and their presence indicates inflammation of the central nervous system) had medium effect, and the number of lesions had the highest impact . In an another study, 2014) recruited 652 patients and studied them for prediction factors that can be classified as disseminated in time (dit), disseminated in space (dis), or both, and concluded that there is a need of more predictive factor combinations to help risk for ms . An analysis of 598 ms patients was performed in norway to determine factors responsible for life expectancy, and the conclusion was that high age at the onset was correlated with unfavorable prognosis (riise et al ., 1988). In another study on early prognostic features on the late course for ms in world war veterans, it was found that pyramidal and cerebellar scores are the best predictors (kurtzke et al ., 1972). Wang et al . (2015) performed a study to combine in vivo biomarker using electrical vestibular stimulation and eye movement recording to measure evoked vestibular - ocular reflexed (evor) in 18 subjects with ms (wang et al ., 2015). The goal was to measure the axonal conduction velocity to understand the myelin process and provide a way for assessing efficacy of novel reparative therapies in ms . There still exists a need to for quantification, combination of various results from studies, and longer longitudinal studies . Lastly, a computational approach will be necessary for various biological, clinical, imaging findings to form an integrative modeling system that encompasses grey matter pathology, myelin sheath aqueous layers, energy metabolism, and perhaps most importantly, multi - scale or integrated modeling . My recent paper (chaubey and goodwin, 2016) is a computational modeling study which makes it possible to make quantitative predictions about the degree to which electrical signals will move more slowly through axonal pathways as a function of how much of their myelin sheath they have lost as a result of ms (using data from diffusion tensor imaging (dti), and other modalities). The paper identifies a new biomarker for network failure in ms that should improve our ability to predict and track loss of sensory, motor and cognitive function in the disease and a better way to measure the efficacy of new treatments . The potential also exists to relate this slow down to disease progression, and eventually, to disease prediction, in which we could use the existing model to predict the disease symptoms in subsequent years . A study in my research center (brain sciences center, va medical center) used magnetoencephalography (meg) resting state recordings as a functional biomarker to classify ms and various other patient populations to their respective groups, by assessing synchronous neural interactions at high temporal resolution as a measurement of the dynamic synchronous neural interactions, an essential aspect of brain function (georgopoulos et al ., 2007). Another study used similar analysis in 50 ms patients (31 rrms, 15 spms, 4 ppms) and 214 healthy controls (carpenter et al ., 2011). Ancova was performed at each of the 30,628 sensor pairs (pccs) using group (ms, control) as a fixed factor and age, gender and handedness as covariates . Similar analyses were done using each ms phenotype (rrms, spms, etc .) As a factor and 300 (out of 30,628) sensor pairs were found to have a significant group differences after correction for multiple comparisons . There is a need to combine such objective measures of brain function with modeling techniques to improve assessment and prediction of the disease over what is possible using established methods . To list few direct advantages of these predictions: 1) these predictions can identify high - risk patients who require early and more aggressive therapies, 2) they can help patients with clinically isolated syndrome (cis, one of the ms disease courses, refers to a first episode of neurologic symptoms, but it is not alone enough to have diagnosis of ms, as there needs to be two episodes disseminated in time or space) to predict how likely and at what time frame the ms diagnosis is likely, 3) they can provide an individual plan for various subjects depending on their current status and symptoms (bergamaschi and montomoli, 2016). There is a need for modeling to help direct research using testable predictions and fixing the current gap in knowledge . There is enormous amount of data available, a lot of which is open access, and it is important to make sense of it all . Computational modeling can be an essential tool to help make use and sense of all this data and lead to better understanding, diagnosis, prediction of various diseases, especially ms . As there is no cure for ms, most treatments typically focus on slowing the progression of the disease and managing symptoms . Since tissue destruction and disease onset begins much earlier than the actual clinical symptoms appear, it will be important to test future therapeutics to detect early axonal loss, axonal dysfunction and efficiency of these treatment methods . Advances in imaging technology and use of new techniques need to be established and validated to determine the success of remyelination and cessation of old damage . Finally, a combination of modeling will allow for prediction and better quality of life for people with ms . There is a huge need for a reliable and personalized disease prediction model of ms . It would benefit patients with ms, as they can plan and select the best individualized treatment option . It would be helpful to the clinician working with patients, especially early stage patients, to help with selection of the best therapeutic treatment options . Integration of modeling, biology, clinical and imaging data can help provide more personalized and reliable monitoring of disease progression and in turn lead to better disease prediction.
Though the pathogens causing aris vary geographically and by season, globally viruses play a major role [13]. In a recent systematic review, the most common respiratory viruses causing acute lower respiratory tract infection (lri) in children under five years of age were respiratory syncytial virus (rsv), influenza virus (ifv), parainfluenza virus (piv) human metapneumovirus (hmpv), and rhinovirus (rv). Besides this, 10%50% of children affected with ari develop secondary bacterial infections, namely, acute otitis media, sinusitis, or pneumonia . Moreover, viruses are the most common pathogen associated with severe respiratory diseases (e.g., bronchiolitis), exacerbation of asthma, or pneumonia in early life and are leading cause of hospitalization in children under two [57]. Although viral etiology of aris and their impact on health care are much studied in developed countries, there is a gap in knowledge regarding the same in developing countries including india . From the public health point of view, it is important to know the most common viral agents causing aris, their manifestations, how often they cause severe disease, and how severe aris can be prevented . In this study, we aimed to characterize the viral spectrum and pattern of upper and lower aris in children under five from eastern part of india . The present study was conducted in the pediatrics department of svppgip, scb medical college, a tertiary care teaching hospital in eastern india over 2-year period (october 2011 to september 2013). Children aged 2 to 60 months with symptoms and signs of acute respiratory tract infections suggested by who were included . Who classification of acute respiratory tract infection in children presenting with cough, difficult breathing, or both is as follows: pneumonia respiratory rate per minute> 50 breaths (211 months of age) or> 40 breaths (1259 months of age); no lower chest indrawing; severe pneumonia symptoms of pneumonia, and lower chest indrawing with or without rapid breathing; very severe disease symptoms of severe pneumonia, inability to drink, convulsions, central cyanosis, being abnormally sleepy or difficulty to wake, stridor in calm child, or clinically severe malnutrition . Those with suspected bacterial etiology (e.g., streptococcal sore throat, lobar pneumonia, pneumatocele, and empyema), underlying chronic conditions, hiv, illness lasting more than a week, severe malnutrition, and those hospitalized in last one month were excluded . Nasal and throat swabs were collected and pooled into tubes containing 2 ml of virus transport medium (vtm, copan, brescia, italy) and kept in refrigerator at 2 to 8c . Then the samples were transported over ice to the laboratory (regional medical research centre, icmr) situated at bhubaneswar, a grade 1 equipped virological laboratory . Total nucleic acids (including dna and rna) were extracted from 200 l of each specimen using a qiaamp minelute virus spin kit (qiagen, mississauga, on, canada) according to the manufacturer's instructions . For all collected specimens, pcr or rt - pcrs were performed to detect infection with rsv, ifv, piv, hmpv, adenovirus (adv), human bocavirus (hbov), human corona virus (hcov), enterovirus (ev), and rv . The method used for each virus is described in the following references: pivs 14, ev, rv, ifv (a, b, and c), and rsv (a and b) were detected by two multiplex nested rt - pcrs; hcov and hmpv by two - step rt - pcr; adv by single - step pcr; and hbov by touchdown pcr . Rt - pcr was performed using a superscript ii one - step rt - pcr platinum taq kit (invitrogen, carlsbad, ca, usa). Typing for ifv, piv, and rsv was performed according to pcr product size . A total of 358 children of 260 mo age and acute respiratory infections were screened and finally 300 patients were included after meeting the eligibility criteria . Following were the reasons for exclusion in 58 children: chest x - ray suggestive of bacterial pneumonia (n = 19), streptococcal sore throat (n = 15), underlying chronic conditions (n = 10), severe malnutrition (n = 7), illness lasting more than a week (n = 3), complicated pneumonia (n = 2), and being hospitalized in last one month (n = 2). The m: f ratio was 1.7: 1 (uri = 1.6: 1; lri = 1.9: 1). Out of 300, 174 (58%) were lri and 126 (42%) were uri cases . Month - wise recruitment of uri and lri cases has been shown in figure 1 . Most lri cases occurred in the month of january, august, november, march, and july . Most children with lri were in the age range of 212 mo (54%) and with uri were in the age range of> 1260 mo (45.2%). Chest x - ray findings were hyperinflation (93%), peribronchial cuffing (60%), interstitial infiltrates (48%), and patchy opacity (46%). Of the 300 nasal and throat swabs, 248 cases were positive for viruses [lri = 142/174 (81.6%), and uri = 106/126 (84.1%)]. Month - wise recruitment of ari cases and samples positive for viruses has been shown in figure 2 . The maximum number of cases recruited and samples positive for viruses were in the months of january, december, and august . The virus positivity in ari was as follows: uri, 65 (61.3%) samples were positive for single virus and 41 (38.7%) were positive for more than one virus, and lri, 86 (61%) samples were positive for single virus and 56 (39%) were positive for more than one virus . The coinfection pattern differed between acute uri and lri, and this has been detailed in table 2 . The most common viruses isolated from uri cases were rv (31.1%), adv (18.9%), rsv (17%), and ifv (17%). The most common viruses isolated from lri cases were rsv (30.3%), ifv (17.6%), rv (14.8%), and adv (13.4%). Rsv, adv, rv, and ifv were the most prevalent viruses isolated from all the ari cases . Ifv - a was predominant over ifv - b, and piv-3 was predominant over other subtypes of piv . Rsv - a subtype dominated over rsv - b in uri cases, whereas rsv - b dominated over rsv - a in lri cases . The detection rates of viruses corresponding to different age groups are shown in table 1 . In case of uris, rv was the most prevalent virus (26 mo age = 2.8%;> 612 mo age = 13.3%;> 1260 mo age = 14.2%). Rsv was the second most common virus detected in age groups of 26 mo and> 612 mo . Adv was the second most common virus detected in age group of> 1260 mo . The least common viruses in all age groups were piv, hbov, and hmpv . Hcov and ev were not detected in any age group . In case of lris, rsv was the most prevalent virus (26 mo age = 13.4%;> 612 mo age = 11.3%;> 1260 mo age = 5.6%). Hmpv, hbov, and hcov were not detected up to 6 mo age . The least common viruses in all age groups were hbov, hcov, ev, and hmpv . Rv was the most common virus detected in uris, but it was fourth most prevalent in lris . It was detected throughout the year, the maximum rate being in december, january, march, and august . Rsv was the most common virus detected in lris, but it was third most prevalent in uris . It was detected throughout the year, the maximum rate being in december, january, and february . Ifv was second most common virus detected in lris, but it was third most prevalent in uris; maximum positivity rate was in august followed by in july, and minimum positivity rate was in november . Piv had a higher prevalence in april to july, and a low prevalence from september to march . There detection rate was lower in lris . Like hmpv, hbov was present throughout the year in lris . The clinical features, demographics, and risk factors of children among viral positive and virus negative group with lris were compared (table 3). It was observed that significantly higher number of children below 12 mo were virus positive (p = 0.01). Children presenting with preceding bronchiolitis were significantly associated with total viral infections (p = 0.003). Rhinorrhea was significantly present in the virus positive group (p = 0.02). Among risk factors, ari in family was significantly associated with virus positivity (p = 0.04). In lris, out of 174 cases (lri = 101; severe lri = 51; very severe lri = 22), 161 were cured and 13 died . Of 13 cases who died, 10 had coinfection with more than 1 respiratory virus (ifv + rsv = 5; rsv + rv = 3; ifv + hcov43 + rv = 2). In uris, in the present study, respiratory viruses were detected in 248 cases (lri = 142; uri = 106). Rsv (25.8%), rv (21.9%), ifv (21%), and adv (12.5%) were the most prevalent single viruses isolated from all the ari cases . The most common viruses isolated from acute uri cases were rv (32.6%), adv (19.6%), rsv (17.4%), and ifv - a (15.2). The most common viruses isolated from acute lri cases were rsv (32.4%), ifv - a (19.6%), adv (18.6%), and rv (15.7%). Most cases occurred in the month of january, december, and august . In uris, the most common age group affected was> 1260 mo, and in lris, the most common age group affected was 212 mo . Present study results are similar to the previous study results from india . In the study by broor et al ., rsv, ifv - a, and piv-3 were important causes of ari in children under five in the rural indian community . In the study by singh et al ., rsv was the most frequently detected virus (21.3%) from hospitalised children presenting as alri followed by ifv (9.0%), measles (8.5%), and adv (5.3%). In the study by bharaj et al ., rsv was the most frequently detected virus (58.1%) from hospitalised children presenting as alri followed by piv (22%), hmpv (10.5%), and ifv - a (9.3%). In the study by maitreyi et al ., rsv was the most frequently detected virus (17%) from hospitalised children presenting as alri followed by ifv (14.5%), piv (11.5%), and adv (1.5%). In the study by yeolekar et al ., rsv was the most frequently detected virus (26%) from hospitalised children presenting as alri followed by ifv (5.4%), piv (2.07%), and adv (0.8%). Panda et al . Recently found rv as the most frequently detected pathogen (24.7%) followed by rsv (4.22%), piv (2.11%), and hmpv (2.11%) in children with ari from eastern india . In the present study, we found rv as the second most important agent to rsv causing ari in young infants and children . The present study findings are more or less similar to the recently published studies from other parts of the globe, though the most prevalent virus may be different [2127]. The viral uri and lri prevalence varied with age, uris being common in children of> 1260 mo and lris being common in infants of <12 mo age . Infants <12 mo age were most commonly positive for rsv and less commonly for ifv, the latter occurring commonly beyond infancy in older children . This is because maternal antibody protection against rsv during infancy remains questionable, and as the child grows, the immunity builds up against rsv due to repeated exposures . Ifv due to marked antigenic diversity evades the herd immunity and does not give long lasting immunity . Therefore, the susceptibility to ifv increases as the child grows . Adv is uncommon during first six months, where maternal antibody confers protection . Previous indian studies [16, 17] have compared the clinical, demographic, and risk factors for alri testing positive for respiratory viruses with those testing negative for respiratory viruses . One study observed that significantly higher number of children below 12 mo of age had rsv infection, those presenting with preceding bronchiolitis were significantly associated with virus positivity, and ari in family was significantly associated with virus positivity . In another study, patients testing positive for viruses showed significantly high percentage of cough, nasal discharge, and diarrhea . We found a significantly higher number of children below 12 mo being virus positive, those presenting with preceding bronchiolitis being significantly associated with virus positivity, rhinorrhea significantly presenting in the virus positive group, and ari in family being significantly associated with virus positivity . The respiratory illness exhibits a clear - cut seasonality, and all the published studies till date support this . Most of the studies report increased incidence of acute respiratory infection (both uri and lri) during winter and fall season [2027]. This is because of the following sequence of events: decrease in air temperature causing decrease in the nasal airway temperature (compromised cooling of the nasal airway) leading to poor defence against infection (because of decrease mucociliary clearance and phagocytosis). In the present study, the incidence of both uri and lri was higher in winter season (as upper airway acts as an entry point to lower airway). Though this cannot be solely explained based on the above hypothesis, it may be because of an increase in the birth cohort during this period . In acute lris, severe clinical phenotypes (severe and very severe pneumonia, acute respiratory distress syndrome) the present study findings throw lights on the management of an individual case as well as the community . First, good hand hygiene, maintenance of warm temperature in the living place / avoidance of exposure to sudden cold temperature, avoidance of taking of freeze dried / cold food items, and wearing of the mask to prevent spread of the infection should be practiced . Second, the nutritional status of the children in the community should be improved as malnutrition predisposes to increase risk of infection and severe illness . Third, vaccines should be developed against respiratory viruses (including against the new viruses) to decrease the morbidity and mortality from respiratory illness . Fourth, any child with ari and viral coinfection should be closely monitored for development of complications / severe illness . Strengths of present study are adequate sample size and children being recruited over 2 consecutive years including all the seasons . Limitations include children presenting to the health facility only were recruited, reinfection rate by the viruses were not considered, and simultaneous / secondary bacterial infections were not studied . Rsv, adv, rv, and ifv were the most prevalent viruses isolated from the ari cases . The epidemiology of respiratory viruses needs to be studied further for vaccine development and implementation purpose . Any child with ari and viral coinfection should be monitored for development of complications / severe illness.
Chondrosarcomas (cs) is a locally aggressive condition resulting in the formation of cartilage rather than bone from the mesenchymal tumor cells . This condition mostly affects femur, humerus, pelvis, and the sacrum accounting for 10 - 12% of all malignant bone tumors . In the maxillofacial region, maxilla is more commonly affected than the mandible, comprising less than 2% of all jaw tumors . Most cs of facial structures occurs after 30 years of age and the frequency increases with advancing age . Cs usually begins with a slow growing mass, with or without pain, associated with displaced and mobile teeth . The areas of occurrence are related to the position of embryonic remnants of cartilaginous tissue such as nasal septal cartilage in the anterior region of the maxilla and meckel's cartilage in the posterior region of mandible . Approximately 90% of cs are slow growing, low grade, non - metastasizing tumors . A 55-year - old female patient reported to us in march 2009 with complaints of a slowly enlarging mass involving the lower jaw for the last 5 years . She had the history of a small swelling in the anterior region of the lower jaw that had been curetted 5 years back . Then she had noticed the slowly enlarging mass, which involved the whole of the lower jaw over time . On examination a firm multilobular mass was found with both buccal and lingual cortical expansion, more on the right side than the left side of mandible [figure 1]. Intraoral examination showed diffuse swelling obliterating the buccal vestibule with multiple displaced and mobile teeth [figure 2]. Preoperative photograph shows diffuse multilobular swelling over the bilateral lower face intraoral examination shows diffuse swelling obliterating the mandibular buccal vestibule with multiple displaced and mobile teeth radiographic study revealed an ill - defined, multilocular radiolucent lesion with massive cortical expansion and multiple displaced teeth (floating teeth appearance) [figure 3a and b]. Computerized tomography (ct) scan showed a large ill - defined homogenous mass measuring 124 77 mm involving the whole of the mandible and extending into the infratemporal fossa region, with no intracranial extension [figure 4]. (a and b) orthopantomogram and posteroanterior view showing an ill - defined, multilocular radiolucent lesion with massive cortical expansion and multiple displaced teeth (floating teeth appearance) computed tomographic scan shows a large ill - defined homogeneous - mass involving the whole of the mandible and extending into the infratemporal fossa region, with no intracranial extension on incisional biopsy, hematoxylin and eosin (h and e) stained sections revealed multiple islands of cartilage arranged in a lobular pattern, with cells that contained large, plump nuclei and were often binucleated or multinucleated . There was an increase in the number of cells, and each lacuna often contained two or more cells . Areas of hemorrhage and sheets of pleomorphic, hyperchromatic spindle cells were present [figures 5 and 6]. A diagnosis of dedifferentiated cs was concluded . Photomicrograph (h and e; 10) reveals island of cartilage with cells containing large, plump nuclei and sheet of spindle cells photomicrograph (h and e; 40) shows increased number of spindle cells with pleomorphism and hyperchromatism, and cartilaginous area having lacunae often containing two or more cells each complete resection of the mandible was planned for the patient . Under general anesthesia, tracheostomy was done to prevent postoperative upper airway obstruction, and the tumor was exposed via apron incision over the bilateral submandibular region . Complete resection of the mandible was done and a few parts of the overlying adherent skin was removed [figures 79]. After complete removal of the tumor, hemostasis was achieved, drains were fixed, and wound was closed in three layers . She was regular to follow - up till 18 months post - surgery [figure 10]. After 5 years, she reported to us with massive recurrence over the bilateral face . The firm, diffuse, nontender swelling involving the malar, preauricular, and temporal regions led to a very horrific facial appearance [figure 11]. A ct scan showed a normal brain, an intensely enhanced soft tissue mass in the masticator space, and infratemporal space on both sides with extension to temporoparietal convexity (extra calvarial) of the left side [figure 12a and b]. She was terminally ill, with difficulty in taking food, and not interested in further treatment . Intraoperative picture shows incision planning the tumor exposed with apron incision over bilateral submandibular region the resected specimen of the involved whole of the mandible after 2.5 years, massive recurrence over the bilateral face (a and b) computed tomographic scan with axial and coronal view shows normal brain, intensely enhanced soft tissue mass in the masticator space, and infratemporal space on both sides with extension to temporoparietal convexity (extracalvarial) of the left side cs arises from mesenchymal stem cells and undergoes a partial differentiation to form chondroblastic differentiation and even definable cartilage . In 1942, lichtenstein and jaffe described cs as a lesion that develops directly from a sarcomatous stroma, developing from full - fledged cartilage and not showing neoplastic osteoid tissue and bone . The mass will emanate from bone as an irregular lytic lesion that will palpate as a firm to hard lobulated soft tissue mass . Most cs will be seen either in the anterior part of the maxilla or in the posterior body region of the mandible . Punctate radiopacities may be present because of dystrophic calcifications or focal ossifications of cartilage . In the tooth bearing areas, a widening of the periodontal ligament space (garrington's sign) may be seen as an early sign of cs, just as it is an early sign of osteosarcoma . Because cartilage itself and cartilaginous tumors are well demonstrated by magnetic resonance imaging (mri), this modality may provide a better delineation of tumor extent than a ct scan . Although metastasis of cs is less frequent than with an osteosarcoma or other sarcomas, a chest radiograph is required to rule out this most likely place for a metastatic focus . Microscopically it is characterized by the formation of malignant cartilage without deposition of osteoids from a sarcomatous stroma . There is an increase in the number of cells, and lacunae often contain two or more cells . Classified cs into three grades i, ii, and iii based on cellularity, nuclear size, and mitotic rate . Grade i tumors tend to have a lobular pattern and two or more cells within a lacuna . Grade ii tumors show an increase in cellularity, with retention of lobules and ossification, while grade iii tumors are markedly cellular, with a proliferation of spindle cells . The lobular pattern is lost . In the jaws, various subtypes were also introduced such as conventional cs, clear cell cs, myxoid cs, mesenchymal cs, and de - differentiated cs . Differential diagnosis based on clinical and radiological features initially suggest a benign odontogenic tumor or a benign tumor of the bone . If the lesion is entirely radiolucent, the clinician may consider an ameloblastoma or odontogenic myxoma . If some punctate radiopacities are identifiable, the lesion will resemble a calcifying epithelial odontogenic tumor, an ossifying fibroma, an immature osteoblastoma, or a cavernous hemangioma of bone . The more obviously aggressive presentations with irregular radiolucencies and perhaps neurosensory loss would be consistent with an intraosseous carcinoma, an osteosarcoma, and a malignant fibrous histiocytoma . Such entities are actually osteosarcomas, which are especially important, since many osteosarcomas of the jaws and facial bones have significant chondroblastic portions within them . However, if tumorous bone arises from the cartilage rather than from the malignant stroma, it remains a true cs . Cs are slow growing tumors with a tendency toward local recurrence after surgery . However, with recurrence they exhibit rapid and aggressive growth . Because of their slow growth and their tendency to undergo neural invasion only later in their course, they are often mistaken for benign chondromas or cartilaginous rests . Several have presented with previous biopsies identifying cartilage where the patient was informed that it merely represented ectopic cartilage . This false impression of a cartilage hamartoma or a benign cartilage forming tumor is often supported by histopathologic features that will appear to be benign because of mature cartilage with little stroma and mostly a single nucleus in each lacuna . The common low grade cs (grades i and ii) of the jaws and facial skeleton are best treated with a local resection using 1.5 cm margins for bone and soft tissue . Neither these lesions may be invasive but they typically grow slowly; lymph node metastasis is therefore rare, and elective neck dissection is not necessarily required . The uncommon high - grade cs (grade iii) is treated with an initial aggressive resection of 3 cm in bone and 2 cm in soft tissue followed by chemotherapy . . However, the less common, high grade (grade iii) cs is associated with a 29% 5 year survival rate . High grade (grade iii) cs more often fail to be cure das distant metastasis (66%), most often to the lungs, sternum, and vertebrae have been reported . However, lymph node metastasis also can develop if the initial therapy did not include a neck dissection . Cs was traditionally regarded as a radio resistant tumor, and radiotherapy was therefore generally reserved for high - grade lesions (as a postoperative adjuvant therapy) and for surgically unresectable lesions . We must state that the appropriate modality in managing cs, whether surgery alone (with or without neck dissection) or adjuvant radiotherapy or chemotherapy, is unclear . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Acute coronary syndrome (acs) is characterized by acute, regional reductions in coronary blood flow and myocardial ischemia . Acs describes a spectrum of clinical manifestations, including unstable angina (ua), non - st - segment elevation myocardial infarction (nstemi), and st - segment elevation myocardial infarction (stemi) [13]. Atherosclerosis is a chronic inflammatory disease of the arterial wall, characterized by endothelial dysfunction, intimal hyperplasia, and smooth muscle proliferation, as well as deposition of lipids and formation of microvessels within the vascular wall [1, 46]. Endothelial dysfunction is accompanied by the expression of adhesion molecules (such as the vascular cell adhesion molecule- (vcam-) 1) [7, 8] and chemokines [4, 9]. The recruitment of inflammatory cells is triggered by the production of cytokines (such as il-1, il-6, il-8, tnf-, and ccl2) within the plaque microenvironment . Chemokines are released from endothelial cells, mast cells, platelets, macrophages, and lymphocytes . The macrophage migration inhibitory factor (mif) is a molecule that consists of 115 amino acids; it was described as the main cytokine involved in attracting immune cells such as macrophages and t and b cells [12, 13]. The secretion of mif by inflammatory cells can be induced by exposure to oxidized low - density lipoprotein or other cytokines, such as tnf- and interleukin - c [13, 14]. Mif activates the expression of various proinflammatory cytokines and chemokines and recruits macrophages to the site of atherosclerosis . Mif participates in the pathogenesis of inflammatory and atherosclerosis processes [12, 13, 1517]. The mif gene is located at 22q11.2 and contains several polymorphisms, including the rs5844572 . This genetic marker is a catt short tandem repeat (str) at position 794, with five to eight length variants (alleles 5 to 8). There is an association between the length of the repeats and the expression of the genetic marker; the higher alleles (catt6, catt7, and catt8) show higher expression of the gene . Since mif is an inflammatory mediator and given the role of genetic factors that modify its expression, mif could contribute to atherosclerosis and susceptibility to acs . The aim of this study was to investigate the association between the 794 (catt)58 mif gene polymorphism and susceptibility to acs in a western mexican population . The study group included 200 acs unrelated patients recruited from hospital de especialidades del centro mdico nacional de occidente del instituto mexicano del seguro social (cmno - imss) and classified according to the criteria of the american college of cardiology (acc). As a control group, 200 unrelated healthy subjects (hs) were recruited from the general population of western mexico . We applied a standardized questionnaire to the hs group and applied routine laboratorial clinical assessments to detect any potential alterations, and those subjects with any clinical alterations were excluded of the study . We considered mexican mestizo subjects, only those individuals who for three generations, including their own, had been born in western mexico . The study conforms to the ethical principles contained in the declaration of helsinki, and ethical approval was obtained from centro universitario de ciencias de la salud, cucs, udeg (c.i . Genotyping of the str 794 (catt)58 polymorphism was achieved by conventional pcr and polyacrylamide gel electrophoresis using the primers reported by radskate et al . . Cycling conditions were as follows: an initial denaturation at 95c for 4 min followed by 30 cycles of 30s at 95c, 30s at 60c, and 30s at 72c and then a final extension of 2 min at 72c . Allele identification was done by using a 10-bp (invitrogen) and homemade allelic ladders containing pooled samples . In addition, as genotyping control, automatized sequencing of one random sample of each homozygote genotype allowed confirmation of results (abi prism 377 genetic analyzer, applied biosystems, foster city, ca, usa). The hardy - weinberg equilibrium test and genotype and allele frequencies were calculated by the chi - square test or fisher's exact test, when applicable . Odds ratios (or) and 95% confidence intervals (95% ci) were calculated to test the probability that the genotype and allele frequencies were associated with acs . A p value <0.05 was considered to be statistically significant . Study the sample size was calculated according to the minor allele frequency of the 794 (catt)58 mif gene polymorphism reported in mexican mestizo population by llamas - covarrubias et al . It means that in order to detect differences, we needed at least 82 individuals each group with 95% confidence interval and statistical power of 80% . In this respect 200 individuals were included exceeding by far the required sample size . The median age of hs and acs groups was 61 and 63 years, respectively . The most prevalent clinical diagnosis in the acs group was stemi (76%), while obesity was the most common risk factor (61%), followed by high blood pressure (hbp: 60%). Also, in the acs group other clinical parameters such as troponin i (tni), troponin t (tnt), creatine phosphokinase (cpk), and creatine kinase (ck) were evaluated in order to find a possible genetic association . However, we did not find any statistical association with 794 (catt)58 mif gene polymorphism . No deviation from the hardy - weinberg equilibrium was detected in the 794 (catt)58 mif polymorphism (p = 0.16). The allele and genotype frequencies in both the acs and hs groups are shown in table 2 . Allele 6 was the most frequent in both groups (acs: 54% and hs: 57%). The most common genotypes in acs and hs subjects were 6/7 and 6/6, respectively . A significant association was found between the 6/7 genotype and susceptibility to acs (68% versus 47% in acs and hs, resp ., we also stratified the main risk factors for development of acs by genotype (table 3). Despite no significant differences, we observed that the 6/7 genotype was the most frequent in each subgroup and that the 5/5 genotype was the less common . Acute coronary syndrome is a multifactorial disease arising through a combination of both environmental and genetic risk factors . Several studies have reported a relationship between atherosclerosis and acs [8, 22]. Activated endothelial cells express adhesion molecules and favor the recruitment of monocytes to the endothelium . These in turn release proinflammatory cytokines including il-1, il-6, tnf-, and mif . Reported a correlation between the expression of mif and an increased intima - media thickness and also with lipid deposition in carotid artery plaques; white et al . Reported a proinflammatory role for mif in acute myocardial infarction; mller et al . This genetic marker has five to eight variants (catt)58 . A higher repeat number is associated with an increase in the gene expression . In the present research, we reviewed the association of the 794 (catt)58 mif polymorphism with acs in mexican mestizo individuals . The genotype and allele frequencies were distributed in a similar way to a previous study with a mexican mestizo population by llamas - covarrubias et al . . We also compared the frequencies with those of caucasian american and african american populations and observed that the genotype frequencies in our population differed from them; they found that the genotype 6/6 was the most frequent, while the 5/8 and 6/8 genotypes were less frequent in caucasian americans and african americans, respectively . In addition, a statistical difference was found in genotypic and allelic distributions when compared with an african ethnic group where the 5/6 genotype and the 5 allele showed the highest frequency; these differences could be attributed to the ancestry of the population . The mexican mestizo population is a crossbreed of amerindian, european, and african populations, with an estimated contribution of 2125%, 6064%, and 15%, respectively (rubi - castellanos, 2009). In this study, we found an association between the 6/7 genotype of the mif 794 (catt)58 polymorphism and susceptibility to acs in a western mexican population . There is also another single nucleotide polymorphism in the promoter of the mif gene at the position 173 that has been previously analyzed and related to the inflammatory response in coronary bypass surgery and coronary alterations in children with kawasaki disease . Recent studies support the fact that mif is a pleiotropic cytokine mainly released from macrophages that has been shown to be increasingly expressed during atherogenesis, oxldl being a major inducer [14, 23, 24]. Acs is a pathology where inflammation favors the recruitment of monocytes to the atherosclerotic plaque; otherwise, new studies have described the functional effect of mif 794 (catt)58, showing that the catt5 allele has the lowest transcriptional activity and the catt68 alleles increase the expression rate . This evidence validates the association found between the 6/7 genotype of the mif 794 (catt)58 polymorphism and susceptibility to acs . Recently, chen et al . Showed that the transcriptional repressor hbp1 (hmg box - containing protein 1) negatively regulates mif expression, but this is still under investigation . We also stratified the risk factors in the acs group regarding the genotypes of the 794 (catt)58 mif polymorphism . As we mentioned, mif has an important function in inflammatory processes (kleemann et al . ), suggesting that mif controls the development of metabolic pathologies associated with acs risk factors such as obesity and type 2 diabetes mellitus . Nevertheless, we did not find any association between the genotypes of the 794 (catt)58 mif polymorphism and the risk factors in the acs group . However a trend is shown between the 6/7 genotype and obesity, type 2 diabetes mellitus, dyslipidemia, and high blood pressure . We want to highlight that, as a limitation of the study, the sample size in each subgroup is not enough to find an association, but there is a marked tendency with the higher alleles, which are responsible for a higher expression . It is worth noting that the 794 (catt)58 promoter polymorphism of mif has not been studied in relation to susceptibility to acs . The 6/7 genotype of the mif 794 (catt)58 polymorphism is related with susceptibility to acs in a western mexican population.
Granuloma annulare (ga) is a relatively common idiopathic disorder of the dermis and subcutaneous tissue with an estimated incidence of 0.10.4%.1 the lesions typically appear as papules and plaques with annular margins most commonly on dorsal surfaces of hands, arms and feet.2 the disease affects all age groups, and women are affected more commonly than men . Various clinical forms of ga, including localized, generalized, subcutaneous, patch and perforating types, have been described . Localized variant is probably the most common form of ga, but the disease can sometimes become disseminated in larger areas of skin . Generalized ga is defined by the simultaneous involvement of trunk and upper or lower extremities . Some authors believe that extensive involvement of extremities without involvement of trunk can also be classified as generalized form of the disease.2,3 subcutaneous ga is characterized by subcutaneous nodules that are often found on lower extremities of children . In patch ga, lesions are flat rather than being papular and sometimes may cover large areas of skin . Perforating ga is defined clinically by umbilicated papules that may have central crust.47 the natural history of ga is quite variable, but it has a tendency to spontaneous involution in a high proportion of patients.8 diagnosis of ga is based on clinical and histopathological examinations . In histopathological studies, the classic presentation of ga is a palisading granuloma characterized by histiocytes and epithelioid cells surrounding a central zone of altered collagen . In well - developed lesions, there is mucin deposition within the foci of altered collagen.3 ga has been associated with a variety of comorbidities, including diabetes mellitus, thyroid disorders, dyslipidemia, underlying malignancies and tuberculosis.914 there are also reports suggesting the appearance of ga following bacillus calmette gurin vaccination.15,16 although several underlying conditions have been linked to ga, little data from controlled studies are available to validate these associations . Even the relationship between ga and diabetes mellitus (which is probably the most reported comorbidity to occur in ga - affected patients) is still controversial . The aim of this study was to examine the relationship between ga and thyroid disorders, diabetes mellitus and positive tuberculin skin test . This case control study was conducted on 28 consecutive ga patients presenting to the department of dermatology, farshchian hospital, hamadan, iran, between january 2013 and june 2014 as the patient group and 41 age- and sex - matched apparently healthy volunteers as the control group . All patients with clinical diagnosis of ga underwent skin biopsy for histopathological examination . After the confirmation of diagnosis, the ethics committee of hamadan university of medical sciences approved the study protocol, and informed consent was obtained from all participants . The study protocol was also in accordance with the 1975 declaration of helsinki (revised in 2008). Demographic and clinical data such as sex, age and distribution of lesions were recorded . Laboratory tests were performed in all participants (patients and control subjects), including serum thyroid stimulating hormone (tsh), fasting blood sugar (fbs) and tuberculin skin tests . Serum tsh was measured using electrochemiluminescence (ecl) assay (roche diagnostics limited, rotkreuz, switzerland). According to the measurement kit, normal fbs was defined as a value of <100 mg / dl after 8 hours of fasting, impaired fasting glucose (ifg) as 100 to <126 mg / dl and diabetes mellitus as 126 mg / dl . Tuberculin skin test was performed using the mantoux method . In this method, 5 units of ppd (0.1 ml) are injected by a 27-gauge needle intradermally in the volar aspect of forearm, and the diameter of induration is read after 4872 hours . Since iran is a high prevalence country for tuberculosis and our study subjects had no known immunosuppression or recent contact with tuberculosis, an induration higher than 10 mm in diameter was considered positive . The results were analyzed using spss 13.0 software (spss inc ., chicago, il, usa) for windows . Of 28 patients, 24 were female and 4 were male . Of 41 controls, there was no statistically significant difference between cases and controls regarding sex (fisher s exact test: p=0.626). The average age of female and male patients was 53.4115.2 and 2320.9 years, respectively . The average age of female and male controls was 55.1214.1 and 2420.3 years, respectively . There was no statistically significant difference between cases and controls regarding age (independent samples t - test: p=0.732). In this study, 20 patients (71.42%) presented the localized variant of ga, whereas generalized variant was observed in 7 patients (25%) and only 1 patient had the subcutaneous form (3.57%). The most prevalent sites of involvement were upper extremities (88%), lower extremities (8%) and trunk (4%). Mean serum level of fbs in the patient group was significantly higher than mean serum level of fbs in the control group (110.6046.31 mg / dl versus 88.3910.58 mg / dl, respectively, independent samples t - test: p=0.004). Six out of 28 patients with ga (21.4%) and 5 out of 41 individuals in the control group (12.1%) had a fasting blood glucose level> 126 mg / dl . All these hyperglycemic individuals in both groups subsequently proved to have diabetes mellitus according to repeated high fasting blood glucose levels . Miu / l, which was not significantly different from mean serum level of tsh in the control group (3.262.11 miu / l, independent samples t - test: p=0.772). According to tsh reference value (0.46.21 miu / l), 1 patient with ga had lower than normal and 3 patients had higher than normal serum tsh levels . The patient with low tsh level was subsequently proved to be hyperthyroid according to high free thyroxine (ft4) level (5.8 ng / dl). The 3 patients whose tsh levels were elevated proved to have subclinical hypothyroidism according to normal values of ft4 . Five out of 41 individuals in the control group (12.1%) had tsh levels higher than normal, but no lower than normal tsh levels were detected in this group . These 5 control subjects subsequently proved to have subclinical hypothyroidism according to normal values of ft4 . The patient group had more positive tuberculin skin tests than the control group, but statistical analysis could not be performed because of the very small number of positive tests in both groups (4 versus 1). It is characterized by collagen degeneration, palisaded or interstitial histiocytic infiltration and mucin deposition histologically.17 although several hypotheses have been proposed to explain the etiopathogenesis of ga, the exact cause of the disease is not yet recognized . Many precipitating factors such as subcutaneous injection for desensitization,18 octopus bite,19 bacillus calmette gurin vaccination,15 mesotherapy20 and ultraviolet light exposure21 have been reported but never confirmed by controlled studies . Recent studies have pointed to a delayed - type hypersensitivity reaction to an unknown antigen as the probable mechanism underlying the development of ga lesions.22,23 associations of ga with systemic diseases have been described but not yet consistently confirmed . Although there are multiple reports of ga occurring in the setting of diabetes mellitus, dyslipidemia, thyroid disorders, malignancies and infections, little data are available from large controlled studies.24 in accordance with other studies, we observed a female predominance of ga (the female to male ratio was 6:1).2,3 on the basis of available data, the prevalence of ga in the general population is estimated to be 0.10.4%, which also shows predominance for women.7 in this study, the average age of male patients was significantly lower than females . Previous studies have revealed that more than two - third of patients with ga are in their first 3 decades of life.8 in our study, the male patients showed the same pattern, whereas females were mostly in their fourth to fifth decades of life . In accordance with previous studies, we found upper and lower extremities the most prevalent sites of involvement (88% and 8%, respectively).2,6 we also found the localized variant of ga the most prevalent clinical type (71.42%), whereas generalized and subcutaneous variants were less prevalent (25% and 3.57%, respectively). Other studies had previously revealed that the localized variant is far more common than the generalized or perforating type.11,25 we demonstrated that the mean level of fbs is significantly higher in the patient group than in the control group (p=0.004). In a study conducted by studer et al,25 12% of 84 patients with ga (localized or generalized) were diabetic while the prevalence of diabetes mellitus among the regional population was estimated to be 5% . Studer et al25 also demonstrated that diabetic patients were more frequently involved by the chronic relapsing form of ga than nondiabetic patients . Muhlemann and williams9 also found a strong association between localized ga and diabetes mellitus in a large population study . Kidd et al26 demonstrated that glucose tolerance may be reduced and insulin levels increased in patients with ga . In spite of these findings, the relationship between ga and diabetes mellitus is still a matter of debate . Nebesio et al27 did not find any significant relationship between these 2 conditions, and dicken et al28 also failed to show this association . Nonetheless, simultaneous appearance of necrobiosis lipoidica, ga and diabetes mellitus in a same patient has been reported previously.29,30 davison et al30 proposed that ga is in fact associated with diabetes mellitus, but this association may be masked because of not recognizing these 2 conditions at the same time . The present study revealed that the mean level of tsh was not significantly different between patients and controls . In the study conducted by vzquez - lpez et al,10 autoimmune thyroiditis was significantly more common in adult women with localized ga than in the control group . In another study carried out by dabski and winkelmann3 on 100 patients with generalized ga, thyroid disorder was found in 13 patients (hypothyroidism in 6, graves disease in 3, thyroiditis in 3, and thyroid adenoma in 1). Findings of our study and other studies mentioned earlier indicate that the relationship between ga and thyroid disorder cannot be definitely confirmed or denied . We observed that tuberculin skin test was more commonly positive in ga patients compared to controls . Winkelmann14 has previously reported a case of chronic inadequately treated tuberculosis with ga - like lesions . Kakurai et al16 have also reported ga in a 12-year - old japanese boy 5 days after bacillus calmette gurin vaccination . Mycobacterium in fact may play a role in induction of a delayed - type hypersensitivity reaction, which subsequently leads to granuloma formation . Mycobacterium antigen can trigger a relatively small population of t cells, which are capable of recruiting a larger number of t cells nonspecifically, leading to the formation of ga lesions . Gurin vaccination or tuberculin skin test based on a few case reports, and since there are no similar controlled studies focusing on the relationship between positive tuberculin skin test and ga, our findings encourage further studies to be performed on this issue . This study indicates that ga may be associated with diabetes mellitus; however, because of the small number of positive tuberculin skin tests, the relationship between ga and this test could not be confirmed . Regarding thyroid disorders, again small number of patients who had the abnormality did not permit us to confirm any relationship.
Bronchial inflammation represents one of the main pathophysiological changes in bronchial asthma (ba). The intensity of the inflammation influences the frequency and extent of symptoms, as well as the level of disease control . Induced sputum provides direct information about inflammation . The non - invasive character and the possibility to perform this test at any disease stage, including exacerbation period, puts it in a special place in the process of evaluation of the child with bronchial asthma . The method is safe, easy to perform and generally well tolerated by children, helping establish the diagnosis and the long - term management . It is also used for research purposes and may be an alternative to bronchoalveolar lavage . Our study s main objective was the analysis of the sputum cellularity in patients with ba in correlation with the level of disease control and the controlling therapy with / without inhaled glucocorticoid (igc). The second objective was to establish the correlation between sputum cellularity and other indirect parameters used to evidence bronchial inflammation (forced expiratory volume in 1 second the study was carried out in a group of children diagnosed with ba, admitted to the 3rd pediatric clinical hospital of cluj - napoca, romania, during july 2008 march 2012 . The study was performed after obtaining informed consent of the parents, according to the regulations of the ethics committee of the emergency clinical hospital for children . The inclusion criteria were: - certified diagnosis of ba;- age between 6 18 years;- absence of airways acute infection signs in the past 6 weeks;- patient s ability to perform respiratory function test correctly;- parents informed consent . - certified diagnosis of ba; - age between 6 18 years; - absence of airways acute infection signs in the past 6 weeks; - patient s ability to perform respiratory function test correctly; - parents informed consent . We excluded from our study the patients in exacerbation and who could not perform the technique of induced sputum and the spirometric test adequately . Ba diagnosis was established based on the clinical manifestations and at least one of the following criteria: - positive bronchial reversibility: increase of fev1 by over 12% after salbutamol administration;- previous admissions for exacerbations;- at least two presentations to the emergency room due to asthma exacerbations . - positive bronchial reversibility: increase of fev1 by over 12% after salbutamol administration; - previous admissions for exacerbations; - at least two presentations to the emergency room due to asthma exacerbations . In children with ba included in our study, we performed induced sputum along with spirometry and determination of eno . We started sputum induction with ultrasound nebulization of 3% sodium chloride solution for 5 minutes, after which the patient was asked to expectorate . If sputum was not obtained with this concentration, higher concentrated saline solutions of 4% and 5% were nebulized for another 5 minutes each . After the change of every concentration,, the patients were assessed by spirometry and received 200 g salbutamol at the beginning and at the end of the test . When changing the solutions, the patient was asked to perform the peak expiratory flow (pef) using the peak flow meter (figure 1). The sputum sample was weighed and dithiothreitol (dtt) was added in a 4:1 ratio . The sputum - dtt mixture was centrifuged (3001500 x g) for 5 minutes, thus having two phases: the sediment (represented by cells, which was analyzed under the microscope), and the liquid phase . After centrifugation, the cells from the sediment were counted with a hemocytometer, the test being valid for interpretation only if the counting showed at least 400 non - squamous cells on slides . From the centrifugal sediment, a may - grunwald - giemsa stained smear was made . Using the optical microscope the number of eosinophils (eo), neutrophils (n), macrophages and bronchial epithelial cells were counted, each value being expressed as percentage from the total of non - squamous cells . The sample obtained was considered unsatisfactory if the percentage of squamous cells was higher than 80% . According to the literature, we considered the upper limit of normal 2.5% for eosinophils and the determination of eno was performed with an appropriate device (niox - mino sweden), the results being expressed in p.p.b . (parts per billion). For children under igc therapy, high levels of eno were considered to be 20 p.p.b . If under 12 years of age, and 25 p.p.b . If over 12 years . In patients not under igc therapy, high eno values were considered 5 p.p.b .. the degree of bronchial obstruction was established by spirometry . The bronchial reversibility test was considered positive in cases in which fev1 increased by more than 12% after salbutamol administration . Asthma was labeled as atopic if patients presented one or more of the following: associated allergic rhinitis, positive skin test for one or several allergens, high total immunoglobulin e (ige) concentration . We used the prick technique for allergy skin tests and the total ige concentration was determined by elisa . We calculated the statistically significant differences between the means of variables obtained in the study subgroups . When we had 3 subgroups we used the kruskal - wallis test, while for the situations in which we formed 2 subgroups the mann - whitney test was applied . The correlation between the parameters was documented using spearman s correlation coefficient (direct correlation if r>0.6 and indirect correlation if r<0.6), considered statistically significant if p<0.05 . We calculated the statistically significant differences between the means of variables obtained in the study subgroups . When we had 3 subgroups we used the kruskal - wallis test, while for the situations in which we formed 2 subgroups the mann - whitney test was applied . The correlation between the parameters was documented using spearman s correlation coefficient (direct correlation if r>0.6 and indirect correlation if r<0.6), considered statistically significant if p<0.05 . The characteristics of children with ba included in the study are shown in table i. sputum for cellularity assessment was obtained from 55 of the 67 patients studied (82%). During the test only 11 children (16.4%) had mild adverse reactions: rhinorheea (7.4%), sneezing (2.9%), cough (5.9%), fev1 decrease by more than 12% after nebulization (4.4%). Sputum cellularity evidenced the existence of statistically significant differences both for eosinophils (p=0.049) and neutrophils (p=0.004) between patients with controlled ba and those with partial or uncontrolled disease . In patients with controlled ba the eosinophils count was significantly lower than in those with partially controlled or uncontrolled ba . Regarding neutrophils, the highest percentage was found in patients with uncontrolled ba, while the lowest count was observed in patents with controlled ba . The analysis of the sputum s cellularity in relation to the controlling therapy did not evidence statistically significant differences between patients on igc therapy and those without this therapy . Higher neutrophils levels were found in patients with uncontrolled asthma, both in those under igc therapy and those without this treatment (table iv). Analyzing the sputum cellularity in relation to spirometric parameters, we found higher eosinophils counts in patients with fev1 80% as compared with those with normal fev1 (meansd: 40.330.3% versus 10.611.9%, p=0.003) (table v). When analyzing fev1 in patients with high eosinophils values, we observed the existence of a moderate to good indirect correlation (r=0.56, p=0.005), but the correlation was not present for patients with normal eosinophils values (figure 1). We could not establish a correlation between fev1 and the neutrophils count in the sputum, whether the latter was high or normal . Of all 55 patients that provided sputum sample, only 24 underwent determination of eno . Our study demonstrated the presence of a moderate to good correlation, statistically significant, between the value of eno and the neutrophils percentage in the sputum (r=0.67, p=0.0003). Our study evidenced higher eosinophils and neutrophils values in the sputum of patients with partially controlled or uncontrolled ba than in those with controlled asthma . We also obtained a statistically significant correlation between the value of eno and the neutrophils percentage in the sputum, which demonstrates the same involvement of neutrophils in the bronchial inflammatory process . The indirect correlation between the high eosinophils percentage and fev1 and the direct correlation between neutrophils and eno point to the value of induced sputum test when it is associated with other indirect parameters used in the assessment of bronchial inflammation . The success rate of induced sputum test in our study was 82%, similar to the reports in literature . During the test we recorded adverse reactions in 16.4% of the patients, similar to other studies . In 4.4% of the patients in which cough was induced, fev1 decreased by more than 12% after saline nebulization, but it was restored after salbutamol administration . The highest percent of eosinophils was observed in patients with partially controlled asthma (1825.2), comparing with patients with uncontrolled asthma (4.914.4). This finding is not according to most of the studies from literature and the explanation for this result can be the relatively small number of paient included in our study, especially by those with uncontrolled asthma . Induced sputum is considered to be one of the most accurate method of assessment of bronchial inflammation . The technique is non - invasive, cheap and may be performed even in patients during a bronchial obstruction episode . The method is successfully used not only in patients with ba, but also in other diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, chronic cough . Though most studies have considered the eosinophils count in the sputum to be relevant for bronchial inflammation, more recent data suggest the same relevance regarding neutrophils . Various studies evidenced high values of neutrophils in the sputum of patients with ba, especially uncontrolled asthma . A highly increased neutrophils count was evidenced in the sputum of patients during an exacerbation episode, triggered by a viral infection or in which respiratory symptoms were persistent . In the case of these patients, the eosinophils count was generally low, while the response to igc therapy was poor . This explanation is unlikely to be valid for our study, as we excluded patients with signs of acute respiratory infections in the 6 weeks prior the study . Various studies analyzing the response to controlling therapy in patients with a high neutrophils level evidenced a lack of response to igc . The lack of studies on the role of neutrophils in the pathogenesis of ba may be explained by the fact that many researchers did not investigate or report all cell types from the sputum, focusing only on eosinophils . Another explanation may be related to the method of sputum collection, many authors analyzing only the samples rich in mucus and not the whole amount of sputum obtained . Our study supports the theory according to which ba should be also classified according to the inflammatory phenotype of sputum cellularity . The literature describes 3 phenotypes according to sputum cellularity: eosinophilic, neutrophilic and mixed . Certain studies mention the neutrophilic type more frequently in severe asthma, refractory to igc therapy . Although our study showed that in uncontrolled asthma patients the predominant sputum cells are neutrophils and not eosinophils like we used to know, there are some recent ones that demonstrated the importance of this type of cells . This studies highlights that in uncontrolled asthmatic patients that do not respond to the glucocorticoid therapy, the explanation rises from the presence of a high percent of neutrophils in sputum . The results have clinical implications because in this patients the therapy needs to be changed and this is a motive for more studies to be made . Regardless the contradictory results, we all agree in the importance of induced sputum test, especially when it is associated with other indirect parameters used in the assessment of bronchial inflammation.
Alzheimer's disease (ad) is the most common form of dementia and represents a serious economic and social burden worldwide . The familial form of ad (fad) is caused by autosomal dominant mutations that increase levels of the 42 amino acid isoform of the amyloid - beta 42 (a42) peptide [1, 2]. The primary genetic risk factor for ad is inheritance of the apoe4 gene for apolipoprotein e (apoe), compared to apoe3, with apoe2 reducing risk [35]. The mechanism(s) by which apoe and a affect pathogenesis of the disease is unclear (reviewed [35]). However, evidence suggests that apoe may isoform - specifically modulate a-induced neurotoxicity [4, 5]. To address potential mechanisms in vivo, several transgenic (tg) mouse models have been developed to assess the structural and functional interactions between apoe and a. however, each of these models has potential drawbacks that affect the interpretation and physiological relevance of the results . This paper will summarize the current models resulting from the introduction of human - apoe into a-tg mice . The continued development and characterization of both apoe and apoe / a-tg mouse models is critical to understanding the apoe - isoform effects on ad pathology . Traditional diagnosis of ad is based on pathology that includes extracellular amyloid plaques, composed primarily of a42, intraneuronal neurofibrillary tangles, hyperphosphorylated tau, neuroinflammation, and neuronal cell loss . A is a 3943 amino acid (4 kda) peptide produced via sequential proteolysis of amyloid precursor protein (app) by -secretase / bace followed by -secretase (composed of presenilins [ps] 1 and 2), to produce a peptides primarily 40 and 42 amino acids in length . Genetic and experimental evidence indicates that a42 is the cause of ad pathogenesis [1, 2]: (1) fad, although only 35% of all ad cases is caused by autosomal dominant mutations in app, ps1, or ps2 that increase levels of a42 or the a42: 40 ratio; (2) down syndrome is caused by trisomy of chromosome 21 (the location of the app gene), and is characterized by plaque deposition and dementia by the age 40; (3) a42 is neurotoxic in vitro and in vivo . A, particularly the more toxic a42, aggregates to form a variety of higher - order assemblies including oligomers, protofibrils, fibrils, and amyloid plaques . Amyloid plaques themselves exist in different conformations including compact plaques (composed of a dense thioflavin - s- (thios-) positive core), neuritic plaques (identified as thios - positive plaques surrounded by a ring of dystrophic neurites), and diffuse plaques (characterized by amorphous wisps of amyloid that lack a central core and are not neurotoxic) [710]. While total plaque burden does not directly correlate with dementia, it is an indication of increased a42 levels, and compact or dense core plaques may be disease relevant [7, 12]. Apoe is the only apolipoprotein synthesized within the blood - brain barrier (bbb) and is the primary apolipoprotein associated with lipoprotein particles in the central nervous system (cns), as peripheral apoe is unable to cross the bbb or blood - cerebrospinal fluid barrier . While the majority of apoe in the cns is secreted by glial cells, particularly astrocytes, neuronal production of apoe has been observed under specific pathological conditions . Cns lipoproteins are critical for lipid homeostasis, particularly as cholesterol and phospholipids are required for neuronal growth, repair, and synaptogenesis (reviewed [35]). In humans, three apoe polymorphic alleles exist (apoe2, apoe3, and apoe4) which encode three protein isoforms (apoe2, apoe3, and apoe4). Although human - apoe (h - apoe) is a 299 amino acid protein, the three apoe - isoforms differ by a single amino acid substitution at residues 112 or 158: apoe2 contains cys, apoe3 contains cysarg, and apoe4 contains arg . In the general population, apoe3 is the most common allele (77%), followed by apoe4 (15%) and apoe2 (8%). Compared to apoe3, inheritance of one or two copies of the apoe4 allele increases the risk for developing ad by 4- and 12-fold, whereas apoe2 decreases risk by 2- and 4-fold [5, 14]. In addition to ad, apoe4 is a risk factor for cerebral amyloid angiopathy (caa; amyloid deposition in blood vessels) and impairs recovery from cerebral insults such as stroke, cerebral hemorrhage, and brain injury . While apoe knock - out mice (apoe) and h - apoe - tg models demonstrate that apoe affects neuronal viability independent of a-induced neurotoxicity, the focus of this paper is on the synergism between the h - apoe isoforms and a on neuropathology . To address the latter in vivo, apoe / a-tg models were subsequently developed to specifically address the isoform - specific effects of h - apoe on a deposition . Several approaches have been used to make tg mouse models to assess the function of apoe . Apoe tg - mice were initially used to help understand the role of apoe in the brain [15, 16] although the homology between mouse (m-) and h - apoe is 70%, and mice express only a single isoform, comparable to apoe4 at residues 112 and 158 . Apoe mice have been used as the background for several h - apoe - tg mouse lines . Heterologous promoters have been used to drive the expression of h - apoe in glia or neurons . Examples include glial fibrillar acidic protein (gfap; glial) [22, 5557], transferrin (neuronal), platelet - derived growth factor (pdgf; neuronal), neuron - specific enolase (nse; neuronal), and thymocyte differentiation antigen 1 (thy1; neuronal). However, these models have limitations inherent in the use of a heterologous promoter and specific to apoe: (1) the expression of protein by a heterologous promoter is not regulated as it would be by the endogenous promoter; (2) the inserted copy number of the transgene cannot be regulated by a traditional tg mouse approach; (3) while the cell - specific expression of apoe in the brain is controversial [6063], the majority of evidence suggests that glia, not neurons, are the primary cell type to express apoe [13, 6467]; (4) by using the m - apoe background and inducing apoe expression via cns - specific promoters, lack of peripheral apoe becomes a variable of potential importance when interpreting results from these mice; (5) evidence suggests that in both humans and apoe - tr mice, apoe4 levels are significantly lower than apoe2 or apoe3 [6873]. Knock - in or targeted - replacement (tr) mice were developed that express h - apoe under the control of the endogenous mouse promoter and provide an alternative to heterologous expression of h - apoe . In apoe - tr mice, the coding domain for each of the h - apoe isoforms replaces the coding domain for m - apoe . Thus, in apoe - tr mice, glial cells express h - apoe in a native conformation at physiologically regulated levels, and in the same temporal and spatial pattern as endogenous m - apoe . Thus, the interpretation of apoe isoform - specific results is determined by the nature of the apoe - tg mouse model . As discussed, a number of apoe - tg mouse models have been developed with apoe expression under the control of different promoters . The general phenotypes of apoe mice and three examples of the most widely studied apoe - tg mice (gfap - apoe, nse - apoe, and apoe - tr) are briefly discussed (table 1). Compared to wild type, apoe mice have decreased excitatory transmission, spine density, and dendritic length [1618]. These changes may underlie behavioral deficits, as apoe mice are cognitively impaired [15, 19, 20]. However, lack of peripheral apoe can have profound effects on plasma lipid homeostasis, potentially leading to a number of confounding variables, including metabolic disease and increased risk for cardiovascular disease, compromising the ability to compensate for oxidative stress or inflammation, deficits that can effect the vasculature of the brain . In addition, the relevance of these mice is unclear as there are no apoe humans . Apoe provides the background for a number of the h - apoe - tg mice . Gfap is an intermediate filament protein, expressed at high levels by glia in the cns . To target apoe expression to glia, mice expressing h - apoe under the control of the gfap promoter were crossed with apoe mice [17, 22, 23]. At the cellular level, gfap - apoe4 mice show increased ca1 cellular atrophy and decreased spine density compared to gfap - apoe3 mice . In addition, compared to gfap - apoe3 mice, gfap - apoe4 mice demonstrate impaired cognition and increased anxiety; common symptoms in ad patients [17, 23, 24]. Interestingly, cognitive impairment is evident at an earlier age in female compared to male gfap - apoe4 mice [23, 24]. A limitation of this model is that the effect of apoe isoform on neuroinflammatory responses cannot be interpreted, as apoe is expressed under the control of a promoter that is induced by neuroinflammation . Neuronal apoe expression remains controversial, although expression has been identified under conditions of stress . To investigate the role of neuronal expression, apoe - tg mice expressing h - apoe under the control of the nse promoter kainic acid was used to induce apoe expression in these mice and resulted in the protection of nse - apoe3 mice from age- and kainic acid - induced presynaptic and dendritic degeneration compared to nse - apoe4 mice . In addition, female nse - apoe4 mice demonstrate impaired cognition compared to female nse - apoe3 mice . In contrast this is similar to the early development of cognitive deficits in gfap - apoe4 . To investigate the function(s) of apoe in a more physiologically relevant apoe - tg model, apoe - tr mice were developed to express each h - apoe isoform under the control of the endogenous m - apoe promoter [2835]. Although apoe protein levels in brain homogenates of these mice were initially described as being similar for each h - apoe isoform, subsequent analysis demonstrated that apoe4 levels are significantly lower in plasma, csf, and brain homogenates than apoe2 and apoe3 [28, 30], similar to the levels of apoe isoforms in humans [6873]. Compared to apoe3-tr mice, apoe4-tr mice demonstrate decreased spine density and dendrite length, reduced excitatory transmission, and long - term potentiation (table 1). Again, similar to the cognitive deficits observed in female gfap - apoe4 mice and nse - apoe4 mice, female apoe4-tr mice are cognitively impaired compared to female apoe3-tr mice [31, 32] interestingly, both apoe3- and apoe4-tr females are cognitively impaired when compared to apoe - isoform - matched males . Although amyloid plaques are a hallmark of ad, the mechanisms underlying a-induced toxicity remain unknown . To help determine the effects of a and amyloid deposition on neurotoxicity in vivo, a-tg mouse models have been produced that express human fad mutations . These models include but are not limited to: pdapp (app), tg2576 (app), j9 (ps1, app), as well as 5xfad (app, ps1) (reviewed in). These a-tg mice express either app mutations alone (pdapp, tg2576) or in combination with ps mutations (j9, 5xfad). Plaque development generally begins at 6 months of age in these mice (with the exception of 5xfad), and the onset of cognitive deficits is dependent on the model (e.g., pdapp at 6 months, tg2576 at 9 months). Because the app mutation is linked primarily to caa, the tg2576 mice also develop a deposition around blood vessels, particularly leptomeningeal and cortical vessels . However, one limitation of a-tg models is the relative lack of neuronal loss as observed in ad (reviewed) although there are some exceptions (e.g., 5xfad, appps1ki, and tba2 mice). Initially, the effect of apoe on a deposition was investigated using apoe crossed with a-tg mice (table 2). Specifically, apoe mice were crossed with pdapp [7779] or tg2576 mice . In both models, the absence of apoe significantly delayed thios - positive plaque deposition by 6 months and decreased a levels in the hippocampus and cortex, as measured biochemically from brain homogenates and by a immunoreactivity [42, 77] (table 2). Apoe affects plaque deposition in a gene - dose - dependent manner, as plaque levels were intermediate in apoe / pdapp compared with apoe / pdapp and apoe / pdapp mice . In addition, the a40 caa present in the tg2576 mice was absent in the apoe / tg2576 mice . The initial studies demonstrating that the lack of m - apoe delayed plaque deposition in a-tg mice led to the question of what would be the effect of introducing h - apoe into apoe / a-tg mice (table 2). To initially address this issue, gfap - apoe mice was crossed with apoe / pdapp mice [41, 42]. Surprisingly, the introduction of h - apoe did not result in the expected reduction in the age of onset of a pathology, rather the presence of h - apoe further delayed a deposition . Amyloid accumulation is delayed from 6 to 12 months in apoe / pdapp mice, and to 15 months in gfap - apoe / pdapp mice (table 2). Once plaque pathology returned, the greatest plaque burden was found in gfap - apoe4/pdapp mice, compared with gfap - apoe2/pdapp and gfap - apoe3/pdapp mice . One potential confounding factor in these mice, as well as the nse - apoe mice described below, is the equal expression of the h - apoe isoforms, in contrast to human data where inheritance of an apoe4 allele results in lower apoe levels [68, 69, 73]. To determine the effect of neuronal h - apoe expression on a pathology, nse - apoe similar to gfap - apoe / pdapp - tg mice, the introduction of h - apoe to j9 mice delayed plaque pathology from 8 to 19 months, with deposition greater in the nse - apoe4/j9 than nse - apoe3/j9 . The physiologic advantages of using apoe - tr mice to study the function(s) of apoe in vivo led to the generation of apoe - tr / pdapp and apoe - tr / tg2576 mice (table 2). The resulting data confirmed that h - apoe delayed plaque deposition . In apoe - tr / pdapp mice, plaque deposition was delayed from 6 months to 18 months of age . An apoe isoform - specific effect on a pathology was also observed, with thios staining, a immunoreactivity, and a levels in brain homogenates highest with apoe4 . In tg2576 mice, plaque deposition initiates at 9 months of age, while in apoe - tr / tg2576 mice there is minimal plaque staining at 15 months . Interestingly, at 15 months, the isoform effect in these mice is primarily on caa (e4> e3), as amyloid deposition in the parenchyma is minimal . The major drawback to the apoe / a-tg crosses described thus far is the significant h - apoe - induced delay in a pathology . For example, in apoe - tr / pdapp mice, plaque deposition is not identified until mice are 18 months of age (table 2). This substantial delay precludes timely analyses of apoe isoform - specific effects on early aspects of a pathology . One approach to address this temporal delay is to introduce an additional insult, such as traumatic brain injury (tbi), kainic acid, nitric - oxide - synthase-2- (nos2-) knock - out, or by blocking a degradation via neprilysin inhibition . Although no amyloid deposition is present in 12-month - old gfap - apoe / pdapp mice, tbi at 9 months leads to amyloid deposition at 12 months, which is greater with apoe4 compared to apoe3 . Kainic acid decreases synaptophysin and map-2 staining in apoe and nse - apoe tg mice, with the effect more pronounced with apoe4 than apoe3 . Nitric oxide, produced by inducible nos (encoded for by the nos2 gene), is an important signaling and redox factor that plays a key role in neuroinflammation and neurodegeneration . Nos2 mice have been crossed with multiple a-tg mouse models, and results demonstrate increased tau phosphorylation and neuronal loss in nos2/a-tg mice . In the tg2576/nos2 mice, inhibition of neprilysin, an enzyme that degrades extracellular a, with thiorphan, induces fibrillization and deposition of a and in wild - type mice . Thiorphan treatment of apoe - tr mice leads to aggregation of mouse a 1 week after treatment, with higher a deposition in apoe4-tr compared to apoe3-tr mice [84, 85]. Thus, thiorphan treatment of h - apoe - a-tg mice represents a potential method of accelerating human a deposition . An alternative method to address the h - apoe - induced temporal delay in a accumulation is to cross apoe - tr mice with a-tg mice that have rapid - onset a pathology, such as 5xfad, 3xtr [86, 87], appps1ki, appps1, or tgcrnd . For example, because 5xfad mice develop plaques by 2 months, the prediction is that apoe - tr/5xfad - tg mice develop plaques around 6 months and 12 months before other human - apoe / a-tg mice (ladu lab, unpublished observations and [5053]). The approaches described herein will lead to more tractable apoe / a-tg - models to assess the apoe isoform - specific effects on a pathology . Human apoe - tg mouse models demonstrate that, compared to apoe3, apoe4 increases markers of neurodegeneration and cognitive impairment . Initially, to determine the effect of apoe on a pathology, a-tg mice were crossed with apoe mice, resulting in a significant delay in plaque deposition . Surprisingly, the introduction of h - apoe to several apoe / a-tg mouse models further delayed plaque deposition . This temporal delay restricts the usefulness of the current apoe / a-tg mice for investigating the process of a accumulation and the resulting neurotoxicity . To accelerate a deposition, current apoe / a-tg models could be treated with an additional insult such as tbi, crossed with other tg models of neurodegeneration (nos2), or treated with drugs that decrease a degradation . Alternatively, the development of a pathology could be accelerated by crossing a-tg models with a rapid onset of a pathology, such as 5xfad mice with apoe - tr mice . Transgenic models such as these provide tractable models for identifying biomarkers and the efficient initial validation of therapeutic targets.
According to the world health organisation (who), the current estimates are that more than 62 million individuals are affected by diabetes mellitus (dm), with 79.4 million cases expected by 2030 [1, 2]. The success of the treatments such as the one started in south india is closely related to early detection and explicit risk factors of diabetes . This is a public health challenge and a number of diabetic patients (estimated one in two) will remain undiagnosed . There are a number of people who are hesitant in taking the blood test till the disease has advanced and for such cases the treatment options for retinopathy or neuropathy become very limited . There have been successes with population screening for diabetic retinopathy (dr) using digital photography of the retina [4, 5]. Studies have shown that changes to both retinal nonvascular and vascular parameters are associated with different stages of dr . There is also evidence of foveal and macular thickening to be an indicator of minimal nonproliferative dr [7, 8]. Vascular parameters such as venule dilation and larger retinal arteriolar calibre [9, 10], changes to the vasculature shapes and arteriolar branching angle, increased tortuosity [1113], and fractal dimension (fd) of the retinal vascular network [1416] are associated with dr . These methods, however, require significant manual supervision and are not suitable for automatic analysis . This study has identified the association between retinal vascular parameters that can be automatically obtained without manual supervision with type ii diabetes and no diagnosed dr . This work provides an explanatory model of diabetes from retinal vascular parameter and knowledge about candidate clinical information, using retinal images with no sign of dr . Rivas, a retinal imaging software that automatically measures a range of parameters, was used . The parameters that have already been reported in the literature were measured along with introduction of two new parameters: total number and average variability of the acute branching angles . The study has developed statistical models using retinal vascular parameters, patients' demographic information, and clinical parameters as the explanatory variables . This is a follow - up to a recent similar work where only fd of optic disk centred (odc) retinal images was analysed . A potential impact of this study is to allow for development of a predictive mode in the future to identify diabetic patients with no dr during a routine visit to an ophthalmologist or an optometrist . Experiments were conducted using database collected in department of the retina, save sight centre hospital located in delhi . Approval for this study was granted by the human research ethics committee (hrec) of the royal melbourne institute of technology (rmit university), melbourne, australia, and also by the institutional review board at save sight centre hospital in accordance with the declaration of helsinki (1975, as revised in 2004). All participants were respondents to a request advertised in the save sight centre in delhi . The purpose and experimental procedure in plain language all the volunteers self - evaluated themselves to be reasonably active and none of them were pregnant . The participants were classified into two groups of type ii diabetes (case) with no observable retinopathy and nondiabetic (control) patients . The diabetic cases were confirmed by the patients' physician based on either (i) their fasting or (ii) postprandial glucose plasma levels being greater than 126 mg and 200 mg / decilitre, respectively . None of the diabetic patients had any observable intraretinal haemorrhages or venous beading, hard exudates, and neovascularisation according to the classification levels by international clinical disease severity scale for dr . This was confirmed by an ophthalmologist after examination of both eyes . The participants' demographic information, including age, gender, weight (kg), height (m), systolic and diastolic blood pressure (mmhg), skin fold (mm), and cholesterol level (ldl and hdl), was recorded . The participants' age was limited to a narrow range of 40 to 73 years to remove confounding effect of the age factor on the analysis outcome and provide a better balance between the number of diabetic cases and the control groups . All the participants were nonsmoker, did not consume alcohol, had no history of any cardiovascular disease, and did not have any history of antihypertensive and lipid - lowering medications . One odc and one macula centred (mc) fundus images were taken from both eyes of each subject making total of four images for each participant . The photographs were taken in mydriatic mode in a dimmed light room using a mydriatic kowa vx alpha camera (kowa, japan). The original image resolution was 300 dpi (4288 2848 pixels) and the camera was set to an angle of 30. all the images were examined in pairs for quality assurance (i.e., vessel to background contrast and illumination artefacts). After checking the quality discarding ungradable images and age adjustment, 180 retinal images (two (left and right) two (odc and mc) 45 subjects (13 diabetics and 32 nondiabetics)) were obtained . The demographic information of the patients at the baseline has been provided in table 1 . In this study, odc and mc images were analysed separately, but for each image category (i.e., odc / mc) the retinal vascular parameters of the left and right eyes of each subject were averaged . All the original images were first cropped and resampled to identical sizes of 729 485 pixels . Image enhancement and segmentation (binarization) was performed to reduce degrading image background artefacts and improve vessel to background contrast . This process was performed using mlvessel v1.3, software provided online by soares et al . . An example of the enhanced retinal vessels has been shown in figure 1 . For more information on the exact technique retinal vascular geometry features were measured quantitatively using unsupervised retinal image and vasculature assessment software (rivas) v1.0 which has been specifically developed by the authors and described in . In brief some of these are (i) vessel calibre of a specified segment, (ii) simple tortuosity, (iii) number of different fractal dimensions (fd), (iv) vessel - to - background ratio / percentage (v / b (%)), (v) average of acute branching angles (aba) defined as the smallest angle between two daughter vessels, and (vi) the total number of branching angles (tba). The fd measures include binary and grayscale box - counting (also known as differential box - counting (dbc)) and fourier fractal dimension (ffd). In this study, simple tortuosity was measured as the ratio between the actual length of a vessel segment and the shortest (euclidean) distance between the two endpoints within the same segment providing a reflection of the shape / curvature of the vessel . Binary box - counting fd was calculated on skeletonized image as indicator of vascular network complexity without comprising any vessel calibre information . Figure 2(a) shows the thinned vascular network (skeletonized) and the candidate points for location of the branches (red dots). The blue line in this figure is the shortest path connecting the two end points of the vessel segments identified by two consecutive true candidate branch points . Prior to segment identification, the candidate points were automatically labelled in rivas, as true (green circle) and false (red circle) branch points as in figure 2(b). True candidates were defined as the points which correspond to the bifurcations (junction between two daughter vessels and a mother vessel) and false candidates were defined as the points corresponding to other possibilities: crossovers or noise . Vessel diameter summary was also measured using ivan software (university of wisconsin, madison, wi, usa) based on the calibre summary of the biggest 6 arterioles and venules separately and vessel summary represented by central retinal arteriolar equivalent (crae) and central retinal venular equivalent (crve) as well as the ratio of the calibre of arterioles to venules (avr). The measurements were performed within a fixed region and in between the margins of 0.5 to 1.0 disc diameters from the disc margin . Crae and crve were obtained based on the revised knudtson - parr - hubbard formula . Statistical analyses were performed using minitab v.16.1.0 and r studio (r statistical software v.3.3.0). As the number of observations was relatively small, the data was statistically upsampled to the new size of 200 samples (i.e., 58 diabetic cases and 142 nondiabetic) using the bootstrapping technique with sample replacement . The upsampled data was then standardized and centred to decrease the multicolinearity between an interaction term and its corresponding main effects as well as making categorical parameters such as gender, comparable with continuous parameters . Sixteen predictors were used in this analysis: vessel tortuosity (both mean and standard deviation (sd)), aba, sd of angle, tba, crae, crve, avr, fd (binary box - counting dimension of skeletonized images), and vb plus the patient's demographic information (i.e., gender, age, systolic and diastolic blood pressure, body mass index (bmi), and skin fold). Multiple linear regression model was built using all the 16 parameters as dependent variable and diabetes status with two possible categories (i.e., case and control) as independent variable . Analysis of variance (anova) test was performed and f statistic was calculated to check the model fit . R was also calculated to examine if the model is close to the regression line and obtain the percentage from the dependent variable's variation explained by the model . For each predictor in the model the coefficients and their significance level were calculated to identify potential nonsignificant variables and remove them from the model . The test for multicolinearity was performed by looking into the variance inflation factor (vif) for standard error of the regression coefficients . This paper also reports a stepwise regression approach, where the aim was to improve the exploratory stages of model building, but without compromising the physiological understanding of the predictors by using methods such as principal component analysis (pca) for dimensionality reduction . This is essential for medical applications because the clinicians are keen to identify the relevant health parameters along with improved labelling of the data . For this purpose, stepwise regression analysis was performed to select the variables that are significantly important . In this process the most important variables are first selected with a forward searching algorithm followed by a backward elimination process to provide a reduced model with most suitable variables . This method adds and removes the predictors in each step until all the variables used in the model have p value and the ones which are not used in the model have p value> with = 0.05 . For each predictor in the reduced model, vif was calculated to test for the multicolinearity followed by testing the model for fitting performance using anova and r statistics . The demographic information (mean sd) of the patients at the baseline prior to bootstrapping the data has been summarized in table 1 . The bmi was calculated as weight (kg) divided by squared height (m). Data analysis was performed separately on both odc and mc images to identify the relationship between potential predictors and diabetes factor . The result from linear regression analysis and anova test for the first full model has been shown in table 2 . In this model, the r of 96.6% indicates 96.6% from the diagnosis variation is due to the model (or due to change in predictors) and only 3.4% is due to error or some unexplained factors . P values of <0.05 represent the fact that predictors play a significant role in the regression model . The anova test also shows that the multiple linear regression model fits well to the data (f = 3094.49, p <0.001). However, in this model, vif is greater than 5 for almost all the predictors (except diastolic blood pressure) showing that there is problem concerning the predictors' colinearity . A second model was built from the full model using stepwise procedure to reduce the number of parameters and the multicolinearity of the predictors . The result has been provided in table 3 . For the second model, anova test shows that the multiple linear regression model fits significantly with the data (f = 50.11, p <0.001). The vif factor in all cases is smaller than 5 showing negligible colinearity between predictors . In this model the coefficients are weaker than the first model with reduced r. this result is in line with general expectations that there is reduction in model goodness of fit with reduction in number of features; however, this does not represent decline in the explanatory power of the reduced model compared to the full model . Also interpretation of the coefficients in the second model with reduced number of variables and negligible degree of colinearity is more valid and accurate compared to the full model . Comparison between the two models shows that some coefficients have lower magnitude in the second model . Also some predictors (i.e., crae, mean tortuosity, vb, and age) change their sign from the full model to the reduced model, resulting in some degree of uncertainty for the interpretation of the full model with highly colinear predictors . The results also show that the retinal vasculature parameters with level of 0.05 that play a significant role in the reduced explanatory model of diabetes are the crae, mean tortuosity, aba, sd of branch angles, vb, and tba . From the clinical and demographical information, only systolic blood pressure and age were found to be significant predictor . The same analysis as explained above was performed on odc images; however, no model was found to provide adequate fit to the mc data for this database . This research has studied retinal vasculature parameters to detect dm in the indian population with no dr . The analyses were performed on both mc and odc images using a 30 nonmydriatic eye - fundus camera . The significance of this work is that it reports automatic analysis of the eye - fundus images and provides an explanatory model for early changes in some retinal vascular parameters as a result of dm . This study has also introduced a set of new retinal vascular parameters, tba and aba, and employed them together with other features and patients demographic information to create an explanatory model for prediction of diabetes in the absence of evident features of diabetic retinopathy . In this work, linear regression analysis was performed to model association between a large number of explanatory variables as the predictors (i.e., retinal vascular parameters and clinical information) and dm as the response variable . The application of stepwise regression allowed for dimensionality reduction as well as solving the multicolinearity problem making the send model clinically interpretable . It was important that predictors should be clinically relevant and without compromising the physiological understanding of the predictors . The result showed that six retinal vascular parameters of (1) crae, (2) mean tortuosity, (3) aba, (4) sd of angle, (5) vb, and (6) tba are associated with diabetes in indian population when there is no observable dr . The anova test and resulting the significant outcome of this study is that it provides the basis for an alternate technique to detect diabetes among people with minimal or no dr . Another major outcome of this study is that it has introduced two retinal vascular parameters that may find application in a predictive model to predict the risk of developing dr in diabetic patients with no dr . Therefore, conducting longitudinal study is proposed to monitor the progress of the patients and identify changes among those who may later develop dr . The measurements are suitable for automation and for being used with simple eye - fundus imaging, which could be put in form of a modified smart - phone in the future . The limitation of this study is that it was limited in the population type, having been conducted only in delhi in india, and represents narrow demographics with limited number of samples and matching cases . It is essential to include subjects with similar cultural, ethnic, and socioeconomic conditions . There is also the need for conducting similar tests on a larger dataset and for other demographics to observe potential differences and better evaluate the performance of the model.
Nontyphoidal salmonella species are important food borne pathogens and acute gastroenteritis is the most common clinical manifestation accounting for about 70% of cases . Splenic abscess is a very rare complication of nontyphoidal salmonella infections since the presence of antibiotics . Here a 63-year - old woman from eastern part of turkey was admitted with the complaint of back pain, vomiting and nausea since 20 days . Laboratory findings were as follows: white blood cell (wbc) count was 14670/mm (88.9% polymorphonuclear cells), haemoglobin 9.5 g / dl, platelet count 183000/mm, erythrocyte sedimentation rate 76 mm / h, and c - reactive protein (crp) 58 mg / dl (normal value <5 mg / l). An abdominal ultrasound revealed a hypoechogenic cystic structure with a diameter of 6172 mm in the upper part of the spleen with calcifications (figure 1). Salmonella enteritidis was yielded from culture of the samples of the abscess obtained during the operation . It was sensitive to ampicillin, cotrimoxazole, cephalosporins of third generation, and ciprofloxacin . After isolation of the s. enteritidis from abscess, the patient was questioned in detail, it was learned that the patient had diarrhoea and fever existed 2 days and recovered without antibiotic treatment one month ago . The usual clinical presentation of nontyphoidal salmonella infection is self - limited gastroenteritis, however bacteraemia and focal extraintestinal infections may occur . Risk of bacteraemia and focal extraintestinal infections are high in individuals with comorbidities such as malignancy, human immunodeficiency virus (hiv), diabetes mellitus, and patients receiving immunosuppressive therapy . Invasive nontyphoidal salmonellae disease is a major cause of mortality in african children and hiv - infected african adults . Any tissue or organ may be seeded hematogenously by nontyphoidal salmonella and may form a local infection, become obvious months or even years after the initial bacteraemia producing characteristic clinical syndromes . Some serovars of salmonella show a higher tendency for causing bacteraemia and these serovars differ in different countries . Non - typhoidal salmonella serovars s. typhimurium and s. enteritidis are cause of invasive disease in industrialized countries, also they are predominant in african region . S. enteritidis had the highest blood invasiveness among non - typhoidal salmonella species in malaysia . Our patient had diabetes mellitus and probably s. enteritidis bacteraemia occurred during the course of diarrhoea one month ago which lead to bacterial seeding in the spleen . Antibiotic treatment for patients with mild to moderate gastroenteritis due to non - typhoidal salmonella is not indicated in healthy adults . However antimicrobial therapy should be initiated for patients who are severely ill and for patients with risk factors for extraintestinal spread of infection . The symptoms of splenic abscess are usually nonspecific the most frequent symptoms and signs are fever, abdominal pain and tenderness over left upper quadrant, splenomegaly, leucocytosis, and left lower chest abnormalities . Our patient had back pain, vomiting, nausea and leucocytosis which were not specific for splenic abscess . Computed tomography remains the gold standard and the most sensitive tool for the definitive diagnosis of splenic abscess . Ultrasonography has 76%, ct has 96% sensitivity for the detection of abdominal masses . In our patient, diagnosis was confirmed with ct . At present, splenectomy is the gold standard for treatment while ultrasound- or ct - assisted percutaneous drainage and antimicrobial therapy can be considered as therapeutic alternatives especially in the presence of an isolated abscess . We preferred splenectomy for treatment as the causative microorganism was not known initially . In conclusion, although non - typhoidal salmonella gastroenteritis is rarely resulted with splenic abscess, patients with comorbities are at increased risk.
A 60-year - old patient presented in the outpatient department with chief complaints of hematuria with off and on episode of urine retention . There was history of passage of small stones in urine occasionally for last few days . On ultrasonography he was found to have 35 g prostate with slightly thickened bladder wall with a large stone in the urinary bladder . X - ray kub showed a large radio - opaque shadow in the pelvic region [figure 1]. Patient underwent open cystolithotomy and large bladder stone [figure 2] with multiple small stones were retrieved . Patient was started on alpha blockers in the immediate postoperative period and foley's catheter was removed on the eighth postoperative day . At follow - up of three weeks after the surgery the patient was voiding in good stream with minimal post - void residual urine . As the name implies this variety of stone has a characteristic shape resembling a child's toy [figure 3]. These types of stone are commonly described in the veterinary literature with common occurrence in cattle, cats and dogs . Dogs are mostly commonly affected and canine jackstones are usually composed of silica . Depicting child toy called jackstone calcium oxalate is the most common component of urinary calculi . Calcium oxalate monohydrate calculi are usually smooth and black, whereas stones comprising calcium oxalate dihydrate tend to be irregular and yellow . Prostatic diseases, previous lower urinary tract surgery, metabolic abnormalities, upper urinary tract calculi, intravesicular foreign bodies, spinal cord injuries, transplant surgery etc . The presentation of vesical calculi varies from completely asymptomatic to symptoms of suprapubic pain, dysuria, intermittency, frequency, hesitancy, nocturia, and urinary retention . Other common signs include terminal gross hematuria and sudden termination of voiding with some degree of associated pain referred to the tip of the penis, scrotum, perineum, back, or hip . The discomfort may be dull or sharp and is often aggravated by sudden movements and exercise . Assuming a supine, prone, or lateral head - down position may alleviate the pain initiated by the stone impacting the bladder neck by causing it to roll back into the bladder . In our case the prostate is the likely cause of this stone . Enlarged prostate probably restricts the calculus into its eccentric location and contributes to the growth of stone by causing stasis of urine . It is important to recognize the characteristic shape of the jackstones as they are susceptible to lithotripsy . We did not offer lithotripsy as this modality is known to be less efficacious in case of vesical calculus.
This review was based on a systematic comprehensive search of six databases: ovid medline (1950 to october week 4, 2010); pubmed (january 1, 2008, to november 16, 2010); ovid embase (january 1, 1980, to 2010 week 44); ovid psycinfo (january 1, 1987, to november week 1, 2010); cinahl (january 1, 1982, to november 16, 2010); and the cochrane library (searched on november 6, 2011). We considered articles in english, dutch, french, or german that were published or in press between january 1, 1996, and november 16, 2010, for inclusion in this review . A study was eligible for inclusion if it: 1) reported on older patients (mean or median age of study participants 65 years or older) who were diagnosed with cancer (any type of cancer, including hematological malignancies) and being seen in oncology clinics (outpatient oncology or hematology clinics or inpatient oncology or hematology units); 2) reported on cross - sectional, longitudinal, observational, or interventional studies that either assessed the feasibility of the use of tools or instruments or the effectiveness of geriatric assessment tools on any of the aforementioned outcome measures; and 3) was written in english, french, dutch, or german . We excluded editorials, case studies, reviews, and expert opinion papers and studies that were published as abstracts only . The following sets of keywords or free text words were used in combination with subject headings where available: cancer (cancer * or neoplasm * or oncolog ) and geriatric assessment (geriatric or elderly or frailty or aged and assessment or evaluation * or consultation ; or consultation service for senior adults or geriatric oncology module or frailty marker ). In the first step, an initial selection based on titles and abstracts was done independently by two authors (mp and jh) using the inclusion and exclusion criteria . When at least one reviewer was uncertain about whether the article fulfilled the inclusion criteria, the abstract was included for full - text review . In the second step, disagreements between reviewers were resolved by consensus (this process was used for eight studies). If multiple articles reported similar results, only the article with the most complete information was retained . For all studies identified as abstract only (n = 50), we attempted to contact the authors by e - mail to determine whether the full - text study had been published . For eight abstracts, of the 42 authors who were contacted, 19 did not respond, six e - mails were undeliverable, 15 authors responded that the studies and/or manuscripts were still in progress, and two authors informed us that their manuscripts were accepted for publication and were included in this systematic review . We also reviewed the reference lists of all selected articles to identify any additional relevant articles, but no additional studies were identified . When an article referred to additional publications for more details on study methods and design, those publications were also obtained . Data were abstracted by the same reviewers using a data abstraction form that was created with excel software (microsoft corporation, redmond, wa). The abstracted information included the study design, aim of the study, location of the study, sampling method, source of data, recruitment, participant inclusion criteria, the characteristics of included study participants, the name used for the geriatric assessment, the instrument(s) used, instrument feasibility, results of the study, outcomes of the assessment, and details about the statistical analysis . If any aspect of the study design was unclear, we attempted to contact the authors of the study by e - mail . For two of 19 studies, no e - mail address could be found, and for one study, the email was undeliverable, leaving 16 authors that could be contacted . Of the 16 authors contacted, five did not respond whereas 11 responded and provided additional details . The reporting of observational studies in epidemiology (strobe), the meta - analysis of observational studies in epidemiology (moose), and preferred reporting items for systematic reviews and meta - analysis (prisma) guidelines were used by two reviewers (mp and jh) to assess the quality of the included studies (1517). Any disagreement, which involved 421 (18%) of 2324 assessed quality items, was resolved though consensus . However, because this is the first systematic review on the use of geriatric assessment in oncology, no study was excluded based on the quality assessment . This review was based on a systematic comprehensive search of six databases: ovid medline (1950 to october week 4, 2010); pubmed (january 1, 2008, to november 16, 2010); ovid embase (january 1, 1980, to 2010 week 44); ovid psycinfo (january 1, 1987, to november week 1, 2010); cinahl (january 1, 1982, to november 16, 2010); and the cochrane library (searched on november 6, 2011). We considered articles in english, dutch, french, or german that were published or in press between january 1, 1996, and november 16, 2010, for inclusion in this review . A study was eligible for inclusion if it: 1) reported on older patients (mean or median age of study participants 65 years or older) who were diagnosed with cancer (any type of cancer, including hematological malignancies) and being seen in oncology clinics (outpatient oncology or hematology clinics or inpatient oncology or hematology units); 2) reported on cross - sectional, longitudinal, observational, or interventional studies that either assessed the feasibility of the use of tools or instruments or the effectiveness of geriatric assessment tools on any of the aforementioned outcome measures; and 3) was written in english, french, dutch, or german . We excluded editorials, case studies, reviews, and expert opinion papers and studies that were published as abstracts only . The following sets of keywords or free text words were used in combination with subject headings where available: cancer (cancer * or neoplasm * or oncolog ) and geriatric assessment (geriatric or elderly or frailty or aged and assessment or evaluation * or consultation ; or consultation service for senior adults or geriatric oncology module or frailty marker ). The studies were selected in two steps (figure 1). In the first step, an initial selection based on titles and abstracts was done independently by two authors (mp and jh) using the inclusion and exclusion criteria . When at least one reviewer was uncertain about whether the article fulfilled the inclusion criteria, the abstract was included for full - text review . In the second step, disagreements between reviewers were resolved by consensus (this process was used for eight studies). If multiple articles reported similar results, only the article with the most complete information was retained . For all studies identified as abstract only (n = 50), we attempted to contact the authors by e - mail to determine whether the full - text study had been published . For eight abstracts, of the 42 authors who were contacted, 19 did not respond, six e - mails were undeliverable, 15 authors responded that the studies and/or manuscripts were still in progress, and two authors informed us that their manuscripts were accepted for publication and were included in this systematic review . We also reviewed the reference lists of all selected articles to identify any additional relevant articles, but no additional studies were identified . When an article referred to additional publications for more details on study methods and design, those publications were also obtained . Data were abstracted by the same reviewers using a data abstraction form that was created with excel software (microsoft corporation, redmond, wa). The abstracted information included the study design, aim of the study, location of the study, sampling method, source of data, recruitment, participant inclusion criteria, the characteristics of included study participants, the name used for the geriatric assessment, the instrument(s) used, instrument feasibility, results of the study, outcomes of the assessment, and details about the statistical analysis . If any aspect of the study design was unclear, we attempted to contact the authors of the study by e - mail . For two of 19 studies, no e - mail address could be found, and for one study, the email was undeliverable, leaving 16 authors that could be contacted . Of the 16 authors contacted, five did not respond whereas 11 responded and provided additional details . The reporting of observational studies in epidemiology (strobe), the meta - analysis of observational studies in epidemiology (moose), and preferred reporting items for systematic reviews and meta - analysis (prisma) guidelines were used by two reviewers (mp and jh) to assess the quality of the included studies (1517). Any disagreement, which involved 421 (18%) of 2324 assessed quality items, was resolved though consensus . However, because this is the first systematic review on the use of geriatric assessment in oncology, no study was excluded based on the quality assessment . A total of 1308 abstracts were initially identified for possible inclusion (figure 1). Based on the review of the abstracts, of the 83 articles included, three were written in french, and 80 were written in english . The quality of most studies was poor to moderate based on moose and prisma guidelines for reporting (supplementary table 1, available online). Of the 59 studies that were not chart reviews, 51 (86%) did not report a response rate (1870), and all but one (38) also did not report the reasons for refusal to participate in study . Furthermore, of the 73 studies, 13 (18%) did not describe the study design (18,20,27,37,42,53,57,64,65,67,69,71,72), and 12 (16%) did not describe the setting in which the study was conducted (20,2224,33,42,44,49,51,53,61,70). Among the 28 prospective studies, nine (32%) did not describe the method of follow - up (20,22,23,27,33,35,37,3941). The amount of missing data was not described in 41 (67%) of 61 studies (excluding studies that reported having complete data) (2023,25,27,3335,3945,47,51,53,54,6063,66,6871,7384), and 41 of 58 studies (excluding studies that reported no missing data or how missing data were handled) did not describe how the study authors dealt with missing data (2023,25,27,33,3739,4147,49,51,53,54,57,58,6064,6872,7485). For 12 (16%) of the 73 studies, three (4%) of the 73 studies did not describe all of the measurement instruments used in the study (ie, geriatric assessment instruments, outcomes, predictors) (20,42,52). Domains of geriatric assessment included in the 73 studies that examined geriatric assessment in the oncology setting both instruments were used in more than 20% of the studies . Among studies that included the domain . The characteristics of the 73 studies reported by the included articles are presented in supplementary table 2 (available online). Twenty - five studies were conducted in north america: 23 in the united states (25,3033,37,38,43,46,52,54,58,62,64,73,74,77,78,80,81,83,85,86, 8892) and two in canada (9395). Forty - three studies were conducted in europe: 19 in italy (20,26,3436,40,42, 44,51,53,57,59,61,63,67,69,70,76,79), 14 in france (19,2729,39,45,48,72,82,84,87, 9698), three in spain (47,55,56), two in germany (41,68,99), one in the united kingdom (22,23), one in norway (71,75), one in greece (24), one in the netherlands (18), and one in austria (65). Two studies were conducted in asia: one in japan (100) and one in korea (50). One study was conducted in australia (66), and two studies were conducted in multiple countries (21,49,60). Overview of the results of the feasibility of the assessments as reported in the article * na = not applicable; nr = not reported; acga = abbreviated geriatric assessment; adl = activities of daily living; adt = androgen deprivation therapy; ags = american geriatric society; bdi = beck depression inventory; bfi = brief fatigue inventory; bmi = body mass index; bun = blood urea nitrogen; cc = carboplatin and cyclophosphamide; cp = carboplatin and paclitaxel; cga = comprehensive geriatric assessment; cci = charlson comorbidity index; cirs - g = cumulative illness rating scale geriatric; dlcl = diffuse large cell lymphoma; ecog = eastern collaborative group oncology; ps = performance status; esl = english as a second language; fact - b = functional assessment cancer treatment breast; gds = geriatric depression scale; gfi = groningen frailty indicator; hads = hospital anxiety and depression scale; iadl = instrumental activities of daily living; iqcode = informant questionnaire on cognitive decline in the elderly; iqr = interquartile range; kps= karnofsky performance status; mga = multidimensional geriatric assessment; mmse = mini mental state examination; mna = mini nutritional assessment; nsi = nutritional risk screening; oars = older americans resources and services; pace = preoperative assessment of cancer in the elderly; pini = prognostic inflammatory and nutrition index; ps = performance status; ppt = physical performance test; qol = quality of life; spmsq = short portable mental screening questionnaire; sppb = short physical performance battery; tug = timed up and go test; ves-13 = vulnerable elder survey-13 items; sic = satariano comorbidity index . Location = inpatient or outpatient setting; timing of geriatric assessment = before, during, or after treatment . Articles reporting on the same study . Articles reporting on the same study . Articles reporting on the same study . Of the 73 studies that included geriatric assessment, 28 (38%) were prospective observational studies (1841,71,7376,94,95,100), 31 (42%) were cross - sectional observational studies (4268,77,78,85,8890,93,99), and 14 (19%) were retrospective studies or chart reviews (69,70,72,7984,86,87,91,92,9698). None of the reviewed studies was a randomized controlled trial specifically designed to examine the effectiveness of geriatric assessment . In studies that investigated a new drug or treatment regimen (26,28,30,39,59,67,70), geriatric assessment was included in seven nonrandomized clinical drug trials (24,26,28,30,59,67,70) and no randomized controlled drug trial . Most of the studies recruited participants either through convenience sampling (25 studies) (2426,3032,36,37,39,51,52,59,65, 67,69,73,74,7678,84,8890,9395,97,99) or by consecutive sampling (32 studies) (18,19,21,27,3335,40,41,4548,55,57,58,6064,66,68,71,75,79,80,82,85,86,91,92,96,98,100) techniques . Three studies used other sampling methods (29,38,54), and in 13 studies the method used for recruitment was not clear or not reported (20,22,23,4244,49,50,53,56,70,72,81,83,87). However, 11 (15%) of 73 studies failed to report clear and explicit inclusion and recruitment procedures criteria (20,2224,42,44,53,57,65,67,77,89,90). Sample sizes ranged from 10 (36) to 12 480 (54) participants ., patients underwent geriatric assessment during admission or stay at inpatient ward (21,38,41,60,61,63,65,68,69,79,82,86,92), and participants in 11 studies were evaluated during initial or routine clinic visits (33,34,4648,56,62,64,74,77,88). In four studies, the geriatric assessment took place either at inpatient admission or in the outpatient clinic (57,72,9395). Table 1 presents an overview of the domains included in geriatric assessments, and supplementary table 3 (available online) presents the detailed content and domains of the geriatric assessment used in each study . Of the 73 studies, 68 included measures of basic activities of daily living (1841,41,42,4451,5363,6567,6972,7483,85100), and 65 included instrumental activities of daily living (1828,3039,41,4463,6572,7495,9799). Psychometric properties and/or diagnostic accuracy reported acga = abbreviated comprehensive geriatric assessment; auc = area under the curve; bq = barber questionnaire; adl= activities of daily living; bmi = body mass index; cga = comprehensive geriatric assessment; cci = charlson comorbidity index; ci = confidence interval; cirs - g = cumulative illness rating scale geriatric; ecog = eastern collaborative group oncology; fact - g = functional assessment of cancer general; ficsit = frailty and injuries: cooperative studies of intervention techniques; f - sozu = questionnaire for the assessment of social support; ps = performance status; gds = geriatric depression scale; gfi = groningen frailty indicator; gom = geriatric oncology module; hads = hospital anxiety and depression scale; iadl = instrumental activities of daily living; icc = intraclass correlation coefficient; iqcode = informant questionnaire on cognitive decline in the elderly; kps = karnofsky performance status; mace = multidimensional assessment protocol for cancer in the elderly; mga = mini geriatric assessment; mmse = mini mental state examination; mna = mini nutritional assessment; na = not applicable; npv = negative predictive value; nsi = nutritional risk screening; ppt = physical performance test; ppv = positive predictive value; qol= quality of life; rand mos = rand corporation medical outcomes survey; sip = sickness impact profile; spmsq = short portable mental screening questionnaire; sppb = short physical performance battery; tug = timed up and go test; ves-13 = vulnerable elder survey-13 items; sic = satariano comorbidity index; who ps= world health organization performance status . Articles reporting on the same study . A total of 58 studies included a comorbidity domain (1825,2732,34,35,3848,50,51,54,55,57,5961,63,6568, 7177,79,80,82,8487,8991,93100). Assessment of depression, anxiety, or general mood was a component of geriatric assessment in 52 studies (1923, 2633,36,39,42,44,4750,52,53,57,58,6067,69,7177,7985, 8795,97,98). A nutritional assessment was conducted in 40 studies (1820, 25,2729,3236,38,39,43,45,47,48,50,51,53,5557,63,65,66,71, 7476,79,82,84,8790,9297,99,100), and 27 studies assessed the risk of falls (19,25,27,38,42,43,45,47,48,50,52,54,58,61,63,66, 72,7678,80,82,84,8896,98,99). Performance status was assessed in 37 studies (20,21,24,3032,34,35,39,41,44, 4648, 50,51,53,55,56,6071,74,75,8690,9398,100). Information about the use of prescription medications was collected from patients in 22 studies (19,25,28,29,39,47,48,5052,55,56,63,66,71,72,75,78,82,84,85,92,98,99), and in 14 of these 22 studies, the information obtained included the total number of prescriptions (25,29,39,47,48,51,52,63,66, 71,75,78,82,84,85,92). In 24 studies (19,22,23,25,28,38,48,5052,55,56,63,65,66,72,73,78,8486,8890,93,97,98), geriatric assessment included the availability of social support and living arrangements, such as the availability of a caregiver . The most commonly used objective measure of physical function was gait speed, which was included in 15 studies (25,29,43,45,50,52,61,64,65,72,78,84,87,88,9395). Patient characteristics that were less often incorporated into geriatric assessments included symptoms [assessed using a symptom inventory, two studies (22,23,85); fatigue or energy levels, seven studies (21,25,37,43,60,85,9395); pain, three studies (37,66,85); quality of life, seven studies (22,23,31,32,37,50,65,68,74); grip strength, five studies (43,64,87,9395); distress, three studies (66,85,89,90); and self - rated health, two studies (54,73)]. In 30 of 73 studies, the results of geriatric assessment were summarized in a summary score (18,20,24,26,34,40,42,43,47,4951,54,55,66,70,7276,78,79,84,9396,98,99). In 12 of those studies (20,24,40,47,50,54,75,79,84,96,98,99), the summary score used was the classification of fit, vulnerable, and frail developed by balducci and stanta (101). In this classification, frail refers to patients who are generally unfit for cancer treatment (defined as those with any of the following characteristics: older than 85 years, more than two disabilities, multiple comorbidities, or the presence of geriatric syndromes) and should receive best supportive care or palliative treatment; fit (defined as patients who are independent and have no clinically significant comorbid conditions) indicates patients who should receive standard therapy; and vulnerable (defined as patients with one or two clinically significant comorbid conditions and/or instrumental activities of daily living disability but no activities of daily living disability) refers to patients for which the standard treatment should be adjusted . Thirty studies reported some aspect of the feasibility of the geriatric assessment, such as time needed to complete the assessment and/or who (study author, patient themselves, or others) conducted the assessment (21,25,26,32,36,37,40,45,46,50,52,5558,60,66,69,7375,77,78,84,85,8789,93,94,99). In most of these studies, the assessment was done through a face - to - face interview and generally took 1045 minutes . Among studies that reported how many participants refused the assessment (26,74,78,94,95), only a small number of participants refused the assessments (table 2). In six studies (33,66,69,85,88,89), however, only four of those studies (66,85,88,89) reported on feasibility, and each showed that it was acceptable (more than 75% of participants could complete the survey without assistance, and participants were satisfied with length of questionnaires and content). Eleven studies (19,37,46,49,51,55,57,65,78,81,83,99) reported psychometric properties or diagnostic accuracy of the geriatric assessment (ie, validity, reliability, and/or sensitivity and specificity) (table 3). Most of these studies examined diagnostic accuracy of one or more short geriatric assessment tools with those of a full geriatric assessment . However, because these studies compared different screening instruments with different forms of full geriatric assessment or used the same instruments but with different cutoffs, it was not possible to summarize the results in a quantitative manner first, shorter forms of geriatric assessment generally had good diagnostic accuracy compared with a full geriatric assessment . For example, aparicio et al . (19) found that concordance between individual domain scores from mini - geriatric assessment and from comprehensive geriatric assessment ranged from 66% to 83% . Second, four studies that compared the vulnerable elder survey-13 items (ves-13) with a full geriatric assessment found that the former had excellent diagnostic accuracy, with an area under the curve that ranged from 0.83 to 0.90, sensitivity that ranged from 54% to 87%, and specificity that ranged from 70% to 89% (49,51,55,78). In addition, one study (49) compared the groningen frailty indicator to a full geriatric assessment; one study (55) compared the barber questionnaire to a full geriatric assessment; and one study (99) compared expert physician judgment to the balducci classification . Thirty - seven studies (51%) examined at least one of the four a priori specified outcomes presented below . The outcomes use of geriatric assessment (followed by interventions) to avoid complications of treatment and health and functional status were not studied in the included studies . Below, the results for each of the studied outcomes are described . Geriatric assessment and treatment decision . An important goal of geriatric assessment is to distinguish between older patients who are fit to undergo standard cancer treatments and frail older patients who would benefit from modified treatment or best supportive care . Only four studies (19,27,48,98), all conducted in france, examined the impact of geriatric assessment before the start of treatment on the cancer treatment plan (table 4). In two studies (19,98), geriatric assessment did not influence the treatment decision, whereas in the other two studies (27,48), geriatric assessment led to changes in the treatment plan for 40%50% of patients, mostly consisting of changes in the chemotherapy regimen . Of note (48), the final treatment decision (which took into account the results of the geriatric assessment) was made by a doctor or team that was not the original doctor or team that conducted the geriatric assessment . In the study of chaibi et al . (27), patients were rediscussed at tumor board, where the multidisciplinary team decided to change their treatment recommendation based on the results of the geriatric assessment . Impact of geriatric assessment on cancer treatment decision - making process or treatment delivery adl = activities of daily living; bmi = body mass index; cga = comprehensive geriatric assessment; ecog ps = eastern collaborative oncology group performance status; na = not applicable; mga = mini geriatric assessment; mna = mini nutritional assessment; mmse = mini mental state examination; sge = simplified geriatric evaluation . In a small pilot study of 15 breast cancer patients, extermann et al . (74) reported that assessment and interventions influenced the oncological treatment, but it was not clear how or how often they influenced the outcome . The impact of geriatric assessment on the treatment decision was examined by marenco et al . (34) in a prospective study with a variety of cancers and stages (n = 571), and by to et al . (66) in a cross - sectional study with diverse cancers and stages (n = 200). However, it is not clear how treatment decisions were specifically impacted (eg, increase in treatment dose or dose reduction was not reported) in these two studies . Three studies (27,74,84) have shown that geriatric assessment led to geriatric interventions, such as nutritional interventions and treatment of depression before the start of treatment . Geriatric assessment and complications or toxicity of treatment . Table 5 lists all studies that examined complications or toxicity of treatment as an outcome of geriatric assessment . Nine studies (21,3033,35,71,73,75,95,100) that examined the impact of geriatric assessment on complications of any type of cancer treatment did not use multivariable analysis techniques . Complications were generally defined as grade 3 or 4 treatment - related toxicity, treatment interruptions, and postoperative complications, such as wound infections . In five studies with mixed cancer diagnoses and stages and sample sizes that ranged from 60 to 660 participants (21,33,35,71,75,100), impairments in basic and instrument activities of daily living, comorbidity, poor mental health, poor social support, and cognitive functioning were associated with treatment complications . In a prospective observational study that included mixed cancer diagnoses and stages (n = 112), (95) reported that low grip strength was the only frailty marker (of seven measured) to predict treatment toxicity . Two other studies (3032) with sample sizes of 20, 28, and 49 participants (most with breast cancer) showed no difference in treatment toxicities with regard to geriatric assessment variables . These studies may have lacked statistical power to detect statistically significant associations . Predictive validity of geriatric assessment for treatment complications na = not applicable; nr = not reported; hr = hazard ratio; rr = relative risk; or = odds ratio; ci = confidence interval; anova= analysis of variance; asa = american society of anesthesiologists; bfi = brief fatigue inventory; cci = charlson comorbidity index; cirs = cumulative illness rating scale; cmf= cyclophosphamide, methotrexate, and fluorouracil; gecog ps = eastern collaborative group oncology performance status; gds = geriatric depression scale; iadl = instrumental activities of daily living; mhi 5 = mental health index 5 items; mmse = mini mental state examination; npv= negative predictive value; ppv= positive predictive value; ps = performance status . No cross - sectional or retrospective studies examined the predictive validity of geriatric assessment for treatment complications . Articles reporting on the same study . Geriatric assessment and prediction of mortality . Table 6 lists all studies that examined mortality as an outcome of domains of geriatric assessment . Sixteen studies examined the ability of geriatric assessment domains to predict mortality: 13 studies were prospective (18,20,23,24,29,34,35,3941,71,73,95), two were cross - sectional (79,80), one was retrospective (97), and all studies included a variety of cancer diagnoses and stages . The following geriatric assessment variables were associated with increased mortality across multiple studies (18,23,29,34,35,41,71,80): older age, inadequate finances, mental health, comorbidity, high medication use, high groningen frailty indictor score, low mini nutritional assessment score, and impairments in activities of daily living . The majority of these studies adjusted for important confounders, such as sex, age, type of malignancy, stage of cancer, and comorbidity . However, three studies with sample sizes of 54 to 182 reported that none of the geriatric assessment variables were independent predictors of mortality (71,95,97). Predictive validity of geriatric assessment for mortality na = not applicable; nr = not reported; hr = hazard ratio; rr = relative risk; or = odds ratio; ci = confidence interval; cci = charlson comorbidity index; cirs = cumulative illness rating scale; ecog ps = eastern collaborative group oncology performance status; figo = international federation of gynecology and obstetrics; gfi = groningen frailty indicator; iadl = instrumental activities of daily living; mhi 5 = mental health index 5 items; kps = karnofsky performance status; mna = mini nutritional assessment; os = overall survival; pfs = progression - free survival; ves-13 = vulnerable elder survey-13 items; who ps = world health organization performance status . Six studies (20,24,40,42,76,79) examined the survival of patients categorized as frail, vulnerable, or fit rather than according to individual components of the geriatric assessment . These studies used tests such as or log rank tests but did not examine predictive validity using multivariable analytic techniques . Three studies examined overall survival, progression - free survival, and/or response to treatment in relation to geriatric assessment in univariate analyses only (24) found no associations between the ves-13 score and overall survival, progression - free survival, or response to treatment . (79) found that the incidence of treatment interruption was higher and had less benefit in terms of response in patients classified as frail according to the balducci classification . Massa et al . (76) reported better response in fit patients compared with frail patients (how the patients were classified as fit, intermediate, and frail was not described), but it is not clear what analysis was conducted two studies (23,94) examined the association between domains of geriatric assessment and the use of care (supplementary table 4, available online). In a prospective study with 337 colorectal cancer patients that adjusted for age and sex but not for illness severity and comorbidity, bailey et al . (23) found that patients who were older and had poorer mental health had greater use of social resources . In a prospective study that used seven markers of frailty markers and (94) reported that only one frailty marker, cognitive impairment, predicted visits to the emergency department after adjustment for confounders such as cancer type, cancer stage comorbidity, age, and sex . Five studies (22,23,34,66,91,98) reported that components of the geriatric assessment, such as age and functional status, were associated with the receipt of certain treatment modalities or regimens, such as surgery only . Other outcomes studied included changes in functional status, distress, clinical response, and discharge to usual place of residence after hospital admission (supplementary table 4, available online). The quality of most studies was poor to moderate based on moose and prisma guidelines for reporting (supplementary table 1, available online). Of the 59 studies that were not chart reviews, 51 (86%) did not report a response rate (1870), and all but one (38) also did not report the reasons for refusal to participate in study . Furthermore, of the 73 studies, 13 (18%) did not describe the study design (18,20,27,37,42,53,57,64,65,67,69,71,72), and 12 (16%) did not describe the setting in which the study was conducted (20,2224,33,42,44,49,51,53,61,70). Among the 28 prospective studies, nine (32%) the amount of missing data was not described in 41 (67%) of 61 studies (excluding studies that reported having complete data) (2023,25,27,3335,3945,47,51,53,54,6063,66,6871,7384), and 41 of 58 studies (excluding studies that reported no missing data or how missing data were handled) did not describe how the study authors dealt with missing data (2023,25,27,33,3739,4147,49,51,53,54,57,58,6064,6872,7485). For 12 (16%) of the 73 studies, three (4%) of the 73 studies did not describe all of the measurement instruments used in the study (ie, geriatric assessment instruments, outcomes, predictors) (20,42,52). Domains of geriatric assessment included in the 73 studies that examined geriatric assessment in the oncology setting both instruments were used in more than 20% of the studies . Among studies that included the domain . The characteristics of the 73 studies reported by the included articles are presented in supplementary table 2 (available online). Twenty - five studies were conducted in north america: 23 in the united states (25,3033,37,38,43,46,52,54,58,62,64,73,74,77,78,80,81,83,85,86, 8892) and two in canada (9395). Forty - three studies were conducted in europe: 19 in italy (20,26,3436,40,42, 44,51,53,57,59,61,63,67,69,70,76,79), 14 in france (19,2729,39,45,48,72,82,84,87, 9698), three in spain (47,55,56), two in germany (41,68,99), one in the united kingdom (22,23), one in norway (71,75), one in greece (24), one in the netherlands (18), and one in austria (65). Two studies were conducted in asia: one in japan (100) and one in korea (50). One study was conducted in australia (66), and two studies were conducted in multiple countries (21,49,60). Overview of the results of the feasibility of the assessments as reported in the article * na = not applicable; nr = not reported; acga = abbreviated geriatric assessment; adl = activities of daily living; adt = androgen deprivation therapy; ags = american geriatric society; bdi = beck depression inventory; bfi = brief fatigue inventory; bmi = body mass index; bun = blood urea nitrogen; cc = carboplatin and cyclophosphamide; cp = carboplatin and paclitaxel; cga = comprehensive geriatric assessment; cci = charlson comorbidity index; cirs - g = cumulative illness rating scale geriatric; dlcl = diffuse large cell lymphoma; ecog = eastern collaborative group oncology; ps = performance status; esl = english as a second language; fact - b = functional assessment cancer treatment breast; gds = geriatric depression scale; gfi = groningen frailty indicator; hads = hospital anxiety and depression scale; iadl = instrumental activities of daily living; iqcode = informant questionnaire on cognitive decline in the elderly; iqr = interquartile range; kps= karnofsky performance status; mga = multidimensional geriatric assessment; mmse = mini mental state examination; mna = mini nutritional assessment; nsi = nutritional risk screening; oars = older americans resources and services; pace = preoperative assessment of cancer in the elderly; pini = prognostic inflammatory and nutrition index; ps = performance status; ppt = physical performance test; qol = quality of life; spmsq = short portable mental screening questionnaire; sppb = short physical performance battery; tug = timed up and go test; ves-13 = vulnerable elder survey-13 items; sic = satariano comorbidity index . Location = inpatient or outpatient setting; timing of geriatric assessment = before, during, or after treatment . Articles reporting on the same study . Of the 73 studies that included geriatric assessment, 28 (38%) were prospective observational studies (1841,71,7376,94,95,100), 31 (42%) were cross - sectional observational studies (4268,77,78,85,8890,93,99), and 14 (19%) were retrospective studies or chart reviews (69,70,72,7984,86,87,91,92,9698). None of the reviewed studies was a randomized controlled trial specifically designed to examine the effectiveness of geriatric assessment . In studies that investigated a new drug or treatment regimen (26,28,30,39,59,67,70), geriatric assessment was included in seven nonrandomized clinical drug trials (24,26,28,30,59,67,70) and no randomized controlled drug trial . Most of the studies recruited participants either through convenience sampling (25 studies) (2426,3032,36,37,39,51,52,59,65, 67,69,73,74,7678,84,8890,9395,97,99) or by consecutive sampling (32 studies) (18,19,21,27,3335,40,41,4548,55,57,58,6064,66,68,71,75,79,80,82,85,86,91,92,96,98,100) techniques . Three studies used other sampling methods (29,38,54), and in 13 studies the method used for recruitment was not clear or not reported (20,22,23,4244,49,50,53,56,70,72,81,83,87). However, 11 (15%) of 73 studies failed to report clear and explicit inclusion and recruitment procedures criteria (20,2224,42,44,53,57,65,67,77,89,90). Sample sizes ranged from 10 (36) to 12 480 (54) participants ., patients underwent geriatric assessment during admission or stay at inpatient ward (21,38,41,60,61,63,65,68,69,79,82,86,92), and participants in 11 studies were evaluated during initial or routine clinic visits (33,34,4648,56,62,64,74,77,88). In four studies, the geriatric assessment took place either at inpatient admission or in the outpatient clinic (57,72,9395). Table 1 presents an overview of the domains included in geriatric assessments, and supplementary table 3 (available online) presents the detailed content and domains of the geriatric assessment used in each study . Of the 73 studies, 68 included measures of basic activities of daily living (1841,41,42,4451,5363,6567,6972,7483,85100), and 65 included instrumental activities of daily living (1828,3039,41,4463,6572,7495,9799). Psychometric properties and/or diagnostic accuracy reported acga = abbreviated comprehensive geriatric assessment; auc = area under the curve; bq = barber questionnaire; adl= activities of daily living; bmi = body mass index; cga = comprehensive geriatric assessment; cci = charlson comorbidity index; ci = confidence interval; cirs - g = cumulative illness rating scale geriatric; ecog = eastern collaborative group oncology; fact - g = functional assessment of cancer general; ficsit = frailty and injuries: cooperative studies of intervention techniques; f - sozu = questionnaire for the assessment of social support; ps = performance status; gds = geriatric depression scale; gfi = groningen frailty indicator; gom = geriatric oncology module; hads = hospital anxiety and depression scale; iadl = instrumental activities of daily living; icc = intraclass correlation coefficient; iqcode = informant questionnaire on cognitive decline in the elderly; kps = karnofsky performance status; mace = multidimensional assessment protocol for cancer in the elderly; mga = mini geriatric assessment; mmse = mini mental state examination; mna = mini nutritional assessment; na = not applicable; npv = negative predictive value; nsi = nutritional risk screening; ppt = physical performance test; ppv = positive predictive value; qol= quality of life; rand mos = rand corporation medical outcomes survey; sip = sickness impact profile; spmsq = short portable mental screening questionnaire; sppb = short physical performance battery; tug = timed up and go test; ves-13 = vulnerable elder survey-13 items; sic = satariano comorbidity index; who ps= world health organization performance status . Articles reporting on the same study . A total of 58 studies included a comorbidity domain (1825,2732,34,35,3848,50,51,54,55,57,5961,63,6568, 7177,79,80,82,8487,8991,93100). Assessment of depression, anxiety, or general mood was a component of geriatric assessment in 52 studies (1923, 2633,36,39,42,44,4750,52,53,57,58,6067,69,7177,7985, 8795,97,98). A nutritional assessment was conducted in 40 studies (1820, 25,2729,3236,38,39,43,45,47,48,50,51,53,5557,63,65,66,71, 7476,79,82,84,8790,9297,99,100), and 27 studies assessed the risk of falls (19,25,27,38,42,43,45,47,48,50,52,54,58,61,63,66, 72,7678,80,82,84,8896,98,99). Performance status was assessed in 37 studies (20,21,24,3032,34,35,39,41,44, 4648, 50,51,53,55,56,6071,74,75,8690,9398,100). Information about the use of prescription medications was collected from patients in 22 studies (19,25,28,29,39,47,48,5052,55,56,63,66,71,72,75,78,82,84,85,92,98,99), and in 14 of these 22 studies, the information obtained included the total number of prescriptions (25,29,39,47,48,51,52,63,66, 71,75,78,82,84,85,92). In 24 studies (19,22,23,25,28,38,48,5052,55,56,63,65,66,72,73,78,8486,8890,93,97,98), geriatric assessment included the availability of social support and living arrangements, such as the availability of a caregiver . The most commonly used objective measure of physical function was gait speed, which was included in 15 studies (25,29,43,45,50,52,61,64,65,72,78,84,87,88,9395). Patient characteristics that were less often incorporated into geriatric assessments included symptoms [assessed using a symptom inventory, two studies (22,23,85); fatigue or energy levels, seven studies (21,25,37,43,60,85,9395); pain, three studies (37,66,85); quality of life, seven studies (22,23,31,32,37,50,65,68,74); grip strength, five studies (43,64,87,9395); distress, three studies (66,85,89,90); and self - rated health, two studies (54,73)]. In 30 of 73 studies, the results of geriatric assessment were summarized in a summary score (18,20,24,26,34,40,42,43,47,4951,54,55,66,70,7276,78,79,84,9396,98,99). In 12 of those studies (20,24,40,47,50,54,75,79,84,96,98,99), the summary score used was the classification of fit, vulnerable, and frail developed by balducci and stanta (101). In this classification, frail refers to patients who are generally unfit for cancer treatment (defined as those with any of the following characteristics: older than 85 years, more than two disabilities, multiple comorbidities, or the presence of geriatric syndromes) and should receive best supportive care or palliative treatment; fit (defined as patients who are independent and have no clinically significant comorbid conditions) indicates patients who should receive standard therapy; and vulnerable (defined as patients with one or two clinically significant comorbid conditions and/or instrumental activities of daily living disability but no activities of daily living disability) refers to patients for which the standard treatment should be adjusted . Thirty studies reported some aspect of the feasibility of the geriatric assessment, such as time needed to complete the assessment and/or who (study author, patient themselves, or others) conducted the assessment (21,25,26,32,36,37,40,45,46,50,52,5558,60,66,69,7375,77,78,84,85,8789,93,94,99). In most of these studies, the assessment was done through a face - to - face interview and generally took 1045 minutes . Among studies that reported how many participants refused the assessment (26,74,78,94,95), only a small number of participants refused the assessments (table 2). In six studies (33,66,69,85,88,89), however, only four of those studies (66,85,88,89) reported on feasibility, and each showed that it was acceptable (more than 75% of participants could complete the survey without assistance, and participants were satisfied with length of questionnaires and content). Eleven studies (19,37,46,49,51,55,57,65,78,81,83,99) reported psychometric properties or diagnostic accuracy of the geriatric assessment (ie, validity, reliability, and/or sensitivity and specificity) (table 3). Most of these studies examined diagnostic accuracy of one or more short geriatric assessment tools with those of a full geriatric assessment . However, because these studies compared different screening instruments with different forms of full geriatric assessment or used the same instruments but with different cutoffs, it was not possible to summarize the results in a quantitative manner . First, shorter forms of geriatric assessment generally had good diagnostic accuracy compared with a full geriatric assessment . (19) found that concordance between individual domain scores from mini - geriatric assessment and from comprehensive geriatric assessment ranged from 66% to 83% . Second, four studies that compared the vulnerable elder survey-13 items (ves-13) with a full geriatric assessment found that the former had excellent diagnostic accuracy, with an area under the curve that ranged from 0.83 to 0.90, sensitivity that ranged from 54% to 87%, and specificity that ranged from 70% to 89% (49,51,55,78). In addition, one study (49) compared the groningen frailty indicator to a full geriatric assessment; one study (55) compared the barber questionnaire to a full geriatric assessment; and one study (99) compared expert physician judgment to the balducci classification . Thirty - seven studies (51%) examined at least one of the four a priori specified outcomes presented below . The outcomes use of geriatric assessment (followed by interventions) to avoid complications of treatment and health and functional status were not studied in the included studies . Below, the results for each of the studied outcomes are described . Geriatric assessment and treatment decision . An important goal of geriatric assessment is to distinguish between older patients who are fit to undergo standard cancer treatments and frail older patients who would benefit from modified treatment or best supportive care . Only four studies (19,27,48,98), all conducted in france, examined the impact of geriatric assessment before the start of treatment on the cancer treatment plan (table 4). In two studies (19,98), geriatric assessment did not influence the treatment decision, whereas in the other two studies (27,48), geriatric assessment led to changes in the treatment plan for 40%50% of patients, mostly consisting of changes in the chemotherapy regimen . Of note (48), the final treatment decision (which took into account the results of the geriatric assessment) was made by a doctor or team that was not the original doctor or team that conducted the geriatric assessment . In the study of chaibi et al . (27), patients were rediscussed at tumor board, where the multidisciplinary team decided to change their treatment recommendation based on the results of the geriatric assessment . Impact of geriatric assessment on cancer treatment decision - making process or treatment delivery adl = activities of daily living; bmi = body mass index; cga = comprehensive geriatric assessment; ecog ps = eastern collaborative oncology group performance status; na = not applicable; mga = mini geriatric assessment; mna = mini nutritional assessment; mmse = mini mental state examination; sge = simplified geriatric evaluation . In a small pilot study of 15 breast cancer patients, extermann et al . (74) reported that assessment and interventions influenced the oncological treatment, but it was not clear how or how often they influenced the outcome . The impact of geriatric assessment on the treatment decision was examined by marenco et al . (34) in a prospective study with a variety of cancers and stages (n = 571), and by to et al . (66) in a cross - sectional study with diverse cancers and stages (n = 200). However, it is not clear how treatment decisions were specifically impacted (eg, increase in treatment dose or dose reduction was not reported) in these two studies . Three studies (27,74,84) have shown that geriatric assessment led to geriatric interventions, such as nutritional interventions and treatment of depression before the start of treatment . Geriatric assessment and complications or toxicity of treatment . Table 5 lists all studies that examined complications or toxicity of treatment as an outcome of geriatric assessment . Nine studies (21,3033,35,71,73,75,95,100) that examined the impact of geriatric assessment on complications of any type of cancer treatment did not use multivariable analysis techniques . Complications were generally defined as grade 3 or 4 treatment - related toxicity, treatment interruptions, and postoperative complications, such as wound infections . In five studies with mixed cancer diagnoses and stages and sample sizes that ranged from 60 to 660 participants (21,33,35,71,75,100), impairments in basic and instrument activities of daily living, comorbidity, poor mental health, poor social support, and cognitive functioning were associated with treatment complications . In a prospective observational study that included mixed cancer diagnoses and stages (n = 112), (95) reported that low grip strength was the only frailty marker (of seven measured) to predict treatment toxicity . Two other studies (3032) with sample sizes of 20, 28, and 49 participants (most with breast cancer) showed no difference in treatment toxicities with regard to geriatric assessment variables . These studies may have lacked statistical power to detect statistically significant associations . Predictive validity of geriatric assessment for treatment complications na = not applicable; nr = not reported; hr = hazard ratio; rr = relative risk; or = odds ratio; ci = confidence interval; anova= analysis of variance; asa = american society of anesthesiologists; bfi = brief fatigue inventory; cci = charlson comorbidity index; cirs = cumulative illness rating scale; cmf= cyclophosphamide, methotrexate, and fluorouracil; gecog ps = eastern collaborative group oncology performance status; gds = geriatric depression scale; iadl = instrumental activities of daily living; mhi 5 = mental health index 5 items; mmse = mini mental state examination; npv= negative predictive value; ppv= positive predictive value; ps = performance status . No cross - sectional or retrospective studies examined the predictive validity of geriatric assessment for treatment complications . Articles reporting on the same study . Geriatric assessment and prediction of mortality . Table 6 lists all studies that examined mortality as an outcome of domains of geriatric assessment . Sixteen studies examined the ability of geriatric assessment domains to predict mortality: 13 studies were prospective (18,20,23,24,29,34,35,3941,71,73,95), two were cross - sectional (79,80), one was retrospective (97), and all studies included a variety of cancer diagnoses and stages . The following geriatric assessment variables were associated with increased mortality across multiple studies (18,23,29,34,35,41,71,80): older age, inadequate finances, mental health, comorbidity, high medication use, high groningen frailty indictor score, low mini nutritional assessment score, and impairments in activities of daily living . The majority of these studies adjusted for important confounders, such as sex, age, type of malignancy, stage of cancer, and comorbidity . However, three studies with sample sizes of 54 to 182 reported that none of the geriatric assessment variables were independent predictors of mortality (71,95,97). Predictive validity of geriatric assessment for mortality na = not applicable; nr = not reported; hr = hazard ratio; rr = relative risk; or = odds ratio; ci = confidence interval; cci = charlson comorbidity index; cirs = cumulative illness rating scale; ecog ps = eastern collaborative group oncology performance status; figo = international federation of gynecology and obstetrics; gfi = groningen frailty indicator; iadl = instrumental activities of daily living; mhi 5 = mental health index 5 items; kps = karnofsky performance status; mna = mini nutritional assessment; os = overall survival; pfs = progression - free survival; ves-13 = vulnerable elder survey-13 items; who ps = world health organization performance status . Six studies (20,24,40,42,76,79) examined the survival of patients categorized as frail, vulnerable, or fit rather than according to individual components of the geriatric assessment . These studies used tests such as or log rank tests but did not examine predictive validity using multivariable analytic techniques . Three studies examined overall survival, progression - free survival, and/or response to treatment in relation to geriatric assessment in univariate analyses only (24) found no associations between the ves-13 score and overall survival, progression - free survival, or response to treatment . (79) found that the incidence of treatment interruption was higher and had less benefit in terms of response in patients classified as frail according to the balducci classification . (76) reported better response in fit patients compared with frail patients (how the patients were classified as fit, intermediate, and frail was not described), but it is not clear what analysis was conducted . Geriatric assessment and the use of care and other outcomes . Two studies (23,94) examined the association between domains of geriatric assessment and the use of care (supplementary table 4, available online). In a prospective study with 337 colorectal cancer patients that adjusted for age and sex but not for illness severity and comorbidity, bailey et al . (23) found that patients who were older and had poorer mental health had greater use of social resources . In a prospective study that used seven markers of frailty markers and (94) reported that only one frailty marker, cognitive impairment, predicted visits to the emergency department after adjustment for confounders such as cancer type, cancer stage comorbidity, age, and sex . Five studies (22,23,34,66,91,98) reported that components of the geriatric assessment, such as age and functional status, were associated with the receipt of certain treatment modalities or regimens, such as surgery only . Other outcomes studied included changes in functional status, distress, clinical response, and discharge to usual place of residence after hospital admission (supplementary table 4, available online). This is the first review, to our knowledge, to systematically summarize all available evidence with regard to the use and effectiveness of geriatric assessment in the oncology setting . The evidence summarized in this review suggests that it is feasible to conduct a geriatric assessment in a hospital setting in older patients with cancer . The use of a geriatric assessment in the hospital setting can identify many health and functional status issues that might not otherwise be known by the treating oncologist . In addition, several domains of geriatric assessment are associated with oncological outcomes, such as toxicity of treatment and mortality, even in heterogeneous study populations . The factors consistently associated with these outcomes include impairments in activities of daily living, comorbidity, and poor mental health . Because most of the studies included heterogeneous study populations and featured small sample sizes, they had limited ability to conduct subgroup analyses . Thus, it was not possible to compare the results for solid tumors vs hematological malignancies or for cancers with different prognoses or treatment trajectories (eg, adjuvant vs metastatic settings). Future studies in more homogeneous populations are needed to identify populations where geriatric assessment might be particularly useful in helping a physician select the cancer treatment, preventing adverse outcomes of cancer and its treatment . We found that although many studies have incorporated some form of geriatric assessment to describe the patient population, fewer studies have examined the usefulness of geriatric assessment in terms of its ability to identify older adults at risk for adverse outcomes of cancer and its treatment . To date, no randomized controlled trial has been conducted to evaluate the effectiveness of geriatric assessment for distinguishing between fit and frail older adults to improve outcomes of cancer treatment compared with usual care in oncology . Nevertheless, experts in the field and siog (13,102) expect that by distinguishing between fit and more vulnerable and frail patients, treatment regimens can be adjusted to maximize the treatment effectiveness and avoid complications; however, this expectation still needs to be proven in a randomized controlled trial setting . Even though no randomized controlled trial has examined the effectiveness of geriatric assessment in the oncology setting, the general principles of geriatric medicine and geriatric assessment are thought to apply to all older adults, including those with cancer . Published guidelines and the recommendations of groups such as the nccn and the siog suggest that most clinicians accept the applicability of geriatric assessment in the oncology setting . However, we found no high - quality evidence (ie, from randomized controlled trials) that conducting a geriatric assessment and tailoring interventions based on its findings altered important patient outcomes in older cancer patients . Thus, based on the results of this systematic review, firm recommendations for implementing geriatric assessment and the type of geriatric assessment in routine clinical practice await additional study because the effectiveness of geriatric assessment in improving patient outcomes remains unclear . Rather, interventions that can improve patient outcomes are identified based on the geriatric assessment . The aim of the traditional geriatric assessment is to predict functional decline and falls in an older population with cognitive and functional impairments . Therefore, it is not surprising that in many of the reviewed studies, geriatric assessment was not useful in predicting oncology outcomes, such as treatment toxicity . Ceiling effects (ie, when most participants score the maximum score possible on a test because the test is unable to distinguish between individuals at the higher score range of the test), as reported by hurria et al . (32), could explain the null effect of geriatric assessment in predicting outcomes in many of the studies that were included in this review . However, experts have recommended using geriatric assessment in clinical oncology practice because it is expected to improve care for older oncology patients by helping improve treatment selection, avoiding toxicity, and identifying undetected medical problems that can interfere with treatment (11,12,13). Future studies should carefully consider which outcomes are most relevant in this population and how the geriatric assessment can be used to identify opportunities for effective interventions . The necessary next step in geriatric oncology requires intervention studies based on geriatric assessment . A recent meta - analysis of 22 randomized controlled trials that evaluated the effect of geriatric assessment vs usual care on independence and discharge to usual residence after hospital admission for older adults admitted to the hospital showed those who received geriatric assessment prior to interventions were more likely to be alive and in their own homes at the 6-month follow - up and less likely to suffer death or deterioration (103). We used systemic methods to identify all relevant studies, and two reviewers independently assessed the titles and abstracts by following the prisma statement . We also used various published quality assessment criteria to take into account different study designs included in this review . Our search strategy was inclusive: we did not exclude any study based on the methodological quality because this is the first systematic review providing a comprehensive overview of the use of geriatric assessment in the oncology setting . A meta - analysis was not possible because the studies were heterogeneous with respect to geriatric assessment instruments, methods, study populations, and outcomes . Furthermore, the findings are limited by the heterogeneous scientific quality of the studies included . Although we tried to contact all study authors if there were questions regarding the study, we were not successful in contacting all study authors, especially because some studies were published 15 years ago . It is thus possible that we rated some quality criteria of each of the individual studies during the quality assessment as unsatisfactory simply because they were not reported or because reporting guidelines such as strobe and moose for different study types were published more recently than some of the studies . In addition, cancer treatment options for older adults have changed because more elder - friendly treatments are being developed with less toxicity . These changes may have impacted the predictive validity results of the geriatric assessments reviewed in this systematic review . In addition, we did not examine the feasibility or effectiveness of geriatric assessment by cancer type or stage . As more studies are conducted, future systematic reviews should take cancer type and stage into account to examine the effectiveness of geriatric assessment in improving patient outcomes for different tumor types and stages . There are four fundamental barriers to advancing the field of geriatric oncology as identified through this systematic review . First and most important is the conceptual issue of the clinical value of a gold standard for geriatric assessment in the oncology setting . There is also no consensus regarding which domains should be included in geriatric assessment and how the instruments should best be designed and used in the oncology setting . The ability to compare newly developed, abbreviated, or otherwise - modified instruments with an idealized geriatric assessment is limited because the value of geriatric assessment in terms of predictive validity and impact on cancer treatment or patient outcomes is unclear . For example, the value of geriatric assessment has not been rigorously compared with usual care in the oncology setting, particularly with respect to the impact on treatment decision making or patient outcomes . This classification approach recommends standard therapy for fit patients, adjusted therapy for those classified as vulnerable, and best supportive care or palliative treatment for those classified as frail . Most authors have defined impairments in two or more domains of the geriatric assessment as criteria for classifying a patient as frail . These approaches are not necessarily in agreement with the concept of frailty as it is used in the geriatric medicine setting (104). In the latter context, frailty is not considered to be the endpoint of the continuum of fit to completely dependent; rather, it represents a state where an individual is independent but at high risk for developing disability . This inconsistent use of the concept of frailty by oncology and geriatric medicine may lead to confusion and hinder the translation of knowledge from research into clinical practice across different settings . In addition, this varied usage hampers research because study results cannot be compared across studies, both within geriatric oncology and across disciplines . The third barrier is the lack of information about the psychometric properties of the tools used in the geriatric assessment . Most studies have used instruments that have been validated in the traditional geriatric medicine setting . The properties of these instruments may be different in the oncology setting because the psychometric properties are determined by the clinical population studied . The clinical population in the oncology setting might be different from the one in the geriatric medicine setting where the psychometric properties of these tools were studied . Older persons with moderate to severe disability or cognitive impairment are less likely to be referred to oncology clinics due to referral bias (105). Thus, most likely, the population in the oncology setting has less cognitive impairment and better functional status than the population in which these tools were developed and tested . Therefore, the psychometric properties of geriatric assessment instruments should be examined within the geriatric oncology setting . This would better allow clinicians and researchers to select or develop the most appropriate and effective tools to include in their geriatric assessment in the oncology setting . Finally, the quality of reporting for studies in the field of geriatric oncology should be improved . Our quality assessment of the published studies suggests that researchers conducting future studies need to report more details on the study design, setting, response rates, and follow - up so that other researchers and clinicians can better evaluate the generalizability of the findings to their own settings . Randomized controlled trials comparing the effectiveness of conducting geriatric assessment with standard oncological care on relevant oncology outcomes are urgently needed to move the field of geriatric oncology forward . Two studies (27,48) showed an impact of geriatric assessment on the cancer treatment decision, whereas two others did not (19,98); however, none of these studies was a randomized controlled trial . Several studies were published after the search for this systematic review was conducted (106112). Two studies examined the impact of geriatric assessment on the treatment decision and showed that for the majority of patients geriatric assessment had no impact on the treatment decision (108,112). In addition, four studies that evaluated the predictive validity of geriatric assessment showed that geriatric assessment domains were predictive of cancer treatment outcomes, such as chemotherapy (106,109112). Although geriatric assessment is recommended to be used in clinical settings for older adults with cancer by both nccn and siog (11,12,13), in a public health care system with finite resources to allocate to competing health care interventions, showing the (cost-) effectiveness of a geriatric assessment in improving oncology outcomes for older adults is necessary for it to become standard of care . Given that geriatric assessment has been recommended as the standard of care, the broad implementation of geriatric assessment in clinical settings is likely to improve oncology outcomes for older adults affected by cancer . There is a dearth of studies examining the impact of geriatric assessment on the use of care, and this outcome should be included in future studies . In addition, no study has examined the impact of geriatric assessment on quality of life, which, for older adults with cancer, is an important consideration (113,114). Thus, there is a need for studies with improved methodological quality, larger sample sizes, and longitudinal design to obtain evidence for the use of geriatric assessment in older patients who are seen in diverse oncology settings . Furthermore, given the costs of conducting multidisciplinary geriatric assessments and the large number of older adults being seen in oncology, there is a need for a short screening tool with good psychometric properties to identify older adults that can benefit from a more in - depth geriatric assessment . Several such tools have been developed for the geriatric oncology setting, including the g-8 (115) and the instrument developed by hurria et al . The effectiveness of such an approach a screening tool for all older patients followed by an in - depth assessment of those deemed to be at risk is not established and needs to be validated in randomized controlled trials . Of course, such screening tools will only be of value once randomized controlled trials have clearly demonstrated that resource - intensive comprehensive geriatric assessment is effective in improving outcomes compared with usual care in the oncology setting . Furthermore, organizations such as siog and nccn that advocate for some form of geriatric assessment should articulate more clearly the current state of knowledge with regard to the benefits and impact of geriatric assessment on specific outcomes along with highlighting the current large gap in evidence.
In the united states, diabetes mellitus affects 26 million people and its chronic vascular complications are associated with significant morbidity [2, 3], disability, and mortality . Chronic complications from diabetes account for approximately $58 billion in excess medical expenditures per year . Common risk factors for microvascular and macrovascular diabetic complications include age, duration of diabetes, hyperglycemia [79], and high blood pressure; therefore multiple complications commonly develop in the same patient [5, 6]. In addition to the management of cardiovascular risk factors, prevention of diabetes complications also involves diabetes self - care such as diet, exercise, self - monitoring of blood glucose, and self - foot examination . As a result, the american diabetes association (ada) has established clinical practice guidelines regarding standard diabetes care, which include recommendations for diabetes process of care measures (frequency of laboratory testing, clinical goals, and recommended medications) and self - care . A few studies have suggested that adherence with these diabetes clinical practice guidelines varies by sex . Women with diabetes have been reported to have worse blood pressure, lipid, and glycemic control compared to men, even amongst those known to have cardiovascular disease [13, 14]. However, sex differences in laboratory testing and other self - care behaviors have not been explored in detail, nor is it known whether these sex disparities persist in high - risk patients, such as those who already have a history of a diabetic complication . Identification of modifiable factors related to diabetes outcomes is imperative if the rate of adverse outcomes is to be decreased, and evaluation of sex - specific differences provides an opportunity to develop strategies to reduce sex - related health disparities in diabetes care . This study examined the associations between sex and selected diabetes process of care measures and self - care activities in a cohort of primary care patients with diabetes . This study also examined whether sex differences in diabetes process of care measures and self - care activities were detectable in the subgroup of subjects with a history of diabetic complications, a particularly high - risk group for adverse outcomes . We conducted a cross - sectional analysis of baseline data from the pathways study, which has been described previously [16, 17]. In brief, the pathways study is a prospective, observational cohort of the prevalence and impact of depression on patients with diabetes at group health (gh), a large nonprofit health maintenance organization (hmo) in washington and idaho, usa . Nine primary care clinics were chosen for patient recruitment based on the number of diabetic patients, ethnic diversity, and proximity to seattle, wa, usa . For the study, 9,063 potential candidates were identified from the gh diabetes registry (figure 1). In 2001 - 2002, surveys were sent to these patients regarding demographic information, diabetes characteristics, diabetic complications, and self - care . Diabetic complications included retinopathy, nephropathy, neuropathy, cerebrovascular, cardiovascular, peripheral vascular disease, or metabolic (hypoglycemia, diabetic ketoacidosis, or hyperosmolar nonketotic coma). Of those identified, 1,222 patients were excluded from the study due to no diabetes, gestational diabetes, cognitive impairment, severe illness, deceased, disenrollment from gh, language or hearing problems, or other reasons . Of the remaining 7,842 eligible patients for the study, 4,839 (61.7%) returned the survey of which 4,467 (92.3%) gave permission to link survey data with automated data from gh regarding laboratory tests, pharmacy records, hospitalizations, and outpatient visits . Baseline hemoglobin a1c and low - density lipoprotein (ldl) were ascertained closest to the date of the baseline epidemiologic survey, up to 12 months prior to study enrollment . Microalbuminuria was defined as a urine albumin to creatinine ratio (uacr)> 17 mg / g for women and> 25 mg / g for men, based on sex - specific cutoffs; given the large proportion of missing data, microalbuminuria was ascertained up to 24 months prior to study enrollment . Estimated glomerular filtration rate (egfr) was calculated using chronic kidney disease - epidemiology (ckd - epi) equations . Chronic kidney disease (ckd) stage was determined by egfr and microalbuminuria, using the national kidney foundation kidney disease outcomes quality initiative classification system . Computerized pharmacy records were used to identify patients who were prescribed any insulin, oral diabetic medication, hmg - coa reductase inhibitor (statin), angiotensin - converting enzyme (ace) inhibitor, or angiotensin receptor blocker (arb) in the 12 months prior to study enrollment . For simplicity we will use the generic term ace inhibitor to refer to either an ace inhibitor or arb . Self - care activities were assessed using the summary of diabetes self - care activities (sdsca), which is a brief questionnaire that asks how many days per week an activity was performed . The sdsca has been shown to be a reliable and valid measure of adherence to diabetes self - care in observational and interventional studies . For this study, investigators selected five sdsca questions regarding diet, exercise, blood glucose testing, and foot care that were considered the most clinically relevant for analysis (table 1). The primary outcomes of interest were sex - specific differences in the following diabetes process of care measures and self - care activities: (1) history of recommended laboratory testing (hemoglobin a1c, ldl, and microalbuminuria), (2) attainment of clinical targets (hemoglobin a1c <7% and ldl <130 mg / dl), (3) medication use (statins in all subjects and in those with ldl> 130 mg / dl, ace inhibitors in all subjects and in those with microalbuminuria), and (4) compliance with self - care (diet, exercise, and foot examination at least 3 times per week; blood glucose testing at least 3 times a week if on oral hypoglycemic agents only or 5 times a week if on insulin). Statistical analyses were performed using stata version 12 (college station, tx, usa). Significant sex differences in diabetes clinical process of care measures and self - care activities were determined using t - tests for continuous data and tests for categorical data . Logistic regression models were used to calculate adjusted odds ratios (aors) to determine if there were adjusted sex differences in compliance with diabetes process of care measures and self - care activities . Models regarding process of care measures were adjusted for age, race / ethnicity, marital status, education, smoking, body mass index, hemoglobin a1c (except for models where hemoglobin a1c testing and achievement of hemoglobin a1c <7% were outcomes of interest), history of hypertension, and ckd stage (except the model for microalbuminuria testing). Models regarding self - care activities were additionally adjusted for a history of major depression, which was strongly associated with both sex and self - care outcomes . Analyses were also performed on the subgroup of patients with a known history of at least one diabetic complication (n = 3,045), since these patients are at high risk for adverse outcomes . Sensitivity analyses demonstrated similar results in this subgroup as with subgroups of patients with specific diabetic complications (cardiovascular, cerebrovascular, or nephropathy). Of the 4,839 subjects in the total cohort, 48.8% were women (table 2). Men tended to be older, more frequently married, and had higher levels of education and income compared to women . Women had higher mean bmi and greater prevalence of hypertension (45.0% versus 41.0%) and major depression (14.1% versus 9.6%) than men . Men had a greater prevalence of microalbuminuria (46.7% versus 34.4%) and higher mean number of diabetic complications (1.4 1.4 versus 1.3 1.3) compared to women . There were similar patterns of sex differences in cohort characteristics amongst the subset of subjects known to have diabetic complications . Approximately 86.7% of subjects had previous hemoglobin a1c testing and 61.4% had urine microalbuminuria screening; these test frequencies did not vary significantly by sex . In contrast, ldl testing was less frequent in women compared to men; only 52.6% of women had their ldl checked in the previous year compared to 59.0% of men (p <0.001). Both men and women had a mean hemoglobin a1c of 7.8%, and a similar proportion of subjects achieved a hemoglobin a1c <7% . Mg / dl) than in men (107.8 33.6 mg / dl, p <0.001), and a lower proportion of women achieved a target ldl of <130 mg / dl (67.3% versus 75.3% in men, p <0.001). Statins were prescribed less frequently to women than men overall (26.5% versus 35.4%, p <0.001) and in those with ldl levels above 130 mg / dl (24.2% versus 31.3%, p = 0.02). There were no sex differences in ace inhibitor use overall or in the subset with microalbuminuria . In adjusted multivariable logistic regression models (table 3), a greater proportion of women were more likely to be guideline discordant than men for ldl testing (aor 0.73, 95% ci 0.620.85), achievement of ldl target <130 mg / dl (aor 0.70, 95% ci 0.580.86), any statin prescription (aor 0.69, 95% ci 0.580.81), or statin prescription if ldl was greater than 130 mg / dl (aor 0.61, 95% ci 0.410.91) compared to men . Women were more likely to achieve hemoglobin a1c target (aor 1.19, 95% ci 1.021.41). There were no differences in the odds of hemoglobin a1c testing, microalbuminuria screening, or ace inhibitor prescription by sex . Women reported more frequent consumption of 5 servings of fruits or vegetables per day and less frequent consumption of high fat foods compared with men . In the week prior to study assessment, a lower proportion of women (55.3%) exercised at least 3 times a week compared to men (63.9%, p <0.001). In adjusted logistic regression models of self - care activities (figure 2(a)), women were more likely to report high fruit and vegetable consumption (aor 1.36, 95% ci 1.151.61), blood glucose testing (1.27, 95% ci 1.041.55), and self - foot examination (aor 1.32, 95% ci 1.111.57) but less likely to report fatty food consumption (aor 0.69, 95% ci 0.590.80) and regular exercise (aor 0.72, 95% ci 0.620.85) compared to men . Based on adjusted logistic regression models of 3,045 patients with at least one known diabetic complication, men and women had similar odds of hemoglobin a1c and microalbuminuria testing (table 4). Women in this subgroup had decreased odds of ldl testing (aor 0.65, 95% ci 0.540.77), achievement of ldl goal <130 mg / dl (aor 0.63, 95% ci 0.510.78), statin use overall (aor 0.67, 95% ci 0.560.79), and statin use if serum ldl was> 130 mg / dl (aor 0.64, 95% ci 0.420.97). Women with at least one diabetic complication had greater odds of achieving target hemoglobin a1c <7% compared to their male counterparts . There were no sex - specific differences in ace inhibitor use . Amongst those with at least one diabetic complication, women had greater odds of high fruit and vegetable consumption (aor 1.61, 95% ci 1.341.93), blood glucose monitoring (aor 1.26, 95% ci 1.011.55), and self - foot examination (aor 1.31, 95% ci 1.081.58) compared to men (figure 2(b)). Women in this subgroup were less likely to consume fatty foods (aor 0.69, 95% ci 0.580.81) or to exercise (aor 0.69, 95% ci 0.590.82) compared to their male counterparts . This analysis found significant sex differences in diabetes process of care measures and self - care activities, even amongst the high - risk subgroup of subjects who were known to have chronic complications of diabetes . In general, women tended to have better glycemic control and adherence to recommended self - care compared to men . However, despite having higher ldl levels, women were less likely to be screened for dyslipidemia or to be prescribed statins compared to men . This is the first study to report sex disparities in diabetes processes of care and self - care behaviors in the subgroup of patients with a history of at least one diabetic complication, which is surprising since these patients are at high - risk for additional diabetic complications and warrant aggressive diabetes care . Our results are congruent with previous findings of sex differences in diabetes process of care measures, particularly with respect to management of dyslipidemia . In a cross - sectional study of 3,849 patients with diabetes from five academic medical centers, wexler et al . Found that women had higher cholesterol levels, were less likely to receive lipid lowering therapy, and when receiving lipid lowering therapy, were less likely to reach ldl targets compared to men . Found that, compared to men, diabetic women without cardiovascular disease received less frequent lipid testing and diabetic women with cardiovascular disease were treated less frequently with lipid lowering agents . Although other studies have reported worse glycemic control in diabetic women compared to men [12, 14], we found that women had better glycemic control than men . Diabetic women tended to be more physically inactive than men, which is consistent with findings from the third national health and nutrition examination survey (nhanes iii). This study builds on previous reports by demonstrating sex differences in other diabetes self - care activities . Diabetic men were consistently less adherent to recommendations regarding diet, blood glucose monitoring, and foot care than women . Likewise, these sex differences in diabetes self - care persisted in patients with known diabetic complications . The observed sex differences in diabetes process of care measures and self - care activities are likely multifactorial and may be related to both provider and patient factors . Providers may perceive diabetic women to have a lower risk of cardiovascular disease and other diabetic complications compared to men, which may result in less aggressive monitoring and treatment in women . Whether sex influences management of diabetic patients has not been investigated; however, research in cardiovascular disease revealed that sex influenced how physicians managed chest pain, coronary heart disease, and cardiovascular disease prevention . Since public awareness of cardiovascular disease in women remains suboptimal [29, 30], female patients may themselves underestimate their risk for diabetic complications and either fail to inquire about or decline routine diabetes care . Moreover, men and women may have differing beliefs in the benefit of self - care . In a survey of new patients to a diabetes education center, women were more likely than men to have a history of previous diabetes education and had higher expectations that self - management would improve health outcomes; this may explain our finding that women had better glycemic control and overall better adherence to self - care than men . Finally, biologic differences between men and women, due to estrogen or lack thereof, may result in differential outcomes in care . Sex hormones are associated with glucose tolerance, lipid metabolism, albuminuria, and coronary heart disease . Diabetes has a greater adverse effect on serum triglyceride and ldl levels in women compared to men, and a recent meta - analysis of statin therapy for secondary cardiovascular prevention found that statins reduced all - cause mortality and stroke risk men but not in women . It is important to recognize that the results of this study present an opportunity to improve the quality of diabetes care not only for women, but for men as well . Although women had less adequate screening and management of dyslipidemia and poorer adherence to exercise, they tended to have better glycemic control and adherence to other recommended self - care activities compared to men . Health care providers should pay particular attention to ordering recommended laboratory tests and medications in women and targeting patient education interventions regarding self - care to men . All patients should be encouraged to follow a regular exercise program and this should be heavily emphasized among women . The strengths of this study include the large sample size of study subjects with comparable access to care . The study surveyed patients directly for variables related to diabetes self - care and we were able to adjust for several known confounding variables, including major depression . However, this study does have several limitations that are important to consider . The cross - sectional nature of this study is subject to unmeasured confounding and cannot establish causal relationships . We did not have access to actual blood pressure measurements and therefore could not adjust for the degree of control of hypertension . There was a large proportion of missing data for laboratory test results, particularly for ldl and microalbuminuria, which affects the validity and generalizability of these results . Pharmacy records could only capture medications prescribed within the gh system and do not reflect actual patient usage . Self - care activities were ascertained by self - reported measures rather than actual measurements . Although the sdsca has been shown to be a reliable and valid measure of diabetes self - management, differential misclassification could occur if there were systematic differences in how men and women recall or report self - care . Finally, this study could not account for several factors that may contribute to the observed sex differences, such as differences in patient visit frequency, provider styles of care, or patient preferences by sex . In conclusion, sex is associated with significant differences in diabetes process of care measures and self - care activities, even amongst subjects known to have chronic complications from diabetes . Women may benefit from more attention to dyslipidemia screening, lipid lowering treatment, and regular exercise, whereas men may require more encouragement in diabetes self - care including healthy diet, self - blood glucose monitoring, and self - foot examination . The findings from this study indicate an opportunity for intervention to reduce sex - related disparities in diabetes care . Although further studies are needed to elucidate the causes for these sex disparities, it is important for primary health care providers to be aware of the existence of sex differences in diabetes care such that these disparities may be eliminated.
Culex mosquitoes are vectors of pathogens including the human filarial nematode, wuchereria bancrofti, and encephalitis - causing viruses, such as st . Louis, japanese, venezuela equine, western equine encephalitis, and west nile virus (nasci and miller, 1996).1 given the resistance of culex populations to modern insecticides, alternative methods of controls are sorely needed . Larval development is a particularly vulnerable phase in the life cycle of culex mosquitoes, as eggs are laid in rafts from which hundreds of larvae emerge in confined areas thus facilitating management . Skatole, a natural product found in animal excreta and also a product of fermentation of organic material, has been identified as an oviposition attractant for the southern house mosquito, culex quinquefasciatus (millar et al ., 1992). Field studies have demonstrated that traps baited with optimal doses of skatole collected significantly more eggs (mboera et al ., 2000) and gravid females (leal et al ., 2008) than control traps, thus suggesting that in combination with a biological agent, bacillus thuringiensis var . Israelensis (barbosa et al ., 2010) oviposition attractants may be used in attract - and - kill strategies . Chemical ecology and olfaction are the pillars of these semiochemical - based, environmentally friendly strategies . Therefore, identification of olfactory proteins involved in the reception of these semiochemicals may open the door for development of better oviposition attractants . Recently, we demonstrated by rna interference that an odorant - binding protein (obp) from cx . Quinquefasciatus, cquiobp1, is involved directly in the reception of skatole and other oviposition attractants (pelletier et al ., 2010a). We also have characterized an odorant receptor (or) from this mosquito species, cquior2, which is highly sensitive to indole and moderately sensitive to skatole (pelletier et al ., 2010b). Here, we characterize cquior10 and show this or to be highly sensitive and narrowly tuned to skatole . Expression of cquior10 in the xenopus oocyte system oocytes were prepared as previously described (pelletier et al ., 2010b). Cquior10 and cquior7, initially cloned into pbluescript (pelletier et al ., 2010b), were transferred to pgemhe for use as templates for synthesis of capped crna by using mmessage mmachine kits (ambion). Twenty - five ng of crna encoding each or subunit were injected into stage v vi xenopus oocytes . Oocytes were incubated at 18c in barth s saline (in mm: 88 nacl, 1 kcl, 2.4 nahco3, 0.3 cano3, 0.41 cacl2, 0.82 mgso4, 15 hepes, ph 7.6, and 100 g / ml amikacin) for 25 d prior to electrophysiological recording . Electrophysiology and data analysis odorant - induced currents were recorded under two - electrode voltage clamp from oocytes expressing ors, by using an automated parallel electrophysiology system (opusxpress 6000a; molecular devices). Oocytes were perfused with nd96 (in mm: 96 nacl, 2 kcl, 1 cacl2, 1 mgcl2, 5 hepes, ph 7.5). Odorants were diluted in nd96 and applied for 20 sec at a flow rate of 1.65 ml / min with extensive washing in nd96 (520 min at 4.6 ml / min) between applications . Current responses approached a plateau during the 20 sec application (pelletier et al ., 2010b). Micropipettes were filled with 3 m kcl and had resistances of 0.22.0 m. the holding potential was 70 mv . Current responses were filtered (4-pole, bessel, low pass) at 20 hz (3 db), sampled at 100 hz, and were captured and stored with opusxpress 1.1 software (molecular devices). Initial analysis of electrophysiological data was done with clampfit 9.1 software (molecular devices). Curve fitting of concentration - response data was done with prism 4 (graphpad). In our search for molecular targets that may be used in a reverse chemical ecology approach for the development of better oviposition attractants (leal et al ., 2008), we recently have mined the genome of cx . Quinquefasciatus and identified an or sensitive to indole, cquior2, which also responded to methylindoles, including skatole (iupac name, 3-methylindole). By mapping the antennae of female cx . Quinquefasciatus, we previously observed that a skatole - detecting orn also responded to geranylacetone and ethyl hexanoate, but not indole (syed and leal, 2009). These observations prompted us to examine the odorant response profile of cquior10, an or closely related to cquior2 (pelletier et al . . Full - length coding sequence of cquior10 and the obligatory co - receptor cquior7 (pelletier et al ., 2010b) were cloned in pgemhe for heterologous expression in xenopus oocytes . To identify the best ligand for this receptor, oocytes expressing cquior10 + cquior7 were screened first with a panel of odorants (fig . 1a), each applied for 20 sec at a concentration of 10 m with extensive washing between applications . Skatole (3-methylindole) elicited the largest current responses, but the receptor also responded with lower sensitivity to indole, other methylindoles, and 2-methylphenol . Interestingly, cquior10 was unresponsive to many compounds in the test panel of 23 odorants, including other oviposition attractants such as trimethylamine, nonanal, and the mosquito oviposition pheromone (mop) (leal et al ., 2008). Each odorant was applied at a concentration of 10 m for 20 sec with 10 min washes between applications . All responses are normalized to the response of the same oocyte to 10 m indole (mean sem, n = 45). B oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole (top trace), indole (middle trace) or 2-methylphenol (bottom trace). Each odorant was applied for 20 sec with 520 min washes between applications . Note different scales: from top to bottom 500, 200 and 400 na . C oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole, indole, and 2-methylphenol . Responses were normalized to the response of each oocyte to 10 m indole and are presented as mean sem (n = 35 for each odorant tested). Data were fit to the equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hbox{i}} = {{\hbox{i}} _ {{\max}}} /\left ({{1} + {{\left ({{\hbox{e}}{{\hbox{c}}_{{5}0}}/{\hbox{x}}} \right)}^{\rm{n}}}} \right) $$\end{document} where i represents the current response at a given concentration of odorant (x), imax is the maximal response, ec50 is the concentration of odorant yielding a half maximal response, and n is the apparent hill coefficient . Derived values are: 3-methylindole, ec50 = 90 17 nm, n = 1.0 0.1; indole, ec50 = 2.4 0.3 m, n = 1.1 0.2; 2-methylphenol, ec50 = 41 7 m, n = 1.0 0.1 odorant receptor cquior10 is highly sensitive to skatole (3-methylindole). Each odorant was applied at a concentration of 10 m for 20 sec with 10 min washes between applications . All responses are normalized to the response of the same oocyte to 10 m indole (mean sem, n = 45). B oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole (top trace), indole (middle trace) or 2-methylphenol (bottom trace). Each odorant was applied for 20 sec with 520 min washes between applications . Note different scales: from top to bottom 500, 200 and 400 na . C oocytes expressing cquior10 + cquior7 were challenged with a range of concentrations of 3-methylindole, indole, and 2-methylphenol . Responses were normalized to the response of each oocyte to 10 m indole and are presented as mean sem (n = 35 for each odorant tested). Data were fit to the equation: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\hbox{i}} = {{\hbox{i}} _ {{\max}}} /\left ({{1} + {{\left ({{\hbox{e}}{{\hbox{c}}_{{5}0}}/{\hbox{x}}} \right)}^{\rm{n}}}} \right) $$\end{document} where i represents the current response at a given concentration of odorant (x), imax is the maximal response, ec50 is the concentration of odorant yielding a half maximal response, and n is the apparent hill coefficient . Derived values are: 3-methylindole, ec50 = 90 17 nm, n = 1.0 0.1; indole, ec50 = 2.4 0.3 m, n = 1.1 0.2; 2-methylphenol, ec50 = 41 7 m, n = 1.0 0.1 next, we performed concentration - response analyses for skatole and two other ligands, indole and 2-methylphenol, which were identified as the best ligands among the indoles and phenols, respectively, for the related receptor cquior2 (pelletier et al ., 2010b). Skatole was the most potent of these compounds, activating the cquior10 + cquior7 receptor with an ec50 of 90 nm . Indole and 2-methylphenol were less potent, activating cquior10 + cquior7 with ec50 values of 2.4 m and 41 m, respectively . Interestingly, indole and 2-methylphenol also displayed lower efficacy (maximal response) than skatole (40 2% and 69 3% of skatole, respectively). In addition, the response threshold for skatole was two to three orders of magnitude higher than that observed for indole and 2-methylphenol (fig . Thus, we found that heterologously expressed cquior10 is highly sensitive and narrowly tuned to the oviposition attractant skatole . In female antennae of the southern house mosquito, skatole is detected by a small - spike - amplitude orn housed in a1 sensilla (syed and leal, 2009), which is also sensitive to a lower degree to geranylacetone and ethyl hexanoate, but does not respond to indole or 2-methylphenol [see figs . S5, s6, supporting information in (syed and leal, 2009)]. In the xenopus oocyte system, although expression of ors in heterologous systems, such as the xenopus oocyte system, is an invaluable tool for de - orphanizing and characterizing receptors, it does not completely mimic the insect olfactory system . Typically, these systems are devoid of obps, odorant - degrading enzymes, sensory neuron membrane proteins, and other olfactory proteins that may play a part in the selectivity and sensitivity of the olfactory system . Thus, it is conceivable that heterologously expressed cquior10 and the receptor in its native environment differ in the detection of geranylacetone because the former is devoid of obps . However, one cannot rule out the possibility that a separate orn responding to geranylacetone has the same spike amplitude as the skatole - detecting orn (syed and leal, 2009), thus rendering them indistinguishable by single unit recordings . Alternatively, the same small - spike neuron sensitive to skatole may express another or along with cquior10 . In marked contrast to the mammalian olfactory system, co - expression of ors has been documented in drosophila melanogaster . Co - expression of cquior10 and another or would not be entirely surprising given the number of putative odorant receptors in the southern house mosquito genome (pelletier et al ., 2010b) and the number of orns in their sensory system (syed and leal, 2007, 2008). Future research aimed at testing these three hypotheses might lead to deeper understanding of odorant reception in mosquitoes.
The most common inflammatory bowel diseases (ibds) are crohn's diseases (cd) and ulcerative colitis (uc). It is recognized that aberrant immune response in the mucosa of the gastrointestinal tract plays an important role in pathogenesis of ibds [1, 2]. A large body of evidence from experimental models of ibd has shown that cd4 + t cells (t helper cells th1 and th2) play a major role in initiating and regulating the immunopathologic process, largely based on the production of proinflammatory cytokines, such as tumor necrosis factor (tnf) [3, 4]. More recently, another subset of t helper cells that produces the cytokine interleukin (il-17, also called il-17a) was identified as th17 cells and has gained particular attention due to its proinflammatory role in the mucosal immune response [5, 6]. The number of th17 cells and il-17 expression were found to be significantly enhanced in the inflamed gut of cd and uc patients [7, 8]. In addition to the il-17 isoforms (il-17a and il-17f), th17 cells also secrete il-6, il-21, il-22, and il-26 . Il-17 drives microbial defense, contributes to neutrophil migration and function, and promotes t - cell priming and cellular production of inflammatory mediators, such as il-1, il-6, tnf, granulocyte macrophage colony - stimulating factor (gm - csf), nitric oxide synthase (nos)-2, prostaglandin e2, and metalloproteases . Therefore, control of the differentiation and function of th17 cells is potentially significant for ibd treatment . It has been recently demonstrated that differentiation of th17 cells is initiated by transforming growth factor 1 (tgf1) and il-6 [9, 14]. Both tgf1 and il-6 regulate th17 differentiation by activating the transcription factor signal transducer and activator of transcription 3 (stat3) [15, 16]. A line of evidence has also indicated a significant role of il-23 in th17 differentiation . Il-23 was initially thought to be critical for th17 differentiation; however, it was later found to be rather important for th17 cell expansion, stabilization, and/or conditioning for a fully inflammatory cell phenotype [17, 18]. The il-23/th17 immune axis has thus been shown to play a crucial role in a number of chronic inflammatory diseases including ibd . The lactobacillus species can rebalance homeostasis in gastrointestinal inflammatory diseases and thus have a protective role against ibd . We have previously shown that lactobacillus (l.) acidophilus treatment can efficiently ameliorate dextran sodium sulfate- (dss-) induced experimental colitis in mice . In the present study, we examined whether the therapeutic effect of l. acidophilus is achieved through suppression of the il-23/th17 pathway, including th17 cell differentiation and the expansion factors il-23, tgf1, and stat3, as well as th17 cell - secreted cytokines il-17 and tnf. We found that oral administration of l. acidophilus at all doses induced a significant downregulation of colitis - enhanced expression of all the examined factors . L. acidophilus was isolated from a normal human intestinal tract smc - s095 sample and sequence - verified by our laboratory . After culturing under anaerobic conditions with de man, rogosa, sharpe (mrs) medium for 24 h, the bacteria were collected, quantified by a spectrophotometer, and diluted with normal saline to 10 cfu / ml . Seventy - two female balb / c mice (68 weeks old, 20.0 2.0 g mean body weight) were purchased from hunan agricultural university and housed under standard conditions (50% 10% humidity, 12 h light / dark cycle, and ad libitum access to standard mouse chow). Colitis was established in 64 mice by adding 5% dss (mw 50000; sigma chemical co., st . Louis, mo) to the drinking water and allowing ad libitum access for 7 days . The mice were randomly divided into the following control and experimental model groups (n = 8 each; day 0): the nontreated model group; the vehicle treated model group (oral gavage of 1 ml/10 g normal saline); the l. acidophilus - treated groups c4c8 (oral gavage of 10, 10, 10, 10, or 10 cfu/10 g body weight, resp . ); the prednisone acetate (an anti - inflammatory agent) treated positive control (administered at 45 g/10 g body weight). Eight mice that were given regular water and received no subsequent treatments served as the normal control group . Different doses were applied in order to identify an appropriate condition that can efficiently modulate the expression of factors related to th17 cell function . On posttreatment day 7, all mice were sacrificed with ether and the colon was collected . A 0.5 cm segment of the distal colon that is 1 cm proximal to the anus was excised and fixed with 10% formalin for later paraffin embedding and sectioning . The rest of the distal colon was stored in liquid nitrogen for rna and protein extractions . The colonic tissue was homogenized, and rna was extracted by using trizol (invitrogen, grand island, ny) according to the manufacturer's instructions . First - strand cdna was synthesized using a reverse transcription kit (mbi, ottawa, ca). 5-taaaacgcagctcagtaacagtccg-3 (product length of 349 bp, tm 60c, 30 cycles), il-17 forward primer 5-tcagactacctcaaccgttcc-3 and reverse primer 5-cagtttccctccgcatt-3 (product length of 129 bp, tm 54.5c, 30 cycles), il-23 forward primer 5-aataatgtgccccgtatcca-3 and reverse primer 5-aggctcccctttgaagatgt-3 (product length of 144 bp, tm 58c, 28 cycles), tnf forward primer 5-ggttgtctttgagatccatgc-3 and reverse primer 5-acgtggaactggcagaagag-3 (product length of 411 bp, tm 54.5c, 30 cycles), tgf forward primer 5-gaagtggatccacgagcccaag-3 and reverse primer 5-gctgcacttgcaggagcgcac-3 (product length of 247 bp, tm 60c, 30 cycles), and stat3 forward primer:5-acccaacagccgccgtag-3 and reverse primer 5-cagactggttgtttccattcagat-3 (product length of 192 bp, tm 62c, 30 cycles). Multiplex reactions were run in duplicate, and pcr products were run on agarose gels . The intensity of dna bands was measured with quantity one software (bio - rad, hercules, ca), and the results were normalized to the internal control -actin . The total homogenate was then centrifuged at 12,000 rpm for 30 minutes at 4c . The supernatant was collected, and protein concentration was measured using the dc protein assay method (bio - rad, hercules, ca). Twenty g proteins were separated on sds - page gels and transferred to pvdf membranes . The membrane was then blotted with primary antibodies for 1 h at room temperature . Antibodies to il-17 (h-132), tnf (n-19), tgf1 (v), stat3 (h-190), and -actin were purchased from santa cruz biotechnology (santa cruz, ca), and anti - il-23 (p19) and anti - p - stat3 (y705) antibodies were obtained from abcam (cambridge, uk). Antibody dilution ratios: (1) il-17 and il-23, 1: 400; (2) stat3, p - stat3 and tgf1, 1: 500; (3) tnf, 1: 2000; and (4) -actin, 1: 5000 . After three 10 min washes, membranes were incubated with anti - mouse, -rabbit, or -goat horseradish peroxidase (hrp) conjugated secondary antibodies (zsgb - bio, beijing, china) and washed, and detection was achieved using a dab kit (zsgb - bio). Densitometric analysis was performed using quantity one software, and the results are expressed as the relative intensity (od) of target protein to that of -actin control . Nonspecific blocking was obtained by incubation in phosphate - buffered saline (pbs) containing 5% normal goat serum for 30 minutes at room temperature . Tissue sections were then incubated with anti - p - stat3 (1: 200) antibody for 1 h at room temperature . Sections were washed with pbs, incubated in the abc reagent for 1 hour at room temperature, washed again, and incubated in a peroxidase solution (zsgb - bio). The signal of immunohistochemical staining was analyzed with image - pro plus 6.0 software (media cybernetics, maryland, usa). All statistical analyses were carried out using the spss statistical software suite (version 16.0; spss inc ., single - factor analysis of variance (one - way anova) was used to analyze the differences between groups, with p <0.05 considered as statistical significance . We have recently shown that oral administration of l. acidophilus ameliorates the pathogenesis of colitis in mice . Given the critical role of th17 cells in autoimmune diseases including ibd, we hypothesized that l. acidophilus might improve colitis at least partially through suppression of th17 cell function . We thus initially sought to examine whether colonic expression of il-17, the hallmark cytokine of th17 cells, is modulated by l. acidophilus treatment . The expression level of il-17 was examined in all nine groups at mrna and protein levels . We found that both mrna and protein expression of il-17 were significantly (p <0.05) enhanced in dss - induced colitis (figures 1(a) and 1(b)), which is consistent with the results of a previous report . L. acidophilus application at different doses in the c4c8 groups all significantly (p <0.05) attenuated colitis - increased il-17 expression (figures 1(a) and 1(b)) compared to levels in the nontreated or vehicle treated groups . Of all the l. acidophilus - treated groups, c5 showed the greatest inhibition of il-17 expression at both the mrna and protein levels, and this inhibition was even superior to the therapeutic effect of prednisone treatment (figure 1(c)). The electrophoresis images showing the mrna and protein expression of internal control -actin are presented for il-17 expression in figures 1(a) and 1(b) and are not repeatedly shown for the other examined molecules . Because tnf is another important proinflammatory cytokine secreted by th17 cells, we also examined the effect of l. acidophilus treatment on tnf expression . Similarly to il-17, tnf expression at both the mrna and protein levels was significantly (p <0.05) decreased by l. acidophilus treatment in groups c4c8 compared to the vehicle treated control group (figures 2(a) and 2(b)). However, unlike il-17, tnf expression was lowest in group c6 among all the dss - treated groups (figure 2(c)). Il-23 and tgf1 have been shown to play essential roles in promoting th17 cell differentiation, expansion, and function [9, 14, 17, 18]. We tested whether the inhibitory effect of l. acidophilus on th17-associated cytokine expression is achieved through downregulation of il-23 and/or tgf1 expression, thereby suppressing th17 cell differentiation and stabilization . Interestingly, treatment of colitis with a lower dose of l. acidophilus (c4c6 groups) caused a dramatic decrease in il-23 mrna expression, which reached a level comparable to that of the normal control group (figure 3(a)). Moreover, all l. acidophilus - treated groups showed a significant (p <0.05) decrease in il-23 protein expression (figure 3(b)). Notably, both the mrna and protein levels of il-23 expression were lowest in group c5 among all the colitis groups (figure 3(c)), at a level similar to that observed for il-17 expression . Likewise, l. acidophilus administration at all doses induced marked inhibition of tgf1 mrna expression (figure 4(a)). A dramatic attenuation of tgf1 protein expression was also observed in the low dose groups, c4 and c5 (figure 4(b)). Again, mice treated with 10 cfu/10 g l. acidophilus, group c5, showed the lowest expression of tgf1 (figure 4(c)). Stat3 is an important transcription factor that mediates the establishment of th17 cells in response to il-23 and tgf1 [15, 16]. We therefore examined whether stat3 expression is altered with diminished expression of il-23 and tgf1 upon l. acidophilus treatment . We observed that stat3 expression was upregulated in the colon of dss - mediated colitis, which is consistent with previous reports [22, 23]. Although the decrease in the stat3 mrna level was relatively moderate in the l. acidophilus - treated groups (figure 5(a)), an apparent decrease in stat3 protein level was observed in contrast to the vehicle treated control group (figure 5(b)). Among all the l. acidophilus - treated groups, inhibition of stat3 protein expression was most obvious in c5 (figure 5(c)). Because phospho- (p-) stat3 is the functional form, it is important to understand whether the phosphorylation level of stat3 is altered by l. acidophilus treatment . We observed that p - stat3 was significantly (p <0.05) lower in all groups (c4c8) relative to the untreated or vehicle treated control groups by western blotting analysis (figure 6(a)). Moreover, immunohistochemical staining showed that p - stat3 localized in the nuclei was predominantly expressed in the inflamed area in the dss - induced untreated control group (figure 6(b)). Importantly, l. acidophilus treatment caused a significant (p <0.05) reduction in p - stat3 signal, with a stronger effect in the c5 and c6 groups (figures 6(b) and 6(c)). These findings suggest that administration of l. acidophilus improves colitis by restoring the homeostasis of p - stat3-mediated cellular events, such as th17 differentiation and il-17 secretion . Ibds, including uc and cd, are considered a consequence of aberrant immune response in the gastrointestinal tract . It is traditionally recognized that cd is associated with th1 cytokines and uc is modulated by th2 cytokines . Although controversy remains, th17 cells are generally accepted as the mediators of intestinal inflammation via the secretion of proinflammatory cytokines, such as il-17 [22, 23]. Therefore, targeting th17 cells can be a potential approach for treating ibd . With the accumulation of evidence supporting the significant role of microbiota in gut homeostasis [24, 25], probiotics have nowadays been more and more frequently used in treating ibd, especially uc, through induction of remission and prevention of pouchitis . However, whether and how probiotics modulate th17 cell fate in the gut remains poorly understood . We found the expression of il-17, a hallmark cytokine characterizing th17 cells, was dramatically increased in colitis, which is consistent with previous reports [7, 8, 21]. Moreover, we herein provide the first evidence that oral administration of l. acidophilus, one of the most common natural inhabitants of the human gut, suppressed the upregulation of the proinflammatory cytokine il-17 in colitis . That oral administration of l. gasseri suppresses il-17 induction in allergen - induced airway inflammation in mice . More recently, amdekar et al . Showed that administration of l. acidophilus downregulates the expression of il-17 and to a lesser extent that of tnf in rats with collagen - induced arthritis . Similarly, we also observed an inhibitory effect of l. acidophilus on colitis - mediated tnf expression in mouse colon . The decrease in il-17 expression might be an important contributor to the ameliorative effect of l. acidophilus on the pathogenesis of colitis as observed in our previous report, because inhibition of il-17 function with a chemical inhibitor or anti - il-17 antibody has been extensively demonstrated to be effective in ibd treatment [2830]. The observed downregulation of il-17 further indicates that administration of l. acidophilus may have modulated the fate of th17 cells . In inflammation, th17 cell differentiation is induced by paracrine signaling and th17 cells are further expanded to achieve full involvement . Accumulating evidence demonstrates that tgf1 is critically involved in th17 cell differentiation, whereas il-23 plays an essential role in maintaining proliferation and survival of th17 cells . Moreover, recent studies have shown that the expression levels of tgf1 and il-23 are both enhanced in ibd [3133], thereby promoting the progression of inflammation by activating th17 differentiation and function . In this study, we showed for the first time that l. acidophilus inhibits the colitis - mediated increase in tgf1 and il-23 expression in mouse colon . These results implicate a decrease in tgf1 and il-23 expression upon l. acidophilus treatment might alter th17 cell population . However, this suspection needs be testified by immunohistochemical staining of th17 cell - specific marker, such as il-17, in colitis treated with and without l. acidophilus . Although we did not attempt to elucidate the molecular mechanisms that mediate the inhibitory role of l. acidophilus in il-23 regulation in the present study, we suspect that reduced expression of tnf may be responsible . In fact, previous studies have clearly shown that nuclear factor- (nf-) b, a critical mediator of tnf signaling, regulates the transcription of the il-23p19 gene . A recent finding by the de vrese lab showed that probiotics bifidobacterium breve and lactobacillus rhamnosus gg (lgg) inhibit lipopolysaccharide- (lps-) activated expression of il-23 in cultured intestinal cells via inhibition of histone acetylation and enhancement of dna methylation, providing another potential mechanism for l. acidophilus - mediated downregulation of il-23 . The results of this study further indicate that the efficacy of different concentrations of commensal probiotics on the expression of proinflammatory cytokines and relevant effectors must be carefully determined . We found that the greatest inhibitory effect was achieved in the c5 or c6 group depending on the molecule examined, which is largely consistent with our earlier observation that the optimal therapeutic effect against disease activity was from the c6 group . Probiotic overdose might break the homeostasis of the gut community and cause aberrant immune response of the gastrointestinal defense system and thus altered expression of cytokine profiles . In conclusion, we showed that oral administration of l. acidophilus suppressed colitis - associated hyper - response of the il-23/th17 axis . Specifically, l. acidophilus treatment inhibited the secretion of proinflammatory cytokine il-17 by downregulating il-23 and tgf1, which are required for th17 cell differentiation and stabilization . These findings indicate that the therapeutic role of l. acidophilus in ibd treatment, at least in part, involves modulating the il-23/th17 immune axis.
Toll - like receptor (tlr) agonists such as endotoxins initiate the synthesis of the inactive il-1 precursor (fig . Although most of the il-1 precursor is in the cytosol, a fraction moves into specialized secretory lysosomes (1). The next step is the conversion of the inactive procaspase-1 to active caspase-1 by a complex of proteins termed the current thinking is that in resting cells procaspase-1 is bound to a large inhibitor molecule, which prevents its activation . During initiation of il-1 synthesis, there is activation of caspase-1, which then processes the il-1 precursor into a mature form ready for secretion . Processing and release are closely linked (fig . 1, c and d). Activation of the nucleotide p2x7 receptor triggers the efflux of potassium ions out of the cell, and within minutes the secretory lysosomes begin releasing their contents of processed il-1 into the extracellular milieu . In support of the role of p2x7, overexpression of the receptor increases the secretion of il-1 (3) and the small peptide ll37 released from activated neutrophils and epithelial cells also stimulates the release of processed il-1 via the p2x7 receptor (5). The efflux of potassium ions signals the influx of calcium ions (3), which in turn activate phospholipases (6). It appears that calcium - independent phospholipase a2 is required for caspase-1 processing in the specialized lysosomes, whereas phosphatidylcholine - specific phospholipase c is required for lysosomal exocytosis and release (6). Dysregulation in any of these steps might account for increased secretion of il-1 and for il-1mediated diseases . Steps in the processing and secretion of il-1. (a) tlr ligands such as endotoxin trigger gene expression and synthesis of the il-1 precursor, which remains diffusely in the cytosol . In the same cell, inactive procaspase-1 is bound to components of the il-1 inflammasome, which contains the products of the nalp-3 gene . The il-1 inflammasome is kept in an inactive state by binding to a large molecular weight putative inhibitor . (b) after tlr signals, there is a transient uncoupling of the inhibitor and nalp-3 gene products from procaspase-1, which then colocalizes with the il-1 in secretory lysosomes . (c) activation of the nucleotide receptor p2x7 by atp or ll37 initiates the efflux of potassium from the cell via a potassium channel . (d) the efflux of potassium ions results in the influx of calcium ions, which in turn activate phospholipases . Phosphatidylcholine - specific phospholipase c (pc - pla-2) facilitates lysosomal exocytosis and secretion of il-1 . Sojia (also known as systemic juvenile rheumatoid arthritis) is a devastating, systemic inflammatory disease that affects growing children for which there are few treatment options other than high dose corticosteroid treatment . In this issue, pascual and colleagues show that blocking il-1 activity with an il-1 receptor antagonist (il-1ra, anakinra) resulted in convincing clinical and hematological responses in nine patients with sojia (7). Resolution of clinical symptoms including fever, marked leukocytosis, thrombocytosis, elevated erythrocyte sedimentation, anemia, and arthritis were rapid and sustained . The efficacy of il-1ra in these children contrasts sharply to that of blocking tnf in sojia . Neutralization of tnf, a successful treatment in some patients with rheumatoid arthritis, is now discredited in sojia since the tnf inhibitors etanercept and infliximab are associated with treatment failures, worsening of disease and/or exacerbations of other autoimmune diseases in these patients . Based on the present study and a similar observation (8), blocking il-1 may become the standard of therapy for sojia . At present, only il-1ra is approved for use in humans, but other agents such as anti il-1 monoclonal antibodies or the il-1 trap molecule (9) reduce il-1 activity and are likely to be effective . The rapid resolution of clinical, hematological, and biochemical manifestations of sojia after a few days of il-1ra treatment is reminiscent of the treatment of refractory adult onset still's disease, a systemic inflammatory disease of adults characterized by similar manifestations of disease seen in sojia . Reduction or complete withdrawal of long - term steroid treatment was achieved without a rebound in disease activity, as is also the case in adult onset still's disease patients treated with il-1ra . For growing children, in addition, il-1ra therapy in rheumatoid arthritis patients does not interfere with tetanus immunization, suggesting that this treatment will not interfere with childhood immunizations . Pascual and coworkers took their investigation of disease mechanisms a step further than most studies . They added 20% serum from four sojia patients or from healthy controls to peripheral blood mononuclear cells (pbmcs) of healthy donors, and changes in gene expression were assessed by microarray analysis (7). Why these four sera of the 16 patients available to the authors were selected is not specified, nor is the reason for using 20% serum . Nevertheless, increases in gene expression of cytokines, cytokine receptors, cell adhesion molecules, and other markers of inflammation were observed in pbmcs incubated with patient sera but not control sera . The authors found that incubating pbmcs with 20% serum from sojia patients increased secretion of il-1 compared with autologous sera . Furthermore, it appeared that the sera from patients with systemic disease induced more il-1 secretion compared with patients with only active arthritis . It may be concluded that 20% serum from patients with sojia contains enough stimulant(s) to increase a portfolio of proinflammatory genes as well as induce secretion of il-1 from healthy pbmcs, and that autologous sera does not contain such stimulants . In my opinion, however, such methods do not support the concept that disease is caused by a circulating factor(s); rather, the effects of sera on cultured cells may be an epiphenomenon . Presence of tlr ligands, such as bacterial lipopolysaccharide, in sojia sera would yield the same results, and a combination of particular serum acute phase reactants could also contribute to the observation . If the authors had added il-1ra to the cultures, they could have at least observed whether the serum stimulant(s) was il-1 itself . The authors also examined steady - state gene expression in pbmcs from 16 sojia patients with active disease and compared the results to pbmcs from 12 healthy children . Many of the same genes induced by sojia sera were spontaneously increased in the pbmcs of these 16 patients . Most notable and significant were the genes encoding il-1, the il-1 decoy receptor, cyclooxygenase-2, tlr-2, and the complement receptor c1q . Pentraxin-3, an il-1inducible gene (11), was also highly overexpressed, and being an acute phase protein, it likely contributes to the high sedimentation rate of red blook cells in sojia . Some genes that were up - regulated by the sojia sera were not increased in steady - state mrna from pbmcs of the 16 patients, including several chemokines and fibronectin, casting doubt on the relevance of the high expression level these genes have in serum studies . Another potentially relevant gene overexpressed in sojia pbmcs is a potassium channel gene kcnj15 . As discussed above, influx of potassium ions is a trigger for activation of caspase-1 and secretion of il-1 in response to activation of the nucleotide receptor p2x7 . Measurement of circulating il-1 is not a reliable indicator of a role for this cytokine in disease, nor does it provide rationale for selection of a therapeutic intervention such as il-1ra . Il-1 is a highly active cytokine in humans; injecting a few nanograms per kilogram results in fever, neutrophilia, thrombocytosis, acute phase proteins, and circulating il-6 (for review see reference 12). Thus, circulating levels of il-1 in the picomolar range may easily escape detection by routine elisas or similar methods . Although there are numerous reports that circulating cytokine levels correlate with severity in a variety of diseases, it is only specific blockade or neutralization of a cytokine that provides a convincing case for causation . For this reason, it is a general concept that il-1mediated disease severity is regulated at the level of ligand production and activity, and not at the receptor level . For example, il-1 type i receptors are expressed on all cells in healthy individuals and increases of only two- or threefold occur in disease . On the other hand, in circulating monocytes and bone marrow macrophages from healthy individuals, il-1 gene expression is absent but increases at least 100-fold when stimulated with microbial products or inflammatory molecules, including products of activated t cells . The total amount of il-1 precursor that is synthesized, however, does not necessarily equate to the amount of active il-1 that is produced, as the caspase-1dependent conversion of il-1 precursor to an active secreted cytokine is a tightly controlled event, despite the presence of constitutive procaspase-1 in the same cell . Hence, the increase in il-1 secretion from pbmcs of sojia patients is a highly relevant observation . The study by pascual and coworkers provides evidence for increased secretion of il-1 by freshly cultured pbmcs from these patients compared with pbmcs from healthy subjects (7). In the absence of exogenous stimulation, cultured pbmcs from healthy subjects do not release il-1, but upon stimulation synthesize the il-1 precursor and release the processed form of il-1 into the supernatant . However, more than 50% of the total il-1 precursor synthesized remains inside monocytes from healthy donors . The amount of il-1 released from pbmcs of five sojia patients was more than 10-fold greater than five healthy controls (7). In the same cultures, the release of tnf and il-6 was similar for healthy and affected subjects, suggesting that the elevated release of il-1 was not due to increased monocyte numbers or increased activation of sojia pbmcs . Unfortunately, il-1 secretion was induced using the unorthodox combination of pma plus ionomycin, whereas most studies of dysfunctional il-1 release use tlr agonists . Nevertheless, the disease - related increase in il-1 secretion may explain the role for il-1 and the responses to il-1ra in these patients who failed to respond to conventional treatments . Increased secretion of il-1 from cultured pbmcs has also been reported for a growing number of inherited, chronic autoinflammatory syndromes, each of which responds to il-1ra (1316). In these syndromes, increased secretion of il-1 is due to a single amino acid mutation in the nalp-3 gene, which controls the activation of caspase-1 found in the il-1 inflammasome (17). Similar to the situation in sojia, circulating levels of il-1 are not detected in these patients (13) but increased secretion of il-1 is observed in vitro . The single point mutation in the nalp-3 gene causes the loss of tight control of il-1 processing . As a result, relatively minor stresses such as exposure to cold results in increased secretion of il-1 with consequent systemic disease (13). It appears that the nalp-3 gene provides an important roadblock to control the secretion of il-1 and raises the issue of a defective roadblock in sojia . Although the reason for the increased secretion of il-1 in the sojia patients remains unclear, both sojia patients and patients with nalp-3 mutations share the phenotype of systemic disease and increased secretion of processed il-1 in vitro . There are chronic inflammatory syndromes without mutations in the nalp-3 gene but nevertheless elevated il-1 release in vitro and also respond to il-1ra therapy; presently, these are mutation - negative neonatal onset multisystem inflammatory disease (nomid; reference 18), pyogenic arthritis, pyoderma gangrenosum, acne syndrome (papa; reference 19), and familial mediterranean fever (fmf; reference 20). Both papa and fmf are genetic diseases associated with the intracellular protein pyrin, which participates in maintaining procaspase-1 as an inactive enzyme . Mutations of the pyrin gene in mice, similar to those found in humans with fmf, result in increased caspase-1 activity and increased secretion of il-1 (21). Although these systemic, multisystem syndromes are not common diseases, they reveal a fundamental role for il-1 in systemic inflammation regardless of the cause . As shown in fig . These are recurrent fevers, neutrophilia, thrombocytosis, elevated serum amyloid a and c reactive protein, and anemia . Hearing loss, developmental delay, and aseptic meningitis can also be observed in childhood . The endothelium is a prime target for il-1mediated inflammation as il-1 receptors on the endothelium trigger prostaglandin e production, cause bone marrow release of neutrophils, and induce the production of il-6 . In fact, il-1 induction of il-6 accounts for hepatic acute phase protein synthesis and the thrombocytosis . The finetuning of il-1mediated inflammation is revealed upon stopping and restarting il-1ra . Upon cessation of il-1ra therapy, clinical signs and symptoms of disease as well as biochemical and hematological abnormalities rebound within days and resolve upon resumption of il-1 receptor blockade (10, 1416). In the pascual study, il-1ra therapy was temporarily withdrawn in two patients due to viral - like illness, during which time the erythrocyte sedimentation rate increased, only to fall upon restarting therapy (7). Systemic manifestations of il-1. Active il-1 is secreted by many cell types including monocytes and macrophages (center). Il-1 enters the circulation and triggers il-1 receptors on the hypothalamic vascular network resulting in synthesis of cyclooxygenase-2, which causes brain levels of prostaglandin e2 to rise, thus activating the thermoregulatory center for fever production (reference 27). In the periphery, il-1 activates il-1 receptors on the endothelium resulting in rashes and the production of il-6 . Circulating il-6 stimulates liver hepatocytes to synthesize several acute phase proteins, which accounts for the increase in erythrocyte sedimentation rate in sojia . Il-1 also acts on the bone marrow to increase mobilization of granulocyte progenitors and mature neutrophils, resulting in peripheral neutrophilia although sojia clearly fits with the clinical, hematological, and biochemical manifestations of the autosomal dominant autoinflammatory syndromes, and although sojia shares with these inherited diseases increased secretion of il-1, no genetic mutations have yet been described in sojia patients that relate to il-1 secretion or any other genetic marker . However, to our knowledge no systematic examination of gene defects in the components of the il-1 inflammasome has been made in sojia . There is no clear pattern of inheritance in sojia, and like other autoimmune diseases, the incidence is greater in families but without identification of a specific gene or mutation . It is likely that other agents that prevent il-1mediated disease will be effective in sojia . These are the il-1 trap, il-1specific monoclonal antibodies, il-1 receptor type i specific monoclonal antibodies, and the caspase-1 inhibitor (22). It is also possible that agents that inhibit the nucleotide receptor p2x7 will reduce il-1mediated disease (23). If sojia is due to dysfunctional control of caspase-1 activity or the p2x7 receptor, treatment options may be targeted to the mechanism of il-1 secretion . It is a general principal in therapeutics to target the most distal mechanism of a disease process . Several levels of control of the synthesis, processing, and secretion of il-1 have evolved, and one may assume that these function to limit inflammation . Moreover, once released, il-1 must contend with competition for receptor occupancy with the naturally occurring il-1ra, the binding and neutralization by the il-1 type ii decoy receptor (24), and the formation of inactive complexes with constitutively secreted soluble il-1 receptor accessory protein (25, 26), each of which also limit il-1 responses . Although increased secretion of il-1 may account for il-1 activity in sojia, loss of control of these additional mechanisms may also be disrupted in sojia.
We quantified the results of entry screening for influenza a(h1n1)pdm09 at auckland international airport . Using the information generated during screening, we retrospectively estimated the number of infected travelers who actually passed through the airport . To estimate the sensitivity of screening, we then compared screening findings with the expected number of infected travelers who passed through the airport . Ethical approval was received from the northern x regional ethics committee of the new zealand ministry of health . The numbers of crew members on inbound international aircraft were estimated by using averages for flights into auckland . The number of travelers detected at each step and referred to the next step of the screening process was obtained from auckland regional public health service records . A confirmed case was one that met the current case definition (as published on the ministry of health website, www.health.govt.nz) and one for which rt - pcr result was positive . We estimated the number of infected travelers screened as the total number of confirmed cases in new zealand during this period, multiplied by the proportion of overseas - acquired cases, and the proportion of international travelers arriving at the airport . On april 30, 2009, nonseasonal influenza a (h1n1) was made notifiable, and these data were collated on the national surveillance database (episurv) (www.surv.esr.cri.nz/episurv). The proportion of infected travelers who acquired the infection overseas was extrapolated from ministry of health records of the first 100 cases of pandemic (h1n1) 2009 because this information was not collected for all travelers with confirmed infection . The proportion of travelers who passed through the airport was determined from statistics new zealand (www.stats.govt.nz) arrivals records . Confidence intervals were calculated by using the online calculator for screening on open epi (4). During the screening period, 456,518 international travelers were screened; 406 (0.09%) of these were referred for medical assessment . Of those, rt - pcr results were located for 89 (82%), among which 4 were positive . The expected number of infected travelers estimated to have passed through the border during the screening program was therefore 69, giving an estimated sensitivity of 5.8% (95% ci 2.3%14.0%). This form of border screening is therefore unlikely to have substantially delayed spread of the pandemic into new zealand in 2009 . Limitations of influenza screening include the high proportion of asymptomatic infected travelers (5), incubation of infections acquired before or during a flight (3), reliance on self - identification, limitations of case definitions, and limitations of thermal scanning (6). Modeling data have shown that the ability of border screening to delay global pandemic influenza is closely linked to the effectiveness of the screening process or travel restriction used . To delay influenza spread by 1.5 weeks, border restrictions need to reduce imported infections by 90% (7). The potential effectiveness of screening arriving travelers to prevent or delay influenza epidemics has been debated . Mathematical models and literature reviews have argued for (7,8) and against (911) this approach . Some authors have found that entry screening for respiratory conditions or influenza a(h1n1)pdm09 is insensitive and not cost - effective (12). This study has several limitations, particularly with regard to estimating the number of infected travelers who would have passed through the airport during the screening period . Most cases of illness acquired overseas would probably not have been notified, particularly those in patients with mild illness who did not see a doctor or who saw a doctor but did not receive a diagnosis . The estimated proportion of overseas - acquired cases was based on data from the first 100 cases and would have decreased as the pandemic progressed . The net effect of these factors is unknown, but they would probably have increased the estimated number of undetected infected travelers passing through screening, thereby further reducing the estimated sensitivity of screening . It might provide public assurance and confidence that something is being done (14). The communication of health information and advice on how to seek treatment is consistently recommended as a pandemic prevention strategy (12,15) and is usually delivered as part of border screening programs . These benefits need to be balanced against the considerable resources used, opportunity cost (resources used for this activity and thereby unavailable for other activities), uncertain effectiveness, and inconvenience of border screening . To delay or prevent influenza entry at borders, influenza screening needs to be considerably more effective than the mostly passive program described here . We hope that during this interepidemic period, a major international review of the role of international air travel in the dissemination of emerging infectious diseases will be conducted to identify effective interventions . Such a review should consider systemwide approaches, including exit screening, standardized health declarations, active screening of individual passengers (including use of rapid laboratory tests and thermal scanning), passenger tracking, policies and practices that support sick travelers wishing to defer travel, and circumstances where airline travel should be suspended entirely.
Idiopathic pulmonary fibrosis (ipf) is a chronic progressive disease with unclear etiology and pathophysiology (1, 2). Its prognosis is poor; survival time after initial diagnosis is only 2.5 - 3.0 yr . There is currently no effective therapy known to improve survival times (3, 4). Numerous studies on the etiology of ipf have identified many possible inciting factors including sawdust, metal particles, smoking, gastroesophageal reflux, and viruses (1). Since ipf is more prevalent in the aged population, it has been hypothesized that the development and progression of ipf may be affected by age - related diseases such as diabetes mellitus (dm), metabolic syndrome, obesity, and cardiovascular disease (5 - 7). Several studies have recently reported relationships between the aforementioned metabolic conditions and ipf . The prevalence of dm ranges from 10% and 32.7% in patients with ipf (5 - 10). Our previous study found a dm prevalence of 25.4% in patients with ipf compared with 13.4% in control subjects, suggesting that dm is related to ipf (11). In 2011, the american thoracic society, the european respiratory society, the japanese respiratory society, and the latin american thoracic association published international evidenced - based guidelines on the diagnosis and management of ipf (12). Those guidelines identify high - resolution computed tomography (hrct) as an essential diagnostic method for ipf . Although the relationship between ipf and dm has been extensively studied, a large - scale national survey has yet to be conducted . Since the korean population almost entirely comprises a single ethnicity, it is uniquely suitable for investigation of the clinical characteristics of a disease . The korean interstitial lung disease (ild) research group retrospectively conducted a national multi - center survey to evaluate the prevalence of ipf and the characteristics of patients with the disease ., we aimed to identify possible risk factors and to examine detailed clinical and radiological characteristics of ipf associated with dm . The korean ild study group affiliated with the korean academy of tuberculosis and respiratory diseases analyzed the medical records of patients who had been newly diagnosed with ipf by pulmonology specialists at 54 large university and training hospitals between january 1, 2003, and december 31, 2007 . Patients with the following clinical conditions were excluded from the study: connective tissue diseases, left ventricular heart failure, pulmonary fibrosis induced by occupational or environmental exposure to chemicals or other known etiologies, or adverse drug reactions . Patients diagnosed with ipf confirmed by lung biopsy and those who met the criteria for ipf according to the 2002 criteria of the american thoracic society / european respiratory society (ats / ers) were included (13)., we analyzed patient age, clinical features, smoking history, concurrent diseases including malignancies, diagnostic methods, time point of diagnosis, pulmonary - function test results, hrct findings, symptoms at the time of initial diagnosis, duration of symptoms, arterial blood gas results, follow - up duration, and survival rate . The diagnosis of dm was established by satisfying one of the following criteria: 1) known dm patient or history of dm and 2) newly diagnosed dm patient before the diagnosis of ipf . Patients were classified as current smokers, former smokers (cessation of smoking for 1 yr), and non - smokers . Hrct findings were interpreted as reticular, honeycomb, ground - glass, or nodular patterns . An asymptomatic patient was defined as one who experienced no clinical symptoms despite abnormal findings on thoracic radiographs . Continuous variables were analyzed using the independent t - test, and categorical variables were analyzed using the pearson's chi - squared test . Chicago, il, usa). A p value of <0.05 derived from a two - tailed test was considered statistically significant . This study protocol was approved by the institutional review board of gachon university gil hospital (irb approval number: 2007/3/29-girba 1652), which deemed informed consent unnecessary . Continuous variables were analyzed using the independent t - test, and categorical variables were analyzed using the pearson's chi - squared test . Chicago, il, usa). A p value of <0.05 derived from a two - tailed test was considered statistically significant . This study protocol was approved by the institutional review board of gachon university gil hospital (irb approval number: 2007/3/29-girba 1652), which deemed informed consent unnecessary . Table 1 shows the distribution of ipf patients with and without dm by clinical features . Of these, 39.1% were diagnosed with ipf by lung biopsy, and 60.9% were clinically diagnosed with ipf . Patients were classified into two groups: those without dm (group a, n = 1,386; 82.3%) and those with dm (group b, n = 299; 17.7%). The mean age and duration of cigarette use for group b patients were 68.0 9.4 yr and 38.5 yr, respectively . There were no significant differences in sex and age between the two groups (p = 0.225 and p = 0.744, respectively). There were no significant differences in arterial blood gas analysis outcome and number years of cigarette use between the two groups . Measurements of pulmonary function did not significantly differ between the time of initial diagnosis and 6 months later . There were no significant differences between the two groups in clinical symptoms at the time of initial diagnosis, such as cough, sputum, hemoptysis, and chest pain . Exertional dyspnea occurred in 87.6% of group a patients and 80.1% of group b patients . Coughing was present in 72.6% of group a patients and 76.6% of group b patients . The prevalence of rheumatoid factor (rf) and antinuclear antibody (ana) were not significantly different between the two groups . On hrct, reticular patterns were observed in 58.6% of group a patients and 67.2% of group b patients, a statistically significant difference (p = 0.014). Honeycomb lung patterns were observed in 71.4% of group a patients and 78.9% of group b patients, a statistically significant difference (p = 0.028). However, there were no significant differences in ground - glass and nodular patterns between the two groups . We analyzed data on hypertension, cardiovascular disease, pulmonary tuberculosis, lung cancer, and other malignant disease including stomach cancer (n = 26), bladder cancer (n = 7), colon cancer (n = 4), hematologic malignancy (n = 4), esophageal cancer (n = 3), biliary duct cancer (n = 3), laryngeal cancer (n = 2), and cervical cancer (n = 5) (table 3). Hypertension was present in 18.5% of group a patients and 37.5% of group b patients, and the difference was statistically significant (p = 0.000). Cardiovascular disease was present in 2.3% of group a patients and 10.7% of group b patients, a difference that was also statistically significant (p = 0.000). However, pulmonary tuberculosis was not significantly associated with ipf in either group . As for malignant tumors, lung cancer was not significantly associated with either group, whereas the difference in other malignant tumors was significant: 4.0% of group a patients and 7.0% of group b patients (p = 0.039). Dm was significantly less prevalent in patients diagnosed by lung biopsy (p = 0.039). Multivariate analysis was performed to determine the risk factors for group b patients (table 4). The significant risk factors included hypertension (odds ratio [or], 2.529; 95% confidence interval [ci], 1.920 - 3.325), other cardiovascular diseases (or, 1.904; 95% ci, 1.209 - 3.000), and malignant tumors excluding lung cancer (or, 1.811; 95% ci, 1.060 - 3.093). Approximately 40.3% of group a patients and approximately 41.5% of group b patients survived, a statistically insignificant difference . The mortality rate was 25.3% in group a and 21.4% in group b; the difference was not statistically significant . The survival rate was not significantly different between the two groups, as estimated using the kaplan - meier method (p = 0.207). Although the survival time of patients with ipf is as short as 2.5 to 3 yr, there is no effective therapy that improves survival (3, 4). It has been reported that dm and cardiovascular diseases such as hypertension, the prevalence of which increases with age, particularly beyond 60 years, affect the progression and prognosis of ipf (5 - 7). Many studies have demonstrated a correlation between dm and ipf (7 - 10, 14). However, few studies have investigated the clinical characteristics, pulmonary function, underlying condition, and prognosis of ipf with concurrent dm . It is important to identify the conditions related to ipf and to evaluate its characteristics to improve management of the disease . To the best of our knowledge, this is the first large - scale study of the characteristics of ipf associated with dm and the relationships between ipf and other clinical entities . In this study, 17.8% of ipf cases (7) reported a prevalence of pulmonary fibrosis associated with dm of 32.7%, but this high percentage could be due to the fact that theirs was a single - institution study . The prevalence of dm in ipf patients has also been reported as 11.3% (9) and 10% (10). In the present study, group b patients underwent lung biopsy less frequently than did patients in group a (p = 0.039). This difference may be explained by fewer lung biopsies being taken in diabetic patients due to the higher risk of complications . The 2011 ipf statement recommends that ipf be confirmed by the presence of a uip pattern on hrct . The uip pattern usually consists of reticular abnormalities and honeycombing, with or without traction bronchiectasis . In this study, group b patients showed typical patterns on hrct images; reticular and honeycomb patterns were observed more frequently (p = 0.014 and p = 0.028, respectively). (15) have documented that ground glass patterns are typically noted in patients with non - specific interstitial pneumonia and hyperreactive pneumonitis, with nodular patterns less common in patients with ipf . Previous studies have indicated that reactive oxygen species (ros) and advanced glycation end products (age) produced by hyperglycemia contribute to fibrosis of various organs, such as the lungs and kidneys (17 - 23). In our study, typical patterns were observed more frequently in group b than in group a. this might have been secondary to damage to the lungs caused by dm or hyperglycemia . Such damage may induce destructive lung parenchyma, seen as reticular and honeycomb patterns on hrct . The prevalence of hypertension and cardiovascular disease was significantly higher in group b than in group a (37.5% vs 18.5%, p = 0.000 for hypertension; 10.7% vs 2.3%, p = 0.000 for cardiovascular disease). It has been reported that the prevalence of cardiovascular disease increases in patients with ipf (24), and numerous studies have supported these results (25 - 27). (25) reported that 186 of 630 patients who underwent lung transplantation had concurrent cardiovascular disease . (27) suggested that patients with ipf have a higher risk of cardiovascular disease . Our results indicate that cardiovascular disease is more frequently found in group b patients . In our study, malignant tumors, lung cancer excluded, were more frequently found in group b than in group a. generally, the prevalence of lung cancer in patients with ipf was significantly higher, at 6.6% (28 patients). However, the difference between group a and group b patients was not statistically significant . This result suggests that smoking may be more closely related to lung cancer than ipf is . Clinicians must be aware of the possibility of cancer development in patients with ipf based on the finding that malignant tumors, excluding lung cancer, occurred more frequently in group b patients . In this study, the risk factors for idiopathic pulmonary fibrosis were reticular patterns on ct images (or, 1.888; 95% ci, 1.87 - 3.281), hypertension (or, 2.527; 95% ci, 1.920 - 3.325), cardiovascular disease (or, 1.904; 95% ci, 1.209 - 3.000), and malignant tumors other than lung cancer (or, 1.811; 95% ci, 1.060 - 3.093). The survival rate was not significantly different between the two groups (p = 0.207). In this study, there were no significant differences between the groups in age, smoking history, and lung cancer . These results differ from those of previous studies that did not include ipf patients as controls . In our study, reticular and honeycomb patterns on ct images were more common in group b patients, and the frequencies of hypertension, cardiovascular disease, and malignant tumors other than lung cancer were higher in group b. therefore, early prevention and treatment of concurrent diseases can slow the progression of ipf, improving the prognosis . Since patients treated between 2003 and 2007 were included in our study, not all have been reached for follow - up . Second, this study was based on earlier, 2002 ats - ers guidelines for diagnosis of ipf and on retrospective study . Therefore, this study may have involved atypical ipf features on hrct . Third, the interpretation of hrct images may have differed in quality among centers, as this study was very large and involved many korean domestic hospitals . Further prospective studies are needed to confirm our results using the new ipf guidelines . In conclusion, our study indicates that ipf patients with dm are more likely to have a typical interstitial pneumonia (uip) pattern on hrct, including reticular and honeycomb patterns, than are ipf patients without dm . The prevalence of hypertension, cardiovascular disease, and other malignancies, excluding lung cancer, is significantly higher in ipf patients with dm than in ipf patients without dm.
Perioperative dental damage is one of the most common anesthesia - related adverse events and is responsible for the greatest number of malpractice claims against anesthesiologists.1 dental injuries includes enamel fracture or tooth subluxation to more severe forms of crown fractures and tooth avulsion . Anterior dislocation of mandible condyle leading to a locked position of temporomandibular joint, tongue injuries, and a variety forms of pressure - induced lesions of the oral cavity soft tissues have also been reported.2 as per retrospective data available, perioperative damage to dental tissues varies from 0.02% to 0.07% . Though the incidence is reported much higher in prospective studies . In a study conducted by chen et al ., the incidence of damage to dental structures before or after anesthesia using endotracheal tube was 12.1% . In a survey by lockhart et al ., the incidence of trauma to dentition during tracheal intubation was 1:1000.3 in order to decrease the incidence of dental trauma related to inubation, measures to protect the teeth have been recommended . Dental trauma due to small forces applied at wrong angles during laryngeal blade contact can be prevented by placing the dental shield . Use of dental shields during intubation might become mandatory in the days to come as a standard for ideal clinical practice . As of today, only 2% of anesthesiologists use dental shields.4 most often anesthetist comes to know of the traumatic dental incident immediately . Even the relatives and patient takes a note of the injury as the tooth is placed in a position that is very sensitive and being noticed very easily . It was found by newland et al . That anesthesia provider discovered trauma to teeth in 86% of the reported incidents while only 14% of the patients reported the injury.3 whenever a tooth is fractured during endotracheal intubation, the dental surgeon should be called to ascertain the degree of damage and to make an exact count of the broken tooth fragments . If all the broken pieces are not retrieved, then radiograph of the abdomen and chest should be taken to rule out aspiration into stomach or respiratory passage.5 the present study was taken among anesthesiologists working in private hospitals to evaluate the risk factors for oro - dental injuries to occur during general anesthesia and the measures taken by anesthesiologists to prevent them . A questionnaire was prepared with six questions to explore the most probable risk factors for iatrogenic oro - dental injuries and preventive measures taken by anesthetists to minimize them (table 1). For each question, subject matter expert raters of our institution evaluated the content validity of the prepared questionnaire . They examined if the preventive skills or knowledge of injuries measured by different set of questions is essential for the questionnaire to be effective . The data thus collected were coded and analyzed using statistical package medcalc (version 12.7.0.0). Questionnaire along with response to evaluate the risk factors for oro - dental injuries to occur during general anesthesia and measures taken by anesthesiologists to prevent them . About 65% of the anesthesiologists felt laryngoscope is the main reason for dental trauma which mostly occurs at the time of intubation (80%). Around 40% felt that poor visibility to hypopharynx carries more risk for dental injury . Very few anesthesiologists (5%) were using mouth guards to prevent direct impact on the tooth . Around 65% of anesthesiologists felt the need of pre - operative dental consultation in order to minimize injuries to oral and perioral tissues . Though anesthesiologists always operate intraorally, they might not have extensive education of oral, perioral tissues, and intraoral prostheses . The chances of perioperative dental injury increase with poor dentition along with associated anesthesia - related risk factors.3 in the present study, 80% of anesthetists felt that the dental injury occurs at the time of intubation . Similar results have been found in a study by monaca et al.4 endotracheal intubation with laryngoscope blades was first performed using straight blades . Curved laryngoscope blade was introduced by macintosh in 1943.1 most of the anesthetist marked laryngoscope as the anesthetical equipment which causes dental trauma . High incidence of dental insult following conventional laryngoscopy has also been reported by mouro et al.6 during laryngoscopy, the blades of the laryngoscope might hit against the tooth or anesthetist might use the tooth as fulcrum to depress the tongue thereby causing damage to the teeth.2 around 30% marked oropharyngeal airway as the cause of dental trauma . Oropharyngeal airway can cause injury during extubation stage as patient might involuntarily bite on airway and it might act as a fulcrum to cause insult.3 bite blocks are used during the recovery phase to prevent mouth closure . Ideally bite blocks should be placed in the molar region . Wrong placement of bite block can cause damage to the anterior tooth.5,7 son et al . Had reported a case in which bite block placement during intubation led to intrusive luxation of the tooth.5 in the present study, around 5% had experienced dental trauma due to the use of bite block, most probably due to the wrong use of bite block . Most of the anesthetists marked that maxillary left central incisor is most prone for injury . Some studies have also shown injuries occurring in the lower tooth.6,8 this finding could be attributed mostly to the path of insertion of anesthetic equipment that comes in contact with the tooth . Mouth guards can help in preventing dental injury as it distributes the forces uniformly over the dentition . The limitation with its use is that it the direct view of glottis and makes it difficult to guide the endotracheal tube to larynx.3 this could be the reason that very few anesthetist in the present study opted for mouth guard as a measure to prevent dental injuries . Similarly in a study by tiku et al ., only 6% of anesthesiologist used mouth guard perioperatively.8 people planned for the elective surgical procedure under general anesthesia should be sent for a pre - operative dental checkup if difficult intubation is anticipated or if an anesthetist finds a mobile tooth and/or ill - fitting dental prosthesis . Moreover, the patient should be informed of his poor dental condition.3 in the present study, only 65% of anesthetists felt the need of dental consultation while 35% of did nt ask for a dental opinion . Further studies can be carried out to find relation between different laryngoscopy techniques and dental traumain the present study, only upper anterior tooth were assessed for dental injury, further studies can be done where posterior tooth and mandibular incisors are also examinedstudies can also be planned to evaluate the effectiveness of different commercially available mouth guards in reducing the impact on teeth with a laryngoscope . Further studies can be carried out to find relation between different laryngoscopy techniques and dental trauma in the present study, only upper anterior tooth were assessed for dental injury, further studies can be done where posterior tooth and mandibular incisors are also examined studies can also be planned to evaluate the effectiveness of different commercially available mouth guards in reducing the impact on teeth with a laryngoscope . Further studies can be carried out to find relation between different laryngoscopy techniques and dental traumain the present study, only upper anterior tooth were assessed for dental injury, further studies can be done where posterior tooth and mandibular incisors are also examinedstudies can also be planned to evaluate the effectiveness of different commercially available mouth guards in reducing the impact on teeth with a laryngoscope . Further studies can be carried out to find relation between different laryngoscopy techniques and dental trauma in the present study, only upper anterior tooth were assessed for dental injury, further studies can be done where posterior tooth and mandibular incisors are also examined studies can also be planned to evaluate the effectiveness of different commercially available mouth guards in reducing the impact on teeth with a laryngoscope . A detailed examination of the patient's mouth pre - operatively can help in identifying the teeth at risk for damage . Anesthesiologists must take a detailed history of the dental procedures done in a patient such as prosthesis, veneers or crowns . Maxillary and mandibular anterior are more prone for injuries hence these teeth should be properly inspected for any mobility or crown fractures.9,10 the patient should be made aware of his poor dental status and all the findings should be documented . If the teeth are teeth grade iii mobile, it would be a wise option to get them extracted before any elective surgeries under general anesthesia are planned.
Environmental enrichment (ee) is an experimental protocol classically defined as a combination of complex inanimate and social stimulation and which provides animals with the opportunity to attain high levels of voluntary physical activity on running wheels and to enhance exploration, cognitive activity, and social interaction . Several studies point out that animals reared in ee show marked brain changes at functional, anatomical, and molecular levels [213] and in particular changes in plasticity factors and mechanisms [14, 15]. Ee can indeed be used as a noninvasive strategy to modulate brain plasticity throughout life; ee can accelerate the development of the central nervous system [1618], can reopen plasticity windows in the adult cortex [19, 20] and causes a significant improvement in learning and memory [14, 2123], especially evident in aged animals [22, 2432], or in animal models of neurodegenerative diseases [3335]. Declarative memories depend initially on the medial temporal lobe system, including the hippocampus, but, over days to weeks, as these memories mature, they become increasingly dependent on other brain regions such as the neocortex [3639]. This process of time - dependent gradual reorganization of the brain regions that supports remote memory storage and underlies the formation of enduring memories, is known as system - level memory consolidation or system consolidation [40, 41]. It has been demonstrated that the progressive stabilization of long - lasting memories is due to the reactivation of hippocampal - cortical connections [42, 43] and the strengthening of corticocortical connections, involving cortical plasticity mechanisms [41, 4448]. Despite all the evidences showing that ee enhances cortical plasticity and learning and memory, there is no evidence on whether and how ee could affect the process of time - dependent system consolidation . This study aimed at testing whether ee could affect the system - level memory consolidation process using a spatial memory paradigm and characterizing the time course of hippocampal and of cortical activation following recall of progressively more remote memories . Spatial memory is a declarative type of memory . The hippocampus plays an essential role in the formation of spatial memories [4953]; subsequently, spatial memories become, in a gradual manner, additionally dependent on other cortical regions . Many studies point out that the privileged final storage site for remote spatial memories is the prefrontal cortex (pfc) and in particular the medial pfc (mpfc), including the anterior cingulate, prelimbic, and infralimbic cortices [47, 48, 54, 55]. Recently, however, it has been suggested that remote memories recall involves the coordinated activation of a broader network of cortical brain regions [5658]. We have therefore characterized the time course of activation of a number of cortical areas in addition to the mpfc . We found that ee not only induces an earlier recruitment of mpfc, but also induces the progressive activation of a distributed cortical network that is not activated in standard housed mice . Male and female c57bl/6 mice of 2 months of age were used in this study . The animals were housed in an animal room with a 12 h/12 h light / dark cycle, with food and water available ad libitum . At 2 months of age, the animals were assigned to one of the following rearing conditions for 40 days: environmental enrichment (ee, n = 24) or standard condition (sc, n = 24). The sc rearing condition consisted of a 26 18 18 cm cage housing 3 animals . The ee rearing condition was achieved using a large cage (44 62 28 cm) containing several food hoppers, one running wheel for voluntary physical exercise, and differently shaped objects (tunnels, toys, shelters, stairs) that were repositioned twice a week and completely substituted with others once a week . Two additional groups of control animals, age and gender matched to sc and ee groups, were housed in home cage standard condition (hc - sc, n = 8) or in home cage enriched condition (hc - ee, n = 7), and they did not perform any behavioural task . The hidden platform version of the mwm test was performed . A large water tank of 120 cm of diameter was filled with white opaque water at 22c . An escape platform of 11 cm of side was submerged 1 cm below the water surface and placed in the center of the sw quadrant . The platform was maintained in this position for all the swim trials through the test . Mice were trained to swim to the platform in 4 daily trials, starting in pseudorandomly varied locations, with a 30 min interval, during 7 consecutive days . The trial was complete once the mouse found the platform or 60 sec had elapsed . If the mouse failed to find the platform on a given trial, the experimenter guided the mouse onto the platform . Once reaching the platform, each mouse was allowed to rest for 20 s on it . After each trial each mouse was returned to its home cage where it rested until the next trial . After the completion of training, spatial memory was assessed in a probe test; a recall probe trial was performed after 1, 10, 20, 30, and 50 days after the end of learning . We used an automated tracking system (noldus ethovision xt) for recording behavioural data from training and probe tests . For each trial we measured the latency (in sec) to reach the platform, the total distance (in cm) swam in order to reach the platform, and the average swim speed (in cm / s). For each probe trial we measured the amount of time spent in the target zone (23 cm in radius, centered on the location of the platform during training) and the average time spent in three other equivalent zones in each quadrant [55, 60]. Mice were anaesthetized and perfused via intracardiac infusion with 0.1 m pbs and then 4% paraformaldehyde (pfa, dissolved in 0.1 m phosphate buffer, ph 7.4) 90 min after the completion of behavioral testing . Brains were removed, fixed overnight in pfa, and then transferred to 30% sucrose solution and stored at 4c . Coronal sections were cut at 40 m thickness on a freezing microtome (sliding leica microtome sm2010r, leica microsystems), and free - floating sections were prepared for immunohistochemistry . After a blocking step in 10% ngs and 0.5% triton x-100 in pbs, sections were incubated in a solution containing 1% ngs, 0.3% triton x-100, and anti - c - fos primary rabbit polyclonal antibody (1: 3000 rabbit anti c - fos polyclonal antibody, calbiochem, usa) for 36 h at 4c . Subsequently, sections were transferred in a solution containing 1% ngs, 0.1% triton x-100, and 1: 200 anti - rabbit biotinylated secondary antibody (vector labs) in pbs . This was followed by incubation in abc kit (vector labs) and final detection with dab reaction kit (vector labs). Sections were finally mounted on gelatinized slides, dehydrated, and sealed with dpx mounting medium (vwr international, uk). Counting of c - fos positive cells in different brain areas was performed using a ccd camera (mbf bioscience, germany) mounted on a zeiss axioskop (zeiss, germany) microscope and the stereo- investigator software (mbf bioscience). Brain structures were anatomically defined according to a mouse brain atlas (paxinos and franklin), and the regions of interest selected for measurement of c - fos - positive nuclei were (numbers indicate the distance in millimeters of the sections from bregma) infralimbic cortex (il, + 1.94 mm); secondary motor cortex (m2, + 0.98 mm); anterior cingulate cortex, area 1 and area 2 (acc, + 0.98 mm); dentate gyrus (dg, 1.94 mm); ca1 field of dorsal hippocampus (dca1, 1.94 mm); ca3 field of dorsal hippocampus (dca3, 1.94 mm); posterior parietal association cortex (ppta, 1.94 mm); primary auditory cortex (au1, 3.16 mm); primary visual cortex (v1, 4.16 mm); medial entorhinal cortex (ment, 4.16 mm). The number of c - fos - positive cells was counted at 20x magnification, following a blind procedure, in 1040 fields (50 50 m or 100 100 m) per section according to the size of brain structure and their density calculated (cells / mm), using at least 5 sections for each structure ., and all statistical analysis were performed using statistical software package sigmastat . For mwm performance in the learning phase, a two - way analysis of variance (anova) for repeated measures (rm) was performed, considering both factor housing condition (ee or sc) and factor learning day, with post hoc analysis holm - sidak method . Performance in each probe test was compared with one - way anova across circular zones (target zone versus the average of other zones) for each housing condition . The c - fos protein expression in each area was analyzed with a two - way anova for housing condition factor and probe test day factor, with post hoc analysis holm - sidak method . To test whether ee can affect the system consolidation process, we trained c57bl/6 mice, housed in different conditions (standard condition, sc n = 24 or environmental enrichment ee n = 24), in a spatial learning task, using the morris water maze, and we analyzed the following parameters referred to the average of 4 daily trials, during 7 consecutive days: latency to find the platform (s), total distance swam (cm), and mean swim speed (cm / s). For the distance swam and the latency to reach the platform during acquisition, a significant learning effect for both housing conditions was found, but not a significant difference between the two groups (two - way rm anova, for latency p = 0.016; for distance swan p <0.001). For the latency parameter only, we found a housing condition day interaction: multiple comparisons showed that the main differences resided on days 4 and 5 (figure 1(a)) (two - way rm anova, post hoc analysis holm - sidak method, for day 4 p = 0.005; for day 5 p = 0.001). We also measured the mean swim velocity throughout the test, in order to exclude differences in navigation speed (data not shown): we observed a significant decrease in the mean swim velocity through the test (two - way rm anova, p <0.001), but neither a difference between housing condition (p = 0.276) nor a housing condition day interaction (p = 0.163). Spatial memory was evaluated in a probe test in which the hidden platform was removed . We performed recall probe tests at 1, 10, 20, 30, and 50 days, and we quantified exploration in the target zone, a circular zone (radius: 23 cm) in quadrant where the platform was placed during training, and the average time spent in three other equivalent zones in each quadrant, for sc and ee mice (figure 1(b)) [55, 60]. We found a significant difference between target zone versus the others in probe tests at 1, 10, 20, and 30 days, for both groups (see figure 1(b) for details). After the probe test, mice were sacrificed and the protein c - fos was immunolabeled as an indicator of neuronal activity . C - fos expression was calculated as the density of number of c - fos - positive cells in mm . First we investigated c - fos expression in the hippocampus, the structure that is responsible for the formation of long term spatial memory [4953]. Levels of c - fos expression in the hippocampus of control mice (home cage mice, hc - ee and hc - sc mice) did not differ between housing conditions (hc - sc n = 8; hc - ee n = 7; one - way anova, p = 0.736); levels of c - fos protein for ee and sc mice were significantly greater than those in their home - cage controls at all retention intervals, (two - way anova, post hoc analysis holm - sidak method, all p values <0.05), suggesting that the hippocampus is involved both in the formation and delayed recall of the spatial memory . We found a similar c - fos expression in the ee and sc mice in all probe tests (two - way anova, post hoc analysis holm - sidak method for housing condition, p = 0.731). We then focused on the dorsal hippocampus (dhcp), known to be specifically involved in spatial memory; again, we observed the same c - fos expression pattern in ee and sc group; activation increased with increasing retention interval up to 30 days (see figure 2 for details). The results for c - fos expression in the mpfc, the final memory storage site in the cortex, show that both the acc and the il have the same time course of c - fos protein expression pattern; c - fos expression at 1 day did not differ from that in home cage animals, both for ee and sc mice, and there was no difference between ee and sc or hc - ee and hc - sc mice (two - way anova, post hoc analysis holm - sidak method, all p values> 0.05); however, starting from the probe test at 10 days, c - fos expression was greater in ee group than hc - ee and sc groups, with a further increase at 20 days (see figure 3 for details); only for ee animals did the c - fos protein expression differ from that of hc control animals . For the probe tests at 30 and 50 days we found that the c - fos expression in sc animals differed from that of hc - sc animals; values of sc and ee groups did not differ (two - way anova, post hoc analysis holm - sidak method; see figure 3 for details). To examine the time - dependent reorganization of neuronal activation in a brain - wide manner, we observed c - fos protein expression in other cortical areas that are important for the construction of spatial maps . Using several techniques, wang et al . Determined that distinct areas of extrastriate visual cortex are gateways for ventral and dorsal streams in the mouse the dorsal stream includes the network hippocampus medial entorhinal cortex posterior parietal cortex for spatial navigation; in addition, the dorsal stream is connected to auditory cortex and to frontal areas, such as cingulate cortex, infralimbic cortex, and motor areas . First we investigated c - fos expression in ment and in ppta, and we found that activation at 1 day in both areas was similar in ee mice, sc mice, and did not differ from that in their home - cage controls (two - way anova, post hoc analysis holm - sidak method, all p values> 0.05); however, in the other probe tests performed, significant differences between ee and sc mice and between ee and hc - ee mice were found (see figure 4 for details). Then, we observed c - fos expression in v1 and m2, for they are connected to the dorsal stream and there is a direct monosynaptic connection between motor and visual cortices . We found that ee group was statistically different from sc and hc - ee groups in m2, only in probe test performed at 20, 30, and 50 days; instead, in v1, we did not find any difference between ee and sc group, only an increase in c - fos expression for the late retention delays in both groups (see figure 5 for details). Finally, we investigated c - fos expression in au1, a sensory cortex not supposed to be involved in spatial learning, and we demonstrated that activation in this area was similar in all groups (see figure 6 for details). In this study, we provide the first evidence that ee can affect the time - dependent spatial memory system consolidation . C57bl/6 mice, housed in standard or in enriched condition, were subjected to spatial learning and then tested up to 50 days after learning to evaluate consolidation process . Using the expression of c - fos protein as an indicator of neuronal activity in a brain - wide manner we have found indications for a difference both in the time course and in the network of cortical areas recruited for recent and remote recall between ee and non - ee animals . In agreement with previous studies [47, 55] there was a progressive increase in c - fos protein expression in both acc and il, as consolidation process proceeded . We showed that ee induces an earlier recruitment of acc and il with respect to sc mice; these areas were recruited following recall of spatial memory in the ee group as early as 10 days after learning, while they were recruited only 30 days after learning in sc mice . The final storage site in the cortex could be the acc, while the il could correlate with motivational aspects of performance, encoding other significant aspects of the environment, such as salient landmarks or preferred locations . The acc was found to be activated after remote memory recall in a number of tasks [47, 48, 54, 55], and, conversely, inactivation of the acc disrupted recall of remote five - arm discrimination, contextual fear, and mwm memories . The acc is highly interconnected to other prefrontal regions and is reciprocally connected to sensory, motor, and limbic cortices [67, 68]; therefore, this connectivity places the acc in favorable position, raising the possibility that this region coordinates retrieval of remote memories stored in distributed cortical networks . The earlier recruitment of acc and il in ee animals could imply an earlier independence of spatial memory recall from hippocampal activation in ee animals . Indeed, in animals provided with running wheels, a component of ee, block of hippocampal activation ceased to block recall of contextual fear memory at shorter time distance from learning with respect to sedentary animals . In addition, we demonstrated that ee induces the involvement of a distributed cortical network in supporting remote spatial memory which is not activated in non ee animals . We observed that ment and ppta were activated following memory recall at 10 days in ee group . Both areas were included in the dorsal network for spatial navigation; the entorhinal cortex contains a spatial representation of environment and plays an interface role between the hippocampus and neocortex; instead, the parietal cortex, specifically the multisensory posterior region, translates coordinate information from spatial maps in the entorhinal cortex and hippocampus into egocentric representations [59, 71]. We also investigated c - fos protein expression in v1 and m2, and we found that ee group showed a greater activation in m2 than sc group, for probe test performed at 20, 30, and 50 days . In v1, instead, we did not find any difference between ee and sc group, only an increase in c - fos expression for the late retention delays in both groups; a recent study showed that v1 could belong to the network of fear contextual memory, although its activation did not change between recent and remote memories . In our study, the v1 pattern activation could be induced by its engagement in attentional process on account of the spatial complex task . The involvement of m2 and v1 is not surprising since they are connected to the dorsal stream, and a direct monosynaptic connection between motor and visual cortices was identified . For the hippocampus we found no difference between ee and sc animals . Our results are consistent with the idea that hippocampus is responsible for encoding spatial memory [4953]; its activation in remote spatial memory recall is not in agreement with studies that showed a progressive reduction in hippocampus activation with increasing retention interval [47, 48, 54], though it is in line with the hypothesis that remote memory never becomes totally independent from the hippocampus . In a more recent study, indeed, lopez et al . Demonstrated that hippocampus recruitment in the recall of remote memories was influenced by the environmental conditions, such as cue saliency and complexity of the task in which memories are initially formed and subsequently recalled; thus the rich spatial details and the complexity of the training in mwm could account for the hippocampal activation found also for remote memory recall . Moreover, it has been found that that precise real - time inhibition of the dorsal ca1 region, using optogenetic method, was sufficient to impair remote recall . We found that ee mice were faster, considering latency parameter, in learning the position of the target platform in comparison to sc mice, but there was no significant difference between groups in the probe tests . These results are consistent with other studies in the literature which used, in rodents living in ee or provided with running wheels, intensive learning protocol for the mwm such as that used by us (4 daily trials for 7 consecutive days). We decided to use a behavioural protocol that maximizes the results of spatial learning task because our purpose was to examine possible differences between ee and sc groups in the time course of system consolidation without confounding effects due to differences in the results of learning, that is in the probe test, and also to be confident in the formation of a remote memory . The results of c - fos data are an indication that the same result (a successful probe test) can be obtained through a different balance of hippocampal and cortical activation during system consolidation . Also in the maviel and bontempi paper response accuracy in animals tested on either day 1 or 30 was similar while cortical activation strongly differed . The faster recruitment of cortical areas found in ee animals and the activation of a distributed cortical network, involving prefrontal and other associative cortices, activated in ee but not in sc animals, could suggest that the quality of the recalled memory is different in the two groups of animals; indeed, activation of prefrontal cortex has been correlated with development of the ability to create a memory that is vivid and rich in contextual details in humans and activation of associative sensory cortices supports memory storage and retrieval of sensory stimuli that have acquired a behavioral salience with the experience . How could ee act on the recruitment of cortical networks during system consolidation? It has been proposed that new neurons generated in the dg become functionally integrated into existing neural circuits; in fact the spatial training when new neurons are more receptive to surrounding neuronal activity favored their subsequent recruitment upon remote memory retrieval [77, 78]. Thus, these tagged adult - generated neurons, once mature, are recruited into hippocampal networks underlying remote spatial memory representation . Therefore tampering with the level of hippocampal neurogenesis could interfere in the hippocampus - only dependent period of memory . Indeed, it has been demonstrated that new hippocampal neurons were recruited into neuronal networks supporting retrieval of remote spatial memory and that the enhanced neurogenesis by voluntary running - wheel exercise sped up the disengaging from hippocampus . Since ee was found to increase hippocampal neurogenesis and promote the survival of newly generated neurons, it is plausible that ee may accelerate the recruitment of extrahippocampal areas . In its turn, ee action on hippocampal neurogenesis is likely mediated by neurotrophins, such as brain - derived neurotrophic factor (bdnf), or by insulin growth factor - l (igf-1) [9, 10], which affect hippocampal neurogenesis and hippocampal and cortical plasticity [14, 79, 80]. Igf-1 plays an important role in cell growth and development, and it also upregulates neurogenesis in the adult hippocampus [8183]. In the adult brain, igf-1 has been shown to mediate both the neuroprotective effects of physical exercise and the enhancement caused by exercise in hippocampal plasticity and in learning and memory . Moreover igf-1 mediates the increased expression of bdnf subsequent to ee and physical exercise [8486]. Bdnf has been shown to regulate adult hippocampal neurogenesis, to mediate ee effects on it, to modulate plasticity during learning by activating signaling pathways that modify local synaptic targets and have long - term effects on transcription, and to mediate the expression of hippocampal ltp, in both the early and late phases [80, 83, 87, 88]. Another nonexclusive possibility is that molecular mechanisms could tag the activated synapses in hippocampal and cortical networks at the time of memory encoding; this early tagging could guide the reactivation of proper hippocampal - cortical connections associated with the specific memory . A recent study showed indeed that cortical tagging seems to be highly specific for precise memory trace and the impairment of early cortical tagging tampers with the postacquisition hippocampal - cortical dialogue, preventing the formation of remote memory . Moreover, they demonstrated that early tagging triggers specific signaling cascades, leading to histone - tail acetylation in the cortex and that the histone deacetylase inhibitor improves remote memory retrieval . Thus, early tagging acts on epigenetic modification that could mediate remote memory formation and retrieval, so ee could modulate the long - term memory formation and consolidation through chromatin remodeling, such as histone - tail acetylation, known to be increased in ee.
Anthocyanins are members of the flavonoid group of phytochemicals, a group predominant in teas, honey, wines, fruits, vegetables, nuts, olive oil, cocoa, and cereals . The flavonoids, perhaps the most important single group of phenolics in foods, comprise a group of over 4000 c15 aromatic plant compounds with multiple substitution patterns (www.nal.usda.gov/fnic/foodcomp/index.html). The primary players in this group include the anthocyanins (eg, cyanidin, pelargonidin, petunidin), the flavonols (quercetin, kaempferol), flavones (luteolin, apigenin), flavanones (myricetin, naringin, hesperetin, naringenin), flavan-3-ols (catechin, epicatechin, gallocatechin), and, although sometimes classified separately, the isoflavones (genistein, daidzein). Phytochemicals in this class are frequently referred to as bioflavonoids due to their multifaceted roles in human health maintenance, and anthocyanins in food are typically ingested as components of complex mixtures of flavonoid components . Daily intake is estimated from 500 mg to 1 g, but can be several g / d if an individual is consuming flavonoid supplements (grape seed extract, ginkgo biloba, or pycnogenol; see, eg,). The colorful anthocyanins are the most recognized, visible members of the bioflavonoid phytochemicals . The free - radical scavenging and antioxidant capacities of anthocyanin pigments are the most highly publicized of the modus operandi used by these pigments to intervene with human therapeutic targets, but, in fact, research clearly suggests that other mechanisms of action are also responsible for observed health benefits [2, 3, 4, 5]. Anthocyanin isolates and anthocyanin - rich mixtures of bioflavonoids may provide protection from dna cleavage, estrogenic activity (altering development of hormone - dependent disease symptoms), enzyme inhibition, boosting production of cytokines (thus regulating immune responses), anti - inflammatory activity, lipid peroxidation, decreasing capillary permeability and fragility, and membrane strengthening [6, 7, 8, 9, 10]. The chemical structure (position, number, and types of substitutions) of the individual anthocyanin molecule also has a bearing on the degree to which anthocyanins exert their bioactive properties [11, 12] and the structure / function relationships also influence the intracellular localization of the pigments . The anthocyanin literature includes some controversy over the relative contributions of glycosylated anthocyanins versus aglycones in terms of bioavailability and bioactive potential [7, 13, 14, 15, 16]. Originally, it was assumed that only aglycones could enter the circulation circuit, however, absorption and metabolism of anthocyanin glycosides has now been demonstrated . The nature of the sugar conjugate and the aglycone are important determinants of anthocyanin absorption and excretion in both humans and rats . The roles of anthocyanin pigments as medicinal agents have been well - accepted dogma in folk medicine throughout the world, and, in fact, these pigments are linked to an amazingly broad - based range of health benefits . For example, anthocyanins from hibiscus sp have historically been used in remedies for liver disfunction and hypertension; and bilberry (vaccinium) anthocyanins have an anecdotal history of use for vision disorders, microbial infections, diarrhea, and diverse other health disorders [17, 18, 19]. But while the use of anthocyanins for therapeutic purposes has long been supported by both anecdotal and epidemiological evidence, it is only in recent years that some of the specific, measurable pharmacological properties of isolated anthocyanin pigments have been conclusively verified by rigorously controlled in vitro, in vivo, or clinical research trials . In many other cases, the exact roles of the anthocyanins in human health maintenance versus other phytochemicals in a complex mixture from a fruit extract or whole food have not been completely sorted out . In fact, some reports suggest that anthocyanin activity is potentiated when delivered in mixtures [20, 21, 22]. For example, visual acuity can be markedly improved through administration of anthocyanin pigments to animal and human subjects, and the role of these pigments in enhancing night vision or overall vision has been particularly well documented . Oral intake of anthocyanosides from black currants resulted in significantly improved night vision adaptation in human subjects, and similar benefits were gained after administration of anthocyanins from bilberries . Three anthocyanins from black currant stimulated regeneration of rhodopsin (a g - protein - coupled receptor localized in the retina of the eye), and formation of a regeneration intermediate was accelerated by cyanidin 3-rutinoside . These studies strongly suggest that enhancement of rhodopsin regeneration is at least one mechanism by which anthocyanins enhance visual acuity . In both in vitro and in vivo research trials, anthocyanins have demonstrated marked ability to reduce cancer cell proliferation and to inhibit tumor formation [27, 28, 29, 30]. The capacity of anthocyanin pigments to interfere with the process of carcinogenesis seems to be linked to multiple potential mechanisms of action including inhibition of cyclooxygenase enzymes and potent antioxidant potential . Hou et al revealed that anthocyanins inhibit tumorigenesis by blocking activation of a mitogen - activated protein kinase pathway . This report provided the first indication of a molecular basis for why anthocyanins demonstrate anticarcinogenic properties . In other research, fruit extracts with significant anthocyanin concentrations proved to be effective against various stages of carcinogenesis [18, 28, 31, 32], but the individual role of anthocyanins versus other components was not determined, in part because the anthocyanins were too easily degraded during bioassays if separated from stabilizing cofactors such as other phenolic constituents . The role of anthocyanins in cardiovascular disease protection is strongly linked to oxidative stress protection . Since endothelial dysfunction is involved in initiation and development of vascular disease, four anthocyanins isolated from elderberries were incorporated into the plasma lemma and cytosol of endothelial cells to directly examine the protective roles . These tests demonstrated not only that anthocyanins could be directly incorporated into endothelial cells, but that significant oxidative stress protection was the result . Delphinidin, but not malvidin or cyanidin, provided endothelium - dependent vasorelaxation in the rat aorta, providing a pharmacological benefit comparable to red wine polyphenolics . In a rat model, little influence of feeding purified anthocyanins (cyanidin 3-o - glucoside) or anthocyanin - rich extracts from elderberry or blackcurrant could be detected on cholesterol levels or fatty acid patterns in liver, but the pigments were capable of sparing vitamin e . Crude anthocyanin extracts from bilberry have been administered both orally and via injection to reduce capillary permeability . In other research related to cardiovascular impairment, the roles of anthocyanin pigments versus other flavonoids delivered in the phytochemical extract have not been completely sorted out . Protection from heart attacks through administration of grape juice or wine was strongly tied to the ability of the anthocyanin - rich products to reduce inflammation and enhance capillary strength and permeability, and to inhibit platelet formation and enhance nitric oxide (no) release . Similarly, delivery of a black currant concentrate with intense anthocyanin content caused endothelium - dependent vasorelaxation in rat aorta rings in vitro . The mechanism of vasorelaxation was attributed to increased levels of no production, but the active compounds in the concentrate were not isolated . When rats were pretreated to create increased susceptibility to oxidative damage, then fed anthocyanin - rich extracts, significant reduction in indices of lipid peroxidation and dna damage resulted . Ingestion of these extracts, which contained mixtures of delphinidin, cyanidin, petunidin, peonidin, and malvidin in the 3-glucopyranoside forms, also increased plasma antioxidant capacity . Tsuda et al recently provided evidence that anthocyanins extracted from purple corn, when provided to mice in tandem with a high - fat diet, effectively inhibited both body weight and adipose tissue increases . Typical symptoms of hyperglycemia, hyperinsulinemia, and hyperleptinemia provoked by a high - fat diet did not occur when mice also ingested isolated anthocyanins . The experiments suggest that anthocyanins, as a functional food component, can aid in the prevention of obesity and diabetes . Anthocyanins have been credited with capacity to modulate cognitive and motor function, to enhance memory, and to have a role in preventing age - related declines in neural function . Cho et al reported that administration of isolated semipurified anthocyanins from purple sweet potato enhanced cognitive performance as assessed by passive avoidance tests in ethanol - treated mice, and also effectively inhibited lipid peroxidation in rat brain tissues . By administering blueberry extracts with significant anthocyanin content (but not purified pigments), it was noted that the blueberry - supplemented diets led to effective reversal of age - related deficits in various neural and behavioral parameters (memory and motor functions). Further investigations by this laboratory team demonstrated that anthocyanins (in particular, cyanidin-3-sambubioside-5-glucoside and cyanidin-3, 5-diglucoside) were highly bioavailable in endothelial cells, which was linked to their roles in prevention of atherosclerosis and neurodegenerative disorders [34, 41]. Anthocyanins exerted multiple protective effects against pleurisy in a rat model and were capable of attenuating inflammation . The antimicrobial activity of anthocyanins in general has been well established, including significant inhibition of aflatoxin biosynthesis . The experimental evidence demonstrating anthocyanin benefits for diabetes and pancreatic disorders has also accumulated in recent years, and again the efficacy is attributed to the multiple, simultaneous biological effects these pigments cause in the body, including prevention of generation of free radicals, decreased lipid peroxidation, reduced pancreatic swelling, and decreased blood sugar concentrations in urine and blood serum [43, 44]. An enigma is defined as anything that perplexes because it is inexplicable, hidden, or obscure; something that serves as a puzzle to be solved . The whole realm of anthocyanin consumption and human health fits into this definition, because several aspects of anthocyanin's pharmacological roles have remained elusive to the scientist . In most of the interventions of anthocyanins in human health, details on the mechanisms of action for bioactivity, uptake, absorption, bioavailability, whole body distribution, and tissue localization are still not fully elucidated . There are at least four primary obstacles that have impeded the formulation, by medical professionals, of robust dietary or prescriptive guidelines on consumption of anthocyanins . Probably the most complicated piece of the puzzle is that, in terms of biological activity in the human body, an anthocyanin pigment is (almost) never acting independently . Typically, anthocyanins and other flavonoid components, or anthocyanins and other nonflavonoid phytochemicals, are interacting together in order to provide full potency . Interactions between phytochemicals within a plant are a key evolutionary strategy for the host plant . There are over 4000 flavonoids described, with multiple substitution patterns and often large complex structures in the mixtures . Bioflavonoids like anthocyanins occur in mixtures within edible foods and are ingested in mixtures . The anthocyanins and related flavonoids are secondary products typically produced by plants as defensive, protective, or attractive agents, and it makes good evolutionary sense for the plant to use a variety of strategies and multiple fronts of attack to accomplish these functions, rather than single compounds to which a pathogen or predator could become resistant . This same multiplicity in bioactive phytochemical synthesis is also a bonus for animals and humans who ingest the plant material donors, and benefit from the interaction of the flavonoids with therapeutic targets . When the interactions between co - occurring phytochemicals are positive (eg, additive effects or synergies), they are called potentiating interactions . In other cases, components in the donor plant can actually inhibit the bioactivity of the flavonoid compound (eg, pectin interference with antioxidant capacity in in vitro assays), and in other cases, concomitant compounds which are not themselves bioactive may work together with a bioflavonoid to enhance bioavailability or absorption . Synergy among flavonoids including anthocyanins has been cited as responsible for antiplatelet activity of red wine and grape juice, with strong interactions between components of grape skin and grape seed required to potentiate antiplatelet activity in human and animal systems . Co - occurring flavonoids working synergistically to antagonize hydrogen peroxide formation are most effective in depressing platelet function . Traditional bioprospecting approaches, which search for single purified plant - derived compounds as a means of drug discovery, will not capture the full potency of a plant extract when multiple potentiating interactions are responsible for bioactivity . Another common well - recognized handicap to scientists exploring the bioactive properties of the flavonoids, and the second part of the anthocyanin puzzle, is the fact that these phytochemicals can be of an evanescent nature . The susceptibility of anthocyanins to oxidation and degradation is one of the concerns of food processors who wish to maximize the shelf life of products enhanced with natural pigment colors . In particular, many of the classic phytochemical methods (including column chromatography), used to extract from plant tissues and fractionate components out of a crude extract, can degrade anthocyanins and/or inactivate them during purification steps . As a result, research that aims to identify bioactive entities and gauge potencies of extracts can easily fail to assess the actual sources of biological activity in situ . Strict attention to the ephemeral nature of some flavonoid constituents in berries (especially during extraction / fractionation sequences) led to the adaptation of a vacuum chromatography technique in our laboratory, which was designed to (as much as feasible) preserve the integrity of the compounds and keep natural mixtures intact until final separation for purposes of identification [32, 47]. Using whole individually - quick - frozen berries as a starting point, fruits are extracted in a waring blender with 70% aqueous acetone (2l solvent kg fruit) then filtered through cheesecloth . Acetone is removed from the filtrate under vacuum in a 40c water bath, and water is then removed by lyophilization, resulting in a dark purple powder . A portion of the crude dry extract is then redissolved in water and poured over a toyopearl resin polymer column for vacuum chromatography . Vacuum chromatography on toyopearl with a series of solvents (water, 50% meoh, 100% meoh, 100% acetone, and 50% acetone) elutes 5 tp fractions which are then concentrated under vacuum, and water is removed by lyophilization . Sugars are very quickly and efficiently removed in the first fraction, which greatly simplifies the handling and analysis of remaining fractions . Once bioactive fractions are identified, a second, third, and subsequent rounds of separation are accomplished on silica gel, also by vacuum, sometimes open column gravity chromatography . At each step of the procedure, isolated mixtures are compared using silica gel thin layer chromatography and 2 spray reagents (vanillin - hcl and dichromate reagent) in order to gauge the composition and number of components in each fraction . In general, this fractionation strategy has permitted rapid separation of relatively large volumes of extract, with less exposure to damaging and expensive solvents, less exposure to column support materials and air, minimal losses, and reliable separation of flavonoids . In tandem with all of the fractionations is a consistent sequence of bioassays (for multiple stages of carcinogenesis) because the fractionation scheme is bioactivity - guided . As fractions become more highly purified, analysis with hplc, hplc - ms, and nmr can be used to conclusively determine the origins of the bioactivity . A third piece of the puzzle is the inducible nature of many of the bioactive flavonoids including anthocyanins . As is true of a plethora of secondary plant products, the initial production and accumulation of phytochemicals is triggered by physical or chemical microenvironmental triggers, usually a stress factor . A researcher intent on maximizing production of anthocyanin pigments must recognize the induction factors and deliberately elicit production of bioactive flavonoids by providing these stimuli to the plant material of interest . Elicitation mimics stresses that provoke secondary product formation in nature, and activates otherwise dormant biochemical pathways . This triggering of productivity can, of course, be very difficult to accomplish in a field setting, but can be accomplished reliably in controlled growth facilities . The final puzzle piece in the anthocyanin enigma is the inability of the scientist or medicinal practitioner to track metabolic progress of anthocyanins after ingestion, due to the plethora of metabolic breakdown products that are rapidly produced in situ . There is substantial current interest in the quest to follow the transport of bioflavonoids through the body, to determine absorption and bioavailability, and to see where breakdown products accumulate and for how long . However, since these phytochemicals are highly metabolized after consumption of anthocyanin - rich foods or supplements, metabolic tracking has not been possible . Despite active research and increasing interest in the realm of natural products and health maintenance, there is a paucity of information on the absorption, biodistribution, and metabolism of anthocyanins and interacting flavonoids . Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body . While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present . The development and optimization of plant cell culture systems which reliably and predictably synthesize anthocyanins in a controlled environment has provided a unique and useful model for in - depth research on anthocyanins, and has helped at least in part to circumvent the obstacles presented in all four cases of the anthocyanin enigma as described above . Callus and cell suspension cultures from a wide and diverse range of plant genera have been cultivated to produce anthocyanin pigments in vitro [48, 49, 50, 51, 52, 53, 54, 55, 56]. In most of these past reports, the overall goal of the plant cell culture production system was to explore an alternative resource for natural plant pigments, for possible use as food colorants . More recently, some anthocyanin - producing plant species have been intensively cultured in vitro in order to harvest the bioactive pigments and related phytochemicals as medicinally - active compounds [47, 57, 58, 59]. By controlling both the physical and the chemical microenvironment of the plant cell cultures, anthocyanin production could in many cases be boosted to higher concentrations than available in the parent plant in vivo . Some of the most intensively - researched cell culture production systems used selections from the genus vitis (grape), where scaled - up bioreactor - based systems approached semicommercial productivity [60, 61]. The cell culture systems can be quite stable, and many have been selected for high anthocyanin yield and lack of dependence on irradiance . Anthocyanin profiles from cell cultures do not necessarily mirror the profiles from the parent plant, and isolation of pigments from the simplified cell culture tissues is substantially more streamlined than from complex fruit or vegetative tissues [47, 53, 58]. This simplicity can be a particular advantage for investigation of the health properties of bioflavonoids including anthocyanins . In most cases, the systems begin with vegetative plant materials (leaves, petioles, stems) and not fruit tissues . Explants from in vivo plants are surface - disinfested and introduced into cell culture to produce rapidly proliferating callus, then cell suspension cultures, which are eventually induced to produce flavonoids (usually with a trigger such as light, elevated carbohydrate, changed nitrogen profile, or elicitation with a fungal extract or other chemical elicitor). Because cell culture anthocyanin production systems are comprised of simple tissues which can be engineered to reliably and predictably accumulate pigment, these systems circumvent many of the obstacles in the anthocyanin enigma . Interactions between potentiating phytochemicals are still in force in cell culture systems, but because the tissues are much simpler to extract, the nature of phytochemical interactions is much easier to sort out and to quantitatively test . Cell cultures permit rapid and efficient isolation without many of the interfering compounds (pectins, excess polysaccharides, enzymes) that can complicate extraction or bioassay from fruits . Aqueous extracts of an anthocyanin - producing sweet potato line exhibited higher potency (antiproliferative and antimutagenic potential) than extract from field - grown crops . Similarly, when antioxidant capacity of cell cultures and various fruit extracts were compared side by side in a galvinoxyl free radical assay, the potency of the cell culture extract was substantially higher than that of all fruit extracts, and only grape seed proanthocyanidins exhibited higher activity . Because these other substances are not present, the flavonoids are much easier to isolate without the degradation that can occur rapidly when isolating slowly from complex, recalcitrant fruit or vegetative tissues . While the flavonoid content of a fruit may comprise only 1% or less of the total substance, a cell culture can be crafted to accumulate much higher concentrations of flavonoids, in the range of 20%30% by volume . Many flavonoids, in particular, high - molecular weight proanthocyanidin oligomers or complex anthocyanin isomers, are either not available commercially or prohibitively expensive . By producing these phytochemicals in volume in cell cultures, cell cultures are a superb model system for testing the effects of elicitation on the inducible bioflavonoid genes, which is a means of resolving yet another aspect of the anthocyanin enigma . In fact, elicitation of cell cultures (using chemical or environmental triggers to production) is a recognized and efficient means of maximizing anthocyanin pigment towards commercialization of product recovery [55, 56, 62, 63], and since the in vitro production environment is rigorously controlled, investigators have the opportunity to test multiple elicitation triggers without interference from uncontrolled environmental conditions in field settings . Perhaps the most significant advantage to investigation using in vitro anthocyanin - accumulating cell cultures is that the cultures can serve as a vehicle for delivery of isotopic labels (13c or 14c) to the metabolizing cells while the pigments are being biosynthesized [59, 64, 65]. By using a radioisotope - labeled source of compounds, an administered phytochemical can be included in a defined diet and can be discerned from preexisting, endogenous sources of the same phytochemical or breakdown product . These large molecules must be synthesized in planta . In this emerging research area, radiolabel or isotopic label has been introduced to metabolizing cell cultures using a carbohydrate source (sucrose or glucose) or a precursor which is much further along the metabolic pathway to anthocyanin synthesis, such as phenylalanine . Levels of incorporation range between 15% and 30%, and levels achieved now allow organ and neuronal localization of the c - labeled compounds and monitoring using autoradiography and scintillation counting . Accelerator mass spectrometry (ams) technology will even permit monitoring of small levels in human systems . With these innovations, it is clear that the effective use of cell - culture - produced anthocyanins can now elucidate previously hidden roles of anthocyanin pigments in human health and metabolism.
In a number of reviews on the induction of bone formation, we have often asked how bone differentiation by induction is initiated? Or how the soluble molecular signals of the transforming growth factor- (tgf-) supergene family are deployed so as to initiate de novo bone formation by induction [13]? Somehow more simply but very confusing in a scenario of redundancy of multiple soluble molecular signals initiating bone differentiation by induction in the primate, which are the molecular signals that initiate de novo bone formation by induction [110]? To be truthful, we have always assigned a prominent and pivotal role to the osteogenic proteins (ops) of the tgf- supergene family [4, 5]. There is no bone formation by induction without the osteogenic soluble molecular signals of the tgf- supergene family [110]. We have, however, always assigned additional prominent critical roles to biomimetic matrices capable of delivering the biological activity of the osteogenic soluble molecular signals [110]. To initiate the induction of bone formation and thus to ultimately erect the skeleton, nature has had a powerful lesson to teach . A lesson that is continuously taught to biomaterial scientists, molecular biologists and tissue engineers alike, who wish to design, manufacture and sculpt new tissue constructs for replacement of lost parts in human patients [2, 5, 1117]. The induction of bone formation requires three key components: an osteoinductive soluble signal, an insoluble signal or substratum and responding host s cells . The insoluble signal delivers the osteogenic soluble molecular signal and acts as a scaffold for bone formation to occur after transmembrane serine - threonine kinase receptors phosphorilation of responding host s cells [1, 12, 14, 16, 17]. The osteogenic soluble molecular signals of the tgf- supergene family need thus to be reconstituted, more figuratively perhaps, recombined, with an insoluble signal or substratum to initiate the cascade of bone differentiation by induction [912, 1719]. This fundamental rule in molecular and cellular biology has now become the cardinal rule to initiate tissue morphogenesis after the molecular dissection of the fascinating phenomenon of bone: formation by autoinduction, though regretfully not always completely understood . The classic experiments of the dissociative extraction and reconstitution of the bone matrix components showed that partially purified [1, 2124] or highly purified recombinant human bone morphogenetic proteins (bmps) [25, 26] need to be reconstituted with an insoluble signal or substratum to trigger the cascade of bone differentiation by induction . The experiments were also critical to learn that putative bmps within the bone matrix could be solubilized by the dissociative extraction of the bone matrix [17, 21, 22]. This has set into motion a ripple - like cascade of biochemical, chromatographic and molecular events that ultimately has resulted in the isolation, characterization and molecular cloning of an entirely new family of protein initiators collectively called the bmps / ops [1719, 2527]. When writing about osteoinduction, it is important to properly define the terminology related to bone: formation by autoinduction. The acid test of the induction of bone formation is the de novo generation of endochondral bone after heterotopic extraskeletal implantation of the osteogenic soluble molecular signals of the tgf- supergene family [5, 17, 18]. The heterotopic implantation site avoids the ambiguities of the orthotopic site where some degree of bone formation by conduction may occur from the viable bone interfaces . The classic studies of sacerdotti and frattin, huggins, levander [30, 31], bridges and pritchard, moss, trueta, urist, urist et al . And reddi and huggins, have shown that several mineralized and non - mineralized extracellular matrices of mammalian tissues including uroepithelium, bone and dentine contain morphogenetic signals capable of initiating de novo bone formation by induction [3, 5]. The osteogenic activity, as defined by different authors [31, 37, 38], resides thus within the extracellular matrices of different tissues and when implanted in heterotopic extraskeletal sites of animal models, this osteogenic activity diffuses out of the implanted matrices interacting with transforming resident mesenchymal cells capable of differentiation into chondroblastic and osteoblastic cell lines initiating bone differentiation by induction [3, 5, 17, 19]. The dissociative extraction and reconstitution of the bone matrix components and of the homology of bmps / ops among mammals have provided the key to implement the phenomenon of the induction of bone formation in pre - clinical and clinical contexts [1719]. Before implantation, recombinant human bmps / ops, either hbmp-2 and/or hop-1, [39, 40] need to be reconstituted with a variety of biomaterial matrices to form the osteogenic device for orthotopic orthopedic and craniofacial applications in clinical contexts [25, 17, 3941]. Alternative approaches are continuously under laboratory and pre - clinical evaluation, i.e. Directing the differentiation of stem cells into the osteogenic lineage firstly in vitro and then in vivo when implanted in skeletal defects of animal models [42, 43]. Additional approaches are based on the manipulation of a deliberately created space within the body, such that it serves as an in vivo bioreactor . Osteogenesis forms within the surgically manipulated space of the bone bioreactor, which deploys the essential ingredients to induce bone formation, i.e. Osteogenic soluble molecular signals, responding cells and the extracellular matrix scaffolds of the operated patients . The in vivo bone bioreactor has available a mesenchymal layer rich in pluripotent cells endowed with the potential to rapidly differentiate into osteoblastic - like cells secreting bone matrix remodelled into newly formed bone for autogenous transplantation . Custom - made bone grafts grown in extraskeletal sites in human beings by hop-1 and marrow aspirated from the iliac crest have been implanted in the latissimus dorsi muscle . Went further by demonstrating that with a single recombinant op combined with a porous hydroxyapatite (ha) carrier, a prefabricated heterotopic bone graft could be constructed in a human outside the skeleton without the addition of cortical bone, bone marrow aspirates or any other bone precursors to engineer a custom - made prefabricated bone flap for human mandibular reconstruction (fig . 1). Preparation and surgical harvest of a prefabricated mandibular graft in the chest of a human patient after combining hop-1 with interpore blocks of porous hydroxyapatite . (a) l - shaped blocks of coral - derived porous hydroxyapatite inserted into the pectoralis major muscle . (b) skeletal scintigraphy demonstrating osteoinduction in the l - shaped prefabribated graft (arrow). (c and d) flap design and mobilization of the prefabricated flap raised as a pectoralis major myo - osseohydroxyapatite flap pedicled on the thoracoacromial artery before transplantation into the recipient left mandibular region . Since the purification and cloning of multiple molecularly different bmps / ops, confirmation of osteoinductive activity has been characterized for naturally derived and several human recombinant bmps / ops in different animal models [3, 5, 17, 19, 25, 26]. Importantly and conclusively, in the bioassay for bone induction in rodents, the tgf- isoforms do not initiate the induction of bone formation . In marked contrast, however, the mammalian tgf- isoforms do induce rapid and substantial endochondral bone formation when implanted in heterotopic extraskeletal sites of papio ursinus[3, 5, 18, 48]. In the primate, the tgf- isoforms may act upstream to the bmps / ops and may induce the induction of heterotopic bone by expressing bmps / ops - related gene products resulting in the cascade of bone differentiation by induction [4951]. Heterotopic implantation of the three mammalian tgf- isoforms results in expression of bmp-3 and op-1 as evaluated by northern blot [49, 50] and rt - pcr analyses . The presence of several molecularly related but different proteins with osteogenic activity in the primate [3, 5, 18] poses important questions about the biological significance of this apparent redundancy, additionally indicating multiple interactions during both embryonic development and the induction of bone formation in postnatal life [5, 18]. We have shown a potent synergistic induction of endochondral bone formation after binary applications of hop-1 and tgf-1 in both heterotopic and orthotopic sites of the non - human primate papio ursinus[48, 49]. The level of tissue induction induced by hop-1 was raised several fold by the binary application of comparatively low doses of the tgf-1 isoform implanted in the rectus abdominis muscle of adult baboons . The rapid induction of large corticalized ossicles in the rectus abdominis muscle of non - human primate species using binary applications of osteogenic molecular signals has important clinical implications . In the human, the primate homo sapiens, regenerative phenomena are slower than in several other animals including non - human primate species . In human patients, the rapidity of tissue morphogenesis complete with mineralization of the outer cortex and bone marrow formation by binary application of recombinant hop-1 and relatively low doses of htgf-1 indicates that osteogenesis and bone induction in homo sapiens should rather be engineered by the synergistic induction of bone formation [3, 52, 53]. The latter overcomes the temporally delayed repair phenomena in human patients where healing progresses slower than in experimental animals including non - human primate species [3, 48, 52, 53]. A most fascinating and novel strategy to initiate the induction of bone formation is to construct biomimetic bioactive biomaterial matrices that per se initiate the morphogenesis of bone . This is initiated within the porous spaces of smart self - inducing biomaterial matrices even when implanted in heterotopic extraskeletal sites and without the addition of osteogenic soluble molecular signals of the tgf- superfamily (fig . 2) [2, 3, 5, 7, 27, 54, 55]. Morphology of tissue induction and regeneration by geometric cues of smart biomimetic matrices implanted heterotopically in the rectus abdominis muscle of adult baboons papio ursinus without the addition of exogenously applied osteogenic proteins . (a and b) induction of bone formation (magenta arrows) in concavities of highly crystalline porous hydroxyapatite implanted in the rectus abdominis muscle of adult baboons and harvested on day 30 . (c) to determine thus the critical role of specific geometries of the substratum, slurry preparation of hydroxyapatite powders were sintered to form solid monolithic discs (20 mm in diameter) of highly crystalline hydroxyapatite with concavities of 1600 m in diameter and 800 m in depth (blue arrow). (d and e) tissue sections of harvested specimens on day 30 from the rectus abdominis were used to immunolocalize bmp-3 (d) and op-1 (e) embedded into the biomimetic matrices (blue arrows). Bone formation by induction thus forms by day 30 (f) facing a highly vascular tissue within the concavity; bone formation increases by day 90 (g) with prominent vascular invasion (h) perforating the fibrous tissue enveloping the implanted disc (blue arrows) and targeting the newly formed bone within the concavity of the biomimetic matrix . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . After millions of years of evolution, nature has had the capacity to nucleate and evolve highly sophisticated tissues and organs . A classical example is the evolution of the skeleton, thus providing for the emergence of the vertebrates, deambulation and body erection freeing the upper limbs for superior foraging and more industrious homo like activities including the use of tools for hunting, foraging above all, however, for maternal care contributing thus to the speciation of the genus homo ultimately directing the emergence of homo sapiens . The emergence of osteogenesis and later in evolution of the vertebrates, of the skeleton and homo sapiens after a billion years from drosophila melanogaster has permitted the precision self - assembly of the bone unit or osteosome and the remodelling processes of the skeleton [57, 58]. The concavities as sculpted in the bio - inspired biomimetic bioceramics biomimetize the remodelling cycle of the osteonic cortico - cancellous bone (fig . Biomimetism of the concavity induced by osteoclastogenesis during the remodelling cycle of the osteonic bone with the induction of bone in concavities of the porous biomimetic substratum even if implanted in heterotopic extraskeletal sites and without the addition of exogenously applied osteogenic proteins . (a and b) sculpted concavities by remodelling cycles in ancient hominid skeletal fossilized remains of cortico - cancellous bone of the australopithecus africanus, the man ape of southern africa, temporally confined on faunal and paleomagnetic grounds to 2.5 to 3 million years before the present . Arrows (magenta) indicate the extent of osteoclastic activity within the trabecular bone of the extinct hominid; blue arrows point to calcite crystals occupying the cancellous spaces of the fossilized cortico - cancellous remains of a. africanus . (c, d and e) cortico - cancellous bone remodelling and osteoclastogenesis (blue arrows) in extant non - human primate papio ursinus . Concavities induced by osteoclastogenesis along trabeculae of bone biomimetize the concavities prepared in biomimetic bioceramics setting formation after resorption (magenta arrows in d and e), (f) self - inducing the spontaneous induction of bone formation (blue arrows) in biomimetic matrices when implanted in heterotopic extraskeletal sites of an adult baboon papio ursinus . (f) arrows indicate the rich vascular network and capillary sprouting that always correlate with the induction of bone formation (magenta arrows) within the porous biomimetic matrices . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . Nature has relied on common yet limited molecular mechanisms to direct morphogenesis and the emergence of specialized tissues and organs [3, 5, 1719]. The bmps / ops reflect nature s parsimony in controlling multiple specialized functions or pleiotropy, deploying several osteogenic molecular signals with minor variation in amino acid motifs within highly conserved carboxy terminal regions [2, 5]. The pleiotropic activity of the bmps / ops gene products is vast and spans from neurothropism to nephrogenesis, from cementogenesis to chondrogenesis, from air follicle induction to tooth morphogenesis, from angiogenesis to neurogenesis and from osteogenesis to cardiogenesis . The pleiotropic activity of the secreted proteins indicates that they are critical in development and are involved in several unrelated events that control pattern formation in embryonic development, morphogenesis and regeneration in postnatal life [3, 5, 1719]. The evolutionary conservation of the ops of the tgf- superfamily is superbly demonstrated by the remarkable observation that recombinant decapentaplegic and 60a proteins, gene products of the fruit fly drosophila melanogaster, induce bone formation in mammals . Decapentaplegic and 60a in d. melanogaster are highly homologous to bmp-2, bmp-4 and bmp-5 and bmp-7, respectively, indicating the primordial role of bmps / ops sequences for the emergence of the vertebrates [5, 60]. Nature has thus usurped phylogenetically ancient amino acid sequences deployed for dorsal - ventral patterning in d. melanogaster to set the unique vertebrate trait of the induction of bone and of the skeleton rather than evolving novel gene products initiating the induction of bone formation [5, 60]. The skeleton with its supramolecular assembly of structural proteins, collagens and vascular structures permeating the osteonic walls bathed by the bone marrow organ is a superior example of design architecture and engineering . The skeleton has evolved through millions of years of evolution and the extant skeleton has appeared as a result of expression and secretion of complex soluble molecular signals . Molecular signals interacting with insoluble signals or substrata of the extracellular matrix of bone populated by several different responding cells within the bone bone / marrow organ . The remodelling of the skeleton, the formation of bone by osteoblasts and the resorption of bone by osteoclasts, is a closely integrated homeostatic system . When thinking about the molecular cell biology of bone remodelling and maintenance, it is important to visualize the geometric pattern of the remodelling cycle as initiated in any given time on each trabecula of the cortico - cancellous bone during the remodelling cycle (fig . 3). The sequential phases of bone remodelling in the primate cortico - cancellous bone are quiescence, remodelling activation, resorption, reversal, formation / induction and quiescence again [5658]. Remodelling of the cortico - cancellous bones entails, at any given time along the trabeculae of bone, three fundamental molecular, cellular and morphological processes that characterize the remodelling cycle of the primate cortico - cancellous bone: (i) resting, i.e. Surfaces lined by resting lining cells as yet to be differentiated; (ii) resorption, i.e. Areas of trabeculae actively resorbed by activated osteoclasts . Of critical importance for tissue engineering of bone, the bone resorption lacunae as formed by osteoclastogenesis are in the form of concavities (fig . 3) [8, 9, 5759]. The concavities are thus regulators of bone initiation and deposition during the remodelling processes of the skeleton . Biomimetic matrices of highly crystalline has [27, 55] or biphasic ha/-tricalcium phosphate (tcp) bioceramics constructed with a series of repetitive concavities offer a geometric configuration which vividly reproduces and biomimetizes the bone remodelling processes of primate osteonic bone (fig . 3) [5, 8, 9, 27, 55, 59, 61]. In several experiments in the rectus abdominis of the non - human primate papio ursinus, we have bioassayed discs of highly crystalline ha and biphasic ha/-tcp with concavities of specific dimensions on both planar surfaces (fig . Concavities were thus prepared to mimic the supramolecular assembly of the rigid mineralized extracellular matrix of primate osteonic bone biomimetizing self - assembling geometric cues de novo initiating bone formation by induction [5, 810, 61]. Independently, other research groups using different bioceramics and animal models reported the intrinsic induction of bone formation [6269]. De groot hypothesized that after heterotopic implantation of calcium phosphate ceramics in vivo, there is de- and re - mineralization processes that may concentrate and immobilize the experimental animal s own naturally derived bmp / op complex. Whether bmps / ops attached to biomimetic matrices implanted heterotopically in animal models are originating from systemic circulation and/or extracellular matrices invading the porous spaces and concavities or de novo expressed in cellular elements resting on the biomimetic matrices, has been elucidated by northern blot analyses showing the local expression of mrna of osteogenic soluble molecular signals in tissues formed within the concavities of the implanted substrata . Concavities are endowed with the striking prerogative of de novo initiating bone differentiation by induction in heterotopic extraskeletal sites of adult non - human primates papio ursinus (figs . 2, 3 and 7) [5, 27, 55, 61]. Our systematic studies have shown that the driving force of the intrinsic induction of bone formation by bioactive biomimetic matrices is the shape of the implanted substratum; the language of shape is the language of geometry; the language of geometry is the language of a sequence of repetitive concavities which biomimetizes the remodelling cycle of the primate osteonic bone (figs . 2, 3 and 7) [5, 8, 9, 55, 61, 71]. Biomimetism of the concavity, the shape of life: construction of biomimetic matrices and the induction of bone formation in highly crystalline sintered porous hydroxyapatite matrices when implanted in the rectus abdominis muscle of adult baboons without the exogenous application of osteogenic proteins of the tgf- superfamily and harvested on day 90 after implantation . (a, b, c and d) low - power views of sintered bioceramics with a series of repetitive concavities with substantial bone formation across the porous spaces . (e and f) detail of bone formation by induction within the biomimetic concavities of the highly crystalline sintered biomatrix together with prominent angiogenesis and vascular invasion . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . The term biomimetism has been recently introduced in the lexicon of regenerative medicine and tissue engineering with the intended meaning of the creative imitation of various specific biological systems [7274]. Biomimetism is particularly appealing in the creation and assembly of biomaterials matrices which are endowed with specific smart functionalities as imparted by the biomimetic organization coupled to the bioactive matrices . Biomaterial biomimetic matrices biomimetize specific biological systems with multifunctional shape memories with self - assembly capacities initiating and promoting angiogenesis and osteogenesis . The induction of angio - osteogenesis exploits and recapitulates events that initiate the induction of bone formation . Bone forms in the concavities as initiated by osteoclastogenesis during the remodelling cycle of the osteonic cortico - cancellous bone of non - human and human primates (figs . 2 and 3) [5, 9, 17, 61, 71]. In collaboration with the council for scientific and industrial research division of materials science and technology we have thus encapsulated into solid matrices repetitive sequences of concavities as biologically programmed morphogenetic cues that result in the differentiation of resident mesenchymal cells into osteoblastic - like cells expressing, secreting and embedding soluble osteogenic molecular signals into the concavities initiating bone formation by induction as a secondary response (fig . We have proposed that there is a direct spatial and temporal relationship of molecular and morphological events that emphasize the pronounced biomimetism of the geometric induction of bone formation, i.e. The induction of bone in smart concavities assembled in biomimetic matrices with the remodelling cycles of cancellous bone (fig . The basic multicellular unit of the cortico - cancellous bone excavates a trench across the surface rather than a tunnel leaving in its wake (with some degree of geometrical latitude) a hemi - osteon rather than an osteon, i.e. A trench with a cross - sectional geometric cue of a concavity at different stages of osteoclastogenesis, i.e. Concavities with different radii of curvatures and depths as induced by osteoclastic activity . Predating formation during the remodelling cycle of primate cortico - cancellous bone, osteoblasts eventually appear at the resorption site, i.e. Lacunae with a geometric configuration of concavities (fig . 3). Which are the molecular signals and/or physical forces imparted by the geometric topography of the substratum that terminate osteoclastogenesis and recruit osteoblastic - like cells initiating bone formation by induction within the concavities (fig . 3)? The available molecular and morphological data on the subject from several research groups have permitted to formulate the following conceptualization of the spontaneous induction of bone formation within porous biomimetic matrices: the net result of the induction of bone formation in concavities of the substratum is nothing but the language of geometry set by the concavities assembled within the biomimetic biomaterials . In the adult skeleton, the demand of osteoblasts is created by bone resorption, i.e. The concavities induced by osteoclastogenesis, whereas the demand of osteoclasts is governed by the purpose of bone remodelling . The concavities assembled in the biomimetic matrices are endowed with multifunctional pleiotropic self - assembly capacities initiating and promoting angiogenesis and differentiating resident mesenchymal cells into osteoblastic cell lines expressing, secreting and embedding osteogenic molecular signals within the concavities of the biomimetic matrices [27, 61, 71]. The molecular and morphogenetic mechanisms initiating the spontaneous induction of bone formation within concavities of the smart biomimetic matrices originate and progress with blood vessels and capillary sprouting developing within the mesenchymal tissue invading the concavities [710]. The extracellular matrix components of type iv collagen and laminin around the invading capillaries bind morphogenetic proteins involved both in angiogenesis and osteogenesis [59, 71, 7579]. Bound morphogenetic proteins are then presented locally in an immobilized form to responding mesenchymal cells and osteoprogenitors alike to initiate osteogenesis in angiogenesis [5, 9, 59, 71]. Morphogenetic progression is sustained by continuous recruitment of mesenchymal cells and capillary invasion within the concavities . Resting mesenchymal cells attach to the matrix and differentiate into osteoblastic - like cells within the concavities of the biomimetic matrices . Differentiating osteoblastic - like cells express ops of the tgf- superfamily; mrna expression, as evaluated by northern blot analyses, is then followed by secretion and embedding of the expressed gene products within the smart concavities of the biomimetic matrices (fig . The induction of bone formation then follows as a secondary response [2, 5, 27, 55, 71]. Synthetic biomimetic matrices mimic the super - smart functionality of living tissue and allow the engineering of smart self - inducing biomimetic matrices for tissue engineering of bone . The assembly of a series of repetitive concavities within porous biomimetic matrices adds selected functionalities or super - smart functionalities to the biomimetic matrices that intrinsically per se induce the spontaneous induction of bone formation without the exogenous application of osteogenic soluble molecular signals of the tgf- superfamily (figs 2 and 7). A critical step is the embedding of smart biological functions within intelligent scaffolds for tissue engineering of bone, i.e. Embedding biological signals into biomaterials designed with super - smart biomimetic functionalities, ultimately resulting in the intrinsic induction of bone formation [61, 71]. Secreted bmps / ops within the concavities of the biomimetic matrices may be the result of osteoblastic - like cell differentiation or macrophages / osteoclasts expressing selected bmps / ops during the remodelling cycle of the primate cortico - cancellous bone . Macrophages / osteoclastic cells are known to express bmps / ops during the remodelling cycle of the cortico - cancellous bone . Macrophages and osteoclastic cells are continuously involved during the osteointegration processes of implanted biomaterials and are essential for effective tissue regeneration as they regulate the recruitment, proliferation and attachment of fibroblasts, osteoblasts and endothelial cells, significantly contributing to construct osteogenesis in angiogenesis [8185]. Whether bmps / ops are secreted and embedded into the smart concavities by differentiating osteoblast - like cells or macrophages / osteoclastic cells still deserves experimental investigation . The embedded soluble molecular signals will then differentiate osteoblastic - like cells, further secreting bone matrix and the induction of bone formation as a secondary response . Besides bmps / ops [82, 83], macrophages are also known to secrete tgf-1 . While the evidence is still lacking, it is tempting to suggest that the induction of bone within concavities of smart biomimetic matrices recapitulates embryonic development by expressing and secreting bmps / ops synchronously and synergistically with tgf- proteins, initiating the synergistic cascade of bone differentiation [18, 48]. Experiments by reddi and huggins have shown that the temporal sequence of fibroblast chondroblast osteoblast transformation is profoundly influenced by the geometry of the transformant, i.e. Demineralized rodent incisors . The specific geometric configuration of the inductor ultimately results in the induction of endochondral bone either with an anlage of cartilage or bone with bone marrow without the induction of chondrogenesis . Subcutaneous implantation of coarse demineralized bone matrix powders (particle size 420850 m) resulted in the local differentiation of endochondral bone . On the other hand, implantation of fine matrix with particle size of 4474 m did not induce the bone . Since implantation of fine demineralized bone matrix (particle size 4474 m) did not induce endochondral bone differentiation, it was interesting to determine whether extracts of fine bone matrix contain endochondral bone differentiation activity or, if the geometry of the implanted matrix particles solely drives the induction of bone . When protein extracts of fine matrix were combined with coarse inactive collagenous matrix residues, there was restoration of the induction of endochondral bone formation . Fine particles thus contain inductive proteins and the geometry of the matrix carrier is critical for the initiation of the bone induction cascade . These powerful experiments demonstrated that although the fine matrix with particle size 74420 m contains osteoinductive proteins, the geometry of the inductor, i.e. The implanted matrix, is a critical factor to drive the biochemical cascade of bone formation by induction [87, 88]. Several studies have clearly highlighted that tissue induction and morphogenesis can be greatly altered by the geometry of the substratum [27, 54, 55, 61, 8794]. In previous experiments, we have shown that the biological activity of bmps / ops could be expressed and delivered by a substratum other than the insoluble collagenous bone matrix as carrier . The dramatic differences observed between substrata of granular and disc configuration underscored the importance of geometry in bone formation by induction . Substrata of coral - derived has in disc configuration reconstituted with bovine osteogenic fractions purified greater than 50,000-fold with respect to crude guanidinium extract induced heterotopic endochondral bone formation when implanted subcutaneously in rodents . Identical coral - derived has but in granular configuration did not induce bone differentiation even if pre - treated with identical doses of highly purified naturally derived bmps / ops . Remarkably thus, the geometric configuration of the substratum can inhibit and overrule the osteogenic activity of bmps / ops both in rodents and non - human primates papio ursinus[90, 94]. Identical coral - derived porous has with distinct geometric configurations were implanted in the rectus abdominis muscle of adult baboons . As in the rodent bioassay, though without the addition of highly purified osteogenic fractions, implants of particulate granular ha implanted in the rectus abdominis muscle of adult baboons, failed to induce bone differentiation within the porous spaces . Instrumental for our understanding of the critical role of the concavity in driving the cascade of bone differentiation, minimal yet some bone formation was only found in a concavity of a particulate granular coral - derived ha specimen harvested on day 90 from the rectus abdominis muscle (fig . The lack of bone differentiation in implants of granular ha implies a critical role of implant geometry on bone differentiation by induction [90, 94]. This has important implications for the construction of appropriate porous bone substitutes for reconstructive bone tissue engineering in clinical contexts [90, 94]. Effects of the substratum of coral - derived hydroxyapatite biomatrices on tissue induction and morphogenesis within the porous spaces of the biomimetic matrix implanted heterotopically in the rectus abdominis muscle of papio ursinus . (a) heterotopic implantation of a rod of coral - derived porous hydroxyapatite (interpore, usa). (b and c) differentiation of osteoblastic - like cells at the hydroxyapatite interface highlighted in c. osteogenetic vessels as defined by trueta penetrate the mesenchymal condensation seemingly providing migrating cellular progenitors (magenta arrows) capable of osteoblastic cell differentiation when in contact with the hydroxyapatite substratum . (d) alkaline phosphatase staining of invading capillaries, the osteogenetic vessels within the porous spaces of the coral - derived porous hydroxyapatite (blue arrows). (e, f and g) undecalcified sections stained freefloating with a modified goldner trichrome showing mineralization (blue arrows) of collagenic condensations (magenta arrows) surfacing the hydroxyapatite substratum . Mineralized bone (blue arrows) is surfaced by osteoid seams populated by osteoblastic cells . (h) tissue morphogenesis in an implant of particulate granular coral - derived hydroxyapatite harvested on day 90 from the rectus abdominis muscle of an adult baboon . Digital images g and h were instrumental for the understanding of the role of specific geometric configurations in the induction of bone and thus for the preparation and testing of solid discs of highly crystalline hydroxyapatite with concavities on both planar surfaces as shown in fig . 2c . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . Predating the induction of bone formation, and in close proximity to developing bone, there is always a rich capillary network invading the porous spaces of the biomimetic matrix, particularly in concavities of the substratum [27, 54, 55]. Trueta has stressed the importance of the blood vessels in osteogenesis, and defined the vascular invasion during bone formation as osteogenetic vessels, suggesting that the endothelium may be capable of osteoblastic differentiation . While circumstantial evidence is lacking, it is tempting to suggest that osteogenetic vessels, penetrating the porous spaces of the substratum, might have provided a temporally regulated flow of cell populations capable of expression of the osteogenic phenotype (figs 2h and 4c, d) [34, 54, 95]. The implantation of calcium carbonate coral - derived has in the rectus abdominis of adult baboons showed that osteocyte - like cells are embedded within a tissue that had intermediate features between fibrous tissue and bone (fig . The development of mesenchymal condensation at the ha interface is a critical developmental event predating the initiation of spontaneous bone differentiation in porous bioceramics when implanted in the rectus abdominis muscle of papio ursinus[54, 87, 88]. Indeed, mesenchymal condensations are critical for the initiation of skeletal development . By including the analysis of early periods of observation (i.e. Days 30 and 60), morphological and histochemical data have shown that the differentiation of large, hyper - chromatic and intensely alkaline phosphatase positive cells at the ha interface is a critical morphogenetic event preceding the differentiation of bone (fig . 4b and c). A critical step during the developmental cascade of the spontaneous and/or intrinsic bone induction in porous bioceramics without the addition of exogenously applied bmps / ops is the differentiation of resident mesenchymal cells in contact with the biomimetic substrata into osteoblastic cell lines resting upon the surface of the implanted matrices (fig . We now propose the following cascade of molecular, cellular and morphological events culminating in the induction of bone formation in heterotopic sites of non - human primates papio ursinus initiating within concavities of smart biomimetic matrices: 1 . Vascular invasion and capillary sprouting within mesenchymal fibrovascular condensations invading specific geometries of the substratum with capillary elongation in close contact with the implanted biomimetic matrix . 2 . Invading mesenchymal cells attach and differentiate at the ha / soft tissue interface of the concavities . There is no differentiation of osteoblastic cell lines without the driving force of the concavities . This crucial step of differentiation is shown by northern blot and pcr analyses of tissue harvested from the concavities of the biomimetic matrices and by the immunolocalization of bmp-3 and op-1 within differentiating osteoblastic cell lines . 3 . Synthesis and secretion of osteogenic soluble molecular signals of the tgf- superfamily from resident resting and transforming osteoblastic - like cells and/or macrophages / osteoclastic cell lines attached to the concavities of the substratum as shown by intercellular immunolocalization and finally embedding of the secreted gene products into the concavities of the ha biomimetic matrices . 4 . Intrinsic osteoinduction with further differentiation of osteoblastic cell lines; bone formation by induction within the concavities of the biomimetic matrices is dependent on a critical threshold of endogenously produced bmps / ops initiating formation by induction as a secondary response . The intrinsic or spontaneous induction of bone formation in a variety of porous biomaterials is a very interesting phenomenon which originally has been shown by implanting porous polyhydroxyethyl - methacrylate in the sub - cutis of white pigs . In the late 1980s, systematic studies were started in non - human primates papio ursinus after the remarkable finding of intrinsic osteoinductivity in the porous spaces of coral - derived has (figs . . Coral - derived has induced substantial amounts of membranous bone when implanted heterotopically in the rectus abdominis muscle of adult papio ursinus (figs . 5 and 6) [54, 96, 97, 100, 101]. In the same animals, coral - derived (figs 8 and 9) and sintered porous has (fig . 10) implanted in calvarial defects also induced extensive bone deposition culminating in complete calvarial regeneration [5, 7, 55, 101]. The morphogenesis of bone in porous coral - derived hydroxyapatites harvested on day 90 from the rectus abdominis muscle of adult baboons papio ursinus . (a and b) low - power photomicrographs showing substantial bone differentiation within the porous spaces of the biomimetic matrix . (c and d) high - power views of the distribution of the newly formed bone (arrows) within the porous spaces in tight contact with the biomimetic substratum . (d and e) details of previous images showing the biomimetism of the concavity inducing bone formation within specific geometries of the biomimetic matrix (blue arrows). Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . Substantial bone morphogenesis by induction in porous coral - derived hydroxyapatites long - term implanted in the rectus abdominis muscle of adult baboons papio ursinus . (a and c) low- and high - power views of bone induction within the implanted biomimetic matrix harvested 6 month after implantation in the rectus abdominis muscle of adult baboon . (b and d) morphogenesis of bone in coral derived porous hydroxyapatites harvested 9 months after implantation . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . (a, b and c) complete regeneration of bone across the porous spaces of coral - derived hydroxyapatites implanted in non - healing calvarial defects of the adult baboon and harvested 6 months after calvarial implantation . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . (a, b, c and d) remodelling and maintenance of the induced bone across the coral - derived porous hydroxyapatites 9 months after calvarial implantation in the adult baboon . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . Morphogenesis of bone across the porous spaces of highly crystalline sintered hydroxyapatites implanted in calvarial defects of adult baboons and harvested on day 90 . (a and b) low - power view of two specimens of sintered porous hydroxyapatites showing the induction of bone across the porous spaces of the sintered ceramics . (c, d, e, f, g and h) details of previous sections showing the morphogenesis of bone in direct contact with the sintered biomatrix biomimetizing the geometric concavities of the bone remodelling cycle and the induction of bone formation . Arrows (e, g and h) indicate the induction of bone formation as driven by the concavities of the biomimetic highly crystalline substratum . Original magnification: (a) 125; (b) 125; (c) 175; (d) 175; (e) 275; (f) 275 . Importantly for application in clinical contexts, the extent of the spontaneous induction of bone formation in calcium phosphate ceramics varies significantly in different animal models . Using coral - derived ha substrata, minimal, if any, bone formation was shown in dogs and rabbits as compared to adult baboons . In contrast to other studies, calcium phosphate ceramics showed bone formation within the porous spaces of the implanted matrices in canine models [70, 102]. The bone also formed in direct contact of calcium phosphate ceramic particles implanted heterotopically in sheep muscles [66, 103, 104] and in goats . Further studies on the cross - species comparison of heterotopic bone induction in biphasic calcium phosphate ha scaffolds showed that the extent of heterotopic bone formation is controlled by the animal species as well as the nature of the implanted scaffolds . Implantation of calcium phosphate bioceramics in the rat heterotopic bioassay results in lack of bone differentiation although specimens harvested from heterotopic sites of murine models showed the differentiation of bone to a varying degree . Several papers have stressed the importance of biomimetic matrices capable of concentrating several proteins including bmps / ops from the body fluids and/or the extracellular matrix . It has been proposed that ceramics sintered at a lower temperature would have the ability to concentrate more circulating and/or locally produced bmps / ops [62, 67, 70]. The capacity to concentrate bmps / ops as found in the circulation or in the microenvironment of the extracellular matrix suggests that the incidence of the spontaneous induction of bone formation varies with animal species as well as the implanted biomimetic matrix . Additional critical parameters for the spontaneous induction of bone formation in a variety of porous bioceramics are the porosity, pore size and distribution, interconnectivity, fenestration, distribution as well as orientation of pores [62, 67]. Kondo et al . Have suggested that the microporous surfaces of the -tcp constructs enhanced protein adsorption and cell attachment contributing to the osteoinductivity of the tested -tcp biomatrices also showing in vitro that microstructure is a key factor for osteoblastic differentiation and proliferation . Data from several investigations in vivo in canine and ovine models have suggested that the microporosity of the implanted bioceramics play important roles as the storage microenvironment for several extracellular matrix components, including bmps, and are ultimately responsible for the induction of bone formation . The reason why, however, the microstructure and interconnected pores are so critical for osteoblastic - like cells differentiation, expression of ops, and thus the induction of bone formation as a secondary response still remain unclear . Similarly, the reason for the differences in biomaterials - induced bone formation in the tested animal species is as yet to be defined [66, 67, 69, 100]. More importantly perhaps, morphological and molecular experiments are now mandatory to assign to specific cellular elements the expression and secretion of the ops of the tgf- superfamily . Porous biomaterials with a series of repetitive concavities with optimal surface topography and microstructure provide porous spaces that are architecturally conducive to differentiate resident mesenchymal cells into osteoblastic cell lines . Expression, secretion and embedding of osteogenic molecular signals are followed by rapid vessel ingrowths, capillary sprouting and the induction of bone formation as a secondary response . The surface topography and the geometry of the concavity affect cellular morphology, and cellular shape will influence function [105107] during the differentiation of osteoblastic cell lines expressing and secreting osteogenic molecular signals . The differentiation of osteoblastic cell lines expressing and secreting osteogenic soluble molecular signals of the tgf- superfamily is at the crux of the intrinsic osteoinductivity of a variety of porous calcium phosphate and biphasic tcp ha bioceramics in a variety of animal models including man . Morphological analyses of coral - derived has implanted in the rectus abdominis muscle of papio ursinus showed the differentiation of large hyper - chromatic cells arranged in one or two layers directly opposed to the ha substratum . Such cells, interpreted as differentiating osteoblasts, were close to a rich capillary network lined by large and hyper - chromatic endothelial cells . The differentiation of large, hyper - chromatic and intensely alkaline phosphatase positive osteoblastic cells in an intimate relationship with endothelial cells and the implanted biomimetic matrix is a critical morphogenetic event preceding the differentiation of bone [54, 55]. The induction of bone is constructed by regulating the expression of selected mrna of gene products as a function of the structure [2, 9, 10, 61]. Biomimetic matrices of highly crystalline has [9, 55] or biphasic ha / tcp bioceramics constructed with a series of repetitive concavities offer a geometric configuration which vividly reproduces and biomimetizes the remodelling processes of the primate osteonic bone [5, 8, 9, 57, 58]. Ultimately, which are the resident mesenchymal cells capable of transformation / differentiation into secreting osteoblastic - like cells at the ha interface? It is obvious that the rectus abdominis muscle of adult baboons is endowed with a stem cell niche[108, 109] that provides a large number of differentiating cells including osteogenic progenitors which attach and differentiate onto the biomimetic matrices . The presence of a stem cell niche in rectus abdominis muscles where stem cells reside and undergo self - renewal continuously producing large number of progenitor cells [108, 109] is additionally supported by the recent identification of myendothelial cells in human skeletal muscle . Myogenic and endothelial cells may derive from a common somatic precursor, and cells co - expressing myogenic and endothelial cell markers residing in the interstitial spaces of skeletal muscle, i.e. Myoendothelial cells, may contribute to postnatal tissue morphogenesis . Importantly, in the context of the spontaneous induction of bone formation in porous biomimetic matrices implanted in the rectus abdominis muscle, clonally expanded myoendothelial cells differentiate into myogenic, chondrogenic and osteogenic cells under appropriate culture conditions . Multipotent myoendothelial cells residing in stem cell niches within the rectus abdominis muscle do respond to endothelial cell mitogens including angiogenic factors; myoendothelial cells may also respond to bmps previously bound to collagen type iv and other extracellular matrix components further inducing the ripple - like cascade of the induction of bone formation within the porous spaces of the biomimetic matrices biomimetizing the cortico - cancellous remodelling cycle of the primate bone [8, 9, 57, 58]. The final leap into the intrinsic induction of bone formation will rest upon the mechanistic understanding of how microstructured surface areas of biphasic or not calcium phosphate biomaterial matrices differentiate inducible stem cells into osteoblastic - like cells initiating the induction of bone formation as a secondary response . Intrinsic osteoinductivity of biphasic calcium phosphate biomatrices has been elegantly investigated by li et al . . The authors suggested that increased surface areas of calcium phosphate biomaterial matrices are endowed with osteoinductivity; microstructured surfaces with increased surface areas might promote superior protein adsorption; selected adsorbed proteins would directly influence cell attachment and proliferation to further promote the induction of the osteogenic phenotype in attached and resident stem cells into osteoblastic - like cells synthesizing, expressing, secreting and embedding osteogenic soluble molecular signals into the biomimetic matrices inducing the ripple - like cascade of bone differentiation by induction [61, 71]. Tissue engineers, molecular biologists, reconstructive surgeons and developmental biologists alike have learned that tissue engineering in postnatal life recapitulates events that occur in the normal course of embryonic development [2, 5, 17]. More figuratively perhaps, tissue engineering of postnatal tissues recapitulates and exploits the very mechanisms of embryonic development though with different ratios and quantities of synchronously expressed soluble molecular signals and insoluble extracellular matrix substrata that nature has so parsimoniously developed through million years of evolution . The summary of several hundred years of research into the mechanisms of regenerative medicine is surprisingly simple: morphogens exploited in embryonic development are re - exploited and re - deployed for postnatal tissue induction and morphogenesis . Perhaps indeed the different ratios and quantities of synchronously expressed soluble molecular signals interacting with insoluble signals or substrata pinpoints the very different capabilities to regenerate between embryos and adult animals, particularly the regenerative potential of homo sapiens . Embryonic development and postnatal tissue regeneration are equally regulated by selected few and highly conserved families of secreted proteins, members of the tgf- superfamily [2, 17, 18]. We have learned that in primates and in primates only, there is an apparent redundancy of soluble molecular signals initiating the induction of bone formation in heterotopic extraskeletal sites . We have learned though that we still have to grapple with the reality of our discoveries, that when implanted in heterotopic sites of the rectus abdominis muscle of non - human primates papio ursinus, the mammalian tgf- isoforms do induce substantial endochondral bone formation by induction [55, 4851]. We have further learned, and again biomimetizing nature that combines signals to induce tissue morphogenesis, that combining osteogenic soluble molecular signals with relatively low doses of the tgf-1 isoform, the rapidly generated tissue constructs are the result of a synergistic interaction which we have labelled synergistic induction of bone formation[2, 5, 18, 48]. Synergistic induction of bone formation has remarkably showed to us that bone tissue develops as a mosaic structure in which members of the tgf- superfamily singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis [2, 5]. The morphogenesis of structurally organized chondrogenic zones, highly reminiscent of rudimentary embryonic growth plates, is a finding that vividly illustrates the concept that regeneration of cartilage and bone in postnatal life shares common cellular and molecular mechanisms with embryonic bone development, and that the memory of developmental events in embryo can be redeployed postnatally by the application of morphogen combinations . The synergistic induction of bone formation reflects nature s parsimony in deploying low doses of molecularly different molecular signals yet resulting in rapid and complete tissue induction and regeneration [48, 49]. Key is to synchronously and synergistically deploy low doses of molecularly different single gene products during both embryogenesis and tissue regeneration in postnatal life, parsimoniously achieving complete tissue regeneration [18, 111]. Supra - physiological doses of single recombinant human bmps / ops [111, 112] contravene nature s elegant deployment of synchronously active molecular signals; this often generates incomplete and partial tissue induction in clinical contexts . Biominetic matrices of ha/-tcp bioceramics have shown remarkable intrinsic oteoinduction in a variety of animal models [113116]. Long - term experiments in the non - human primate papio ursinus were set to investigate the induction of bone formation by biphasic biomimetic matrices (ha/-tcp) 40 to 60 and 20 to 80, respectively [117, 118]. One year after implantation in orthotopic calvarial sites there was prominent osteogenesis coupled with resorption / dissolution of the implanted biomimetic matrices (fig . Solid discs of biphasic ha/-tcp, with concavities prepared on one planar surface only, were implanted heterotopically in the rectus abdominis muscle of adult baboons [117, 118]. Histological analyses of heterotopic specimens harvested on days 90 and 365 showed the induction of bone formation also on the planar surfaces of the implanted biphasic ha/-tcp discs . Osteoclasts / macrophages excavate resorption lacunae and pits upon which osteoblastic cell lines secrete bone matrix within the microconcavities cut by osteoclastogenesis [117, 118]. Morphological analyses 1 year after heterotopic implantation showed the induction of substantial bone with marrow on both planar surfaces with further dissolution of the implanted ha/-tcp biomimetic matrix with bone formation within the excavated resorption lacunae [117, 118]. Orthotopic calvarial specimens showed complete induction of bone with resorption and dissolution of the implanted biphasic biomimetic matrices (fig . Induction of bone formation by post - sinter 19/81 hydroxyapatite/-tricalcium phosphate hydroxyapatite biomimetic matrices harvested from heterotopic rectus abdominis (a, c, e, f, h, i) and orthotopic calvarial sites (b, d, g, j) from adult baboons papio ursinus on day 365 after implantation . (a) magenta arrows point to substantial bone differentiation in pre - carved concavity of the bioactive biomimetic matrix . Opposite facing the muscle, there is bone differentiation on the planar surface without pre - cut concavities . (c, e, f, h, i) high - power microphotographs detailing the induction of bone formation (magenta arrows) in a continuum of morphological processes of resorption / dissolution and bone formation . (b) low - power view of substantial induction of bone formation throughout the porous spaces of the implanted biomimetic matrices across the defects (blue arrows). (d, g, j) porous spaces filled by newly formed bone tightly attached to the biomimetic matrices showing resorption / dissolution of the implanted scaffolds . Original magnification: (a) 3.7; (b) 2.7; (c) 65; (d) 35; (e, f, h, i)) 175; (g) 35; (j) 75 . Continuum of molecular and morphological processes that ultimately results in significant amounts of bone formation by induction in toto replacing the implanted smart biomimetic matrices [117, 118]. Above all, the overall geometric configuration of novel biomimetic matrices will provide biomimetic constructs to optimally deliver low doses of ops of the tgf- superfamily . The incorporation of specific biological activities into biomimetic biomaterial matrices by manipulating the geometry of the substratum, defined as geometric induction of bone formation[5, 8, 9, 55] is now helping to engineer therapeutic osteogenesis in clinical contexts.
The human abo blood group consists of three major types, a, b, and o . These alleles code for glycosyltransferases, with the terminal sugar chain modifications varying between types . The enzyme encoded by functional alleles of type a and b transfer a galnac or a gal on the precursor oligosaccharides of type h. the nucleotide sequences of the human abo blood group genes have been previously determined and the molecular basis of these differences has been revealed [2, 3]. The alleles a and b differ in exon 7 by four nonsynonymous mutations, and two of which are critical for the sugar specificity (codons 266 and 268 encode l - g for a and m - a for b). The major o allele has a single nucleotide deletion (261) in exon 6 that induces a frameshift, resulting in a truncated protein deprived of any glycosyltransferase activity . Major haplogroups (a101, a201, b101, o01, o02, and o09) exist in the human abo blood group genes [5, 6]. A101 and b101 are the main haplogroups for the a and b alleles, respectively . The activity of a201 is reduced 20- to 50-fold compared to a101, because a201 has a point deletion at nucleotide position 1061 that results in a frameshift adding 21 additional amino acid residues to the protein . A series of nucleotide differences have been observed between o01 and o02 [5, 6, 8]. Although o09 shares 261 with o01 and o02, its sequence is quite similar to a101 . Thus, o09 most likely evolved from an ancestral a101-like common allele by a gene conversion in exon 6, introducing 261 from another o allele [5, 6]. Several studies have examined the evolution of the human abo blood group genes [5, 811]. These studies have identified that the a allele is the most ancient, because the chimpanzee, which is the closest relative of humans, has a and o alleles . The o allele of the chimpanzee has evolved by a different mechanism compared to humans [10, 12]. The b allele diverged from the a allele, with nucleotide substitutions on the two critical residues in exon 7 . The o02 allele diverged from the a allele with a single nucleotide deletion (261) in exon 6, after which the o01 allele diverged from the o02 allele . In contrast, there are some studies [13, 14] that argue transspecies polymorphism of the a and b alleles . In any case recently, a new model for the human abo blood group genes has been developed, using phylogenetic network analysis . They argued that although the b and o alleles diverged from the a allele, the modern human a allele is not a direct descendant of the ancestral a allele . The modern human a allele emerged through a recombination between the b and o alleles, where the intact exon 6 from the b allele and two critical a type sites in exon 7 from the o allele were jointed less than 300,000 years ago . Since the previous study used a restricted dataset, that is, seattle snps project data, which is a set of 90 sequences in european- and african - americans, it is necessary to analyze a more comprehensive dataset to corroborate this hypothesis . Therefore, in the present study, we used snp data from the hapmap project to examine the evolution of the human abo blood group genes . We retrieved two kinds of phased haplotype datasets for the abo blood group genes from the hapmap project . The first was the three population dataset (3pop_data) that included the yoruba in ibadan, nigeria (yri), ceph (utah residents with ancestry from northern and western europe) (ceu), japanese in tokyo, japan, and han chinese in beijing, china (jpt + chb) (hapmap data rel 24/phaseii nov08, on ncbi b36 assembly, dbsnp b126). The other dataset contained eleven populations (11pop_data) including african ancestry in southwest usa (asw), utah residents with northern and western european ancestry from the ceph collection (ceu), han chinese in beijing, china (chb), chinese in metropolitan denver, colorado (chd), gujarati indians in houston, texas (gih), japanese in tokyo, japan (jpt), luhya in webuye, kenya (lwk), mexican ancestry in los angeles, california (mex), maasai in kinyawa, kenya (mkk), toscans in italy (tsi), and yoruba in ibadan, nigeria (yri) (hapmap data phaseiii / rel#3, may 10, on ncbi b36 assembly, dbsnp b126). Since haplotypes from most populations in the hapmap project are estimated from genotypes, there is a possibility that the data might contain erroneous haplotypes . To reduce the possibility of artificial recombinants the datasets of 3pop_data_1 and 11pop_data_1 consisted of haplotypes from homozygous individuals and individuals carrying only one heterozygous site . Meanwhile, the 3pop_data_2 and 11pop_data_2 datasets consisted of haplotypes observed more than two times from the all populations . They showed the relationship between a recombinant and its two parental alleles in a phylogenetic network . We used model data to explain how to infer a recombination event from a phylogenetic network (figure 1). First, an ancestry sequence (o) produces two different sequences (p1 and p2) (figure 1(a)). The p1 has five substitutions at sites 2, 4, 5, 9, and 15 (bold red), and the p2 has four substitutions at sites 1, 6, 8, and 11 (bold blue), from sequence o. then, if recombination occurred between sites 6 and 7 for p1 and p2, two recombinants (r1 and r2) exist . After the recombination, three nucleotide substitutions at sites 7 (purple), 12 (blue), and 13 (red) accumulate to p1, p2, and r1, respectively, and three nucleotide substitutions at sites 3, 10, and 14 (gray) also accumulate to produce an outgroup (o) from sequence o. assuming that r1 and r2 were produced by a single recombination event, transmission of both recombinant alleles to the next generation is highly improbable . Figure 1(b) is the phylogenetic network represented by figure 1(a) at the time . The phylogenetic network (figure 1(b)) indicates the relationship between the extant alleles (p1, p2, and r1) and an outgroup (o). Two parental alleles (p1 and p2) are located on opposing vertices of the rectangle and have longer (compared to that of the recombinant allele) external branches (sites 13, 9, and 15 for p1 and sites 12, 1, and 6 for p2), while the recombinant allele (r1) is located on the vertex opposing the outgroup allele (o) and has a shorter (compared to those of parental alleles) external branch (site 7). The pnarec (phylogenetic network - based recombination detection) method, a general application of, was applied for selected haplotypes (they are representative haplotypes from haplogroups) using the pnarec program (available from supplementary material of). Fifty - four and 36 snps for 3pop_data and 11pop_data, respectively, were retrieved from the hapmap project (figure 2). We predicted abo types for each haplotype (see supplementary material available online at http://dx.doi.org/10.1155/2013/406209) by using haplotype - specific snps, following data of . Since insertion and deletion variations are not contained in the data, a201 and o09 could not be distinguished from a101 . These haplogroups are treated as the a101-a201-o09 haplogroup, because it is highly likely that a201 and o09 evolved from a101 [5, 6]. Figure 3(a) describes a phylogenetic network of the human abo blood group genes using 3pop_data_1 . Since the o01 haplogroup has a shorter external branch and is located on the vertex opposing the outgroup, the o01 haplogroup is thought to be a recombinant lineage . Its parental allele lineages are expected to be the o02 and b101 haplogroups, because the o02 and b101 haplogroups are located on opposing vertices of the rectangle with longer external branches (figure 3(b)). Twelve sites (11, 19, 20, 21, 23, 25, 28, 29, 30, 31, 32, and 54) support o01 and b101 haplogroup clustering, while four sites (42, 47, 48, and 49) support o01 and o02 haplogroup clustering . This corresponds to a region between intron 1 and intron 3 (figure 2). If we cannot expect to have a recombination event, we need to assume four (42, 47, 48, and 49) parallel substitutions at the o01 and o02 lineages or 12 (11, 19, 20, 21, 23, 25, 28, 29, 30, 31, 32, and 54) parallel substitutions at the o01 and b101 lineages . In the phylogenetic network, the a101-a201-o09 haplogroup is also thought to be a recombinant lineage, and its parental lineages are the o01 and b101 haplogroups (figure 3(c)). Three sites (9, 13, and 15) support a101-a201-o09 and o01 haplogroup clustering, while seven sites (36, 37, 46, 50, 51, 52, and 53) support a101-a202-o09 and b101 haplogroup clustering . Thus, the recombination point is estimated to be between sites 15 and 36, which correspond to a region between intron 2 and exon 6 . Since a101 and a201 do not have 261, we can expect that the recombination point is located in exon 6 (between sites 15 and 261). If we do not assume a recombination event, we should assume three parallel substitutions (9, 13, and 15) at the a101-a201-o09 and o01 lineages or seven parallel substitutions (36, 37, 46, 50, 51, 52, and 53) at the a101-a201-o09 and b101 lineages . The o47 haplogroup is thought to be a recombinant between o47 and some other haplogroups . However, in the phylogenetic network, the o47 haplogroup is not located on the vertex opposing the outgroup, probably because its parental o47 haplogroup lineage is not included in the data . The haplotype xv is thought to be a recombinant between the o02 and b101 haplogroups [5, 6]. Since this haplotype has 261, the recombination point should be located between 261 and site 12 (table 1). Thus, in contrast with other o type alleles which have two critical a type sites in exon 7, this o type allele has two critical b type sites . The o02/b101 haplotype is observed only in african populations (yri of hapmap data and african americans of the seattle snps data). We reconstructed a phylogenetic network excluding the haplotype o02/b101 (figure 5(a)), which shows similar results compared to figure 3 . The o01 haplogroup is thought to be a recombinant lineage, where the o02 and b101 haplogroups are its parental allele lineages (figure 5(b)). Seven sites (19, 20, 21, 23, 25, 29, and 32) support o01 and b101 haplogroup clustering, while five sites (36, 42, 47, 48, and 49) support o01 and o02 haplogroup clustering . This corresponds to a region between intron 2 and intron 3 (figure 2). It is also thought that the a101-a201-o09 haplogroup is a recombinant lineage, where its parental lineages are the o01 and b101 haplogroups (figure 5(c)). We can expect that the recombination point is located in exon 6 (between sites 32 and 261), because a101 and a201 should not have 261 . If we do not assume a recombination event, we should assume three parallel substitutions (9, 13, and 15) at the a101-a201-o09 and o01 lineages or seven parallel substitutions (37, 46, 50, 51, 52, 53, and 54) at the a101-a201-o09 and b101 lineages . These results strongly support the hypothesis that the modern human a allele is derived from a recombination event between the o01 and b101 lineages . We also used 11pop_data, which contains data from 993 individuals from the world, but the number of snps is lower at 36 compared to 54 in 3pop_data . This phylogenetic network indicated similar results to 3pop_data; the o01 and a101-a201-o09 haplogroups are recombinant lineages (figures 6(b) and 6(c)). In addition, since each haplogroup consists of individuals from several populations, it is suggested that they had been formed before divergences of human populations, that is, prior to migration out of africa . Some minor haplotypes (table 2), which probably occurred by recombination or gene conversion, were not included in the phylogenetic network to prevent construction of a complex multidimensional phylogenetic network . This phylogenetic network showed similar results with the above phylogenetic networks; the o01 and a101-a201-o09 haplogroups are recombinant lineages (figures 7(b) and 7(c)). Three o47 haplotypes (r, p, and w) were separated from each other in the phylogenetic network . This result is not unexpected, because o47 haplotypes are thought to be recombinants between o47 and some other haplogroups, and these three haplotypes (r, p, and w) share only one o47 specific site (site 1 in figure 2; see also supplementary material). The v haplotype was assigned to the haplogroup x tentatively, because this haplotype could not be predicted as a known haplogroup (see supplementary material). We used five haplotypes (i, ii, iii, v, and vii) from 3pop_data_1, five haplotypes (i, ii, iii, vii, and xv) from 3pop_data_2, and four (a, e, f, and g) from 11pop_data_1, as representative haplotypes . It is suggested that the a101-a201-o09 haplogroup is derived from a recombination event in all the datasets, though o02 is assigned as a parental allele instead of o01 . Step a: count the numbers of singleton sites for each sequence, step b: choose all possible quartet sequences, including the outgroup and count the three types of phylogenetically informative sites, step c: choose quartets in which one of the splits had no phylogenetically informative site, step d: examine the distribution of the remaining two phylogenetically informative sites for each quartet, and choose ones whose site distributions do not overlap each other, and step e: choose the quartet whose recombinant descendant lineage sequence candidate has the smallest number of singleton sites . If there is more than one candidate with the smallest number of singleton sites, choose the quartet in which the two parental descendant lineage sequence candidates have the largest sum of singleton sites . Until step d, o01 remains as a candidate of a parental allele . At step e, o02 is chosen as a parental allele instead of o01, because o02 has a longer external branch than o01 . The pnarec method is still under development, and further improvements to this the method are required . O01 was eliminated from recombinant candidates at step c, because o01 has parallel changes with the chimpanzee outgroup . Meanwhile, it was suggested that o01 appears to resemble a mosaic of b101 and o02 by gene conversions rather than simple recombination . The haplotype xv is clearly detected as a recombinant between the o02 and b101 haplogroups . Figure 8 depicts a possible evolutionary scheme of haplogroups for the human abo blood genes . We assume a as an ancestor in humans, because chimpanzees mainly have a alleles [10, 12, 19]. B101 then diverged from a, followed by substitutions for the two critical sites, and o (o02) diverged from a with 261 . O01 might be formed by a recombination coupled with gene conversions (including transfer of 261) between b101 and o02 . A101 is the recombinant product with the intact exon 6 from b101 and two critical a type sites in exon 7 from o01 that had been joined to form the functional a allele . The results from this study, using the hapmap data, mirror the results of . A101 should be distinct from the ancestral a allele, which produced the b101 and o02 haplogroups . It is not clear whether the ancestral a allele coexisted with other haplogroups in modern humans . If the ancestral a allele exists in the human population, it should be located near the common ancestral position with a longer external branch in a phylogenetic network . We observed the v haplotype (x haplogroup), which is located near the common ancestral position in a phylogenetic network (figure 7). Since this haplotype consists of individuals from asw, ceu, gih, mex, and tsi, it seems to have been formed prior to migration out of africa . Thus, it is possible to expect that the x haplogroup may be the ancestral a haplotype, that is, the most ancient haplotype . Further studies are needed to clarify whether the x haplogroup is indeed the ancestral a allele.
People whose rights are not respected on the basis of their gender identity or sexual orientation lack protection within families and the community at large . This places a constraint on their ability to seek health services and information . At the same time, for economic reasons or due to low self - esteem, they may engage in risky behavior which can predispose them to infection with human immunodeficiency virus (hiv) and hepatitis virus . The lack of opportunities for people experiencing social exclusion pushes them toward financial poverty, worsens their physical and mental health status, and perpetuates this vicious cycle.1 according to research conducted by anthropologists, pockets of areas existed in africa before the colonial era where homosexuality was practiced . It was not openly acknowledged because issues related to sexuality were taboo and not intended for public discussion.2 open acknowledgment of same sex relationships is perceived as a threat to the traditional african culture and religion because it contradicts their definition of what constitutes family and community . It is also perceived to be the negative influence of westernization (globalization) and should therefore be repressed or curtailed . Several actions are undertaken to achieve this, including bullying, taunting, hostility, discrimination, and violence.3 stigma, discrimination, and human rights violation has trailed the hiv epidemic and had posed a significant challenge hampering efforts geared toward prevention, care, and support . From the report of a survey conducted between 2008 and 2011,4 a high proportion of men and women living with hiv in nigeria reported verbal and physical abuse, experiencing stigma within the family and community, as well as loss of job or income.5 achieving universal access will remain a dream if people most at risk, infected, or affected cannot access information and services . This can be as a result of punitive laws, economic circumstances, religious beliefs, sociocultural prejudices, and norms that pose structural barriers.4 human rights violations, including extrajudicial killings, torture, detention, and even rape, have been perpetuated against individuals on the basis of their sexual orientation and gender identity . To protect the human rights of individuals with respect to this issue, a set of guidelines, ie, the yogyakarta principles, were developed to guide the application of international human rights.6 as of may 2013, most of the un member countries were not conforming to the yogyakarta principles; 65 countries and 85 entities have anti - discriminatory laws compared with the 31 countries and 35 entities that recognize same sex unions . Same sex activities and relationships in the northern sharia - implementing states of nigeria, somalia, and another five countries in the world attract the death penalty . In 71 countries and five entities, the punishment is imprisonment.1,7 according to chapter 21 articles 214 and 217 of the country s criminal code, same - sex sexual activity is a felony in nigeria, and punishable by imprisonment of up to 14 years.1,6,7 greater experiences of family rejection, a traumatic form of emotional abuse as a result of gender identity and sexual orientation, has been linked with poor mental health outcomes (attempted suicide, high levels of depression, and substance use problems among adolescents).8 one of the recognized coping mechanisms seen upon exposure to a stressful environment is alcohol and drug use aimed at numbing the pain experienced . This can lead to alcohol dependency and drug abuse, creating a situation whereby men will engage in risky sexual behavior . Globally, the number of new hiv infections and acquired immune deficiency syndrome (aids)-related deaths has been dropping over the years . An estimated 20% reduction in the number of people newly infected with hiv and 24% fewer deaths from the disease was observed between 2001 and 2011.5 in nigeria, available reports indicate an increase in the number of people newly infected with hiv from an estimated 250,000 in 2005 to over 388,000 in 2012 . Men who have sex with men (msm) and their partners, according to the report, were responsible for 10% of all new infections that occurred in the country within that time period.9 globally, the prevalence of hiv has been observed to be higher among msm than in the general population in most countries.5 in nigeria, 17.2% of msm were living with hiv in 2010 compared with the national prevalence of 4.1%.10 this study examined the occurrence of human rights violations among msm living in lagos in 2012 before the same sex marriage prohibition bill was signed into law by the president of the federal republic of nigeria . The study site was lagos, a densely populated cosmopolitan city in south western nigeria . A list of community centers frequented by msm in lagos state was collected from the lagos state aids control agency . Only seven of the 20 local government areas (lgas) in the state had community centers for msm . Their activities are sponsored by partners and donor agencies through grants, provision of behavioral communication materials, training, and capacity development of staff . Msm go there to access services through referrals and by one - on - one introduction . Activities are carried out indoors; there are no banners or signpost advertising the venue or its activities . It is a close - knit setting; some of the staff manning the centers are females, but only msm can access services at these centers . Three of the seven lgas were selected through simple random sampling; each of the selected lgas had only one community center, so all three were used . Advocacy visits were subsequently conducted to the three community centers, and the stakeholders and networks manning the centers were duly briefed on the study and its purpose . The benefit of the study was also highlighted, and verbal informed consent was given by the authorities manning the three centers . Participants recruited for the study were men over 18 years of age who had had sexual relationships with members of the same sex and visited the community center for recreational activities or hiv prevention information and services between 9 am and 6 pm from monday to saturday in august 2012 . Heterosexuals who visited the community centers for whatever reason during the period of data collection were excluded from participation by staff and peers . Only msm who agreed to participate in the study participation was voluntary and participants were assured that all information supplied would be anonymous and kept in a confidential manner . The formula for calculating sample size in a cross - sectional descriptive study was used based on the proportion of msm reporting risky sexual behavior in the 2010 integrated biological and behavioural surveillance survey (25.5%).10 the significance level was set at 0.05 and a calculated minimum sample size of 291 was obtained, which was increased by 10% to 320 to allow for nonresponse . The snowball sampling method was used to recruit 107 participants from each of the centers . The msm operating the centers were recruited as the initial seeds, and were then asked to recruit peers who visit the centers . These next groups of respondents were similarly asked to identify peers, and this went on until the desired sample size was attained . None of the respondents received any form of incentive or reimbursement for participation in this research . Three research assistants were recruited, trained, and paid a stipend to assist in the distribution and collection of the questionnaires in the field . Ethical approval was obtained from the lagos university teaching hospital health research and ethics committee (reference adm / dcst / hrec / vol . A semistructured pretested questionnaire was used to collect information on demographics, alcohol and drug use, hiv risk behavior, and experience of human rights violations . Aggression was described as unprovoked attacks, confrontation, and violent behavior as a result of sexual orientation . Alienation was described as a perceived feeling of isolation by family or friends from activities which they would have normally been involved in . The data analysis was done using statistical package for the social sciences version 20 software (ibm corporation, armonk, ny, usa) and presented as means and percentages . Only 291 questionnaires were analyzed because some of the respondents did not complete all items in the questionnaire . Bivariate analysis was performed to test the relationship between dependent and independent variables at p<0.05 . A logistic regression model was used to assess the relationship between the outcome variables, namely acts of aggression and sexual and physical abuse, and sociodemographic variables that were statistically significant on bivariate analysis with a 95% confidence interval and p<0.05 . Three of the seven lgas were selected through simple random sampling; each of the selected lgas had only one community center, so all three were used . Advocacy visits were subsequently conducted to the three community centers, and the stakeholders and networks manning the centers were duly briefed on the study and its purpose . The benefit of the study was also highlighted, and verbal informed consent was given by the authorities manning the three centers . Participants recruited for the study were men over 18 years of age who had had sexual relationships with members of the same sex and visited the community center for recreational activities or hiv prevention information and services between 9 am and 6 pm from monday to saturday in august 2012 . Heterosexuals who visited the community centers for whatever reason during the period of data collection were excluded from participation by staff and peers . Only msm who agreed to participate in the study were given questionnaires to complete . Participation was voluntary and participants were assured that all information supplied would be anonymous and kept in a confidential manner . The formula for calculating sample size in a cross - sectional descriptive study was used based on the proportion of msm reporting risky sexual behavior in the 2010 integrated biological and behavioural surveillance survey (25.5%).10 the significance level was set at 0.05 and a calculated minimum sample size of 291 was obtained, which was increased by 10% to 320 to allow for nonresponse . The snowball sampling method was used to recruit 107 participants from each of the centers . The msm operating the centers were recruited as the initial seeds, and were then asked to recruit peers who visit the centers . These next groups of respondents were similarly asked to identify peers, and this went on until the desired sample size was attained . None of the respondents received any form of incentive or reimbursement for participation in this research . Three research assistants were recruited, trained, and paid a stipend to assist in the distribution and collection of the questionnaires in the field . Ethical approval was obtained from the lagos university teaching hospital health research and ethics committee (reference adm / dcst / hrec / vol . A semistructured pretested questionnaire was used to collect information on demographics, alcohol and drug use, hiv risk behavior, and experience of human rights violations . Aggression was described as unprovoked attacks, confrontation, and violent behavior as a result of sexual orientation . Alienation was described as a perceived feeling of isolation by family or friends from activities which they would have normally been involved in . The data analysis was done using statistical package for the social sciences version 20 software (ibm corporation, armonk, ny, usa) and presented as means and percentages . Only 291 questionnaires were analyzed because some of the respondents did not complete all items in the questionnaire . Bivariate analysis was performed to test the relationship between dependent and independent variables at p<0.05 . A logistic regression model was used to assess the relationship between the outcome variables, namely acts of aggression and sexual and physical abuse, and sociodemographic variables that were statistically significant on bivariate analysis with a 95% confidence interval and p<0.05 . The study participants had a mean (standard deviation) age of 25.34.6 years, close to two thirds (59.1%) had at least a secondary school education, and 29.9% had post - secondary education . Two thirds (66.0%) of the men were currently single and not in a steady relationship, while 19.6% were cohabiting or in a steady relationship and only 12.0% were married . Regarding sexual orientation / identity, 50.5% self - reported as gay, 47.8% as bisexual, and 1.7% as transgender (table 1). Alcohol use was quite common, with 56.7% of the msm consuming alcohol; in this group, more than half (57.6%) reported drinking alcohol to the point of intoxication . Only 11.1% of the men reported using hard drugs, ie, marijuana, amphetamines, and cocaine, and none reported using more than one type of hard drug or injecting drugs (table 1). Recreational activities include visiting hotspots (reported by 43.6%), partying (38.8%), clubbing (35.7%), watching movies (35.7%), and surfing the internet (39.2%); 29.9% of the men sought sexual partners at the community center, 21.0% from hotels / brothels, 39.5% at parties, 39.9% when they visited private homes, and 48.1% from bars / clubs . One third (33.0%) had disclosed that they had same sex relationships to either a family member / friend or both . Homosexual / transgender respondents were more likely to have made such a disclosure than bisexual respondents (51.3% versus 15.8%; p<0.001, see table 1). About a third of the men reported at least one act of human rights violation, ranging from aggression 35.7%, alienation 29.9%, discrimination in the workplace 8.2%, eviction 2.1%, or refusal of service 4.1% . Another 20.3% reported at least one incident of violence, including rape by a man 16.8%, verbal abuse 19.2%, physical abuse 17.9%, and psychological abuse 20.3% (table 2). On bivariate analysis, a higher proportion of msm who were single and not in a steady relationship were found to report acts of aggression compared with those who were married / in a steady relationship (p=0.003). . A higher proportion of young msm (<30 years) and men with a low level of education reported experiencing acts of aggression, respectively (p=0.020 and p=0.029; table 3). Homosexuals were more likely to report experiences of social oppression, ranging from acts of aggression (p=0.002) to physical (p=0.036) and sexual (p=0.044) violence . Disclosure of sexual orientation was associated with acts of aggression (p<0.001) and physical abuse (p<0.001). Alcohol consumption was associated with reports of physical (p=0.020) and sexual (p=0.001) violence . A higher proportion of msm who solicited for sexual partners from brothels, clubs, and community centers reported instances of aggression (p=0.003, p=0.014, and p=0.003, respectively). The relationship between age and reported acts of aggression was not maintained in multivariate analysis . Msm with less than a secondary school education were twice as likely to report acts of aggression (adjusted odds ratio [aor] 2.6; p=0.019). Homosexual / transgender respondents were also twice as likely to report acts of aggression (aor 1.9; p=0.017), as were unmarried msm not in a steady relationship (aor 2.3, p=0.005). Men who consumed alcohol were three times as likely to report sexual abuse (aor 3.4; p=0.001) and twice as likely to report physical abuse (aor 2.3; p=0.013). Homosexual / transgender men were twice as likely to report sexual (aor 2.2; p=0.021) and physical abuse (aor 2.1; p=0.022). The respondents in this study were mostly young men under the age of 30 years with a fair level of education for a low income country . It has been observed that young msm are especially vulnerable in this era of hiv infection.11 decreasing or stable rates of infection have not been achieved in this group . Globally, less than 10% of msm are estimated to have access to hiv prevention services at an individual level, within a social context, within communities, or from health service providers.12 further, a high prevalence of hiv infection was recorded among people less than 30 years of age in the 2010 national seroprevalence study conducted among the general population in nigeria.9 bisexuality has been found to be common among african msm . The proportion of the young people in this study who described themselves as bisexual is much higher than the 17% recorded in cape town.13 bisexuality was described as an adaptation to escape the social stigma associated with gender orientation or identity by msm in senegal.14 alcohol consumption was more common than tobacco use or substance abuse, and like the study conducted among msm in northern thailand, alcohol use and substance abuse was not significantly associated with sexual orientation.15 in african countries where same sex relationships have been criminalized or where msm experience social stigma, more and more msm are turning to the internet to meet their peers as well as to solicit for sexual partners.16 studies carried out in various african countries have reported human rights violations among msm . In cape town, almost a quarter of respondents in one study had been subjected to either blackmail, physical / sexual abuse, or denial of services.13 in lesotho, about three quarters of msm in a study reported having experienced beatings, rape, blackmail, or harassment as a result of their sexual orientation.17 respondents in a multicountry study carried out in malawi, namibia, and botswana mentioned violence as a threat to their life as msm, with more than 40% having been raped by men, beaten up, blackmailed, or denied health care.16 experiences of msm in this study with regard to this issue were similar, although sexual orientation was a predictor of social oppression . Social stigma puts msm at greater risk of developing mental health problems, thereby adding to the disease burden . In most sub - saharan african countries, the expression of masculinity is that men should be strong and not display signs of weakness or vulnerability . This leads to poor health - seeking behavior by men, resulting in a low turnout for preventive, curative, and rehabilitative care until disease has progressed or complications arise resulting in premature death.18 an hiv prevention, care, and treatment program carried out in senegal between 2004 and 2007 was able to achieve a reduction in individual risk - taking behavior (unprotected sex) among msm . This can only be carried out in an environment free of violence, blackmail, and acts of discrimination . With the criminalization of same sex relationships in the same country, there was a substantial reduction in the provision of services by health workers and the uptake of these services . The majority of the targeted group went into hiding as a result of increased violence and discrimination.14 similarly, a high proportion of msm in other african countries reported being afraid to seek health care services because they had been victims of blackmail on the basis of their sexual orientation.19 bisexual men in hiding will not access services from community centers and networks linked to msm because of fear of exposure . At the same time, they are not adequately catered for in national programs, which focus more on women and children . They are therefore at the risk of being missed, excluded, or side - lined.19 recruitment of msm occurred at a community center which is most probably frequented by msm from a particular network / lga . Access to the study population would have been extremely difficult if the researchers did not actively engage the management team of the centers . The msm operating the centers were the ones recruiting their peers into the study, which could have introduced some degree of selection and response bias . A nonprobability sampling method was used to recruit participants for the study, even though the lgas where the centers were located were selected using a simple random sampling method . This study was carried out among a subset of the key population in need of intervention in the hiv epidemic, and reveals that msm in lagos state have been victims of various acts of human rights violation and abuse . There is an urgent need to document and quantify these happenings in all the states of the federation . It will also be necessary to investigate how human rights violations of msm in nigeria impacts on access to hiv care and prevention of transmission.
Toxoplasmosis, a zoonosis caused by obligate intracellular protozoan toxoplasma gondii [1, 2], occurs in about one third of world population . Having the cats as definitive hosts, especially the domestic cat, it shows a wide range of intermediate hosts, including humans . In immunocompetent individuals, severe forms are secondary to infection in immunocompromised patients or when acquired during pregnancy, when it may cause congenital disease with severe visual and neurological impairment . Transplacental transmission of t. gondii occurs during the period of acute maternal infection [57]. The rate of congenital toxoplasmosis with risk for severe fetal varies from 15 to 68%, depending on gestational age; the transmission rate is highest in the later stages of pregnancy [8, 9]. The congenital disease is characterized by a broad spectrum of clinical manifestations with neurological, ophthalmological, and systemic involvement . Although most newborns with congenital infection show no signs of infection at birth, up to 85% of these will develop visual impairment in their lives, and 55% will present neurological disorders [1012]. The prenatal care, when done properly and from the beginning of the pregnancy, allows early diagnosis of infection, allowing the team more time and resources to treat the fetus . There are few studies addressing the degree of knowledge on toxoplasmosis of the health professionals . In the few surveys carried out among obstetricians, a deficit in the knowledge about the diagnostic, clinical, and epidemiological aspects of toxoplasmosis nevertheless, similar surveys were not developed in countries with high prevalence of toxoplasmosis as brazil [1820], where a previous study observed little knowledge on the subject from pregnant women, in particular regarding the infection prevention . In a previous work, the authors observed that the majority (81.3%) of the women sent to a referral service with a diagnosis of toxoplasmosis were coming from public health units and that for only 16% of them treatment was initiated at the origin unit . This study aims to assess the knowledge about toxoplasmosis by doctors and nurses involved in prenatal care at public health units in brazil . We conducted a cross - sectional study of 118 physicians and nurses who provide prenatal care at public health units of a midsize brazilian city . We chose the city of juiz de fora, in minas gerais (214551s and 432101w), with 500,000 inhabitants and altitude tropical climate . This city was selected because it is a located in a region of high prevalence of toxoplasma infection and offers the following services for the care of pregnant women: basic health units with the family health program (fhp) and three public hospitals . All physicians and nurses providing care in all basic units of the family health program (fhp), prenatal clinics, and public hospitals were invited to participate . The study excluded the professionals who did not provide prenatal care last year, who works exclusively in rural areas, and those who did not agree to participate . Data was collected through an anonymous and self - completed questionnaire, including questions related to the professional profile and nine items about toxoplasmosis equally distributed among diagnosis, clinical issues, and prevention (table 1). Epidata version 3.1 was the software used with double capture and control for data entry . The analysis was performed in the statistic software statistical package for social sciences (spss) version 16.0 . In exploratory analysis of the data, we described the frequencies of qualitative variables and summary measures, such as mean and standard deviation for continuous variables . In continuous variables, we checked the data normality (total number of correct answers in each of the variables: diagnosis, clinical issues, and prevention) by the kolmogorov - smirnov test . At the 5% level of significance, the hypothesis of normally distributed data was rejected, which indicated the use of nonparametric tests . To check for differences in the number of correct answers and the different professional performances (fhp or hospital) p values <.05 indicated significant differences, as demonstrated by the comparison of medians (md) and their respective interquartile ranges (iqrs). In checking the association between qualitative variables (sex, time from graduation, type of graduation, profession, expertise, previous experience with pregnant women with toxoplasmosis, areas of expertise) and the total number of correct scored (up to four correct answers and over four correct answers) the independence pearson chi - square test was used . For tables with low counts (less than five) fisher's exact test was used . The cutoff of the total number of correct answers was categorized according to the median of total correct answers . The study was approved by the ethics in research committee from the institute of clinical research evandro chagas (instituto de pesquisa clnica: ipec)-fiocruz under the protocol 0029.0.009.000 - 09 . Of the 118 professionals who participated in the study, 112 respondents completed the questionnaire (61 physicians and 56 nurses), and six of them did not agree to respond the questionnaire . The population consisted mostly of women (80.5%), age 39 9.7, with a degree in public schools (88.2%) and postgraduation stricto and lato sensu . Seventy percent (83) of professionals worked at primary fhp and 30.0% in public hospitals . Fifteen percent also worked in private hospitals, 13.7% in private clinics, and 12% in educational and/or research institutions . Fifteen percent of the respondents reported having provided care for pregnant women with toxoplasmosis in the past 12 months . In the block of questions related to the diagnosis, most professionals said that repeated serology for toxoplasmosis in pregnant women is needed for those who are not immune . Regarding prevention, 97.4% of professionals recognized the cat as the animal that eliminates the parasite in the feces, but 51.7% said the dogs also eliminate oocysts . The largest number of errors was evident in relation to the education of nonimmune pregnant women in regard to not ingesting raw vegetables (table 1). The overall median from the hospital was significantly higher than the fhp's median . Comparing the number of correct answers according to the area of the professional expertise, the professionals who worked in hospitals showed the total number of correct answers on the issues of diagnosis, significantly higher than those who worked in the fhp . Both professionals in hospitals and the fhp had more correct answers to questions about clinical issues and fewer correct answers on prevention (table 2). In the comparison according to the professions, the physicians had the highest number of correct answers in the diagnostic and clinical issues . Regarding prevention, no significant differences were found between the two professional categories . The issues of prevention had lower scores both among physicians and nurses (table 3). Table 4 presents the results of the analysis of the association of the total number of correct answers classified (up to four or> 4 correct answers) with several characteristics of the professionals . Greater number of correct answers was observed in professionals with time of graduation up to 10 years and among physicians . The basic health units (bhus) are the main gateway to the health system in brazil, and the professionals who work at the family health strategy have a wide work area . Therefore, it is necessary that health professionals have a general education as the proper conduct of health problems depends crucially on the degree of the professional's knowledge . As it is a mother - to - child-(mtc-) transmitted disease, early diagnosis and treatment can prevent serious and irreversible fetal damage [13, 23]. Thus, it is critical that doctors and nurses who provide prenatal care are appropriately trained on the prophylactic, diagnostic, and clinical aspects of the mtc - transmitted diseases . In relation to t. gondii infection, its complex life cycle, the variable clinical spectrum including mostly oligosymptomatic presentation, and no consensus on guidelines contribute to the ignorance or mistaken notions of professionals with regard to this zoonosis . Regarding the contents of the questionnaire, the block of questions concerning diagnosis, most professionals have responded correctly that it is necessary to repeat serology for toxoplasmosis in pregnant women who are not immune . The periodicity of this screening was not asked as the authors opted for a question of minor complexity . Less than half of professionals recognized low avidity as an indicator of recently acquired infection, and an even smaller proportion still knew that igm anti - t . Gondii may remain positive for more than a year . The greatest number of correct answers concentrated on clinical issues, although the responses showed a misunderstanding between the period of greatest transmissibility and the period of increased severity of congenital injuries [25, 26]. About prevention, although 97.4% of professionals recognize the cat as the animal that eliminates the parasite in feces, professionals attributed this role, which is unique to felidae [27, 28], to other animals as well, especially dogs (51.7%) and pigeons (21.6%). The largest number of errors was evident in relation to the orientation of non - immune pregnant women as to not eating raw vegetables . As oocyst has a major importance in the epidemiology of toxoplasmosis, and taking into account water as an important font of infection and oocyst resistance to chlorine and acetic acid, the authors recommend that eating raw salads and unpeeled fruits should be avoided in nonimmune toxoplasma pregnant women in brazil [18, 2931]. The fact that the overall median number of correct answers and diagnosis from professionals working in hospitals were higher than those of the fhp may in part be explained by the fact that professionals working in hospitals have more training in high - risk prenatal care . However, a higher number of correct questions of prevention among professionals fhp were expected, since the basic health unit has as one of its main missions educational activities aimed at preventing health problems . Likewise, the median number of correct answers among physicians higher than that of nurses is expected in the diagnosis and clinical issues, as these issues are the object of focus in medical schools than in schools of nursing . However, the basic knowledge of nurses in issues related to prevention is inconsistent with the role of nurses as health educators . Eleven (15.3%) respondents reported having provided care to pregnant women with toxoplasmosis in the last 12 months, two times the percentage already considered high in a study conducted in the united states, implying that a considerable proportion of doctors and nurses encounter over their lives with at least one case of a pregnant woman with toxoplasmosis . The greatest number of correct answers among the professionals with less training time is consistent with the literature that indicates an inverse correlation between knowledge and years of professional practice, justifying the need for recertification exams in some countries [31, 32]. Continued education seems especially useful when targeted to specific groups and disciplines like our target population . The low ratio observed (44.4%) of correct answers on questions about global toxoplasmosis reinforces the need for action and continuing education programs on this zoonosis . Finally, the authors suggest that similar surveys shall be conducted in other endemic regions and with larger populations . The establishment of a cutoff for the number of correct answers can be a useful tool for a possible intervention, providing support to actions for continuing education about pregnancy and congenital toxoplasmosis for these occupational categories.
Cardiovascular diseases are among the most frequent causes of death worldwide [1, 2]. Heart failure is an enormous medical and societal burden and a leading cause of hospitalization . It is estimated that 2.6 millions hospitalizations annually in the usa are due to heart failure as a primary or secondary diagnosis . In the last 19 years the role of several immunological, metabolic, and neurohormonal abnormalities has been recognized in the pathophysiology and progression of the congestive heart failure (chf) [4, 5]. Among them, the cardio renal anemia syndrome (cras) represents a pathological triangle in which the primary failing organ is the heart or the kidney and the dysfunction of one organ leads to dysfunction of the other . The presence of anemia or renal dysfunction increases morbidity and mortality in patients with heart failure . It seems that there is an impaired mechanism operating between congestive heart failure, chronic kidney disease (ckd), and anemia, where each might cause or worsen the other . Therefore, correction of anemia would be a major part of this vicious circle in the reduction of the severity of the heart failure . This could be explained by the fact that a significant feature of the congestive heart failure is impaired energy metabolism and therefore the failing heart is an energy - starved heart . Oxygen delivery through hemoglobin (hb) is essential for energy production and improvement of hb levels could also improve energy production in cardiomyocytes . Using the historical definition by the world health organization, anemia is defined when hb concentration is less than 13 g / dl for men or less than 12 g / dl for women . However, particularly in the setting of heart failure, this definition has not been subjected to rigorous clinical validation and its appropriateness and clinical applicability continues to be debated . Therefore, some investigators use more conservative definitions (e.g., <12 g / dl for men and <11 g / dl for women) to ensure a higher confidence in capturing the affecting population . A recent meta - analysis of 153,180 patients with chf, reported in 34 published studies from 2001 to 2007, estimated the prevalence of anemia to be 37.2% (1049%). Similarly, the latest prospective stamina - hfp (study of anemia in a heart failure population) registry estimated a prevalence of 34% . The variability in the estimated prevalence of anemia is partly attributable to use of different definitions of anemia, whereas patients in the acute decompensated states experience more dilutional anemia and therefore the prevalence may be increased . Patients with chf and anemia tend to be older than their nonanemic counterparts, whereas, in patients less than 55 years, the age of anemic and nonanemic patients does not appear to differ . Concerning the gender, in studies of chf and anemia enrolling a preponderance of men, the proportion of women steadily increases as hb concentration falls to the point that women can predominate among patients with chf and severe anemia . One of the most frequent comorbid conditions in patients with chf is ckd (as defined by an estimated glomerular filtration rate (egfr) <90 ml / min/1.72 m). Chf and ckd share some common causes (e.g., hypertension), features (e.g., malnutrition, impaired performance status), and risk factors (e.g., older age). In a meta - analysis of 16 studies, it was found that 63% of 80,098 patients with chf had some degree of concomitant impaired renal function and 29% of them had severe ckd . This is associated with an increased risk of adverse outcome, being probably a stronger predictor of mortality than ejection fraction (ef) or new york heart association (nyha) functional classes . Anemia is more prevalent when chf and ckd coexist in both ambulatory and hospitalized settings . In large chf registries the degree of anemia closely parallels to egfr, although primary renal disease is relatively uncommon in chf [17, 18]. This justifies that kidneys play a major role in the pathophysiology of anemia in chf . It is important that patients with chf and ckd develop anemia in higher values of egfr than patients with ckd alone . This provides indirect evidence that other factors than ckd are involved in the pathophysiology of anemia of chf . There is an impaired mechanism in which tissue hypoxia and release of nitric oxide (no) cause decreased arteriolar resistance and peripheral vasodilatation . These in turn lead to decreased blood pressure, increased sympathetic activation, renal vasoconstriction, reduced renal function, and activation of renin - angiotensin aldosterone system . The results are production of antidiuretic hormone, fluid retention, left ventricular (lv) hypertrophy and dilation, worsening of heart failure, release of brain natriuretic peptide (bnp), and signs from stress on myocardium . This, however, implies that in the presence of volume overload there might be a decrease in hb concentration and also oxygen content, although red cell mass remains stable . The patient complains of shortness of breath, tachycardia, dizziness, faintness, and fatigue . Thus, there is a greater prevalence of anemia in hospitalized patients than ambulatory ones . On the other hand, the presence of more advanced nyha functional classes has been associated with greater prevalence of anemia [21, 22]. Furthermore, anemic patients with chf have more commonly diabetes mellitus and more advance disease, with higher nyha class and more severe symptoms . Those symptoms include lower exercise capacity, worse quality - of - life scores, greater peripheral edema, lower dry weight and blood pressure, higher use of diuretics and other cardiovascular medications, and worse neurohormonal profile (such as renal dysfunction, high bnp and c - reactive protein, low serum albumin) (range, 30% to 61% versus range, 4% to 23% for less symptomatic ambulatory patients) [23, 24]. It is remarkable that anemia does not seem to be related to lv dysfunction, whereas in few studies hb levels were inversely related to ef . That means that patients with lower values of hb had higher ef, whereas increase of hb could decrease lvef, especially in ckd, in a dose - dependent manner [22, 2527]. Finally, most studies indicate that the prevalence of anemia is increased in patients with chf who also have co - morbid kidney disease, advanced age, and more severe symptoms when compared to less symptomatic ambulatory populations . The major factors contributing to chf - related anemia involve ckd, renin - angiotensin system, hematinic abnormalities, mainly iron deficiency, chronic inflammation, and hemodilution (figure 1). A major factor contributing to anemia of chf is kidney dysfunction, being associated with the cardiac disorder . The renal damage in the chf is mainly hypoxic, due to reduced renal flow, caused by the reduced cardiac output [14, 24]. Hypoxia induces erythropoietin (epo) production by peritubular fibroblasts, although renal blood flow in chf is relatively maintained until the late stages of the syndrome, especially when receiving angiotensin - converting enzyme (ace) inhibition . This suggests that renal dysfunction plays a role in the blunted epo production in anemic patients with chf, resulting in increased epo levels but not as expected for the degree of anemia, suggesting that in chf there is both blunted epo production and resistance to epo . Furthermore, the coexistence of chf with ckd is associated with reduced epo production from the kidney, as well as with urinary losses of serum epo and transferrin [29, 30], which further deteriorate the anemia . The renin - angiotensin system seems also to be involved in the control of erythropoiesis . Angiotensin ii reduces renal blood flow, increases the oxygen demands, and thereby stimulates epo production . It also stimulates the proliferation of normal bone marrow early erythroid progenitors in a direct manner . Both ace inhibition and angiotensin receptor blockade decrease erythropoiesis, causing a modest reduction in hb, up to 0.3 g / dl [14, 32]. This suppression is attributed to a mild reduction of epo production and also to prevention of hematopoiesis inhibitor n - acetyl - seryl - aspartyl - lysyl - proline (acsdkp) breakdown . Ace inhibition should be expected to cause a mild reduction of erythropoiesis, although various knockout mice models, involving different ace components, do not support this theory . Iron deficiency is common in patients with chf especially when accompanied by ckd, whereas vitamin b12 and folic acid deficiencies or iron overload are not . It is of interest that the incidence of iron deficiency is increasing with the severity of heart failure . In half cases, iron deficiency is absolute (with low transferrin saturation and serum ferritin, usually associated with decreased iron stores and reduced iron deposits in the bone marrow). In the other half cases iron deficiency is functional - relative (with low transferrin saturation and normal or elevated serum ferritin, usually associated with normal or elevated iron stores and iron deposits in the bone marrow). It has been reported that in 17% of anemic patients with chf the iron deficiency is both absolute and functional . There are many causes of absolute iron deficiency associated with chf, especially in coexistence with ckd . These include low iron intake (due to low protein diets and anorexia), gastrointestinal blood loss (due to platelet dysfunction or coagulation abnormalities; caused by platelet inhibitors, anticoagulants, or uremia), iron malabsorption (due to either chf or uremia related - bowel edema, causing intestinal cell dysfunction, or to proton pump inhibitors or to phosphate binders, that also bind iron), removal of blood for tests . In chf the functional iron deficiency is related to iron disuse, resembling anemia of chronic disease, as evidenced by iron acquisition by the reticuloendothelial system . In patients with severe chf, elevation of several inflammatory cytokines serum levels has been found . Among them, interleukin-1 (il-1), interleukin-6 (il-6), tumor necrosis factor- (tnf-), and less frequently interleukin-18 (il-18) seem to be the most important, whereas il-6-induced hepcidin expression also participates in the phenomenon [14, 38, 39]. This inflammatory process causes reduced epo production, through activation of gata 2 binding protein and nuclear factor-b, and impaired response to bone marrow erythroblasts . It also causes hepcidin - induced blockade of iron absorption from the gut and iron trapping in reticuloendothelial system's stores . Hepcidin is an acute phase antibacterial protein, induced by il-6, through jak / stat3 pathway, released from the liver and excreted from the urine . Therefore, in chf with a concomitant ckd, there is a reduced hepcidin removal from the kidney, implying a further increase of its levels . Hepcidin inhibits ferroportin, a protein expressed on intestinal cells, macrophages, and hepatocytes that releases the iron from those cells into the blood . Hepcidin - induced inhibition of ferroportin causes decreased iron absorption from the gut and blockade of iron release from its stores, in hepatocytes and macrophages, into the blood . This implies inadequate iron delivery to the bone marrow erythroblasts, although the total stores may be adequate, causing a functional iron deficiency . Iron metabolism is crucial for energy production in the body and most importantly for cells with high energy demands, such as cardiomyocytes . Iron plays a crucial role in oxygen transportation (as a component of hb), oxygen storage (as a component of myoglobin), oxidative metabolism (as a component of oxidative enzymes and respiratory chain processes), and in metabolism of lipids, carbohydrates, nucleic acids, collagen, tyrosine, and catecholamines [41, 42]. In chf, an energy - starved situation, iron deficiency, absolute or functional, can impair oxidative metabolism, cellular energetic, and cellular immune mechanisms . Iron deficiency in rat hearts causes mitochondrial ultrastructural aberrations, irregular sarcomere organization, and release of cytochrome c . In addition, experimental animal models with severe iron deficiency have major disruption in energy production causing cardiac damage, with diastolic dysfunction and heart failure, accompanied by reduced epo and increased tnf- serum levels and worsening of molecular signaling pathways . Those defects may participate in the transition from adaptive cardiac hypertrophy to permanent cardiac impairment in chronic iron deficiency . On the other hand it has been shown that hemodialysis patients receiving epo with iv iron supplementation had lower inflammation markers (lower levels of proinflammatory tnf- and free radicals, as expressed by total peroxides, and higher levels of anti - inflammatory interleukin-4) compared to patients receiving epo alone . Furthermore iron deficiency anemia seems to enhance red cell oxidative stress and has been associated with lower peak oxygen consumption and higher ratios of ventilation to carbon dioxide production . In a recent study the disordered iron homeostasis has been identified as an independent risk factor for death . Chf is a hypercoagulable state, where the co - existence with iron deficiency - related thrombocytosis increases the risk of thrombosis and the mortality rate . Furthermore, it has been shown that the concomitant administration of iv iron with epo (which can cause iron deficiency) in hemodialysis patients significantly reduces the platelet counts, compared to patient receiving epo alone . It has also been shown that the use of erythropoiesis - stimulating agents (esas) in iron - deficient patients increases the risk of thrombocytosis and thrombosis . Except iron disuse it has been suggested that there is a diminished responsiveness of erythroid cells to epo, being accompanied by increased levels of inflammatory cytokines, such as il-6, soluble tnf receptor 2, and tnf- levels [52, 53]. Their activation does not result in epo receptor downregulation, but in blunted epo - induced jak - stat signaling . This is confirmed by the partial abrogation of the inhibitory effects of anaemic sera on erythroid colony growth by anti - tnf- neutralising antibodies . Endothelial dysfunction associated with heart failure may alter endothelial no synthase activity, hence further augmenting myocardial dysfunction due to increased oxidative stress . Paralleling these cardiodepressive actions, no seems to have a direct inhibitory effect on bone marrow hematopoietic activity [55, 56]. Furthermore, it is of interest that no inhibits blood cell formation in nonischemic murine chf, whereas inflammatory cytokines, such as tnf-, impair hematopoiesis in chf following myocardial infraction . A recent study suggests that vitamin d deficiency is independently associated with anemia in end - stage heart failure, based on the fact that vitamin d may stimulate erythropoiesis . On this study circulating 1,25-dihydroxyvitamin d was a better predictor of anemia than circulating 25-hydroxyvitamin d. prospective randomized studies with administration of vitamin d will have to clarify if the association of vitamin d deficiency with anemia is causal . In conclusion anemia in chf two major factors contributing and exacerbating to its appearance are kidney dysfunction and iron deficiency . In 2003 this abnormality was described as cardio renal anemia syndrome (cras), whereas the correction of anemia could play a major role in this vicious circle in improving the severity of chf . In the last years, the role of iron has been recognized, as a major component of many energy - producing systems . In view of a possible independent association of iron deficiency and cardiac failure, renal failure and anemia, the same authors rename the syndrome as cardiorenal anemia iron deficiency (craid) syndrome . It is the final common pathway of other conditions, where renal failure and anemia contribute to the progression to a more severe disease status . Since the two major components of chf anemia are iron deficiency and reduced epo activity (absolute or functional in both cases), the main goals of intervention would be to increase their levels . Anemia treatment strategies in heart failure patients include erythropoiesis - stimulating agents (esas) and red blood cell transfusions . Iron replacement in iron - deficient patients with or without anemia has also been investigated . Before starting any treatment for anemia in chf, it is necessary to exclude and treat, if possible, any other causes of anemia, such as active bleeding, hemolysis, vitamin b12 or folate deficiency, or even more chronic situations such as myelodysplastic syndromes and other malignancies . Health services research & development service's evidence - based synthesis program has collected the literature from 1949 until november 2010 and published a review regarding the treatment of anemia in chf and coronary heart disease patients . Despite the association with poorer outcomes, it remains unclear whether treating anemia or iron deficiency may improve outcomes . It has been suggested that a small reduction of hb levels may worsen the outcomes and symptoms of chf, whereas the correction of anemia may improve nyha classification, lvef, lvh, and diuretic response [6063]. Treatment has been centered on administration of erythropoiesis - stimulating agents (esas) and parenteral iron supplementation, but most studies are poorly powered and therefore with limited validity . On the other hand, the only way to cause a rapid increase of ht and therefore tissue oxygenation is the blood transfusion . Nevertheless, the only recommendations referring to anemia of chf suggest treating any correctable causes of anemia, if this is evitable, such as iron, folate, or vitamin b12 deficiencies . The goal of hb correction is also transfusion of packed red cells in hb values lower than 9 g / dl or ht less than 30% may be suggested, but in the setting of acute coronary syndromes this has been associated with higher mortality . There are many data supporting that iron deficiency may contribute to the increased mortality in chf patients . It has also been shown that correction of iron deficiency could improve symptoms and status of the syndrome . Nevertheless, this has not been adequately confirmed . There are several studies in chf, with iron deficiency, with and without anemia, suggesting that correction of iron deficiency, using iv preparations, could improve symptoms and signs of chf, such as s-wave, e / e ratio, peak systolic strain rate, nyha class, 6-minute walk distance, even without improvement of ef or rise of hb levels, without major side effects [6670]. It is suggested, with high evidence, that correction of iron deficiency can improve exercise tolerance and duration, as well as the quality of life, in patients with stable chf and mild ckd . There are also other studies suggesting that anemia in chf can be corrected only with iv iron and not the oral forms [7174], whereas long - term oral iron does not seem to improve any chf parameters [75, 76]. This seems reasonable since in chf there is increased hepcidin expression that blocks iron use, even if it is absorbed . In general terms it is preferred to administrate iv instead of oral iron, even though it can cause oxidative stress . When using intravenous preparations, the majority of the dose is deposited in long - term storage . There is a small portion of this iron that is rapidly bound to transferrin and available for transport to the bone marrow, bypassing the restrictions on iron release imposed by hepcidin . The use of large amounts of iron, delivered over minutes or hours as pulse therapy, could lead to poor utilization of this iron with tissue deposition, free radical formation, and increased risk of infection, because of a decrease in cellular immunity and promotion of bacterial growth . Other concerns of iron administration have to do with an increased risk of coronary heart disease, but still have not been confirmed . In conclusion, there are not efficient data proving evidence that iron administration in non - iron - deficient patients with chf could improve the cardiac disease . It is however of great value to recognize iron deficiency, even in the absence of anemia and to correct it . Regarding esas, there are only pilot studies for their use in the treatment of anemia of chf . They are commonly used with simultaneous administration of iv iron, giving promising results in ameliorating symptoms and improving cardiac function, but only with very limited evidence [77, 78]. Moreover, the epo receptor is present in many nonerythropoietic tissues, including myocardium, endothelium, vascular smooth muscle cells, and neurons, where epo has shown tissue protective properties, because of its antiapoptotic action . Treatment with epo in excitable murine and human left ventricular muscle preparations have resulted in an increase in twitch tension and in peak sarcomere shortening . This suggests that epo exhibits direct positive inotropic and lusitropic effects in cardiomyocytes and ventricular muscle preparation, being mediated through pi3-k and pkc isoform signalling, to directly affect both calcium release dynamics and myofilament function . Therefore, there could be an extra role of epo in ameliorating symptoms, in addition to improving hb levels . The major concern of epo administration, based on hemodialysis experience, has to do with a possible increase of thrombotic risk, especially by overzealous correction of anemia or when iron - deficiency - associated thrombocytosis coexists . At present, there are not clearly defined targets of hb in ckd anemia, although an hb level of 10 g / dl seems to be a widely accepted goal . Recently, there have been many studies of anemia in chf patients with an effort to improve hb levels . There have been used esa and oral or iv iron and even iv iron without esa, that have shown a positive effect on hospitalization, nyha functional class, cardiac and renal function, quality of life, exercise capacity, and reduced bnp, without any increase in cardiovascular damage related to the therapy . However, adequately powered long - term placebo - controlled studies of esa and iv iron in chf are still needed and are currently being carried out . Nevertheless, until recently, there is high evidence that the use of esa in stable chf patients with serious renal disease did not have any particular effect on cardiovascular events, whereas there is moderate evidence that their use might have a negative impact on the survival . Recent studies have recognized that dilutional anemia is highly prevalent in chf patients . In this regard, arginine vasopressin antagonists might be an attractive treatment option, by increasing aquaresis . Therefore, future clinical research should explore the role of arginine vasopressin pathway activation in determining dilutional anemia and, ultimately, assess it as a therapeutic target . The evidence base to date does not convincingly support a role for esas for anemia correction . On the other hand, iron treatment may help ameliorate symptoms over the short term in patients with symptomatic heart failure . It seems that anemia exacerbates chf, causing a vicious circle, where renal dysfunction and neurohormonal and proinflammatory cytokine activation participate in the development of anemia . On the other hand, so, it is important to recognize any possible causes of anemia . It would also be beneficial to treat them, if possible . Administration of iron and esas seems to be promising, since they can both also improve factors other than anemia, but still there are many questions to be answered . These mainly concern their safety, the goals in hb elevation, and probably their cost . Further studies are required to understand the association of anemia with chf outcomes, to recognize the impact of anemia improvement, to asses when to initiate and when to cease the treatment, and finally to estimate the safety of these interventions.
Neurofibromatosis type 1 (nf-1) is an autosomal dominant disorder affecting 1 in 25003500 individuals . The fact that it is a progressive disorder and that almost 50% of cases are sporadic mutations multiplies its clinical magnitude several folds . Discuss a report in which they described and classified seven different types of nf . Of these, nf-1 and nf-2 it is a distinct relative of nf-1 but is 10%20% rarer, accounting for approximately one case in 36,00080,000 individuals . We present a case; there was nf-1 with an extra calvarial plexiform neurofibroma (pnf) in occipito - cervical region with intracranial extension . A large portion of the visible swelling consisted of a neurofibroma and multiple caf au lait spots on the left upper and lower trunk [figure 1]. Swelling was excised completely . (a) clinical photographs showing the occipito - cervical swelling . (b) clinical photographs showing caf au lait spots on the left side of upper and lower trunk this 5-year - old boy presented with history of a gradually progressive painless swelling in the occipito - cervical region since birth . On examination, he had a large (10 cm 10 cm 5 cm) occipito - cervical swelling, that was soft and nonfluctuant, and transillumination was negative . The boy had no neurological deficits and no signs or family history of nf-1 . In neuroimaging studies, the lesion was seen as an isointense on t1-weighted and heterogeneously hyperintense mass on the t2-weighted image and heterogeneous enhancement mass on postcontrast study [figure 2]. There were multiple small underlying bone defect in the right occipital region [figure 3]. The excision was total, and cerebrospinal fluid (csf) leak was seen on bone defect site, so periosteum was buried on bone defect site for preventing the csf leak . Histopathological findings in hematoxylin and eosin staining were showing focal myxoid generation and spindle cells, with serpentine nuclei and wispy cytoplasmic border suggestive of pnf . T1-weighted with postcontrast sagittal magnetic resonance image shows heterogeneous enhancement lesion in the scalp of the right occipital region and nape of neck (a and b) both photographs showing three - dimensional computerized tomography skull showing thing of occipital bone right - sided with multiple bone defects after that pnf of scalp reported by ohaegbulam . In general, pnfs are found in association with nf (in 26.7% of patients). Pnf is usually found along the course of a major nerve trunk, the ophthalmic division in the face . It is an unencapsulated lesion and infiltrates the surrounding soft tissue to produce a fusiform appearance . It usually involves one large segment of the body and may involve both sides of the body either symmetrically or asymmetrically . The patient in this report had a pnf with nf and with telangiectatic discoloration of the overlying swelling, with multiple caf au lait spots on the left upper and lower trunk . Only nine reports were found in the literature, of these nine, one case had nonoperative management, and two had only a biopsy . In the initial description by helmholtz and cushing, as detailed by scott, the location of the pnf was at the forehead and temporal region . It was suggested then that there was a propensity for the forehead and the temporal region, especially the area of the scalp innervated by the trigeminal nerve . Although the forehead and the distribution along the course of the trigeminal nerve are the most common location, there have been a few reports of the pnf occurring at the occiput detail given in table 1; pnf occurs with slight male preponderance (male / female 7:5). Most reported cases have been congenital, but presented later in life due to apprehensions of a social or psychosocial nature, mostly previous reported cases, there was only thinning of the occipital bone with no true dysplasia . Review of literature of occipital neurofibroma nf is generally considered to be a neurocutaneous disorder of neural crest origin with very little emphasis on osseous abnormalities, although osseous dysplasia is one of the seven criteria for diagnosing nf-1 . Most of the osseous lesions are thought to be secondary to the altered functioning of the nf-1 gene . Besides true dysplasias, the secondary involvement of the bones may be due to compression of the malignant tumors seen in association with this disorder, such as malignant peripheral nerve sheath tumors and rhabdomyosarcomas . Osseous manifestations in nf-1 are relatively common and occur in up to 50% of patients with nf-1 . There are only a few cases reported, and in most, it is difficult to ascertain whether the osteolysis was a result of the erosion secondary to the giant tumor or was merely associated as part of the disease syndrome . Surgery for pnf has been described as being difficult because these tumors are usually very vascular, and torrential hemorrhage has been reported . There have been earlier reports of tumor firmly adhering to a dominant transverse sinus precluding complete excision . Even in patients in whom there was no involvement of the sinuses, some authors have avoided reconstruction due to the theoretical risks of csf fistulas, the risk of malignancies in nf-1 is approximately 2.7% more than in the general population . There is approximately a 10% chance of malignant transformation reported in an existing case of nf . Early surgical management should be the treatment of choice, not only just for cosmetic considerations but also for the theoretical risk of malignant transformation . However, the occurrence of osteolysis may also be prevented if it is indeed secondary to the pressure effects of the lesion, which cannot be disproven conclusively.
Advances in our ability to identify and quantify complex glycans and glycoconjugates has led to an increasing awareness of the key roles that these molecules play in a wide range of physiological and pathological processes, including cell adherence, cell cell interactions, molecular trafficking, biosynthetic quality control, signal transduction and host pathogen recognition . Various types of glycans and glycoconjugates are thus becoming recognized as essential participants in almost all biological processes . Structural analysis of glycoconjugates is technically challenging, requiring sophisticated analytical and computational techniques applied at the interface of biology and chemistry . Although recent technical advances in this area have led to the emergence of glycomics as a distinct discipline, progress is slowed by the unavailability of robust, generally applicable software tools required to process, annotate, archive and mine the data now being generated in this domain . The complexity of glycans and the diversity of their structures and molecular contexts have necessitated the development of a wide range of experimental techniques and instrumentation for their analysis . Although mass spectrometry (ms) is the most frequently applied methodology for glycan analysis, array - based ligand - binding assays, high - performance liquid chromatography, capillary electrophoresis (ce), nuclear magnetic resonance (nmr) and several other techniques are now being routinely used for this purpose . Recent advances in analytical methodology and instrumentation make it possible to produce glycomics data with increased depth, speed and efficiency, resulting in the generation of extremely large and diverse datasets . However, the reporting and/or distribution of information obtained during a glycomics experiment pose unique challenges to the analyst . This includes the identification and presentation of relevant metadata that allows the results to be objectively evaluated and interpreted in a biological context and reproduced in the laboratories of other scientists . It is important to note that glycomics cannot be viewed as a straightforward extension of proteomics . Glycomics and proteomics experiments share the same basic goal, i.e. The identification and quantification (where possible) of specific molecular structures in a particular biological context . However, the complex and often branched structures of glycans, in combination with the non - template - driven mechanisms leading to their biosynthesis, have made the emergence of glycomics as a discipline dependent on the ongoing development of new analytical approaches and computational tools that are not required for proteomics . The quality and information content of the annotated data generated by such tools can vary considerably, depending on the exact experimental conditions used to generate primary data, the suitability and configuration of the computational tools used to process this data, the quality of any databases that are invoked during the data processing and the validity of any assumptions that are made when assigning glycan structures in the presence of incomplete analytical data . The validity of glycan structure assignments can be assessed only if the relevant experimental parameters, computational methods and underlying assumptions used to make the assignments are described . In addition glycan analysis is often performed not just using one method or technique but by utilizing several orthogonal methods including array - based ligand - binding assays, liquid chromatography (lc), ce, nmr, various types of ms such as mass profiling and tandem ms or hyphenated analytical methods such as gc therefore, any information derived from each technique used has to be reported to provide a comprehensive and meaningful overview on the structure assignment, since each technique will provide additional information consolidating the structural assignment or illustrating exclusion of alternative structures . The mirage guidelines provide a framework that allows this information to be identified and presented in a consistent manner in order to enhance the value of structural analyses that are disseminated by both scientific journals and databases . Scientific journals can use the mirage guidelines as the foundation for developing their own checklists and guidelines for publishing glycomics data . In fact, the recently published mirage guidelines for glycan analysis by ms (kolarich et al . 2013) were evaluated by members of the glycobiology community (including representatives of several journals such as glycobiology and molecular and cellular proteomics (mcp)) at a workshop in athens, georgia in august 2012 before establishing guidelines for publishing mass spectral (ms) based glycomics data, later called athens guidelines (wells and hart 2013) (see also instructions to authors for glycobiology). The mirage guidelines are not strictly enforced by the editors of mcp or glycobiology, as the mirage guidelines are considerably more detailed than the athens guidelines . However, the two sets of guidelines are mutually consistent and complementary . The mirage guidelines provide reviewers with specific, technical descriptions of the metadata required to evaluate the glycomics analysis, and can thus be used by a qualified reviewer as a basis for making judgments regarding the validity of specific conclusions in the manuscript . However, they are not intended to substitute for the review process itself or to define acceptance criteria for submitted manuscripts . The criteria for acceptance of glycomics data for inclusion into a database are likely to be somewhat different than those for acceptance in a scientific journal . For example, the curatorial process for inclusion of a dataset in a database may not include review by an expert . In such cases, rigorous guidelines for the inclusion of metadata are crucial, as these metadata are a necessary prerequisite for the (automated or manual) selection of relevant, trusted data from the database for comparison to new datasets or inclusion in data - mining investigations . We expect that the mirage guidelines (developed by analysts, biologists and bioinformaticians with glycomics expertise) will serve as a convenient foundation to define the information that has to be stored in a database to provide comprehensive and reproducible datasets to its users . In 2006, participants at the workshop on analytical and bioinformatic glycomics, agreed that there is an urgent need to develop infrastructure, including standardized protocols for the exchange and reporting of structural glycan data and metadata to implement a worldwide network of databases containing experimental and analytical data relevant to the structures and functions of glycans 2008). A subsequent nih workshop organized by the consortium for functional glycomics in 2009 extended and refined these requirements, emphasizing the need to define specific criteria that make it possible for experts and non - experts to rapidly assess the depth and quality of a structural characterization that is described in a publication or structural database entry . (workshop on analytic and bioinformatic glycomics 2009). In july 2011, an international group of scientists attending the 2nd beilstein symposium on glyco - bioinformatics in potsdam (germany) established the mirage initiative under the auspices of the beilstein - institut (http://www.beilstein-institut.de). Mirage (i.e. The minimum information required for a glycomics experiment) integrates worldwide efforts to develop reporting guidelines for glycomics analytic data with the goal of facilitating the interpretation, evaluation and reproduction of these data . These guidelines are intended to improve the quality of glycomics datasets published in journals and stored in databases . Glycomics analysis offers unique challenges that necessitate reporting and archiving standards that are distinct from those already established for proteomics (such as the miape standard; taylor et al . 2007, 2008). The mirage standards thus serve as critical elements of the infrastructure required to integrate relevant scientific knowledge into a worldwide glycomics bioinformatics system capable of addressing diverse needs of the scientific community . In this context, success of the mirage project represents a critical step toward fulfilling the recommendations of the nas committee on assessing the importance and impact of glycomics and glycosciences (walt et al . 2012), which include the development of a well - documented glycan structure database that can be linked back to the original experimental data . The mirage project is steered by an international panel of scientists with expertise in diverse disciplines, including medicinal and developmental glycobiology, carbohydrate chemistry, glycoanalytics and glyco - bioinformatics, ensuring that the guidelines will encompass the most frequently used and relevant technologies . The mirage project incorporates three organizational components: the working group, the coordinating group and the advisory board (figure 1). The working group defines the tasks, makes general decisions and integrates detailed guidelines, which are developed by subgroups focusing on structural analysis, interaction analysis and bioinformatics . 1.mirage incorporates three organizational components: the working group, the coordinating group and the advisory board . Mirage incorporates three organizational components: the working group, the coordinating group and the advisory board . The efforts of the structural analysis subgroup have already led to the publication of guidelines for reporting glycoanalytic mass spectral (ms) data (kolarich et al . This guideline has an organizational structure similar to that of the miape - ms guideline for proteins (taylor et al . 2007) but the content is significantly different, addressing the specific requirements of glycan analytics . Future work will encompass diverse sample preparation techniques and alternative techniques for the identification of glycan structures, such as lc, hplc, ce or nmr . The interaction analysis subgroup considers methods to define the biological interactions of glycans and glycoconjugates with other macromolecules, such as glycan - binding proteins, glycan - potentiated signal receptors or microbes such as bacteria and viruses . Initial efforts have focused on evaluating and describing glycan microarray analyses and the data generated by these experiments . Future work will encompass diverse technologies such as surface plasmon resonance, flow cytometry, enzyme - linked immuno - sorbent assays, saturation - transfer difference nmr and isothermal titration calorimetry . The bioinformatics subgroup is charged with integrating data processing parameters into the guidelines for each laboratory method and defining separate guidelines for exchanging the information defined in the guidelines between different software systems and databases . The second component of mirage is the co - ordination group, which is concerned with the general organization of meeting, community participation and dissemination of the documents to both the members of mirage and the broader scientific community . The third component of mirage is the advisory board, which is composed of internationally recognized glycoscientists who support mirage and have agreed to devote the time required to oversee the efforts of the first two groups . The aim of reporting guidelines is to support the scientific community in publishing experimental data of high quality with respect to integrity, comprehensiveness and reproducibility by providing a framework to recommend details that should be reported along with the data . It is critical for the success of these guidelines that they do not appear to dictate how experiments and analysis should be designed or implemented . In order to gain general acceptance, mirage thus shares several essential prerequisites with other guideline initiatives such as miape (taylor et al . These are: sufficiency: the guidelines should adequately describe information about the experimental data and the experimental conditions and methods used to generate the data to enable individuals to understand, critically evaluate, interpret and reproduce the data.practicability: the guidelines should be concise, understandable and limited to specific parameters that have a significant effect on the outcome of an experiment, facilitating compliance by scientists who use them.stability: the guidelines must be stable over a time period that is adequate to ensure consistency and comparability in data reporting . Nevertheless, the guidelines must accommodate technical and scientific advances that should be considered when a new technique is sufficiently mature and robust for widespread use . Sufficiency: the guidelines should adequately describe information about the experimental data and the experimental conditions and methods used to generate the data to enable individuals to understand, critically evaluate, interpret and reproduce the data . Practicability: the guidelines should be concise, understandable and limited to specific parameters that have a significant effect on the outcome of an experiment, facilitating compliance by scientists who use them . Stability: the guidelines must be stable over a time period that is adequate to ensure consistency and comparability in data reporting . Nevertheless, the guidelines must accommodate technical and scientific advances that should be considered when a new technique is sufficiently mature and robust for widespread use . Scientific journals will play a crucial role in encouraging acceptance of the guidelines by recommending that authors refer to the guidelines when submitting their data . In many cases, the status of the guidelines may change from a simple recommendation to a requirement that must be considered by all participants in the publication process (including authors, reviewers, curators and editors). Maturation of the document through this process increases its visibility and encourages consensus among stakeholders, facilitating its acceptance by the community . Although this multistep development is time consuming and laborious, it has been proven to be an effective process for developing the ms guidelines (kolarich et al . 2013), which have gained broad support from the ms and scientific publishing communities . The successful development and administration of any reporting standard involves balancing specifications that are sufficient to ensure the integrity and reproducibility of the reported data with the need for flexibility in designing and implementing experiments and the free dissemination of valuable experimental results (klipp et al . 2007). Any restriction of scientific freedom regarding the generation or description of data should be avoided . Although scientific advances become valuable to the community only when new techniques and the data they generate are effectively communicated to others, innovation required for such advances must not be stifled by standards aimed at improving the information content of scientific reports . With these human aspects in mind, it is clear that development of the mirage guidelines should be a highly transparent process that respects the viewpoints of the many different stakeholders and thereby encourages maximum participation and continued support by the community . The parallel development of similar guidelines by different groups would lead to confusion and must be avoided . Therefore, once a new mirage guideline has been reviewed by the advisory committee, it is made available to the general public via the mirage project web site (http://glycomics.ccrc.uga.edu/mirage/index.php). This site also provides information about the mission and history of the mirage group, along with descriptions of the diverse projects and their progress . In addition, several hundred international researchers working on glycan analysis, structure and function are proactively approached via email for advice, for example, to obtain feedback on draft versions of the guidelines . The mirage committee recognizes the need for broadly based input, and welcomes all comments, advice and help in developing the guidelines . The mirage project has been established to define data reporting guidelines for glycomics databases and publications . They describe the essential information necessary to understand and reproduce a glycomics experiment without dictating how the experiment itself should be performed . Adoption of these guidelines by scientific journals in the form of checklists or additions to the author instructions will increase the quality and reproducibility of the published results . In addition, adoption of the guidelines by glycomics databases will facilitate methods for processing and mining of the data produced by glycoanalytic experiments . At the moment of writing this paper a guideline for glycomics further guidelines for other techniques used for glycomics experiments, such as glycan array experiments or lc separation in combination with or without ms, are in currently in preparation and will be finished in summer 2014 . Future activities of the mirage project will be not just to create guidelines for other types of experiments but also to promote existing guidelines with scientific journals to accomplish the integration of these guidelines by the journals . The beilstein - institut, a non - profit foundation established under civil law and located in frankfurt am main, germany, has provided funding for advancement of the mirage initiative . This work has also been supported by several other funding agencies: dk and er are both supported by the max planck society, and in addition, dk is funded by the marie curie grant pcig09-ga-2011 - 293847 . Both, er and pmr have a grant from the european union's seventh framework programme (fp7-health - f5 - 2011, grant agreement no 278535 highglycan); in addition, pmr has a grant from the european union's seventh framework programme (fp7/2007 - 2013, grant no . 259896); ad and sh are supported by the biotechnology and biological sciences research council (grants bb / k016164/1 and bb / i017011/1); mc and np are supported by the australian government project nectar financed by the education investment fund; jz and cc are supported by the nih / nigms grant p41gm104603; wy, rr, mt and lw are supported as part of the nih / nigms funded national center for glycomics and glycoproteomics (8p41gm103490). Nk's contribution to this initiative is supported by the swedish foundation for international cooperation in research and higher education . Tf and yl are supported by a wellcome trust biomedical resource grant wt099197ma . Funding to pay the open access publication charges for this article was provided by beilstein - institut.
A large increase in fusion rates for spine surgery has been observed in the last 20 years9), and the frequency of revision spine surgery continues to increase . The most common reasons for additional surgery7) following fusion surgery are implant complications and pseudoarthrosis . . They may be in the shape of a male or female rectangle or hexagonal, octagonal, or star - shaped . Therefore, it is necessary to have the instrument set used during the first operation or to have an appropriate screwdriver available . This is not always possible and in some circumstances the implant may no longer be in production . When revision surgery studies were examined5,12), methods to remove broken screws are reported but very little information related to removing normal screws has been provided . No previous report has described a single - piece screw remover to remove normal screws . A small part of the rod is cut and placed in the screw when a removal set is not available . After tightening the screw head, mobile screw's head this screw is a single piece that can be removed by hand or with pincers10). The polyaxial remover presented here requires only a cap - screw screwdriver to remove the polyaxial screws . Due to the structure of the remover and the method, existing screws can be removed with minimal dissection of surrounding soft tissue . Debridement is important to remove scars, envision the bone elements, and prepare a new fusion bed during revision surgery . Additional procedures applied to remove screws cause the surgeon to expend extra effort, which may lead to loss of time . Our tool simplifies and accelerates the screw removal process; thus, shortening operating time . The aim of our polyaxial screw remover design is to facilitate screw removal and thereby reduce blood loss, infection risk, cost, and effort expended by the surgeon . The patient demographic data and reasons for primary and revision surgery are given in table 1 . The aim when removing polyaxial screws is to stabilize the polyaxial head so the screw becomes monoaxial and can be removed by turning . The method described by kose et al.6) was of benefit when we designed our instrument, which has not been described previously in the literature . In the method of kose et al ., the screw becomes monoaxial with a rod and cap screw, whereas we used a u - shaped end section in our instrument without the need for a rod . The polyaxial remover consists of a single - piece shaft, sleeve, and handle (fig . The shaft section is hollow, and the screw cap is tightened with a screwdriver . Thus, a spine screw enters easily, and the surgeon avoids damaging surrounding tissue . The head section of a polyaxial screw was removed, and the screw was placed in the u - section (fig . The cap screw was tightened on the screw, and the screw was fixed in the end section of the apparatus . The polyaxial screw that became monoaxial can be removed by turning the handles on the shaft . This method also removes the need to clean the fibrous tissue covering the internal screws and to see the screw shape . A total of 42 polyaxial screws were removed from five patients with the new single - piece screw remover (fig . The polyaxial cap screws were 4.5 hexagonal screws in four patients and star - shaped in one patient . After removing the cap screws and rods, all of the polyaxial screws were made monoaxial with the instrument described above and were removed rapidly in a minimally invasive way with no complications . We have described a screw removal instrument designed to overcome the problems of screw removal often encountered during revision surgery . Our clinical application shows that these screws can be removed without the need for additional materials or soft tissue debridement within or below the screw head . The correct screwdriver for the materials used cannot always be obtained to remove existing implants during revision surgery because of the different designs . This explains why revision surgery can be a longer and more fatiguing operation with increased blood loss . Of the several known spine surgery problems, the most accepted and most widely used method for repair is pedicle screw fixation1,2,3,4,5,6,7,8,9,10,11). After fixation, the most common reasons for revision surgery7) are implant problems and pseudoarthrosis . Methods and instruments to remove broken screws have been described in the literature5,12). However, no additional methods6,10), other than the rod techniques for removing normal screws, have been described . In the u - rod technique described by kse et al.6), the cap screws are removed with an allen key, and the screw is placed in the u - shaped rod after removing the rods . After tightening the screw head, the screw with a mobile head becomes a monoblock screw . This single - structure screw can be removed by hand or with pincers . In addition, large metal cutters are required to cut the rods, a rod bender is needed to shape, and pincers are needed to turn the rod placed on the screw head . Our screw remover design is a single piece and will not damage surrounding tissue or the pedicle (fig . There is no need for metal cutters or a rod bender; thus, reducing the time and effort required . The tool also removes the need to clean the fibrous tissue covering the internal screws to determine screw shape . Second, a polyaxial remover was used after removing the cap screws, so it was necessary to have a screwdriver suitable for the cap screws . However, cap screws are generally hexagonal or star - shaped and there are few varieties . These screws are generally found in sets, or the screwdriver in the revision set can be used . Another limitation was that the polyaxial remover could not be used if the cap screw is damaged or broken . The screw removal technique using the polyaxial remover is practical and easy and requires a minimal amount of time . The instrument described here can be used to remove all types of polyaxial and monoaxial screws used in spine surgery . Here is no requirement for additional screw removal tools or materials and unnecessary debridement is avoided.
Ureteropelvic junction obstruction (upjo) in children most commonly occurs due to adynamic segment of ureter . Previously, ureteral polyp causing upjo in children has been reported in as low as 0.5% of pyeloplasties in children.1 however, in recent studies the incidence reported was around 5%.2 3 4 5 as li et al mentioned in their report, authors are unsure on whether it is due to more careful patient data documentation or it is about the increase of incidence.5 fibroepithelial polyps are more common in boys than girls.1 2 3 4 5 it is predominant at the left side in up to 70% of the patients.1 6 7 moreover, bilateral cases were reported as well.8 even if many phenotypes of fibroepithelial polyp (fep) were defined, there is no consensus on etiology such as chronic irritation, infections, hormonal imbalance, allergy, or developmental defects.9 10 hydronephrosis with ureteral polyp requires surgical treatment, due to the risk of malignity1 11 and obstruction . Excision of the polyp can be performed by laparoscopy, retroperitoneoscopy, transurethral ureteroscopy, or percutaneous access.12 13 14 15 16 17 overall, kojima et al described performing endoscopy via a working port while laparoscopic exploration.4 in this case report, we present laparoscopic technique in a patient who had ureteral polyp close to the left upj . A-10-year old boy was admitted at our pediatric emergency department with complaints of abdominal pain and vomiting . In his medical history, he had episodes of abdominal pain without hematuria or urinary tract infection . The abdominal pain was located on the left upper abdomen and lumbar region . Although previous ultrasound reports revealed only mild hydronephrosis, the last one showed hydronephrosis related to a polyploid lesion, which was located near upj and protruding into the proximal left ureter (fig . 1). A tc diethylenetriamine pentaacetic acid (dtpa) renography revealed nonobstructive hydronephrosis with late drainage after diuretic injection . Umbilical 10 mm optic port (10 mm reusble port, karl storz gmbh & co. kg) and two 5 mm (disposable) working ports, one on the epigastric region and the other on the left lower quadrant were introduced . Pneumoperitoneum was created at a pressure of 13 mm hg with the flow of 3.5 l / min . Transmesocolic laparoscopic exploration showed a protruding mass on upj and hydronephrosis (figs . 2 and 3). Following the transmesocolic dissection of upj, a stay suture passing through the abdominal wall was placed to keep the left ureter hanging . Pelvis, close to the upj, was incised and a cauliflower - shaped polyp that was about 20 mm in diameter with a wide base protruding (fig . Excision of the mass was performed including the regions of pelvis and ureter close to the mass (fig . Dismembered pyeloplasty was performed with interrupted 50 polydioxanone sutures and double j stent was placed . The total operative time was 200 minutes, with an estimated operative blood loss of 20 ml . After a follow - up of 2 years, patient did well with normal dtpa renography . Feps of the ureter are of mesoderm origin, rare benign tumors in children.18 they may arise in any location from renal pelvis to the urethra in the order of decreasing frequency.19 on the other hand, feps may have varied sizes and shapes . They might be 1to 50 mm long or have shapes such as pedunculated with a smooth surface or with multiple finger - like projections.1 10 20 21 age of the patient, structural features of polyps, and experience level of the surgeon determine treatment preferences and consequences of surgery . However, although there are few studies, transitional cell carcinoma arising from fibroepithelial polyp and recurrence due to incomplete resection has been reported.11 22 on the other hand, adey et al stated in 2003 that only 22% of feps were diagnosed preoperatively and li et al mentioned in 2014 that none of the feps in their study were detected before surgery.1 5 in our case, fep was detected after a follow - up of 4 years . So we believe the delay in diagnosis might be due to intermittent obstruction and slow growing nature of polyp . Although malignity, recurrence, and any other associated polyp is extremely low, surgeon should ensure not to leave remnant of feps during excision . In the past, open surgery was the only option, however, recently minimal invasive approaches have been recommended in children and adults.4 13 14 15 23 furthermore, several authors have described their successful experience in using holmium laser resection, percutaneous, and/or ureteroscopic approaches in the treatment of feps.13 14 15 conversely, sun et al figured out in their study that strictures might be cause of over vaporization and kiel et al mentioned the risk of recurrence due to incomplete resection of tumor.4 15 24 bartone et al speculated that resecting the polyp without base might cause obstruction and recurrence.25 on the other hand, adey et al underlined the importance of dismembered pyeloplasty whereas polyps might have been missed, if spiral flap or y v pyeloplasty is performed.1 moreover, iwatsuki et al and tsuzuki et al published their successful laparoscopic treatment of fep either in child and adult patients.16 26 since fep was located close to upj, we preferred to perform laparoscopy . It should be noted that, despite postoperative controversies of endoscopic resection or fulguration of a wide base polyp; local resection, fulguration, or pyeloplasty can be performed easily during laparoscopic approach . The overall laparoscopic exploration shows the exact location and size of the polyp, neighboring organs, and structures . Luminal mucosal evaluation with exposure of proximal and distal resection margins could be performed well with optic magnification . There are several options for laparoscopic approach to renal pelvis and upj region on the left side . Depending on the patient and experience of the surgeon retroperitoneoscopy, transabdominal retrocolic or transmesocolic approaches can be performed.12 13 14 15 16 17 to our knowledge, in pediatric age group, retroperitoneoscopy and endoscopy may not provide enough surgical working space . So we suggest transabdominal transmesocolic approach and believe that exploration and surgical manipulation might be easier compared to retroperitoneoscopy or endoscopy . Pediatric surgery is a specialty that has its own characteristics owing to relevance of congenital anomalies . Time to time, it is hard to estimate the real anomaly although advanced diagnostic tools are in use, as was in our case . And to me, a pediatric surgeon should be always alert, for concomitant pathologies, in any anomaly . Besides open access, both endoscopic or laparoscopic approaches should be in practice of surgeon, for being minimally invasive.
Colorectal cancer (crc) is one of the most commonly diagnosed cancers in the world, with over 600,000 deaths per year . Unfortunately, the morbidity of crc has been rapidly rising in asian countries in recent years . However, the multiple known carcinogenic factors and complex genetic backgrounds make it difficult to estimate the key factor in crc progression . At the present time, the basic prognostic biomarker in crc is the clinicopathologic tumor staging, based on the tumor - node - metastasis (tnm) system . Nevertheless, the tnm stage is not an ideal biomarker for crc outcomes . Patients at the same tnm stage may have different progressions and clinical outcomes due to their various genetic and epigenetic backgrounds . Therefore, it is necessary to identify sensitive and specific molecular biomarkers for crc clinical outcomes . Long non - coding rnas (lncrnas) transcribed by rna polymerase ii lack open reading frames (orf) longer than 200 nucleotides . Although the functions of most lncrnas are unknown, more and more lncrnas are characterized and many of them are reported to regulate gene expression in the development and differentiation of diseases [811]. For example, lncrna cche1 promotes cervical cancer cell proliferation, hnf1a - as1 regulates proliferation and metastasis in lung adenocarcinoma, and malat1 is associated with poor prognosis of glioma . Spry4-it1 is significantly increased in plasma samples of nsclc patients and can act as a biomarker in nsclc . Trpm2-as can act as a novel biomarker and therapeutic target in prostate cancer . In this study, we focused on lncrna spry4 intronic transcript 1 (spry4-it1), which is located within an intron of the spry4 gene . Spry4-it1 was previously reported to be up - regulated in melanoma, gastric cancer, breast cancer, and esophageal squamous cell carcinoma [1720]. However, the effect of spry4-it1 in crc prognosis is unknown . In the present study, we applied different methods in analyzing the association between spry4-it1 expression and clinical features, aiming to determine the clinical influences of spry4-it1 in crc patients and to discover a reliable predictor for crc . In this study, 106 crc patients confirmed by pathological and clinical diagnoses at the pla general hospital were enrolled from october 2008 to january 2014 . This study was approved by the ethics committee of pla general hospital, and written consent was obtained from all the patients . Tumor and adjacent normal tissues were obtained from the crc patients before they received any chemotherapy or radiotherapy . All the tissue samples were stored in liquid nitrogen until they were utilized . In order to observe the results of the surgery, follow - up was performed every 3 months in the first 2 years and then every 6 months until the end of the study . Total rna was extracted from all the tissues using trizol reagent according to the manufacturers instructions . The extracted rna was dissolved in diethyl pyrocarbonate (depc)-water and then treated by dnase to remove dna . The concentration of the total rna was detected by uv absorbance at 260 nm and 280 nm (a260/a280). Fluorescence quantitative real - time pcr (qrt - pcr) was used to assess the relative expression levels of spry4-it1 in pathologic and adjacent normal tissues of crc patients . The complementary dna (cdna) temples enrolling in the qrt - pcr were from the primescript rt reagent kit (takara, china). The qrt - pcr was performed with sybr green assay (takara, china). Student s t - test was used to estimate the different expression levels of spry4-it1 and the data are shown as mean standard deviation (sd). The association between the clinical features and spry4-it expression was evaluated by chi - square method . Kaplan - meier method with log - rank test was applied to analyze the overall survival of the crc patients, and univariate and multivariate cox regression analysis were used to evaluate the prognostic value of spry4-it1 . In this study, 106 crc patients confirmed by pathological and clinical diagnoses at the pla general hospital were enrolled from october 2008 to january 2014 . This study was approved by the ethics committee of pla general hospital, and written consent was obtained from all the patients . Tumor and adjacent normal tissues were obtained from the crc patients before they received any chemotherapy or radiotherapy . All the tissue samples were stored in liquid nitrogen until they were utilized . In order to observe the results of the surgery, follow - up was performed every 3 months in the first 2 years and then every 6 months until the end of the study . Total rna was extracted from all the tissues using trizol reagent according to the manufacturers instructions . The extracted rna was dissolved in diethyl pyrocarbonate (depc)-water and then treated by dnase to remove dna . The concentration of the total rna was detected by uv absorbance at 260 nm and 280 nm (a260/a280). Fluorescence quantitative real - time pcr (qrt - pcr) was used to assess the relative expression levels of spry4-it1 in pathologic and adjacent normal tissues of crc patients . The complementary dna (cdna) temples enrolling in the qrt - pcr were from the primescript rt reagent kit (takara, china). The qrt - pcr was performed with sybr green assay (takara, china). Statistical analysis was completed in spss 18.0 software . Student s t - test was used to estimate the different expression levels of spry4-it1 and the data are shown as mean standard deviation (sd). The association between the clinical features and spry4-it expression was evaluated by chi - square method . Kaplan - meier method with log - rank test was applied to analyze the overall survival of the crc patients, and univariate and multivariate cox regression analysis were used to evaluate the prognostic value of spry4-it1 . The 106 crc patients enrolled in this study included 52 men and 54 women with an average age of 55.02 years old . Qrt - pcr was used to evaluate the relative expression of spry4-it1 in crc tissues and normal tissues . The results indicated that the relative expression of spry4-it1 in pathologic tissues was significantly higher than that in the adjacent normal tissues (p=0.000, figure 1). To evaluate the association between spry4-it1 expression and clinical features, the crc patients were divided into high and low expression groups on the basis of their average expression of spry4-it1 . The chi - square results are shown in table 2, which shows that the expression levels of spry4-it1 were associated with the tumor size (p=0.013), the depth of invasion (p=0.004), lymph node metastasis (p=0.017), distant invasion (p=0.012), and tumor stage (p=0.015). However, there was not significant relationship between the expression levels and sex, age, tumor location, histological differentiation, venous invasion, or nerve invasion (p>0.05). The results suggest that the expression level of spry4-it1 might be associated with the development of the crc . Overall survival analysis was conducted by kaplan - meier method, showing that the crc patients with high expression of spry4-it1 had low overall survival (the average overall survival was 39.3 months), while the low expression patients had an average overall survival of 49.3 months (figure 2). In other words, differences between the 2 groups were significant (log - rank test, p=0.016). In this study, we used cox regression analysis to estimate the prognostic value of spry4-it1 . The results of univariate analysis show that the levels of spry4-it1 were significantly associated with the poor prognosis in crc patients (p=0.021). Multivariate analysis suggests that spry4-it1 is an independent factor for crc prognosis (hr=2.341, 95% ci=1.1364.826, p=0.021). The 106 crc patients enrolled in this study included 52 men and 54 women with an average age of 55.02 years old . Qrt - pcr was used to evaluate the relative expression of spry4-it1 in crc tissues and normal tissues . The results indicated that the relative expression of spry4-it1 in pathologic tissues was significantly higher than that in the adjacent normal tissues (p=0.000, figure 1). To evaluate the association between spry4-it1 expression and clinical features, the crc patients were divided into high and low expression groups on the basis of their average expression of spry4-it1 . The chi - square results are shown in table 2, which shows that the expression levels of spry4-it1 were associated with the tumor size (p=0.013), the depth of invasion (p=0.004), lymph node metastasis (p=0.017), distant invasion (p=0.012), and tumor stage (p=0.015). However, there was not significant relationship between the expression levels and sex, age, tumor location, histological differentiation, venous invasion, or nerve invasion (p>0.05). The results suggest that the expression level of spry4-it1 might be associated with the development of the crc . Overall survival analysis was conducted by kaplan - meier method, showing that the crc patients with high expression of spry4-it1 had low overall survival (the average overall survival was 39.3 months), while the low expression patients had an average overall survival of 49.3 months (figure 2). In other words, differences between the 2 groups were significant (log - rank test, p=0.016). In this study, we used cox regression analysis to estimate the prognostic value of spry4-it1 . The results of univariate analysis show that the levels of spry4-it1 were significantly associated with the poor prognosis in crc patients (p=0.021). Multivariate analysis suggests that spry4-it1 is an independent factor for crc prognosis (hr=2.341, 95% ci=1.1364.826, p=0.021). Despite great progress in early diagnosis, surgical techniques, and chemotherapy, the prognosis of patients with crc is still unsatisfactory . Prognostic factors are helpful in determining the therapeutic regimen in cancers . Due to the complex genetic background and epigenetic factors, there is still no highly sensitive and specific biomarker for crc clinical outcomes, so the prognosis of crc patients is poor . Recently, many lncrnas have been reported to play significant regulatory roles in human diseases . Cancer - specific lncrnas have been proven to contribute in tumor progression and serve as prognostic factors in many types of cancer . Wu et al . Reported that lncrna uca1 could be a promising biomarker for early detection and prognosis in gastric cancer . Lncrna bancr was proved to regulate the growth and metastasis of the retinoblastoma cells and act as a prognostic target . Chen et al . Demonstrated that lncrna hottip promoted pancreatic cancer cell proliferation, survival, and migration . As a result, the association between crc and lncrnas may also provide significant information about the development of cancer and clinical outcomes . Lncrna spry4-it1 is a novel lncrna, which was first reported to be associated with molecular etiology in human melanoma . It is a 687nt unspliced, polyadenylated transcript, localized at chromosome 5q31.3 . In this study, we detected the relative expression levels of spry4-it1 in crc patients using qrt - pcr (gapdh as normalized control), aiming to estimate the clinical significance of spry4-it1 in crc . The results showed that the relative expression of spry4-it1 was much higher in diseased tissues than in the corresponding adjacent normal tissues and there were significant differences between them . We also analyzed the relationship between the spry4-it1 expression and the clinical characteristics by chi - square method . The results indicated that the relative expression levels of spry4-it1 were related to the tumor size, the depth of invasion, lymph node invasion, distant invasion, and tumor stage . Spry4-it1 expression was not relevant for sex, age, tumor location, histological differentiation, venous invasion, or nervous invasion . These data suggest that spry4-it1 may play a role in the tumorigenesis and progression in crc . The prognostic value of spry4-it1 in crc was also analyzed in this study . Over - expression of spry4-it1 was correlated with low overall survival and the cox regression analysis showed that spry4-it1 could be an independent prognostic biomarker for crc . Similar results were also found in other types of cancer, and higher expression of spry4-it1 predicted poor prognosis in many cancers, such as gastric cancer, esophageal squamous cell carcinoma, clear cell renal cell carcinoma, and non - small cell lung cancer . These studies indicated that spry4-it1 might be useful for providing insights into mechanisms of cancers development . This study proves that spry4-it1 expresses aberrantly in diseased tissues of crc patients compared with the adjacent normal tissues . Moreover, the expression level is associated with tumor size, the depth of invasion, lymph node invasion, distant invasion, and tumor stage . Additional, spry4-it1 can act as an independent biomarker for crc prognosis and over - expression of spry4-it1 predicts poor prognosis in crc.
Patient 1, a 55-year - old man who had received a diagnosis of ankylosing spondylitis 7 years previously, was admitted to laikon hosptal, athens, greece, in may 2005 for evaluation of encrusted vesicular lesions on the face . The patient had been receiving nonsteroidal antiinflammatory agents until 12 months before admission, when his medications were changed to infliximab (3 mg / kg) plus methotrexate (10 mg weekly) because of his deteriorating clinical condition . He was living in a leishmaniasis - endemic area in athens, had no pets in his house, and had no history of recent travel abroad . The central scale was removed from one of the lesions, and scrapings from the base of the lesion were stained with giemsa stain, which showed intracellular amastigotes with peripheral nuclei and rod - shaped kinetoplasts . Results of indirect immunoflorescent antibody (ifa) testing were positive for leishmania parasites (titer 6,400). Infliximab and methotrexate therapy was discontinued, and treatment with liposomal amphotericin b was started at a dose of 3 mg / kg, for days 1 to 5, and 2 additional doses (3 mg / kg) on days 14 and 21 . Eighteen months later, treatment with etanercept was begun due to the patient s severe spondyloarthritis; 2 years after the new anti - tnf treatment, he is well, with no signs or symptoms of leishmaniasis . Patient 2, a 71-year - old woman who had giant cell arteritis, was admitted to the euroclinic hospital, athens, in may 2005 with a high fever and fatigue . The patient had been treated with infliximab (0.25 mg / kg) and variable doses of methylprednisolone for the previous 2 years . She was also living in an athens suburb, which is leishmaniasis - endemic, and had 4 dogs . Laboratory tests showed a high level of c - reactive protein (163 mg / 06 mg / l), high erythrocyte sedimentation rate (77 mm / h), pancytopenia (hemoglobin level 12.5 g / dl, leukocyte count 3,300/mm, platelet count 122,000/ mm), and diffuse hyperglobulinemia . The examination of giemsa - stained smears from bone marrow aspirate demonstrated abundant leishmania parasites, and ifa was marginally positive for leishmania antibodies (titer 400). Pcr was positive for the detection of the leishmania genome in peripheral blood . Infliximab and methotrexate treatment was discontinued, and treatment with intravenous liposomal amphotericin b was started at a dose of 3 mg / kg for 5 days . Two days later, the fever subsided, and within the next few days, the patient recovered from pancytopenia, while the inflammatory markers showed a gradual decrease . She received 2 additional doses of liposomal amphotericin b (3 mg / kg) on days 7 and 14, and by that time, she exhibited no signs or symptoms of visceral leishmaniasis . We then searched medline, embase, and current contents databases for all reports on leishmaniasis in europe and the mediterranean area among patients with autoimmune rheumatic diseases, which are often treated with anti - tnf agents . In our search strategy, we used medical subject heading terms and text words, including rheumatoid arthritis, juvenile rheumatoid arthritis, still s disease, seronegative arthritis, psoriatic arthritis, behet s disease, ankylosing spondylitis, reactive arthritis, vasculitis, giant cell arteritis, wegener s granulomatosis (anca [anti - neutrophil cytoplasmic antibody]associated vasculitis), panarteritis nodosa, leishmaniasis, leishmania, and anti - tnf . We found 13 additional cases of leishmaniasis in patients with autoimmune rheumatic diseases (214), all published after the introduction of anti - tnf agents in 1998 (table). All 15 patients (including our 2 patients) were treated in the past or at the time of the diagnosis of leishmaniasis with> 1 standard immunosuppressive agents, including corticosteroids (11/14 [78.5%]) patients for whom treatment details were reported), methotrexate (9/14 [64.3%]), cyclosporine (3/14 [21.4%]), cyclophosphamide (3/14 [21.4%]), azathioprine (2/14 [14.3]), and chlorambucil (1/14 [7.1%]). Seven (46.6%) patients received an anti - tnf agent along with standard immunosuppressive agents . Two of the 15 reported patients had been treated with a recombinant interleukin1 receptor antagonist (anakinra; amgen inc ., thousand oaks, ca, usa) (5,13). In most of the patients, visceral leishmaniasis developed (13 patients, 86.6%), while cutaneous leishmaniasis developed in 2 patients (1 was receiving an anti - tnf agent). All patients were living in leishmaniasis - endemic areas of europe (figure). * reference; anca, anti - neutrophil cytoplasmic antibody; na, not applicable . All biologic treatments had been terminated 6 mo before leishmaniasis occurred . Reported cases of leishmaniasis in patients with autoimmune rheumatic diseases in europe, indicated by stars (1 case from israel not shown). Dark gray shading, distribution of leishmaniasis; light gray shading, distribution of leishmaniasis vector sandfly . The anti - tnf agents were introduced into clinical practice in 1998, and the first case of leishmaniasis associated with anti - tnf blockade occurred in 2001 (4). During the 6-year period (19982003), a total of 6 reports were made of leishmaniasis in patients with rheumatic diseases; 1 (16.6%) occurred in a patient treated with an anti - tnf agent . During the ensuing 5 years (20042008), 9 cases of leishmaniasis were reported, 6 (66.6%) in patients receiving anti - tnf agents . The median duration of previous immunosuppressive therapy, before the diagnosis of leishmaniasis, was 60 months (range 2360 months) for all 15 patients (able). For the 7 patients who received anti - tnf agents, the median duration of anti - tnf treatment was 18 months (range 960 months). Six of these 7 patients were receiving anti - tnf agents when symptoms and signs of leishmaniasis occurred . In 1 patient (5), biologic treatments had been discontinued 6 months before the diagnosis of leishmaniasis (table). Only 1 patient had been tested for antibodies against leishmania spp . Before immunosuppressive therapy was begun, and the results were negative (6). Therefore, this is the only case with compelling evidence that leishmaniasis was a primary infection and not reactivation of a latent infection . Our data suggest that the introduction of tnf blockade into the clinical practice is associated with increasing reports of leishmaniasis in patients with autoimmune rheumatic diseases who live in leishmaniasis - endemic areas of europe . Notably, in most reported cases, patients had not received anti - tnf agents but other immunosuppressants . However, all cases of leishmaniasis in patients with autoimmune rheumatic diseases were reported after 1998, the year of introduction of anti - tnf agents, and most (9/15) of the reported leishmaniasis cases occurred during the past 5 years (20042008), mainly among patients receiving anti - tnf agents (6 of the 9 patients with leishmaniasis; 66.6%). This increase coincides with the increasing use of anti - tnf agents during the same period, as prescription practice started changing toward treating patients with lower disease activity (15). Another indirect piece of evidence that tnf blockade may increase the risk for leishmaniasis is that the median duration of previous anti - tnf treatment before the diagnosis of leishmaniasis was significantly shorter than the median duration of immunosuppressive therapy for all 15 patients (18 vs. 60 months). It is unclear for all cases (with 1 exception) presented in this article whether leishmaniasis was primary infection or reactivation of latent disease . We cannot also exclude the possibility that the concomitant, long - term use of other immunosuppressants, and not the anti - tnf agents per se, played a crucial role in the development of leishmaniasis . Different prescribing patterns of anti - tnf agents might influence the number of cases reported from each disease - endemic european country . However, the small number of reported cases and the lack of data on differences in the anti - tnf prescribing policies do not allow any conclusions to be reached . Finally, due to underreporting, the reported cases may underestimate the real incidence of leishmaniasis among patients with autoimmune rheumatic diseases . Prospective studies to estimate the incidence of the disease, the impact of risk factors and the need for serologic screening for leishmaniasis before initiation of anti - tnf agents or any other immunosuppressive treatment are clearly needed . This is particularly important since currently only a few patients with autoimmune rheumatic diseases receive anti - tnf agents (15). Therefore, the use of anti - tnf treatment is likely going to increase, possibly causing a parallel increase in opportunistic infections such as leishmaniasis.
Osteoporosis is defined by the world health organization (who) as having a bone mineral density (bmd) of more than 2.5 standard deviations below the norm for healthy young adults (1). Bmd, measured by dual - energy x - ray absorptiometry, is used as a bone mass index to predict the risk of fracture (1). Osteoporotic bones are porous and fragile, thus making them more susceptible to fracture than healthy bones . In order to maintain a healthy skeleton, a steady - state of bone remodeling occurs in adults in which new bone is generated at the same rate that old bone is removed . The remodeling process is important for maintaining bone strength because it adapts the structure of bone to mechanical loads (2). In osteoporosis, the rate of bone resorption exceeds that of the formation, resulting in a progressive deterioration of bone mass and microarchitecture (2). There are the two kinds of bone that comprise the skeleton are compact and cancellous (i.e. Trabecular). Compact bone predominates in the appendicular skeleton and is designed to resist bending forces . On the other hand, cancellous bone is concentrated in the axial skeleton and is structured to resist compressive forces, including gravity . Since it is lighter and more porous than compact bone, cancellous bone provides more surface area for bone remodeling and is more metabolically active (2). Bisphosphonates, including alendronate and risedronate, are antiresorptive agents which are stable analogues of inorganic pyrophosphate and have a high affinity for hydroxyapatite crystals . They bind selectively to mineralized surfaces of bone to disrupt degradative osteoclast activity and promote apoptosis of mature osteoclasts (4). Bisphosphonates are also thought to prolong the life of osteoblasts by inhibiting apoptosis (5). Alendronate and risedronate are highly effective in decreasing the risk of vertebral and hip fractures (68). But, it was also demonstrated that the observed increase in bmd in bisphosphonate - treated patients does not entirely account for the reduction in fracture risk (6). In fact, it is hypothesized that bisphosphonates may act by increasing bone mass, improving trabecular microarchitecture, and/or increasing secondary mineralization of the calcified matrix (9). Recently, more emphasis has been placed on the concept of bone quality which encompasses the microarchitectural and biomechanical properties of bone (10, 11). It is estimated that osteoporosis is responsible for about 300 000 hip fractures per year in the united states (12). Recent studies have shown that hip - fractured patients rapidly lose bone density within 6 months after fracture, possibly because of the absence of weight bearing for 6 weeks post - operatively and the fact that peri - prosthetic bone loss rapidly occurs following hip arthroplasty (13). Since there is no standard of care for the prevention and the treatment of osteoporosis in these patients, our interest is to determine the best approach to prevent bone loss in this group . As the effect of bisphosphonates on the femoral neck and surrounding areas has not yet been assessed, we will analyze the bone quality of proximal femurs from treated patients that are discarded after hip replacement surgery . Prior to undertake these clinical trials, however, protocols must be established in an untreated population in order to properly assess the quality of bone . This project aims to determine whether imaging and compression testing of cancellous bone from the femoral neck can accurately assess the bone s microarchitectural and biomechanical properties, respectively, and if together they will serve as a good index of bone quality . Twenty - two bovine (calf) femurs were obtained from a butcher (boucherie charcuterie rivire des prairies, montreal, qc). William fisher (montreal general hospital, montreal, qc) from total hip replacement orthopedic surgeries . A rochester bone biopsy trephine (item #87400 - 000, gauthier medical inc ., rochester, mn) 7.5 mm in diameter was used to extract cylindrical cores of trabecular bone from the centre of the femoral necks . Both ends of the cores were cut at right angles to the long axis in order to minimize shearing during biomechanical compression testing (15). The dimensions of the trabecular bone cores were 7.5 mm in diameter by 14 mm in length, thereby maintaining a valid aspect ratio between 1.5 and 2 (16). The trabecular bone cores were scanned using a lunar piximus densitometer (lunar corp, madison, wi) at the bone centre (mcgill university, montreal, qc) to measure the bone mineral density (bmd) for a region of interest inside the specimen (11.3 mm x 5 mm). They were subsequently scanned with a microcomputed tomography (ct) instrument (model 1072, skyscan, aartselaar, belgium) at the bone centre (mcgill university, montreal, qc). The cross - sections along the specimen axis were reconstructed using cone - beam reconstruction software (skyscan), with a distance between each cross - section of 18.25m . The ct - analyser software (skyscan) was used to analyze the trabecular bone of the sample using segmentation method . Specimens were mounted on the testing machine (instron 5569, buckinghamshire, uk) so that the long axes of the cylinders were perpendicular to the lower anvil . A compressive force was then applied to the upper surface of the samples using a 50kn load cell at a constant crosshead displacement rate of 1 mm / min until failure occurred . The compressive load and the sample length were recorded at 0.1 second intervals during compression . Using this data, the stress (force / area) and strain (length / original length) were calculated and plotted against one another . The elastic modulus, an indication of the extent of compressive deformation in a specimen for a given load, was obtained from the linear portion of the stress versus strain curve (15). Initially, it was imperative to demonstrate that the protocol used to retrieve cylindrical cores of cancellous bone would provide valid samples for biomechanical testing . Twenty two cores of cancellous bone were extracted from the femoral neck of calf femurs and subjected to compression testing . The compressive strength and the elastic modulus were calculated for each sample and plotted against each other . It should be noted that the compressive strength is a measure of the stress within a sample immediately before the yielding point, while the elastic modulus is a measure of the rigidity of a sample . In concordance with previous findings, the compressive strength correlates strongly to the modulus (r = 0.98) in the calf femur samples (see figure 1) (18). Furthermore, these parameters fall within the range of published bovine values (19). Upon validating our technique, the same procedure was applied to the human femoral neck samples obtained from hip replacement surgeries . Comparable to the bovine samples, the compressive strength and the modulus of the human samples are strongly correlated (r = 0.99), as demonstrated in figure 2 . Moreover, the values for these parameters fall within the range of published human values for the femoral neck (20). Prior to subjecting the human cancellous samples to biomechanical testing, they were scanned using three dimensional ct and densitometry . As demonstrated in figure 3, the osteoporotic bone appears much more porous than the unaffected bone . The compressive strength was plotted against the trabecular thickness, number and separation, as shown in figure 4 . The latter parameters failed to show any statistically significant relationship to the biomechanics of bone . The trabecular thickness and separation remained fairly constant despite the strength of the bone, whereas the number of trabeculae appeared to increase with the compressive strength although not well . Needless to say, the trabecular data proved to be poor indicators of bone quality . Further investigation of the imaging data revealed that the relative porosity, calculated as the complement of the percent bone volume (i.e. 1 -% bone volume), and the bmd were the best predictors of bone quality . In fact, the relative porosity and the bmd correlated strongly to one another (see figure 5, r = 0.97). Upon analyzing the data, the compressive strength and the modulus were shown to vary exponentially with relative porosity and the bmd . That is to say, the strength and the modulus of the samples increased at a faster rate as the relative porosity decreased or the bmd increased . Thus, the latter were plotted against the natural logarithms of the compressive strength and the modulus for each bone sample (see figures 69). The relative porosity and the bmd correlated equally well to the compressive strength and the modulus (ln(compressive strength) vs. relative porosity, r = 0.90; ln(modulus) vs. relative porosity, r = 0.88; ln(compressive strength) vs. bmd, r = 0.94; ln(modulus) vs. bmd, r = 0.92). The assessment of bone quality is of the utmost importance in osteoporotic patients as it correlates well to the risk of fracture (1) and indicates when it is appropriate to initiate the proper preventative measures (68). Bisphosphonates have proven to be quite successful in deterring the progression of osteoporosis (6). Recently, there have been trials that have studied the effect of risedronate in osteoporotic patients using ct (11). These studies have indirectly assessed the drug s effect on bone quality using a remote site (i.e. The iliac crest) rather than the site of interest (e.g. Vertebrae, femoral neck). Understandably, the analysis of the iliac crest is convenient because it is easily accessible from the surface and poses minimal risk to the patient . There has been, however, no evidence to indicate whether any correlation exists between the microarchitecture at the iliac crest and the site of interest . To our knowledge, the effects of bisphosphonates on bone quality have not yet been studied in humans at sites than at the iliac crest . Prior to embarking on a hip fracture study, we wanted to establish reliable and reproducible procedures for biomechanical testing to ensure that the data were valid and representative of bone quality . Intuitively, bone quality should diminish as the bone becomes more porous and/or less dense with mineral . Indeed, we found the relative porosity and the bmd to be equally strong indicators of bone quality as both of these parameters correlate strongly to the biomechanical properties of bone, though in opposite fashion (see figures 69). A potential limitation to this experiment is the inherent design flaw that results from the extraction of trabecular bone . As the cylindrical core of bone is removed from the sample, the cross struts from the trabeculae along the periphery are severed . This unavoidably compromises structural integrity, especially since the compact bone significantly contributes to the overall strength of the femur (21). Hence, the biomechanical studies will not be entirely representative of the properties of trabeculae within the bone . However, taking into consideration that all the samples are prepared identically before testing, the data analysis remains valid . We have successfully established a protocol that will be used in a trial setting to analyze the bone quality in human femoral necks using ct and biomechanical compression testing . Using cores of cancellous bone from human femoral necks, the relative porosity and the bmd were shown to be excellent predictive markers of fracture resistance . As this trial evolves in its early stages, additional samples collected from hip replacements will be tested and the data compiled to construct a reference database for comparison to bisphosphonate treatment cohorts.
The extensive loss of the alveolar ridge and teeth in the posterior mandible presents a complex case for reconstruction . Several augmentation techniques are currently utilized to create sufficient bone volume for predictable placement of endosseous implants in such cases . The numerous surgical approaches proposed consist of autogenous bone grafts, alloplastic materials 18, and recently, alveolar distraction osteogenesis 9 . After tooth loss, the alveolar ridge undergoes a continuous resorptive process that is severely accelerated by denture wear 10 . This process is the most pronounced during the first 12 months after the tooth extractions 11,12 . Extensive resorption of the alveolar ridge in a vertical direction may compromise implant placement and prosthetic rehabilitation . The continuing resorption of the alveolar ridge will eventually result in insufficient bone height superior to the ian, making dental implant placement impossible without performing augmentation of the alveolar bone in terms of height . The augmentation procedure above the ian provides sufficient bone for implant placement and allows for long - term successful restoration of missing teeth with implant - supported prosthesis . All the methods suggested should take into consideration patient - related issues, which consist of pain, swelling, sensory nerve disturbances, incidence of graft failure and resorption, and functional long - term restoration . The reconstruction of vertically atrophic posterior mandibles with onlay bone grafts has been well documented, but the results have not been promising . Different donor sites (symphysis menti, calvaria, iliac crest) have been used as sources of autogenous bone . Vermeeren and associates 13 demonstrated bone resorption up to 50% even when autogenous onlay grafts were used . Rigid fixation of the graft material is imperative to prevent microrotation, which can result in nonunion or fibrous union of the graft material . The use of expanded polytetrafluoroethylene membranes is a treatment option for posterior mandibular reconstruction that has been used with varying degrees of success, as reported by various authors 15,16 . Tinti and coworkers 17 commented that vertical augmentation is a highly sensitive technique, predictable only when the surgical protocol is followed strictly . Vertical ridge augmentation of the atrophic maxilla and mandible by means of a titanium mesh and autogenous bone grafts has been used successfully and has gained popularity since its introduction 18,19 . The titanium mesh used must be fixed by titanium screws, and infection is a common complication 18 that may cause loss of grafted bone, resulting in failure . Visor osteotomy was first described in 1975 by harle 20 to increase the absolute height of the atrophic edentulous mandible . In this technique, when the procedure is applied to vertical ridge augmentation in the posterior mandible, the mandible is split vertically and, unfortunately, the width of the ridge is reduced . The sandwich technique, which uses bone block graft positioned between osteotomized bony segments, was developed by schettler 21 in 1974 stoelinga and colleagues 22 combined the visor osteotomy and sandwich techniques to augment the severely atrophic edentulous mandible with success 22 . A 49-year - old female patient was presented with a bilaterally atrophic mandible and a need for implant therapy . A thorough radiographic examination using cone - beam tomography revealed mandibular ridges that were not suitable for immediate implant placement in terms of height (6.2 mm on the left side and 7.2 on the right side . The patient was suggested the augmentation of the ridge using an interpositional block of allogeneic bone under local anesthesia . The patient gave her written informed consent, and a preoperative radiograph (fig.1a), computerized tomographic (ct) scan and cone - beam tomography of the left and right mandibular ridges were obtained (fig.1b). The mucoperiostal flap was raised to expose the mental foramen, and the mental nerve was identified, this helps to design the horizontal bone cut . Then, two other vertical bone cuts were performed on the extremities of the horizontal cut . The more mesial vertical cut was performed 2 mm away from the adjacent tooth . The bone segment was then raised upward to leave space for the bone graft (fig.2), with no disturbance of the lingual periosteum . An allogeneic bone block was inserted interpositionally and placed in the middle of the space formerly created without any fixation between the basal segment and the cranial segment (fig.3a) the remaining spaces in both ends were filled with particular bone graft (fig.3b). The wound was then closed primarily with 4 - 0 vicryl u - shaped suture . A postoperative cone - beam x - ray and ct scan were obtained to assess the new vertical height of the mandible (fig.4a and b). After 3 months of healing, a crestal incision on the attached gingiva was made . The mucoperiostal flap was detached and endosseous implants were inserted using the classical approach, two into the right side, and three in the left side of the mandible, measuring 4 mm in diameter and 10 mm in length . The primary stability was relatively high (fig.5), and allowed for placement of the healing abutments (fig.6). Post surgical follow - up visits were carried out at months 1, 3, 6, 12, 18, and 24 after the surgical procedure . At each follow - up procedure, after 2 years of follow - up, the patient s conditions were optimal, the hard and soft tissue did not show any changes . (a) allogenic bone block inserted interpositionally and placed in the middle of the space between the two bone segments . (b) cone - beam tomography of the new mandible heights after the surgical procedure . Moderate to severe posterior mandibular atrophy was successfully treated with interpositional sandwich osteotomy bone grafts . This led to the successful placement of implants and fixed prosthetic implant restorations, thus allowing ever more patients to be considered for implant treatment . The technique, which has been recently revisited, permits dental rehabilitation in terms of raising the bone above the nerve, reshaping the alveolar crest, and normalizing the interocclusal distance and the crown - implant ratio.
Septic arthritis is a well recognized occurrence in patients with steroid dependent rheumatoid arthritis.1 treatment includes broad - spectrum antibiotics usually accompanied by surgical or needle drainage of the joint.2 while pericardial effusions are common in patients with rheumatologic disorders, the development of purulent pericarditis with pericardial tamponade is rare . We report a case of purulent pericarditis with pericardial tamponade masquerading as septic shock related to proteus mirabilis septic arthritis . A 53-year - old man with long - standing, steroid - dependent rheumatoid arthritis complained of a painful, swollen, left elbow with purulent drainage emanating from what appeared to be a small ulceration . The patient was admitted to the hospital, and blood and wound cultures were obtained; empiric cefepime and vancomycin were started . Because of persistent fever and continuing joint drainage, he was transferred to a tertiary facility for surgical treatment of presumed septic arthritis . The early postoperative course, however, was remarkable for persistent fever, hypotension, and tachycardia . On the first postoperative day, he was transferred to the medical service for the management of presumed septic shock . On physical examination the temperature was 39c, blood pressure was 100/70 mmhg, heart rate was 130 beats per minute, and oxygen saturation was 96% on 3 l oxygen per nasal cannula . Physical examination was remarkable for: marked jugular venous distention; clear lungs; normal heart sounds without heart murmurs or rubs; and a benign abdomen . 2) showed diffuse st elevation, pr depression, and lateral t - waves inversion . A subxiphoid pericardiocentesis yielded 500 ml of purulent fluid with prompt normalization of the blood pressure . Pericardial fluid ph was 8.0; wbc, 126,000/l (100% neutrophils); rbc, 180,000/l; lactate dehydrogenase (ldh), 43,300 u / l; protein, 4.8 g / dl; triglycerides, 18 mg / dl . A pericardial window was later placed surgically with drainage of an additional 375 ml of purulent fluid . Figure 1portable chest x - ray depicting an enlarged cardiac silhouette and mediastinum.figure 2admission electrocardiogram depicting diffuse st elevation with t - wave inversion and pr depression.figure 3transthoracic two - dimensional echocardiogram . While multiple blood cultures were negative, articular and pericardial fluid cultures grew staphylococcus epidermidis and proteus mirabilis . While candida albicans was present in the urine, there was no evidence of a proteus urinary tract infection . Head, chest, abdomen, and pelvis computerized tomography scans were performed to exclude possible sites of metastatic infection . A radiolabeled leukocyte scan was positive in the left elbow and other areas of joint replacement . There was no recurrence of the pericardial effusion by echocardiography 8 weeks post - discharge . In the setting of an infected joint prosthesis, fever, and immunosuppression, this patient s hemodynamic instability was initially ascribed to septic shock and not to pericardial tamponade . The usual signs and symptoms of pericardial inflammation were absent . The expected chest pain and a pericardial friction rub were missing and were, no doubt, attenuated by the chronic steroid use . Despite a large and grossly purulent pericardial effusion, the electrocardiographic findings were quite subtle . Only a high index of suspicion prompted the consideration of pericardial tamponade . A careful physical examination and critical interpretation of the chest radiograph were essential steps in establishing the correct diagnosis . In this case, prompt consideration of pericardial tamponade led to a life - saving pericardiocentesis . The abnormally high ph of the pericardial fluid presaged the possibility of a proteus infection . Pericardial involvement is usually a result of contiguous spread of an adjacent intrathoracic infection.3 blood borne infections account for less than 30% of the cases of purulent pericarditis . Gram - positive organisms continue to be the most commonly observed pathogens, although, there is evidence that the increased utilization of antibiotics may favor the emergence of gram - negative organisms.4proteus mirabilis, a common urologic pathogen, is a distinctly uncommon cause of purulent pericarditis . Is rare and has been previously reported in association with pneumonia4 and coronary bypass surgery.5 septic arthritis leading to pericardial infection is equally uncommon . An association between septic arthritis and purulent pericarditis has been observed with hemophilius influenza,6neisseria meningitides,7 and neisseria gonorrhoeae.8 the simultaneous occurrence of proteus arthritis and pericarditis in this patient is unique and has not been previously reported . It is highly likely that a single pathogen was responsible for both the articular and pericardial infections . Based on antibiotic sensitivities, the proteus isolated from the joint space and pericardial fluid appears to be identical . An extensive evaluation confirmed that the source of pericardial infection was the infected prosthetic elbow . The resulting articular infection then spread hematogenously to the pericardial space without any evidence of additional intrapulmonary or intracardiac infection . The initial treatment with oral cephalexin may have been inadequate for septic arthritis in an immunocompromised patients . Likewise, early failure to drain and debride the infected joint may have facilitated the systemic spread of the infection . While pericardial effusions are commonly present in patients with rheumatologic disorders, pericardial infections and pericardial tamponade are not . This case illustrates how, in the setting of a chronic pericardial effusion and chronic immunosuppressive therapy, a blood borne infection may seed the pericardial space . It further demonstrates how rapidly, a closed space bacterial infection may then rapidly progress to pericardial tamponade, masquerading as septic shock . It emphasizes the importance of a careful physical examination and the thoughtful synthesis of ancillary studies . In immunocompromised patients, the typical signs and symptoms of pericarditis may be absent, and the clinical presentation of pericardial tamponade may be misinterpreted as one of septic shock . It further illustrates the potential for atypical transmission, to the pericardium, of gram - negative pathogens in patients with a high likelihood of preexisting pericardial disease . Knowledge of this possibility should facilitate prompt recognition and management of this life - threatening condition . This case also underscores the importance of appropriate antibiotic selection in the initial treatment of immunocompromised patients with infected prosthetic joints . This would include the need for the administration of intravenous broad - spectrum antibiotics with surgical lavage and debridement of the infected space.
Emergency is a condition determined clinically or considered by the patient or his relatives as requiring urgent medical, dental or allied service, failing, which it would result in loss of life or limb.1 the identification of epidemiological trends in hospital admissions are essential for health - care planning and resource allocation . Non - communicable diseases particularly cardiovascular diseases, diabetes mellitus (dm), cancer and respiratory diseases are the major causes of morbidity and mortality in the developed world and are emerging as an important component of the burden of diseases in developing countries.2 there are reports on studies carried out in nigeria and other african countries on the pattern of medical cases admitted to the emergency unit, but none to our knowledge on the pattern of endocrine - related diseases presenting as an emergency among adult patients in sub - saharan africa.3456 in a study carried out in port - harcourt, nigeria, on the medical ward admissions over a 4-year period; non - communicable diseases constituted 56.2% of all medical admissions, where cardiovascular, endocrine and renal systems were most commonly seen, accounting for 35.7%, 18.5%, and 16.8% respectively.6 knowledge on admission and mortality patterns of endocrine - related diseases will give insight into the magnitude of the condition and provide effective tools for planning, delivery and evaluation of health - care needs . This study aimed to determine the pattern of admission and mortality of endocrine - related diseases in the emergency unit of the lagos university teaching hospital (luth). This retrospective study was carried out at the adult emergency unit of the luth, situated in lagos state in south western nigeria . The hospital occupies 92 acres of land and is an over 700 bed facility, making it one of the largest teaching hospitals in nigeria . It serves as a referral hospital for lagos and its adjoining states . The emergency unit we retrieved medical records of patients that visited the emergency unit of the hospital over a year period (march 2011 to february 2012) from the hospital admissions and death registers . Information retrieved included: age, gender, diagnosis at admission and death, co - morbidities . Data were entered into excel work sheet for cleaning and then transferred to statistical package for social sciences (spss for windows version 17) for analysis . Quantitative data were expressed as mean standard deviation while qualitative variables were expressed as percentages . Data were entered into excel work sheet for cleaning and then transferred to statistical package for social sciences (spss for windows version 17) for analysis . Quantitative data were expressed as mean standard deviation while qualitative variables were expressed as percentages . A total of 1703 adult cases were seen in the emergency unit from march 2011 to february 2012 . Of the 1703 cases, 174 were endocrine - related, accounting for 10.2% of the total emergency unit visitation / admission in the hospital [figure 1]. Males were 81 (46.6%) and females 93 (53.4%) females, giving a male to female ratio of 1:1.2 . The mean age of the patients was 47.8 14.2, with an age range of 18 - 78 years . Percentage distribution of endocrine and non - endocrine admissions out of the 174 endocrine - related cases, 75 (43.1%) had hyperglycaemic crises (diabetic ketoacidosis, 36% and hyperosmolar hyperglycaemic state 64%); 33 (19.0%) had diabetes mellitus foot syndrome (dmfs); 23 (13.2%) had hypoglycaemia; 3 (1.7%) had dm hand syndrome; 1 (0.6%) had conn's syndrome; 1 (0.6%) had cushing's syndrome; 5 (2.8%) had thyroid diseases, and 33 (19.0%) had dm related co - morbidities [tables 1 and 2]. Distribution of endocrine - related admissions contribution of dm co - morbidities to endocrine - related admissions a total of 39 endocrine - related deaths were recorded in the emergency room within this period [table 3]. There were 18 (46.2%) males and 21 (53.8%) females (= 0.002, p = 0.964). About 35.8% (14 out of 39) of the mortalities were sepsis - related . The fatality rates were higher in patients who presented with thyrotoxic crises, 60% (3 out of 5) and hypoglycaemic coma, 39.1% (9 out of 23). This study has attempted at showing the pattern of admissions and mortality of patients with endocrine - related conditions in the emergency setting in a resource poor tertiary health facility of a developing country . The diagnoses were mainly based on clinical and laboratory findings as autopsy could not be carried out for the majority of the patients on cultural and religious grounds . Global projections of disease burden and mortality have indicated a significant shift from infection / communicable to non - communicable diseases world - wide, and this transition is expected to affect developing countries like nigeria.7 it has been projected that by the year 2000, the prevalence of non - communicable disease will parallel that of the communicable diseases in developing nations, which will have to contend with the double burden of the two groups of diseases.8 in our study, endocrine - related diseases accounted for a significant proportion of the total adult medical emergency unit admissions, with dm emergencies predominating . This finding is in agreement with documentations of the preponderance of chronic non - communicable diseases including endocrine diseases in various hospitals across developing countries especially nigeria.391011121314 the preponderance of diabetic emergencies may be associated with the increasing prevalence of dm across the world, particularly in sub - saharan african where nigeria has the highest number of people living with dm . The reported prevalence of dm across nigeria varies from 0.65 in rural mangu village to as high as 11.0% in urban lagos where this study was carried out;15 this may also be linked to the increasing urbanization, reduced physical activity and the epidemic boom of obesity especially in the developing world . This finding is similar to results of a study carried out in ilorin, nigeria, in which the highest numbers of admissions were due to diabetic hyperglycaemic emergencies, septicaemia, and diabetic foot syndrome.16 the mean age of the subjects was similar to that seen in other related studies in nigeria.6 gender disparity was observed in the overall distribution of endocrine - related admissions, with more females compared to males . This finding is in variance with reported gender distribution of non - communicable diseases reported in previous studies.617 however, chinenye et al . Reported a female preponderance in their study on the profile of dm patients, and suggested it may be a reflection of the health - care financing pattern in the country with females likely to be supported by relations and loved ones with financial assistance toward hospital visits than males.15 the mortality rate was also highest in patients who presented with hyperglycaemic emergencies, sepsis, thyroid, and hypoglycaemic crisis . This is also similar to the reports on mortality patterns in dm in other studies.1416 a greater proportion of those who presented with thyroid crises and hypoglycaemia died . Late presentation and prolonged neuroglycopenia with irreversible brain damage is likely responsible for the non - response to treatment in hypoglycaemic patients . Thyroid storm and thyrotoxic heart disease were the causes of death in patients who presented on account of thyroid crises . The prognosis for thyrotoxic crisis remains poor with high mortality recorded in untreated cases and mortality rates of 20 - 30% reported even with treatment.1819 this finding is similar to those cited by ogbera.20 most of the mortalities recorded were complicated by sepsis . This alluded the findings of a huge burden of infectious diseases on the outcome of medical admissions in a similar study.621 from this study, death from hyperglycaemic crises appears to be mostly associated with sepsis . The immunosuppressive state associated with dm may be responsible for the high rate of infection among this group of patients; and sepsis may also be the precipitant for the hyperglycaemic crisis in them . Endocrine - related diseases are common in the emergency room of this health facility, with diabetic complications accounting for most of the admissions and mortality . Surveillance for infection is recommended for patients presenting to the emergency room with endocrine - related conditions, especially hyperglycaemic emergencies.
Every year more than 14 million cases of obstetric hemorrhage occur, resulting in an estimated 127,000 deaths.1 postpartum hemorrhage (pph), blood loss of 500 ml or more, accounts for the majority of these hemorrhage deaths.2 pph is the leading cause of maternal death in low income countries and is the primary cause of approximately one - quarter of global maternal deaths.3 among pph survivors, an estimated 12% will suffer from the consequences of severe anemia.2 several factors contribute to the high pph mortality estimates . In most developing countries, 50% or more of deliveries are attended by unskilled providers at home.4 in addition, health facilities are often not adequately staffed or lack medicines that can address pph.5 these structural barriers are further complicated by difficulties in predicting who will develop pph . Many women who develop pph do not present with any of the risk factors typically associated with the complication.6 consequently, pph is an obstetric complication that requires effective preventive interventions, tailored to the diverse needs of women and providers in resource - poor settings . The purpose of this paper is to present current perspectives on the prevention of pph, particularly in resource poor - settings, where pph is the leading cause of maternal mortality.3 we reviewed historical events and the current evidence related to pph prevention and highlight the progress in policy and program implementation to reduce this disease . We reviewed the current strategies being implemented to prevent pph, ranging from active management of the third stage of labor (amtsl) in health facilities to the use of misoprostol in home births, which was given particular attention . In addition, we looked at challenges to the implementation and scale - up of these interventions, as well as examples of ongoing efforts that could be positions as opportunities to increase access to pph prevention interventions . Using pubmed, we conducted a review of the literature on randomized controlled trials (rcts) of interventions to prevent pph . We then searched pubmed and google scholar for nonrandomized field trials of interventions to prevent pph . We limited our review to interventions that are discussed in the current world health organization (who) recommendations for pph prevention, which we consider to be key interventions, and present evidence on the use of these interventions from 2000 to 2013 . The following search terms were used, among others, in varying combinations: postpartum hemorrhage, pph, pph prevention interventions, active management of the third stage of labor, amtsl, controlled cord traction, cord clamping, uterine massage, oxytocin, ergometrine, misoprostol, systematic review, cochrane review, randomized controlled trial, rct, operations research, low - resource settings, and developing countries . We assessed the evidence from rcts of pph prevention interventions, as well as evidence from field trials and implementation programs . We focused our review on comparisons of nondrug pph prevention interventions versus no intervention and of uterotonics versus placebo; this review was not intended to decipher the relative effectiveness of uterotonic drugs . The interventions and conventional uterotonic drugs reviewed are those presumed to, either alone or in combination with other drugs, prevent pph . The nondrug interventions included were amtsl and the specific components (ie, controlled cord traction, cord clamping, and uterine massage). The conventional uterotonics included were oxytocin, ergot - based alkaloid (ergometrine), and misoprostol . To avoid duplication, we started with systematic reviews (often cochrane reviews) conducted since 2000 and then added individual studies conducted after the review . We also searched for other studies not included in the most recent reviews that would meet our search criteria, including peer - reviewed articles, documents in the gray literature, manuals, reports, clinical guidelines, and relevant publications from organizations working to promote pph prevention, such as the who, the international federation of gynecology and obstetrics (figo), the international confederation of midwives (icm), as well as the work of many other nongovernmental organizations . We recognize the barriers to implementation that developing countries may face and have described the challenges to and opportunities for scaling up recommended interventions . We focused specifically on scalability in light of limited access to services and shortages in skilled health care workers and commodities . Figure 1 presents a timeline of the important milestones related to pph prevention, including discoveries, research publications, policies, and programs . To provide a historical perspective, we started in 1953 with the elucidation of the amino acid sequence of oxytocin, followed by its biochemical synthesis.710 following these discoveries, a landmark in the history of pph prevention occurred when the three components of amtsl were first described in 1962: the administration of a prophylactic uterotonic drug, early cord cutting and clamping, and controlled cord traction.11 however, it was not until the 1980s that data from an rct of amtsl revealed a significant reduction in the incidence of pph compared with expectant management of the third stage of labor.12 more than two decades later, figo and icm released their first statement on amtsl.13 then, recognizing that amtsl could only be provided by skilled attendants, thus excluding women who deliver at home and limiting the coverage of this intervention, researchers were encouraged with the discovery and potential of a prostaglandin analogue in tablet form (ie, misoprostol).14 in 2005, the first placebo - controlled trial of misoprostol use for pph prevention at home births was carried out, with promising results.15 the findings paved the way for women with limited or no access to health facilities to have a pph prevention intervention delivered at home, but the use of misoprostol for this purpose also introduced policy and program - related challenges . A considerable impediment was that at the time, misoprostol was an off - patent drug that was only on the market for the treatment of gastric ulcers, and in many developing countries, a product needs to be registered for the specific indication for which it is marketed . In 2006, the who organized a technical consultation meeting to review the evidence for interventions to prevent and treat pph; the following year, the who published recommendations based on this meeting, which included support of the use of misoprostol for pph prevention in the absence of oxytocin but not at home births.16 by that time, individual country efforts to take advantage of misoprostol for pph prevention had already started . In 2006, nigeria was the first country in the world to register the use of misoprostol for pph prevention.17 five years later, another important landmark policy reform occurred, which was the inclusion of misoprostol in the who model list of essential medicines in 2011,18 followed by a revision of the recommendations for the prevention and treatment of pph, in 2012.3 around this time, the evidence regarding the impact of misoprostol in reducing pph became clear, and its inclusion in the united nations commission on life - saving commodities for women and children in 2012 was a testament to its contribution.19 the nonprofit organizations and professional associations with maternal health programs that attend largely to rural populations have recently issued a call for the scaling up of pph prevention programs that include the use of misoprostol.20,21 in addition, programs to strengthen labor and delivery practices continue to take place.22 table 1 presents a description of the key pph prevention interventions and the who recommendations with respect to each . Amtsl is considered the gold standard strategy to reduce the incidence of pph . The amtsl preferred uterotonic is oxytocin,3 and the relative importance of other components has changed over time . Even though the who strongly recommends amtsl, it also provides recommendations on the relative importance of each component . For example, the practice of controlled cord traction has a weak recommendation level, only to be practiced if small reductions in blood loss or durations of the third stage of labor are perceived to be beneficial (table 1).3 cord clamping is now subdivided into late and early cord clamping; the latter is no longer recommended . Similarly, sustained uterine massage is no longer recommended in women who receive prophylactic oxytocin, although it was initially a common component of amtsl . Instead, it is recommended that abdominal tonus assessment be conducted by a skilled provider, for all women (table 1). However, the figo guidelines (2012) for the prevention of pph in low - resource settings defines amtsl as: administration of oxytocin, controlled cord traction, and uterine massage after the delivery of the placenta.23 uterotonic drugs, such as oxytocin, ergometrine, and misoprostol, are strongly recommended (table 1). Some researchers feel that oxytocin, which has minimal side effects and is safe to use among women with hypertension and preeclampsia, is all that is needed.24 ergometrine, which has proven to be a powerful drug in reducing pph, especially when combined with oxytocin, is known to be associated with serious side effects, such as severe vomiting and hypertension, and retained placenta when given intravenously.25 misoprostol is similarly associated with side effects, but none have been shown to threaten the life of the mother or newborn.26 its use is associated with significantly higher incidences of shivering and fever among mothers compared with placebo.26 misoprostol has had recent success because of its heat stability and multiple routes of administration,27 which is critical in resource - poor countries . It is important to note that all of these recommended interventions, with the exception of misoprostol administration, are to be provided by skilled providers; as a result, most of them are available only to women who attend facility deliveries . Large numbers of women living in countries with high maternal mortality deliver at home.4 thus, the recent recommendation supporting the inclusion of community health workers in the provision of misoprostol for the prevention of pph was welcomed by the safe motherhood community.3,28 the current recommendations with regards to pph prevention are based largely on rct evidence . The clinical evidence of the initial amtsl package from the 1980s showed that pph could be reduced by 70% compared with expectant management.29 the most recent cochrane review of active versus expectant management of the third stage of labor includes seven studies (table 2), and the results indicate a significant reduction in the risk of pph.29 controlled cord traction was an initial component of amtsl, but since 2000, three rcts assessing amtsl with and without cord traction were published, all of which found a nonsignificant difference in the risk of pph (table 2).3032 regarding cord clamping, it was initially thought that early cord clamping was best, but recent findings indicate that the timing is not critical.3 in 2013, the cochrane collaboration researchers updated a previous review on the subject, and their findings confirmed that there are no significant differences in the risk of pph between early and late cord clamping.33 uterine massage, although not officially in the initial amtsl package, was often included.3 however, a cochrane review of two rcts comparing uterine massage after birth and before or after the delivery of the placenta for the prevention of pph found the differences between groups to be insignificant.34 in contrast, the efficacy of lower uterine segment compression versus nothing, in addition to oxytocin, cutting and clamping of the umbilical cord, and controlled cord traction, was found to be associated with a significantly lower incidence of pph.35 table 2 also presents the recent clinical evidence on the conventional uterotonics, demonstrating their importance in the prevention of pph . A recent review of seven rcts investigating the efficacy of oxytocin, three with intramuscular (i m) and four with intravenous (iv) preparations, showed that this uterotonic can significantly reduce the risk of pph.36 in addition to the i m and iv preparations, two rcts investigating the intraumbilical administration of oxytocin also found significant reductions in blood loss when compared with a control saline solution.37,38 collectively, the results from these studies make it clear why oxytocin is the preferred uterotonic for pph prevention . In terms of the other uterotonics, a 2011 review of ergot alkaloids containing six studies (rcts and quasi - rcts) found a significant reduction of pph when these were administered in the third stage of labor.39 regarding misoprostol, three recent reviews of rcts comparing it to a placebo were identified, two published in 2012 and one in 2013.4042 results of these reviews showed both significant reductions and nonsignificant reductions of pph . The inconsistencies in the results could be due to the fact that misoprostol was assessed in different doses and routes and also that the individual studies inclusion criteria varied . The cochrane review included 72 rcts that assessed the effects of prophylactic prostaglandin use versus a placebo, eight of which assessed 600 g oral or sublingual misoprostol (table 2). Based on data from one trial, the authors of the review concluded that sublingual misoprostol was associated with significant reductions in the incidence of pph . The data from seven trials on oral misoprostol were not totaled due to heterogeneity, but the authors concluded that compared with a placebo, oral misoprostol shows promising results.42 another 2012 meta - analysis of three rcts investigating oral or sublingual misoprostol sound significant reductions in both acute and severe pph.40 an even more recent meta - analysis of three rcts showed that misoprostol does not provide significant reductions in the incidence of pph when compared with placebo.41 of the three rcts included in these two latest reviews from 2012 and 2013, two of the rcts were the same,15,43 while one was different.44,45 two seminal studies that were included showed a reduction in pph of 24%45 and 47%15 in home births . Thus, factoring in the findings from the abovementioned reviews, all of which showed reductions in the risk of pph, even if nonsignificant, it is understandable that the who ultimately supported the use of misoprostol in the absence of other uterotonics . Assessment of the nonrandomized data on the interventions to prevent pph supports the clinical evidence from the rcts . A quasi - experimental study of amtsl in vietnam (table 3) showed similar levels of reduction in pph to those found in the rcts.46 an analysis of the independent or combined effect of uterotonic agents, controlled cord traction, and uterine massage from hospital - based data in four countries also showed similar results to the rcts.47 no recent nonrandomized or field trials were found reporting on the individual components of amtsl, nor for oxytocin or ergometrine use in pph prevention . On the contrary, evidence was found on the use of misoprostol to prevent pph in facilities and home births . All of the nonrandomized studies with blood loss and/or acute or severe pph as outcomes showed that misoprostol use in the third stage of labor can significantly reduce pph (table 3). The evidence demonstrates that misoprostol can be effective in the prevention of pph in home births.4850 these findings are especially important for developing countries, which contribute the majority of pph morbidity and mortality, where skilled attendance at delivery is limited, and where the use of the other two uterotonics is more challenging.51 table 1 presents a description of the key pph prevention interventions and the who recommendations with respect to each . Amtsl is considered the gold standard strategy to reduce the incidence of pph . The amtsl preferred uterotonic is oxytocin,3 and the relative importance of other components has changed over time . Even though the who strongly recommends amtsl, it also provides recommendations on the relative importance of each component . For example, the practice of controlled cord traction has a weak recommendation level, only to be practiced if small reductions in blood loss or durations of the third stage of labor are perceived to be beneficial (table 1).3 cord clamping is now subdivided into late and early cord clamping; the latter is no longer recommended . Similarly, sustained uterine massage is no longer recommended in women who receive prophylactic oxytocin, although it was initially a common component of amtsl . Instead, it is recommended that abdominal tonus assessment be conducted by a skilled provider, for all women (table 1). However, the figo guidelines (2012) for the prevention of pph in low - resource settings defines amtsl as: administration of oxytocin, controlled cord traction, and uterine massage after the delivery of the placenta.23 uterotonic drugs, such as oxytocin, ergometrine, and misoprostol, are strongly recommended (table 1). Some researchers feel that oxytocin, which has minimal side effects and is safe to use among women with hypertension and preeclampsia, is all that is needed.24 ergometrine, which has proven to be a powerful drug in reducing pph, especially when combined with oxytocin, is known to be associated with serious side effects, such as severe vomiting and hypertension, and retained placenta when given intravenously.25 misoprostol is similarly associated with side effects, but none have been shown to threaten the life of the mother or newborn.26 its use is associated with significantly higher incidences of shivering and fever among mothers compared with placebo.26 misoprostol has had recent success because of its heat stability and multiple routes of administration,27 which is critical in resource - poor countries . It is important to note that all of these recommended interventions, with the exception of misoprostol administration, are to be provided by skilled providers; as a result, most of them are available only to women who attend facility deliveries . Large numbers of women living in countries with high maternal mortality deliver at home.4 thus, the recent recommendation supporting the inclusion of community health workers in the provision of misoprostol for the prevention of pph was welcomed by the safe motherhood community.3,28 the clinical evidence of the initial amtsl package from the 1980s showed that pph could be reduced by 70% compared with expectant management.29 the most recent cochrane review of active versus expectant management of the third stage of labor includes seven studies (table 2), and the results indicate a significant reduction in the risk of pph.29 controlled cord traction was an initial component of amtsl, but since 2000, three rcts assessing amtsl with and without cord traction were published, all of which found a nonsignificant difference in the risk of pph (table 2).3032 regarding cord clamping, it was initially thought that early cord clamping was best, but recent findings indicate that the timing is not critical.3 in 2013, the cochrane collaboration researchers updated a previous review on the subject, and their findings confirmed that there are no significant differences in the risk of pph between early and late cord clamping.33 uterine massage, although not officially in the initial amtsl package, was often included.3 however, a cochrane review of two rcts comparing uterine massage after birth and before or after the delivery of the placenta for the prevention of pph found the differences between groups to be insignificant.34 in contrast, the efficacy of lower uterine segment compression versus nothing, in addition to oxytocin, cutting and clamping of the umbilical cord, and controlled cord traction, was found to be associated with a significantly lower incidence of pph.35 table 2 also presents the recent clinical evidence on the conventional uterotonics, demonstrating their importance in the prevention of pph . A recent review of seven rcts investigating the efficacy of oxytocin, three with intramuscular (i m) and four with intravenous (iv) preparations, showed that this uterotonic can significantly reduce the risk of pph.36 in addition to the i m and iv preparations, two rcts investigating the intraumbilical administration of oxytocin also found significant reductions in blood loss when compared with a control saline solution.37,38 collectively, the results from these studies make it clear why oxytocin is the preferred uterotonic for pph prevention . In terms of the other uterotonics, a 2011 review of ergot alkaloids containing six studies (rcts and quasi - rcts) found a significant reduction of pph when these were administered in the third stage of labor.39 regarding misoprostol, three recent reviews of rcts comparing it to a placebo were identified, two published in 2012 and one in 2013.4042 results of these reviews showed both significant reductions and nonsignificant reductions of pph . The inconsistencies in the results could be due to the fact that misoprostol was assessed in different doses and routes and also that the individual studies inclusion criteria varied . The cochrane review included 72 rcts that assessed the effects of prophylactic prostaglandin use versus a placebo, eight of which assessed 600 g oral or sublingual misoprostol (table 2). Based on data from one trial, the authors of the review concluded that sublingual misoprostol was associated with significant reductions in the incidence of pph . The data from seven trials on oral misoprostol were not totaled due to heterogeneity, but the authors concluded that compared with a placebo, oral misoprostol shows promising results.42 another 2012 meta - analysis of three rcts investigating oral or sublingual misoprostol sound significant reductions in both acute and severe pph.40 an even more recent meta - analysis of three rcts showed that misoprostol does not provide significant reductions in the incidence of pph when compared with placebo.41 of the three rcts included in these two latest reviews from 2012 and 2013, two of the rcts were the same,15,43 while one was different.44,45 two seminal studies that were included showed a reduction in pph of 24%45 and 47%15 in home births . Thus, factoring in the findings from the abovementioned reviews, all of which showed reductions in the risk of pph, even if nonsignificant, it is understandable that the who ultimately supported the use of misoprostol in the absence of other uterotonics . Assessment of the nonrandomized data on the interventions to prevent pph supports the clinical evidence from the rcts . A quasi - experimental study of amtsl in vietnam (table 3) showed similar levels of reduction in pph to those found in the rcts.46 an analysis of the independent or combined effect of uterotonic agents, controlled cord traction, and uterine massage from hospital - based data in four countries also showed similar results to the rcts.47 no recent nonrandomized or field trials were found reporting on the individual components of amtsl, nor for oxytocin or ergometrine use in pph prevention . On the contrary, evidence was found on the use of misoprostol to prevent pph in facilities and home births . All of the nonrandomized studies with blood loss and/or acute or severe pph as outcomes showed that misoprostol use in the third stage of labor can significantly reduce pph (table 3). The evidence demonstrates that misoprostol can be effective in the prevention of pph in home births.4850 these findings are especially important for developing countries, which contribute the majority of pph morbidity and mortality, where skilled attendance at delivery is limited, and where the use of the other two uterotonics is more challenging.51 when considering scaling up pph prevention interventions, it is important to consider where deliveries are occurring . Approximately 46% of all births worldwide take place outside of an institutional setting, attended by a traditional birth attendant, a relative, or no one.52,53 as previously mentioned, all who - recommended interventions other than misoprostol administration require a skilled birth attendant, and many require a facility - based delivery.3 one strategy for increasing access to these life - saving interventions is to encourage facility - based delivery, especially during prenatal care . However, one must keep in mind the limited number of skilled providers in settings where the most at - risk women deliver.4 in fact, it is in these high maternal mortality settings where the shortage of trained health providers is most acute . For example, 57 countries, many of which are among the least developed countries, have a shortage of approximately 2.4 million physicians, nurses, and midwives . In sub - saharan africa specifically, their health care workforce shortage is so severe that they have only 1.3% of the world s skilled providers, but 25% of the global burden of disease.54 most mortality from pph would be eliminated if women had access to a skilled birth attendant, yet only 35% of births are attended by a skilled health worker in the least - developed countries.55 it is estimated that the coverage of skilled attendance at birth is improving at a rate of less than 0.5% per year,56 thus it will be many years until we see adequate coverage at delivery; alternative solutions are needed . For many developing countries, poor storage conditions and deficient public sector supply chains also contribute to the limited access to and utilization of uterotonic drugs . The storage of oxytocin and ergometrine can be particularly challenging, due to their instability in high temperatures and sensitivity to light.51,57 additionally, because these two drugs are only available in injection form, their administration is limited to skilled providers . Finally, in some settings where oxytocin and ergometrine are accessible, procurement of unregistered drugs may be prolific and the quality of the drugs may be compromised . For example, in ghana, researchers found 89% (n=90) of all ampoules of oxytocin and ergometrine tested did not meet the specifications for the active ingredient, which was not a result of being expired; this problem was present in both the public and private sector.58 on the other hand, misoprostol is reportedly more stable, available in tablet form, and can be provided through multiple routes of administration.59 however, all uterotonics can be exposed to health system failures, such as the case of oxytocin in tanzania and ethiopia, where the drugs were available but not properly distributed to health facilities.60,61 even when available, uterotonics are not always used consistently to prevent pph . A survey of 15 tertiary level facilities (including 452 vaginal deliveries) conducted by the global network for perinatal and reproductive health showed that prophylactic oxytocin was used in 44% of the cases and was the least used of the three components of amtsl assessed (use of early cord clamping was 79% and use of controlled cord traction was 70%).62 for home births without a skilled provider, misoprostol has been distributed using a variety of approaches with or without a safe delivery kit, including distribution at antenatal care visits, at household visits by community health workers during pregnancy, at home births assisted by traditional birth attendants, and in some instances, through a combination of these methods.63 none of these approaches are without challenges related to access and utilization of the drug . For example, antenatal care distribution is only effective in increasing access to this drug in those settings where the majority of women who will deliver at home also attend antenatal care . Furthermore, if advanced distribution of misoprostol requires a specific gestational age among eligible women (eg, in mozambique, this is 28 weeks gestation; in bangladesh, this is 32 weeks),64,65 only those attending antenatal care after the required gestational age would receive the drug and be able to use it in home births . Separate from the interventions themselves is the health care system in which these interventions are being implemented . The continuum of care is an important consideration because these interventions in isolation will likely not be enough to prevent maternal morbidity and mortality . These interventions are not a replacement for a weak health care infrastructure and limited health care personnel, which must not be forgotten in this discussion of pph prevention interventions . The implementation of effective approaches is also dependent on the timely translation of research findings into policies and programs, which remains a considerable barrier in accelerating pph prevention efforts . In general, the translation of research into clinical practice is often conceptualized as proceeding from awareness through acceptance to adoption.66 however, decades may pass before research findings are integrated into guidelines and routine clinical practice (see figure 1). There is no agreement or set of rules among policy makers regarding the amount of evidence needed (ie, number and type of studies) to prompt changes in policies and programs . As mentioned before, by 2007, there was only one rct published on the use of misoprostol in home births, which prompted mathai et al to express, after the 2007 release of the who guidelines, that there was insufficient evidence for the safe use of misoprostol by lay providers in nonfacility settings.67 nonetheless, based solely on the evidence of its efficacy in clinical settings and encouraged by the potential impact that misoprostol could have in the absence of oxytocin, many countries had already begun conducting operations research to test its safety, feasibility, and acceptability for home births, even before consensus was reached and the official guidelines were in place.68,69 eventually, a placebo - controlled rct with traditional birth attendants was conducted, from 2006 to 2008, in pakistan.45 similar to the history of misoprostol, but with less debate, amtsl was first described in 1962,11 but the first rct comparing amtsl with expectant management was not done until 1988,12 and the official guidelines and policies were not adapted until later,13 after many countries had already begun to incorporate this practice . The evidence of the relative importance of its components has only become available in the last 3 years, prompting the who to review its recommendations (see table 1). Finally, the challenges in the use of evidence to recommend improvements in the quality of care are further complicated by the delays in provider uptake of practices based on new knowledge . Even when health providers recognize and accept guidelines, they may fail to adopt them,70 and pph prevention efforts are no exception.62 as noted, the translation of research into policy is often a long and inconsistent process, and the prioritization of a health intervention based solely on costs or cost - effectiveness is an equally challenging proposition . However, in resource - poor settings, the reduction of maternal mortality in the most cost - effective way possible should be a priority to policy makers.71 in a modeling exercise, the prevention of pph with advanced distribution of misoprostol was found to be very cost - effective in resource - poor settings, relative to other maternal - health interventions included in the who mother - baby package, only preceded by family planning and safe abortion.72 the cost of each intervention recommended by the who might differ due to the cost of each uterotonic.67 the costs of services also depend on the level of health facility and provider involved . However, given that pph is the main cause of maternal death, saving lives with any pph prevention intervention is an effective way to reduce overall maternal mortality, which is the goal of every developing country, in accordance with the united nations millennium development goal (mdg) 5 . An economic assessment of the reduction of pph in developing countries estimated that the consistent use of a conventional uterotonic in every birth could avert 41 million cases of pph, resulting in an estimated 1.4 million lives saved.73 specifically with regard to misoprostol, an article utilizing modeling techniques determined that misoprostol use for pph prevention is a cost - effective intervention that could reduce maternal deaths by approximately 38%.74 one of the greatest opportunities in pph prevention is the fact that the currently available evidence is sufficient to establish policies and programs that will increase access to effective interventions in low - resource settings . In order to leverage this opportunity, countries must establish a supportive national policy, which starts with the adoption of national guidelines for pph prevention that reflect the latest research and the most recent who recommendations.3,20 early on, it is important to identify local champions who will engage policy makers and clinicians.75 with respect to misoprostol, countries must register it for pph prevention and ensure its inclusion on the national essential medicines list.20 the next step for any of the interventions is to secure adequate funding in the national budget to ensure the consistent availability of the drugs and the training of health care providers.20 with regards to misoprostol, correct use by community health workers and women could be increased if misoprostol is procured in indication - specific packaging, with 600 g packets for pph prevention,69 and if pph prevention information is included as part of information, education, and communication (iec) mass media campaigns . Another crucial step is the building of community awareness and demand for services and drugs, which will help to ensure the success of any of the pph prevention interventions, home- or facility - based . Specifically related to the community - based distribution of misoprostol, several countries have conducted successful pilot projects, and some, including ghana, nepal, niger, and bangladesh, are currently working to scale - up misoprostol access.20,28,76,77 country - to - county regional exchanges involving countries that have already begun to change and implement national pph prevention policies would provide a great opportunity to share experiences and best practices.75 moving from evidence to action, particularly with regard to misoprostol, is an important next step in improving pph prevention that needs to be prioritized in low - resource countries.75 involving community - based and lay providers, to the extent the evidence allows, should be an integral part of this step.28 community health workers are being engaged more regularly in task - shifting strategies to provide basic health services, including the prevention of pph.63,78,79 in a recent integrative review of 18 programs using lay health care workers to provide misoprostol via advanced distribution or at - delivery distribution, smith et al found that high coverage and use of misoprostol can be achieved via multiple routes of distribution.63 in addition, very low rates of incorrect use were found.63 other opportunities are on the horizon with regard to new drug formulations / drugs and delivery mechanisms . Oxytocin is the preferred drug for preventing and managing pph.3 however, in its current formulation, oxytocin is not heat stable and therefore an impractical intervention in many low - resource settings where extreme heat is coupled with limited access to refrigeration.51 yet it is in these low - resource settings that we see the majority of maternal deaths; therefore, research on oxytocin formulations that are heat stable is paramount . Two research teams are currently leading efforts in this field.80 a team at monash university, australia is working to develop oxytocin for aerosol delivery,81 and this formulation would allow women to inhale oxytocin immediately after childbirth, with no refrigeration of the product required . A nonprofit pharmaceutical development group in the netherlands is attempting to stabilize oxytocin under tropical conditions.82 another way to potentially increase access to oxytocin is to diversify its route of delivery . Oxytocin in the uniject auto - disable injection system (uniject; bd biosciences, franklin lakes, nj, usa) is comprised of a plastic, nonreusable, disposable syringe that is prefilled with a single dose of 10 international units (iu) of oxytocin in 1 ml . Given its simple design and safety features, uniject can be used by lay health workers, which is the reason oxytocin in uniject is an important innovation for resource - poor settings.83 oxytocin in uniject is produced by an argentine pharmaceutical distributor that has regulatory approval in eight countries in latin america . Though it is not yet broadly available, access to oxytocin in uniject would be particularly important in countries where human resources are limited and where task - shifting to lower cadres of health professionals is necessary.84 field studies in several countries have demonstrated the acceptability of oxytocin in uniject among health workers with less training, due to its ease of use.8587 the oxytocin in uniject is not heat stable, but the product packaging provides a straightforward time - temperature indicator to allow health workers to monitor heat exposure.84 given the early success of oxytocin in uniject, the who has amended its model list of essential medicines to include oxytocin in uniject . However, the path to the expanded availability of oxytocin in uniject will require a concurrent increase in demand and supply to counter the challenges of low - volume / high - price production.84 another recent innovation in the prevention of pph is carbetocin, a long - acting oxytocin agonist, which mimics the action of oxytocin and helps to reduce blood loss.88 carbetocin is currently indicated for the prevention of uterine atony after delivery by cesarean section in spinal or epidural anesthesia in 23 countries, but it is not approved for vaginal births . However, it has had proven success in the prevention of pph, due to its longer duration of action and demonstrated fewer side effects in several studies.88 further research is necessary to determine the cost effectiveness of carbetocin as a uterotonic agent.88 it is also important to assess the feasibility and acceptability of carbetocin in the prevention of pph in vaginal deliveries in low - resource settings . Fortunately, in the 2012 annual technical report of the who special program of research, development and research training in human reproduction, plans were announced for a multicenter, controlled trial in 2014 that will compare a new heat - stable formulation of carbetocin with oxytocin, for use in low- and middle - income countries.89 another intervention that presents an opportunity for preventing pph in home births but that has limited evidence of effectiveness is the home - based life - saving skills (hblss) package . A community- and competency - based program that aims to reduce maternal and neonatal mortality by increasing access to basic life - saving measures within the home and community and by decreasing delays in reaching referral facilities where life threatening problems can be managed.90 hblss is implemented by hblss guides who are selected by the community and who are then trained using a modular design that focuses on the prevention, recognition, and initial home management of life - threatening maternal and newborn problems and referral to a facility, where possible.91 these guides then share their hblss knowledge and skills with women, family caregivers, and homebirth attendants (ie, people involved in delivery care and decision making) by way of group discussions, demonstrations, and use of pictorial learning cards.90,91 when implemented within an existing health care infrastructure, the instruction of family and community members in techniques such as uterine fundal massage and emergency preparedness has the potential to reduce maternal morbidity and mortality due to pph.23 the potential effectiveness of this approach in relation to pph relies on early identification of hemorrhage and quick initiation of treatment . The findings from an evaluation of a field test of hblss in ethiopia were promising.91 pre- and posttraining tests of hblss guides pph knowledge demonstrated a statistically significant increase, and although lower, the knowledge remained much higher than the pretraining levels at 1 year posttraining.91 in addition, the management of pph (according to postpartum interviews) was significantly better among women who delivered with an hblss guide compared with another unskilled attendant.91 other similar programs implemented in low - resource settings have shown success in increasing the coverage of uterotonics and/or reducing pph by actively engaging women, the community, and traditional birth attendants in community - based interventions and using locally produced materials to gauge blood loss.9294 public private partnerships need to be further developed to ensure better collaboration in the procurement, distribution, and marketing of uterotonics . For example, local pharmaceutical manufacturers, distributors, or mobile network providers can be partners in creating demand through their extensive network of product retailers, that could support the dissemination of information about misoprostol.95 in addition, new mobile technologies are being tested to support supply chain management, provide training and diagnostic assistance for health workers, and disseminate information in hard - to - reach communities . Text messaging is being used to collect and transmit a wide range of information, from the documentation of stock levels of commodities to the circulation of information to women about where and how to access maternal health care.96 one of the greatest opportunities in pph prevention is the fact that the currently available evidence is sufficient to establish policies and programs that will increase access to effective interventions in low - resource settings . In order to leverage this opportunity, countries must establish a supportive national policy, which starts with the adoption of national guidelines for pph prevention that reflect the latest research and the most recent who recommendations.3,20 early on, it is important to identify local champions who will engage policy makers and clinicians.75 with respect to misoprostol, countries must register it for pph prevention and ensure its inclusion on the national essential medicines list.20 the next step for any of the interventions is to secure adequate funding in the national budget to ensure the consistent availability of the drugs and the training of health care providers.20 with regards to misoprostol, correct use by community health workers and women could be increased if misoprostol is procured in indication - specific packaging, with 600 g packets for pph prevention,69 and if pph prevention information is included as part of information, education, and communication (iec) mass media campaigns . Another crucial step is the building of community awareness and demand for services and drugs, which will help to ensure the success of any of the pph prevention interventions, home- or facility - based . Specifically related to the community - based distribution of misoprostol, several countries have conducted successful pilot projects, and some, including ghana, nepal, niger, and bangladesh, are currently working to scale - up misoprostol access.20,28,76,77 country - to - county regional exchanges involving countries that have already begun to change and implement national pph prevention policies would provide a great opportunity to share experiences and best practices.75 moving from evidence to action, particularly with regard to misoprostol, is an important next step in improving pph prevention that needs to be prioritized in low - resource countries.75 involving community - based and lay providers, to the extent the evidence allows, should be an integral part of this step.28 community health workers are being engaged more regularly in task - shifting strategies to provide basic health services, including the prevention of pph.63,78,79 in a recent integrative review of 18 programs using lay health care workers to provide misoprostol via advanced distribution or at - delivery distribution, smith et al found that high coverage and use of misoprostol can be achieved via multiple routes of distribution.63 in addition, very low rates of incorrect use were found.63 other opportunities are on the horizon with regard to new drug formulations / drugs and delivery mechanisms . Oxytocin is the preferred drug for preventing and managing pph.3 however, in its current formulation, oxytocin is not heat stable and therefore an impractical intervention in many low - resource settings where extreme heat is coupled with limited access to refrigeration.51 yet it is in these low - resource settings that we see the majority of maternal deaths; therefore, research on oxytocin formulations that are heat stable is paramount . Two research teams are currently leading efforts in this field.80 a team at monash university, australia is working to develop oxytocin for aerosol delivery,81 and this formulation would allow women to inhale oxytocin immediately after childbirth, with no refrigeration of the product required . A nonprofit pharmaceutical development group in the netherlands is attempting to stabilize oxytocin under tropical conditions.82 another way to potentially increase access to oxytocin is to diversify its route of delivery . Oxytocin in the uniject auto - disable injection system (uniject; bd biosciences, franklin lakes, nj, usa) is comprised of a plastic, nonreusable, disposable syringe that is prefilled with a single dose of 10 international units (iu) of oxytocin in 1 ml . Given its simple design and safety features, uniject can be used by lay health workers, which is the reason oxytocin in uniject is an important innovation for resource - poor settings.83 oxytocin in uniject is produced by an argentine pharmaceutical distributor that has regulatory approval in eight countries in latin america . Though it is not yet broadly available, access to oxytocin in uniject would be particularly important in countries where human resources are limited and where task - shifting to lower cadres of health professionals is necessary.84 field studies in several countries have demonstrated the acceptability of oxytocin in uniject among health workers with less training, due to its ease of use.8587 the oxytocin in uniject is not heat stable, but the product packaging provides a straightforward time - temperature indicator to allow health workers to monitor heat exposure.84 given the early success of oxytocin in uniject, the who has amended its model list of essential medicines to include oxytocin in uniject . However, the path to the expanded availability of oxytocin in uniject will require a concurrent increase in demand and supply to counter the challenges of low - volume / high - price production.84 another recent innovation in the prevention of pph is carbetocin, a long - acting oxytocin agonist, which mimics the action of oxytocin and helps to reduce blood loss.88 carbetocin is currently indicated for the prevention of uterine atony after delivery by cesarean section in spinal or epidural anesthesia in 23 countries, but it is not approved for vaginal births . However, it has had proven success in the prevention of pph, due to its longer duration of action and demonstrated fewer side effects in several studies.88 further research is necessary to determine the cost effectiveness of carbetocin as a uterotonic agent.88 it is also important to assess the feasibility and acceptability of carbetocin in the prevention of pph in vaginal deliveries in low - resource settings . Fortunately, in the 2012 annual technical report of the who special program of research, development and research training in human reproduction, plans were announced for a multicenter, controlled trial in 2014 that will compare a new heat - stable formulation of carbetocin with oxytocin, for use in low- and middle - income countries.89 another intervention that presents an opportunity for preventing pph in home births but that has limited evidence of effectiveness is the home - based life - saving skills (hblss) package . A community- and competency - based program that aims to reduce maternal and neonatal mortality by increasing access to basic life - saving measures within the home and community and by decreasing delays in reaching referral facilities where life threatening problems can be managed.90 hblss is implemented by hblss guides who are selected by the community and who are then trained using a modular design that focuses on the prevention, recognition, and initial home management of life - threatening maternal and newborn problems and referral to a facility, where possible.91 these guides then share their hblss knowledge and skills with women, family caregivers, and homebirth attendants (ie, people involved in delivery care and decision making) by way of group discussions, demonstrations, and use of pictorial learning cards.90,91 when implemented within an existing health care infrastructure, the instruction of family and community members in techniques such as uterine fundal massage and emergency preparedness has the potential to reduce maternal morbidity and mortality due to pph.23 the potential effectiveness of this approach in relation to pph relies on early identification of hemorrhage and quick initiation of treatment . The findings from an evaluation of a field test of hblss in ethiopia were promising.91 pre- and posttraining tests of hblss guides pph knowledge demonstrated a statistically significant increase, and although lower, the knowledge remained much higher than the pretraining levels at 1 year posttraining.91 in addition, the management of pph (according to postpartum interviews) was significantly better among women who delivered with an hblss guide compared with another unskilled attendant.91 other similar programs implemented in low - resource settings have shown success in increasing the coverage of uterotonics and/or reducing pph by actively engaging women, the community, and traditional birth attendants in community - based interventions and using locally produced materials to gauge blood loss.9294 public private partnerships need to be further developed to ensure better collaboration in the procurement, distribution, and marketing of uterotonics . For example, local pharmaceutical manufacturers, distributors, or mobile network providers can be partners in creating demand through their extensive network of product retailers, that could support the dissemination of information about misoprostol.95 in addition, new mobile technologies are being tested to support supply chain management, provide training and diagnostic assistance for health workers, and disseminate information in hard - to - reach communities . Text messaging is being used to collect and transmit a wide range of information, from the documentation of stock levels of commodities to the circulation of information to women about where and how to access maternal health care.96 as pph is the main cause of maternal mortality worldwide, pph prevention interventions need to be prioritized as an essential way to improve maternal health . Each country must develop its own context - dependent policies and programs, incorporating myriad approaches that combine the most recent recommendations and reflect the experiences of other countries . Though oxytocin is the recommended uterotonic, it is not readily available in settings with the highest risk for mortality and morbidity from pph, due to its sensitivity to heat and need for provision by a skilled provider . Yet increasing access to prophylactic uterotonics, regardless of where deliveries occur, should be the primary means of reducing the burden of this complication . There is still some debate as to whether misoprostol is effective in pph prevention,97 and some have called for additional high - quality studies that demonstrate significant reductions in pph.48 but at the present time, based on the evidence available, the best way to reduce pph deaths in low - resource settings where women continue to deliver without access to a skilled birth attendant is to make misoprostol widely available . Therefore, efforts need to be directed at increasing misoprostol supplies and supporting correct and consistent utilization by providers and by women themselves, in the case of home births.
Mast cells (mcs) belong to the innate - compartment of the immune system and are widely known for their role in allergic reactions via their binding to ige receptor (alvarez - errico et al ., 2009). Mcs are a common cellular component of both connective and mucosal tissues (kitamura and ito, 2005). Beside this, mcs contain a wide range of biologically active molecules, including biogenic amines, heparin or heparan sulfate proteoglycans, neutral proteases, and neuropeptides . In addition, upon stimulation, they also produce and eject a large number of factors (wilhelm et al ., 2000). Taking these characteristics together, it is clear that even a small number of such potent unicellular glands have a significant effect on different physiological processes . In addition to the very well known and described mechanism of mc activation and posterior degranulation throughout ige receptor, several other alternative but not redundant mechanisms of mc activation have been described (mousli et al ., 1994; bradding, 2005; kim et al .,, 2005; vasiadi et al ., 2006; narita et al ., 2007; zaitsu et al ., 2007; jensen et al ., 2010; jing et al ., 2011; walter et al ., we will discuss in this review the current bibliography evidences about the effect of female sex hormones on mc functionality . Female sex steroid hormones act primarily via their receptors: estrogen via estrogen receptor er or er, progesterone via progesterone receptor pr - a or pr - b (carey et al ., 2007). Steroid receptors are best described as nuclear receptors acting as transcription factors on gene expression . However, in the past decade abundant evidences accumulated showing addition binding sides localized at the plasma membrane (levin, 2011), whose activation is more often involved in the rapid effects of steroids occurring within seconds to minutes (watson et al ., 1999; watson and gametchu, 2003). In this regard, it has been shown that classical er at the membrane but not in the nucleus mediates 17-estradiol (e2)-induced rapid signaling to kinase activation (levin, 2011). Similarly, extra - nuclear pr induces activation of erk / mapk kinases, which lead to cell surviving as well as cells migration (levin, 2011). We and other authors have demonstrated the expression of, estradiol and progesterone receptors in human, mouse, and rat mcs (theoharides et al ., 1993; (2007) have shown mrna expression of er but not er in human and mouse mcs . Alongside the authors have also shown that e2 rapidly stimulated mc degranulation which could be blocked by tamoxifen, a tissue specific er antagonist, clearly indicating that estradiol - induced mc degranulation throughout one of its receptors . Bone marrow - derived mcs (bmmcs) isolated from er knockout animals did not degranulate in response to e2 treatment confirming that the e2 effect on mcs is more likely mediated by the er (zaitsu et al ., 2007). Due to the rapid onset of e2 effect on mc activation the authors concluded that e2 in this context does not function through the classical (genomic) mechanisms, which require enhanced mrna and protein synthesis over 2 h or longer period and proposed that the effect is mediated by a membrane - associated (non - genomic) form of er (zaitsu et al . We were additionally able to show that the human mast cell line (hmc-1) treated in vitro with physiological concentration of e2 and p4 significantly increased the synthesis of -tryptase, which is a serine proteinase abundantly produced by mcs, and is a marker of mc maturation . Beside, e2 and p4 treatment induced degranulation of hmc-1 in vitro (jensen et al ., 2010). Supporting the idea of female sex hormones having an effect on mc function, kirmaz et al . (2004) have demonstrated that allergen skin prick tests (spt), a very sensitive and specific tests to detect allergic sensitization in atopic patients, is altered in women upon hormonal changes during the menstrual cycle . In addition to female sex hormone receptor expression, mcs have been also shown to express androgen receptor (chen et al ., 2010). However, testosterone treatment had no effect on mc degranulation (chen et al ., 2010). The idea that female sex hormones, e2 and p4, may affect mc functionality and therefore have an influence on the symptoms of mc - associated disorders has long been suggested . Asthma and other allergic diseases of the airway are up to three times more common in women than in men during the early to middle adulthood and remains so through the reproductive years (de marco et al ., 2002; mannino et al ., 2002; schatz and camargo, 2003). A number of clinical and epidemiological studies suggested that female sex hormones are accountable for these differences . Beside this, postmenopausal women taken hormone replacement therapy had higher risk of new onset of asthma (barr et al ., 2004). Furthermore, 3040% of women who had asthma, experience a worsening of their symptoms during the perimenstrual phase of the menstrual cycle (perimenstrual asthma) being the time point when e2 and p4 concentrations are changing rapidly (vrieze et al ., 2003). In this context, it is of great importance to mention that the prevalence and morbidity of asthma and other allergic diseases have increased dramatically during the last 30 years, particularly in developing countries (burr et al ., 2006). (2007) have nicely demonstrated that this may be related to the increase of low concentrations of environmental like - estrogen compounds . These estrogen - like compounds, called xenoestrogens, are present in the environmental pollutants mainly in water and food . They are able not only to activate mcs but enhance mc degranulation upon allergen cross - linking of ige which may explain the above described increment of allergic diseases in the last years in developing countries (narita et al ., 2007). In an animal model of allergic disease, the role of female sex hormone was tested . Female mice have reportedly an increased susceptibility to allergic airway disease in compared with male mice (reviewed in carey et al ., 2007). Levels of ige are much higher in allergic female mice compared to their syngeneic male (corteling and trifilieff, 2004). Female rats that underwent ovariectomization developed less airway inflammation compared with sham controls animals (ligeiro de oliveira et al . However, estrogen replacement in the ovariectomized animals re - established airway inflammation levels of intact females (ligeiro de oliveira et al ., 2004). Treatment of intact female rats with the selective estrogen receptor antagonist tamoxifen also reduced the development of allergic airway disease (ligeiro de oliveira et al ., 2004). Thus, the direct effect of these hormones on disease development is hereby demonstrated . Beyond the well - documented effects of estradiol and progesterone on mc function in mc - associated diseases, for instance, estradiol was showed to be a potent inducer of ovarian mc degranulation, which seems to be a necessary factor during the process of oocyte ovulation (jaiswal and krishna, 1996; tamura and kogo, 1999). The presence of mcs in the uterus has been already described in many species including human (drudy et al ., 1991), mouse (padilla et al ., 1990), rat (aydin et al ., 1998), hamster (harvey, 1964) as well as goat (karaca et al ., 2008). Besides, the number of mcs in the uterus was shown to fluctuate during estrous cycle suggesting an influence of female sex hormones on mc recruitment to the uterus (aydin et al ., 1998). Ovariectomized mice, in which estradiol and progesterone are almost absent, have less number of uterine mcs compared to control, non - ovariectomized animals (jensen et al ., 2010). Hormonal replacement, estradiol alone or in combination with progesterone, restored the number of uterine mcs after ovariectomization, which was comparable to the levels observed in control mice (jensen et al ., 2010). Hormonal replacement additionally induced an augmentation in the levels of mc - related proteases expression in the uterus as well as boosted mc degranulation (jensen et al ., 2010). This is of particular importance because upon degranulation, mcs release several molecules (histamine, proteases, metalloproteinases, pro - angiogenic factors), all very well known to account for the process of embryo implantation . Mast cells, the so - called unicellular glands, once solely known as effectors cells of the innate immune system only activated by ige cross - linking to the ige receptor upon allergen stimulation are now known to be much more plastic and susceptible to be activated by several factors including female sex hormones, estradiol and progesterone . Strong data in the last years reinforced the idea that these hormones are crucial component of mc behavior not only in physiological conditions but also in several mc pathological situations . Deciphering the mechanisms by which female sex hormones activate mcs and under which conditions these happens, alongside with explanation why female sex hormones have these effects is of crucial interest for a better understanding of the physiology of these cells . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The structures of acl and mcp in the gas phase were optimized at the b3lyp/6 - 31 g * level . The structures of the water clusters were determined with classical molecular dynamics trajectories (see details in the si). The cluster models include solvents that have at least one of the atoms within 3 and 4 from acl and mcp, respectively . The geometry of the acl and mcp in the water clusters was further optimized at the b3lyp / cc - pvdz and cam - b3lyp / cc - pvdz levels, respectively . In the optimization, the water molecules were fixed at the md structure and replaced by point - charges (tip3p charges). We also fixed the c atom next to the o atom of acl . In mcp, the atomic coordinates of the central c atom were fixed . The hf orbitals were transformed into mos localized within each fragment (solute and solvent). Our transformation uses reference orbitals (rmos) obtained with external calculations for isolated molecules ., we show the populations at the fragments . In the perturbation - selection step of the sac - ci calculations, a set of threshold, levelfour, as a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors
A 19-year - old male presented to the casualty with a history of injury to his left eye by a palm leaf, which fell from a height of about 10 meters . A piece of the stalk had been removed from the wound at a local hospital earlier in the day . On examination, his vision was 20/20, the globe was intact and ocular movements were full . The wound was dressed and the patient was asked to review in the outpatient department . The patient returned 18 months later with a swelling at the same site . On examination, there was a non - axial proptosis of the left eye with limitation of elevation . The globe was pushed 3 mm forwards and 2 mm upwards . A 2 1 cm firm, non - tender mass was present at the infraorbital margin computed tomography (ct) scan of the orbit obtained in axial and coronal planes depicted a medium - sized soft tissue density mass in the inferomedial aspect of the left orbit . The mass involved the retrobulbar, intra and extraconal spaces, abutting and slightly displacing the optic nerve superiorly [fig . 2a, b]. As the results of the investigations did not suggest the presence of a retained intraorbital foreign body, we considered other possible diagnoses like tuberculosis, sarcoidosis and idiopathic orbital inflammation . However, systemic examination did not reveal any clinical evidence of tuberculosis or sarcoidosis . Complete blood count, esr, mantoux test and chest x - ray were within normal limits . As the patient was not very symptomatic at the time, we decided to keep him under observation for some time . He presented two years later with a discharging sinus at the site . At this point, we strongly suspected the presence of a residual foreign body and advised a magnetic resonance imaging (mri) scan of the orbit . It grew enterobacter species, sensitive to ciprofloxacin, norfloxacin, lomefloxacin and gentamicin . Ciprofloxacin 500 mg twice daily) and daily dressing of the sinus was continued . One week later, a foreign body, measuring 1.2 cm, was seen in the gauze when the dressing was removed . On follow - up so further active intervention was deferred at that point and the patient was advised regular follow - up . He said that he had been having recurrent discharge from the sinus, which was being dressed at a local hospital . Another piece of the wooden foreign body was seen on the gauze when the dressing was removed that day . 3]. On follow - up, the swelling resolved and the sinus healed completely . We repeated a ct scan of the orbit a year later, which showed complete resolution of the mass [fig . Most metallic foreign bodies remain quiescent for a long period of time without causing any problems . So the general recommendation is to leave them alone in the absence of specific indications for removal.4 however, organic foreign bodies like wood have a much higher incidence of potentially sight - threatening and life- threatening complications.5 they may remain dormant for a variable period of time and manifest with delayed - onset orbital granuloma, cellulitis, abscess or chronic draining sinus.1 hence, surgical removal of organic intraorbital foreign bodies is recommended.5 in our case, a piece of the foreign body had been removed after the initial trauma . When seen in our casualty on the evening of the same day, the wound looked superficial and there was no clinical evidence of a residual foreign body . It is important to remember that wooden foreign bodies often break during attempted removal.1 the associated wound may be small and self - sealing.6 therefore, if there is recurrence of clinical symptoms, especially, if there is a discharging sinus, the possibility of a retained foreign body should be considered . Imaging and prompt exploration of the sinus may help in localizing and removing the foreign body . A ct scan is the standard diagnostic test, because it demonstrates most intraorbital foreign bodies and is safe in the presence of metallic foreign bodies.5 in our case, however, the ct scan did not show any evidence of the foreign body . Review of previous reports suggests that wood is often not detected on ct scan.7 computed tomographic imaging relies on the differing radiodensities of tissues for their differentiation . The radiodensity of wood is variable and may be similar to that of the orbital tissues, which may account for the potential difficulty of recognition . The ct appearances seem related to the interval between injury and examination.8 in the acute stage, the very low density of wood can be confusing with low window settings, mimicking air bubbles . In the subacute stage, wood assumes a moderate density and may be difficult to distinguish from surrounding orbital fat . In the chronic stage, the density of wood can become higher than that of orbital muscle . It may be associated with a foreign - body reaction, which appears as a homogenous mass surrounding the dense wooden foreign body, with a density similar to the adjacent extraocular muscles . Spiral ct scanners have improved resolution and faster speed of acquisition of images.9 the fast image capture allows the scanning to occur with minimal motion artifact . In spite of these advantages, wooden intraocular foreign bodies could only be described as probable intraocular foreign bodies by neuroradiologists in a pilot study using a spiral ct scanner.9 the mri scans are better at demonstrating wooden foreign bodies . Magnetic resonance imaging depends on the density of protons in the tissue and their different relaxation times . These properties of wood are dissimilar enough from those of the soft tissue to allow differentiation.7 therefore, it is recommended that mri scan should be performed after a negative ct scan if there is a possibility of a wooden intraorbital foreign body . An mri scan may be performed as the primary imaging modality if there is a definite history of a wooden intraorbital foreign body.5 several aspects of this case are interesting . The long latent period between injury and development of the orbital mass and sinus, the apparent removal of the foreign body from the wound after the initial trauma, the spontaneous extrusion of pieces of the foreign body twice with an interval of 18 months and complete resolution of the orbital mass on ct scan are distinctive features of this case . The entire sequence of events spanned over five years . This was partly due to the frequent defaulting on the part of the patient . In conclusion, we would like to emphasize that intraorbital foreign bodies may often present a confusing clinical picture . It is important for the ophthalmologist, radiologist and pathologist to include foreign body granuloma among the differentials of an intraorbital mass . It is imperative to seek past and recent history of trauma and maintain a high index of suspicion in all such cases, regardless of the interval between the trauma and current presentation.
Secondary sjgren's syndrome (sss) is classified as a connective tissue disease characterized mainly by xerophthalmia and xerostomia, and accompanied by various autoimmune diseases such as rheumatoid arthritis (ra), systemic lupus erythematosus, scleroderma, vasculitis, mixed connective tissue disease, primary biliary cirrhosis or autoimmune thyroiditis [1, 2]. To recognize the sss presence, one subjective symptom of sickness and positive results of two objective tests for dryness of eyes and mouth are required . The sss can also be diagnosed without any sicca symptoms, but then the following three criteria should be met: positive results of both objective tests, the presence of antibodies (anti - ss - a / ro and anti - ss - b / la), and inflammatory infiltration in biopsy of minor salivary glands . Prevalence of sss in ra varies considerably depending on the geographic region and duration of ra . For instance, data from norway show the lowest prevalence, i.e. 7%, whereas data from spain reveal a cumulative prevalence of 17% and 25% after 10 and 30 years of the disease, respectively . According to andonopoulos et al ., 5% of ra patients had clinical manifestations of sss and 20% demonstrated sub - clinical sss . Patients with secondary ss and ra require special attention regarding evaluation of specific systemic complications like inflammatory pulmonary disease or polyneuropathy . Das28 is an important outcome measure of ra activity . In a number of european countries, the aim of this study is to evaluate whether the difference in the values of the das28 calculated taking into account diverse parameters of inflammatory activity (esr or crp) is clinically relevant . In case the discrepancy is relevant, the study should indicate which parameter is more valuable for assessing activity of ra with sss . To capture data regarding the study we queried our medical records database of the connective tissue disease department in the institute of rheumatology in warsaw from 2009 through 2011 seeking to identify all patients with ra and sss . Initially, 1980 patients were identified with ra who had been diagnosed according to the american college of rheumatology criteria laid down in 1987 . We have separated 119 patients with ra . After a review of medical records, only patients with ra and sss who had been diagnosed based on the 2002 revised version of the american eventually, only 60 subjects registered with the icd-10 code for sss and ra (m35.0 and m05) were included in the research group . The data were extracted from medical records retrospectively based on their fulfilment of inclusion criteria for our study . The inclusion criteria have been specified as follows: radiological confirmation of ra (criteria for ra),presence of data on das28-crp and das28-esr, presence of at least one subjective symptom of dry eyes or dry mouth (criteria for ss),one objective symptom of dryness, presence of specific antibodies ss - a and ss - b, in biopsy at least focus score of one . Radiological confirmation of ra (criteria for ra), presence of data on das28-crp and das28-esr, presence of at least one subjective symptom of dry eyes or dry mouth (criteria for ss), one objective symptom of dryness, presence of specific antibodies ss - a and ss - b, in biopsy at least focus score of one . Subjective ocular symptoms were usually described in the medical records as present or absent, and included symptoms of dry eyes lasting over 3 months, foreign object sensation in the eyes, or subjective oral symptoms such as symptoms of dry mouth lasting over 3 months or swollen salivary glands . Schirmer's test without anaesthesia consists in corneal staining assessment during over 5 minutes and is performed with eyes closed by inserting a schirmer filter paper strip into the eye and reading the result after 5 minutes . The schirmer test result is abnormal when during 5 minutes the tears flow is less than 5 mm . Histopathological examination of lymphocytic infiltration at salivary glands with at least focus score of one was performed . The evaluated medical records contained basic clinical laboratory tests and included rheumatoid factor (rf), anti - cyclic citrullinated peptide (ccp) antibodies, as well as morphology, inflammatory markers (esr and crp), and proteinogram . Das 28 embraces swollen joint counts (sjcs) and tender joint counts (tjcs), a self - determined assessment of patient's general health on visual analogue scale (vas) from 0 to 10, and acute - phase response factors esr or crp . According to eular recommendations, das28 scores represent the following disease activities: remission (<2.6), mild (2.6, <3.2), moderate (3.2, <5.1) or severe (5.1). Das28 values were calculated using the following equations: das28-crp = 0.56_ (tjc-28) + 0.28_ (sjc-28) + 0.014_gh+ 0.36_ln (crp + 1) + 0.96, das28-esr = 0.56_ (tjc-28) + 0.28_ (sjc-28) + 0.014_gh+ 0.70_ln (esr). The following methods were used for statistical analysis: statistica release 10 from stat soft poland, sas system 10.0 sas institute inc . Normal distribution data have been presented as mean and standard deviations, whereas abnormal distribution data as median and interquartile range . Depending on their distribution, data were analyzed using student t - test or mann - whitney u test . Cohen's coefficient with linear weighting was used to measure agreement between das28-esr and das28-crp values . In order to assess the variation between das28-esr and das28-crp, the bland - altman plot was used . To capture data regarding the study we queried our medical records database of the connective tissue disease department in the institute of rheumatology in warsaw from 2009 through 2011 seeking to identify all patients with ra and sss . Initially, 1980 patients were identified with ra who had been diagnosed according to the american college of rheumatology criteria laid down in 1987 . We have separated 119 patients with ra . After a review of medical records, only patients with ra and sss who had been diagnosed based on the 2002 revised version of the american eventually, only 60 subjects registered with the icd-10 code for sss and ra (m35.0 and m05) were included in the research group . The data were extracted from medical records retrospectively based on their fulfilment of inclusion criteria for our study . The inclusion criteria have been specified as follows: radiological confirmation of ra (criteria for ra),presence of data on das28-crp and das28-esr, presence of at least one subjective symptom of dry eyes or dry mouth (criteria for ss),one objective symptom of dryness, presence of specific antibodies ss - a and ss - b, in biopsy at least focus score of one . Radiological confirmation of ra (criteria for ra), presence of data on das28-crp and das28-esr, presence of at least one subjective symptom of dry eyes or dry mouth (criteria for ss), one objective symptom of dryness, presence of specific antibodies ss - a and ss - b, in biopsy at least focus score of one . Subjective ocular symptoms were usually described in the medical records as present or absent, and included symptoms of dry eyes lasting over 3 months, foreign object sensation in the eyes, or subjective oral symptoms such as symptoms of dry mouth lasting over 3 months or swollen salivary glands . Schirmer's test without anaesthesia consists in corneal staining assessment during over 5 minutes and is performed with eyes closed by inserting a schirmer filter paper strip into the eye and reading the result after 5 minutes . The schirmer test result is abnormal when during 5 minutes the tears flow is less than 5 mm . In addition, histopathological examination of lymphocytic infiltration at salivary glands with at least focus score of one was performed . The evaluated medical records contained basic clinical laboratory tests and included rheumatoid factor (rf), anti - cyclic citrullinated peptide (ccp) antibodies, as well as morphology, inflammatory markers (esr and crp), and proteinogram . Das 28 embraces swollen joint counts (sjcs) and tender joint counts (tjcs), a self - determined assessment of patient's general health on visual analogue scale (vas) from 0 to 10, and acute - phase response factors esr or crp . According to eular recommendations, das28 scores represent the following disease activities: remission (<2.6), mild (2.6, <3.2), moderate (3.2, <5.1) or severe (5.1). Das28 values were calculated using the following equations: das28-crp = 0.56_ (tjc-28) + 0.28_ (sjc-28) + 0.014_gh+ 0.36_ln (crp + 1) + 0.96, das28-esr = 0.56_ (tjc-28) + 0.28_ (sjc-28) + 0.014_gh+ 0.70_ln (esr). The following methods were used for statistical analysis: statistica release 10 from stat soft poland, sas system 10.0 sas institute inc . Normal distribution data have been presented as mean and standard deviations, whereas abnormal distribution data as median and interquartile range . Depending on their distribution, data were analyzed using student t - test or mann - whitney u test . Cohen's coefficient with linear weighting was used to measure agreement between das28-esr and das28-crp values . In order to assess the variation between das28-esr and das28-crp, the bland - altman plot was used . 119 out of 1980 patients registered in the institute of rheumatology database who suffer from ra have fulfilled inclusion criteria for the research study . One group comprised 60 patients with ra and sss, second the control group patients with ra alone . In the patients with ra and sss group, there were 56 female participants (93%) and only 4 male (7%). Mean age of the control group patients was 51.6 (range: 23 - 77) years . Standard deviation in the group of patients with ra and sss, 50 out of 60 patients (83%) have complained of dry eyes, of whom 15 (25%) have complained of dry mouth also . Moreover, 30 out of 52 patients (87%) have obtained a positive result of schirmer test, i.e. Less than <5 mm, 17 (33%) demonstrated border tears flow, while only 9% normal tears flow . With regard to 8 patients, schirmer's test data were unavailable . With reference to 52 out of 60 patients (87%), data of collected ana antibodies indicated that 28 of them (54%) show positive ana antibodies, 13 (26%) ssa / ro antibodies, and 6 (12%) ssb / la antibodies . The salivary gland biopsy has been performed in 50 of our patients (83%), whereas 47 out of 50 (94%) obtained focus score of one and more than one . A group of 46 patients with ra and sss (82%) demonstrated a positive rf, however, for the remaining 4 patients there were no data . Out of 31 patients (52%) with available data of anti - ccp antibodies, 28 (90%) presented positive anti - ccp antibodies . Divergently a group of ra patients from the control group reported a positive rf in 51% and positive anticcp antibodies in 78% . It should be noted that in patients with ra and sss, the mean value of esr was 39 mm / hr, whereas of crp, 25 mg / l . On the contrary in ra patients the mean value of esr was 15 mm / hr, and almost similar crp 16 mg / l . Linear regression analysis revealed a significant correlation between das28-esr and das28-crp pearson correlation coefficients r = 0.9, p <0.0001 (fig . 1) in the first group of patients with ra and sss . In the control group, there was the same correlation between das28-esr and das28-crp pearson correlation coefficients r = 0.91, p <0.02 (fig . Moreover, agreement between das28-esr and das28-crp was good; cohen's kappa coefficient with linear weighting = 0.60; 95% ci: 0.45 - 0.75 in the ra and sss group, and bland - altman analysis (fig ., we found that in patients with ra and sss, das28 calculated from esr was significantly higher than calculated from crp . A relevant difference was identified with p <0.0001 . On the other hand, in the control group (patients with ra), mean das28-esr= 4.7 while mean das28-crp = 4.6; no significant difference was identified with p> 0.05 (p = 0.91). Scatter plot of das28-crp (y axis) vs. das28- esr (x axis) values with a regression line in the group patients with ra and sss . Each point corresponds to a single patient's data scatter plot of das28-crp (y axis) vs. das28- esr (x axis) values with a reggression line in the group patients with ra . The ordinate scale represents the difference between das28-crp and das28-esr (das28-crp minus das28-esr). The abscissa scale represents the average das28- esr and das28-crp (das28-esr plus das28-crp divided by 2). The middle dashed line indicates the bias (mean difference), and the upper and lower dotted lines represent the limits of agreement (mean 2 sd). The solid red line represents the regression line of the average difference the patients were divided into 4 groups: with remission, low, medium and high disease activity score (see table 2). In our study, in ra patients with sss, das28-esr 5.1 has been reported in 33 patients (55%), while das28-crp 5.1 in 23 patients (38%). Full agreement has been found for das28 values calculated from crp; for every das28-crp 5.1, the das28-esr is also 5.1 . On the other hand, 10 patients for whom das28-crp ranged from 3.2 to 5.1 had the das28-esr value over 5.1 . As a result, the biggest discrepancies were found for high das28 values, especially in the range from 3.2 to 5.1, and over 5.1 . Compared to the control group, there are no large differences in the value of das28-crp and das28-esr between 2.6 and 5.1 . Das28-esr 5.1 was measured in 25 patients (42.7%), while das28-crp 5.1 in 29 patients (49%). Concordance between das28 values using crp and esr in ra patients with ss and the control group the objective of the study was to find a relationship between higher esr and hypergammaglobulinemia in patients with ra and sss . No statistically significant results were obtained in the case of higher esr and hypergammaglobulinemia, however, connections were found between higher esr and dysproteinaemia . Patients with ra and sss who presented dysproteinaemia obtained median esr over 42 mm / h, whereas participants without dysproteinaemia had lower median esr, only 22 mm / h (p <0.011) (table 3). Differences in esr and crp values in groups of patients with ra and sss, dysproteinaemia and hypergammaglobulinemia; all values are median and iqr interquartile range this study evaluating 60 patients with ra with sss revealed that values of das28-crp are significantly lower than those of das28-esr . Discrepancies were found in patients with moderate disease activity (das28 from 3.2 to 5.1) (das28-crp 30 patients, das28-esr 23 patients) and a high disease activity score (das28 above 5.1) (das28-crp 23 patients, das28-esr 33 patients). These observations have implications for clinical evaluations since a high disease activity score qualifies patients for biological treatment . The afore - mentioned findings change the therapeutic approach, hence, this study is aimed to prove that patients with ra and sss have different outcomes in assessment from das28-esr than from das28-crp and, therefore, evaluations from das28-crp are recommended . In several previous studies [69], reported that not only the das28-crp 4.38 [sd 1.47] and das 28 esr 4.41 [sd 1.27] are similar, but also that crp could be replaced by esr . According to wells et al ., most patients were classified as having the same eular state regardless of which das28 definition has been employed . Moreover, inoue et al . Have shown that das28-crp can be used as an alternative to the das28-esr given the strong linear relationship between das28-esr and das28-crp values (correlation coefficient 0.946). By contrast, in our research group, the said correlation coefficient is equal to only 0.73 . In comparison to his predecessors, matsui et al . Demonstrated that das28-crp significantly underestimated disease activity and overestimated the improvement in disease activity compared with das28-esr . Our study has revealed that the mean value of das28-crp (4.71, sd 1.2) was significantly smaller than the mean value of das28-esr (5.27, sd 1.32) (p <0.0001) in a group with ra and sss (table 1). There were no significant statistical correlations in the control group with ra . Nevertheless, the changes of das28 values in das28-esr and das28-crp in both groups were very similar and closely correlated . Although the values of das28-esr and those of das28-crp were comparable in the reports of wells et al . And fransen et al . In contrast with the previous research, this study covers a different group of patients than the statistical rheumatoid patients . In our research, that syndrome is responsible for changes in immunoglobulin, especially activation of polyclonal b - cell leading to chronic hypergammaglobulinemia and increased levels of 2-microglobulinemia which affect the esr and cause higher esr . Apart from the aforementioned, esr can also be affected by such factors as sex, age, immunoglobulin levels, and abnormal size or shape of red blood cells . In chronic diseases, the esr - elevation is considered to be caused by fibrinogen, mono- or polyclonal increase in igg, iga, and igm, alone or in combinations . Normal values of esr for females are higher than for males and increase with age . Due to the fact that the study group was dominated by women (93%), that factor could possibly have influenced the results . In an inflammatory process, the high proportion of fibrinogen in the blood causes red blood cells to stick to each other . Fibrinogen is considered to be responsible for the rouleaux phenomenon, however, it has a co - influence on immunoglobulin to induce the rouleaux type of aggregates and high esr . High esr is a result of either rouleaux type aggregates where fibrinogen is dominant, or immunologic type aggregates where igg, iga or igm are the dominant proteins [12, 13]. On the other hand, total leukocyte and absolute neutrophil counts are not correlated with either esr or haematocrit standardised esr . A strong correlation (r = 0.75) this correlation has not been measured . However, in our study polyclonal gammopathy and dysproteinaemia were reported . Serum protein electrophoresis is a laboratory examination that is commonly used to identify two major types of proteins: albumin and globulins . The five components (globulins) are labeled 1, 2, 1, 2, and . Dysproteinaemia in our study means an abnormality of the protein content of the blood, involving the immunoglobulins (, and globulins). Polyclonal hypergammaglobulinemia and elevated 2-globulins are often seen . In our study 46% of patients demonstrated hypergammaglobulinemia and 69% dysproteinaemia, hence, higher esr can be determined by this pathology . A retrospective cohort study of 148 patients from the mayo clinic showed that in patients with ra, globulin levels are elevated in 37% of patients, and are reported to correlate with disease activity . Our study also revealed that patients with dysproteinaemia have higher esr, what proved to be a significant correlated factor . Furthermore, esr tends to show the disease activity over the course of a few weeks, whereas crp can develop more short - term changes in the disease activity . Moreover, crp is less dependable on sex, age, and fibrinogen and immunoglobulin levels . However, our study provides evidence that in patients with ra and sss, das28 should be evaluated based on crp . Although our results are based on small numbers, they confirm that in patients with ra and sss, das28 should be evaluated from crp.
Metastatic squamous cell carcinoma (scc) of the colon is rare, but colonic metastases from primary tumors of the lung, esophagus and uterine cervix have been reported [14]. Carcinoma of unknown primary site is a well - recognized clinical disorder, accounting for 35% of all malignant epithelial tumors . Scc is the most common histologic type of carcinoma of unknown primary site found in cervical lymph nodes [1,2,]. However, it is extremely rare for carcinoma of unknown primary site to metastasize to the gastrointestinal tract . We describe a case of synchronous metastatic scc in the colon and cervical lymph nodes from a carcinoma of unknown primary site . A 75-year - old woman with a mass in the left submandibular region consulted the department of otorhinolaryngology . She required assistance in everyday life because of chronic rheumatoid arthritis and a previous cerebral hemorrhage . Fine - needle aspiration showed class v cytology, but the exact histologic type was unclear . Left submandibular gland resection with cervical lymph node dissection was performed . Histological examination showed poorly differentiated scc in the lymph nodes located at level ii (fig . The patient was considered to have a carcinoma of unknown primary site because no primary lesions were found on detailed examinations of the esophagus, lung, uterine cervix and skin . A preoperative computed tomographic (ct) scan showed a small lesion with contrast effect in the transverse colon, which was difficult to distinguish from inflammation . Three months after operation, it was noted that the colonic lesion had grown considerably (fig . Fluorodeoxyglucose positron emission tomography showed abnormal uptake in the transverse colon, but there was no accumulation at other sites (fig . The specimen shows poorly differentiated scc similar to that seen in the cervical lymph nodes (h&e stain, 40). Figure 2:a preoperative ct scan showed a small lesion with contrast effect in the transverse colon, which was difficult to distinguish from inflammation (a). Three months after operation, it was noted that the colonic lesion had grown considerably (b). Figure 3:fluorine-18 fluorodeoxyglucose positron emission tomographic scan shows positive findings in the transverse colon, with no other positive lesions . The specimen shows poorly differentiated scc similar to that seen in the cervical lymph nodes (h&e stain, 40). A preoperative ct scan showed a small lesion with contrast effect in the transverse colon, which was difficult to distinguish from inflammation (a). Three months after operation, it was noted that the colonic lesion had grown considerably (b). Fluorine-18 fluorodeoxyglucose positron emission tomographic scan shows positive findings in the transverse colon, with no other positive lesions . Colonoscopy revealed a 40-mm submucosal tumor with an inflamed surface in the transverse colon . The appearance of this lesion suggested a metastatic lesion from another site, and biopsy was performed, but did not provide a definitive diagnosis (fig . Histological examination of the resected specimen showed features of poorly differentiated scc, similar to that previously seen in the cervical lymph nodes (fig . Figure 4:colonoscopy reveals a 40-mm submucosal tumor with an inflamed surface in the transverse colon . Chromoendoscopy shows a tumor covered with normal mucosa . This lesion was a solid mass with a negative cushion sign (pressure applied to the tumor with a closed pair of biopsy forceps). Colonoscopy reveals a 40-mm submucosal tumor with an inflamed surface in the transverse colon . Chromoendoscopy shows a tumor covered with normal mucosa . This lesion was a solid mass with a negative cushion sign (pressure applied to the tumor with a closed pair of biopsy forceps). The lesion seen in the transverse colon on preoperative ct scans was in fact a small metastatic scc . This report documents the clinical course of a patient with synchronous metastatic scc to the colon and cervical lymph nodes from a carcinoma of unknown primary site . This patient demonstrates two important clinical issues: (i) carcinoma of unknown primary site can present as colonic metastasis and (ii) colonoscopy is useful for establishing the diagnosis . First, carcinoma of unknown primary site can present with synchronous metastatic scc to the colon and cervical lymph nodes . Scc from an unknown primary lesion with cervical lymph node involvement is found in ~5% of all head and neck cancers . Treatment of patients who have metastatic scc with cervical lymph node involvement from a carcinoma of unknown primary site should be similar to that given to patients with locally advanced carcinoma . Visceral metastases from a carcinoma of unknown primary site are rare and generally have poor prognoses . The most commonly involved metastatic site is the liver, followed by lymph nodes, lungs, bones and brain . Squamous cell histology is a very rare subset of visceral metastases from carcinoma of unknown primary site . Recently, patients with carcinoma of unknown primary site have been divided into subsets with favorable (20%) and unfavorable (80%) prognoses . Scc involving the cervical lymph nodes is classified as belonging to the favorable subset, whereas colonic metastases belong to the unfavorable subset . In our patient, synchronous metastatic scc to the colon and cervical lymph nodes was completely resected . Postoperative adjuvant chemoradiotherapy was not given because the patient s general condition was poor . As of 1.5 years after surgery, the patient is alive with no evidence of recurrence . The second important clinical feature in our patient is that colonoscopy was useful for diagnosis . Colonic lesions often represent direct invasion from adjacent organs or peritoneal dissemination, but lymphatic and hematogenous metastases from distant organs can also occur . A submucosal tumor in the gastrointestinal tract is one feature of lesions caused by lymphatic or hematogenous spread from other sites . Endoscopic examination in our patient revealed a gently elevated submucosal tumor with an inflamed surface . These findings are compatible with colonic metastasis from a carcinoma of unknown primary site . Shimada et al . Reported a synchronous asymptomatic colonic metastasis from a primary esophageal scc . Several reports have documented colonic metastases from the primary scc of the lung . Because endoscopic findings differ between epithelial tumor - like lesions and submucosal tumors, colonoscopy was consistently useful for establishing the diagnosis . A definitive diagnosis was possible after biopsy in some previously reported cases . In conclusion, we reported a rare case of synchronous metastatic scc to the colon and cervical lymph nodes in a patient with carcinoma of unknown primary site.
The white matter tracts that are the last to myelinate are the first to degenerate . Superficial white matter (swm) is located beneath the infragranular layer of the cerebral cortex and these tracts are some of the last fibers to myelinate, often occurring in the fourth decade of life . Therefore, swm represents a population of tracts that may be most vulnerable to ad . Swm consists of u fibers of meynert, crown fibers and merging deep white matter fibers . U fibers originate from layers iii and v of the cortex, exit and follow concave surfaces of a sulcus and re - enter the cortex at distances up to 3 cm . Merging fibers originate within the deep white matter, cross other tracts in swm and extend towards the cortex . Perhaps due to the complex architecture and significant inter - subject variability of swm, a magnetization transfer ratio magnetic resonance imaging (mri) study found evidence of demyelination of swm in ad and an association with lower mini - mental state examination scores . A diffusion tensor mri study found decreased white matter integrity of swm in ad, particularly in the frontal and temporal lobes and associations with lower mini - mental state examination scores . Additional imaging studies are needed to characterize the alterations of swm in ad and its cognitive correlates . Tractography is an application of diffusion tensor mri that reconstructs white matter tracts based on restricted diffusion of water molecules along myelinated axons . Disruption of tracts can be detected as increased mean diffusivity (md, degree of water diffusion), increased axial diffusivity (axd, largest eigenvalue), increased radial diffusivity (rd, average of the two smaller eigenvalues) or decreased fractional anisotropy (fa, directional diffusion). A tractography study in healthy older adults demonstrated an association between lower swm fa and lower scores on cognitive testing . Earlier tractography studies in ad assessed lobar white matter that likely consisted of both swm and deep white matter, but none assessed only swm . The aim of this study was to use tractography to characterize alterations in swm associated with ad . We used this method to reconstruct each participant's unique network of swm tracts . Since ad has differential effects across cerebral lobes, we separately assessed frontal, occipital, parietal and temporal swm . In each lobe, we compared the diffusion characteristics (md, axd, rd and fa) of swm tracts between healthy older adults and persons with ad and correlated it with the performance on neuropsychological testing . We hypothesized that ad would be associated with worse swm diffusion characteristics (increased md / axd / rd and decreased fa). We also hypothesized that worse swm diffusion characteristics would be associated with decreased cognitive function . The study was approved by the queen's university research ethics board . Prior to entering the study, all participants provided written informed consent . The neuropsychological battery consisted of the following cognitive tests: global cognitive function (montreal cognitive assessment, moca and mini - mental state examination), selective attention (stroop test), processing speed (trail making test b, trail), focused attention (wechsler memory scale - iii longest span forward) and working memory (wechsler memory scale - iii longest span backward and letter number sequencing). Participants with ad were within the mild stages of the disease, as measured by a moca score of 18/30 . Exclusion criteria were the presence of metallic objects, devices or conditions unsafe for mri . Twenty - four cognitive normal controls and 16 participants with ad were included in the study . Brain imaging was acquired in a single session on a 3 tesla siemens magnetom trio mri system (siemens medical systems, erlangen, germany) with the use of a 12-channel head coil . An anatomical scan was acquired with a sagittal t1-weighted 3-dimensional magnetization prepared rapid gradient echo sequence [field of view (fov) 256 mm, spatial resolution 1 1 1 mm, repetition time (tr) 1,760 ms, echo time (te) 2.2 ms, flip angle 9, number of slices 176]. An axial t2-weighted 2-dimensional fluid - attenuated inversion recovery sequence (flair) interleaved scan was acquired (fov 250 mm, voxel size 1 1 3 mm, tr 9,000 ms, te 79 ms, flip angle 180, number of slices 40). Diffusion tensor imaging (dti) data were acquired in 30 directions using a single - shot echo planar imaging sequence with 31 volumes of 60 axial slices (b - value 1 = 0 s / mm, and b - value 2 = 1,000 s / mm), slice thickness 2 mm, tr / te = 7,800/95 ms, fov 256 256 mm and an acquisition matrix of 128 128, resulting in a resolution of 2 2 2 mm . Dti reconstruction was completed with diffusion toolkit 0.5 (ruopeng wang, van j. wedeen, martinos center for biomedical imaging, massachusetts general hospital, www.trackvis.org). Tracts were created in the diffusion toolkit by the fiber assignment by continuous tracking (fact) method with an angle threshold of 35 . The participant's t2 flair mri was registered by the affine method using 12 degrees of freedom to the diffusion - weighted image map with slicer 3d 4.1 (www.slicer.org). White matter hyperintensities (wmh) were manually segmented on axial t2 flair as described in an earlier study . Swm was defined as tracts originating within 5 mm of the cortical surface, and it was manually segmented into frontal, occipital, parietal and temporal lobe swm based on anatomical landmarks [landmarks are described in ref . ]. Within each lobe, region of interests (rois) rois were manually traced for each lobe, slice by slice, on the 40 axial t2 flair slices for each patient using the trackvis mouse - controlled interface . Frontal, occipital, parietal and temporal lobe swm was segmented by selecting tracts that crossed through the respective lobar rois . The area of roi as well as fa, md, axd and rd of the lobar swm were measured . The fa, md, axd and rd of segmented swm tracts were compared between controls and participants with ad while adjusting for the effects of age and the wmh volume using multiple linear regressions . Cognitive scores were not available in all participants, so participants were included in the analysis only when their scores were available (sample sizes are presented in tables 3 and 4). We presented unstandardized regression coefficients for the associations between cognitive test scores with tracts md, axd, rd and fa and number using multiple linear regressions, adjusting for the effects of age and wmh volume . To account for comparison of the 4 swm lobes, a bonferroni correction was used, and p values <0.0125 were considered statistically significant . The study was approved by the queen's university research ethics board . Prior to entering the study, all participants provided written informed consent . The neuropsychological battery consisted of the following cognitive tests: global cognitive function (montreal cognitive assessment, moca and mini - mental state examination), selective attention (stroop test), processing speed (trail making test b, trail), focused attention (wechsler memory scale - iii longest span forward) and working memory (wechsler memory scale - iii longest span backward and letter number sequencing). Participants with ad were within the mild stages of the disease, as measured by a moca score of 18/30 . Exclusion criteria were the presence of metallic objects, devices or conditions unsafe for mri . Twenty - four cognitive normal controls and 16 participants with ad were included in the study . Brain imaging was acquired in a single session on a 3 tesla siemens magnetom trio mri system (siemens medical systems, erlangen, germany) with the use of a 12-channel head coil . An anatomical scan was acquired with a sagittal t1-weighted 3-dimensional magnetization prepared rapid gradient echo sequence [field of view (fov) 256 mm, spatial resolution 1 1 1 mm, repetition time (tr) 1,760 ms, echo time (te) 2.2 ms, flip angle 9, number of slices 176]. An axial t2-weighted 2-dimensional fluid - attenuated inversion recovery sequence (flair) interleaved scan was acquired (fov 250 mm, voxel size 1 1 3 mm, tr 9,000 ms, te 79 ms, flip angle 180, number of slices 40). Diffusion tensor imaging (dti) data were acquired in 30 directions using a single - shot echo planar imaging sequence with 31 volumes of 60 axial slices (b - value 1 = 0 s / mm, and b - value 2 = 1,000 s / mm), slice thickness 2 mm, tr / te = 7,800/95 ms, fov 256 256 mm and an acquisition matrix of 128 128, resulting in a resolution of 2 2 2 mm . Dti reconstruction was completed with diffusion toolkit 0.5 (ruopeng wang, van j. wedeen, martinos center for biomedical imaging, massachusetts general hospital, www.trackvis.org). Tracts were created in the diffusion toolkit by the fiber assignment by continuous tracking (fact) method with an angle threshold of 35 . The participant's t2 flair mri was registered by the affine method using 12 degrees of freedom to the diffusion - weighted image map with slicer 3d 4.1 (www.slicer.org). White matter hyperintensities (wmh) were manually segmented on axial t2 flair as described in an earlier study . Swm was defined as tracts originating within 5 mm of the cortical surface, and it was manually segmented into frontal, occipital, parietal and temporal lobe swm based on anatomical landmarks [landmarks are described in ref . ]. Within each lobe, region of interests (rois) were outlined paralleling the cortex within 5 mm of the cortical surface . Rois were manually traced for each lobe, slice by slice, on the 40 axial t2 flair slices for each patient using the trackvis mouse - controlled interface . Frontal, occipital, parietal and temporal lobe swm was segmented by selecting tracts that crossed through the respective lobar rois . The area of roi as well as fa, md, axd and rd of the lobar swm were measured . The fa, md, axd and rd of segmented swm tracts were compared between controls and participants with ad while adjusting for the effects of age and the wmh volume using multiple linear regressions . Cognitive scores were not available in all participants, so participants were included in the analysis only when their scores were available (sample sizes are presented in tables 3 and 4). We presented unstandardized regression coefficients for the associations between cognitive test scores with tracts md, axd, rd and fa and number using multiple linear regressions, adjusting for the effects of age and wmh volume . To account for comparison of the 4 swm lobes, a bonferroni correction was used, and p values <0.0125 were considered statistically significant . There was a trend for increased age and increased wmh volume in participants with ad compared to controls; however, it was not statistically significant (table 1). As expected, participants with ad had statistically significantly lower scores on all cognitive tests compared to controls (table 1; all tests p <0.001). Compared to controls, the area of swm was smaller in participants with ad (frontal swm: 5,576 4,556 vs. 1,119 517, p = 0.00073; occipital swm: 1,627 1,211 vs. 316 155, p = 0.00025; parietal swm: 3,652 2,747 vs. 687 161, p = 0.00015; temporal swm: 2,083 1,706 vs. 391 227, p = 0.00029). Compared to controls, in participants with ad, there was increased md (percentage difference 12%), increased axd (9%) and increased rd in temporal swm (14%; table 2). There were no statistically significant differences in the fa of swm between controls and participants with ad (table 2). Among controls, in frontal swm, increased rd and decreased fa were associated with worse moca scores (table 3). In occipital swm, increased rd was associated with worse moca and trail scores, and decreased fa was associated with worse trail scores . In temporal swm, decreased fa was associated with worse moca scores, and increased md and rd were associated with worse stroop scores . In participants with ad, the fa, md, rd and axd of swm was not associated with the performance on the cognitive tests (table 4). This is the first tractography study to detect alterations in the swm in ad . Compared to controls, the retrogenesis hypothesis predicted that, as late myelinating fibers, swm would be vulnerable to degeneration in ad . In support of the retrogenesis hypothesis, we detected disruption of swm in ad . Our results are in agreement with an earlier magnetization transfer ratio imaging study and the dti study that found evidence for disruption of swm in ad . The pattern of swm tract changes observed in this study was similar to earlier reported patterns of cortical neurodegeneration in ad . Based on the braak staging system, neurofibrillary degeneration develops in the temporal lobe and spreads to the rest of the neocortex in later stages of the disease . Consistent with its involvement in early stages of ad, in this study, we found the largest diffusion abnormalities in the temporal lobe . The large increases in md, axd and rd within the temporal swm ranged from 9 to 14% and are consistent with the 10 - 25% increase reported by an earlier dti study . It is believed that increased md represents tissue atrophy, increased axd represents wallerian degeneration, and increased rd reflects myelin disruption . Based on these associations, our study suggests that in ad, there is prominent tissue atrophy, wallerian degeneration and myelin disruption in temporal swm . Consistent with earlier studies, there were greater differences in md, axd and rd than in fa between controls and participants with early - stage ad . Both axd and rd were increased in participants with ad, which may have attenuated changes in fa in ad . In healthy older adults, decreased global cognitive function was associated with worse diffusion characteristics of swm in several lobes . Decreased performance on the moca was associated with increased rd / decreased fa in frontal swm, increased rd in occipital swm and decreased fa in temporal swm . Decreased cognitive function in specific cognitive domains was associated with worse diffusion characteristics of swm in specific lobes . Decreased selective attention assessed by the stroop test was associated with increased md / rd in temporal swm . Decreased processing speed assessed by the trail test was associated with increased rd / decreased fa in occipital swm . Cognitive test scores were only available in a subset of participants leading to smaller sample sizes for cognitive correlations . The small sample size may have limited the ability to detect diffusion differences between participants with ad and controls or detect cognitive associations with swm . Alternatively, the disruption of swm in ad may have contributed to the lack of cognitive associations for swm in ad . Additional limitations of this study are the multiple comparisons, which may have led to false - positive results, and the accuracy of tractography . The complex architecture of swm makes it difficult to follow the course of tracts through crossing or abutting fibers . Although some tracts may not have been tracked at all or not correctly, this study and earlier ones have demonstrated that it is possible to perform tractography for swm . With tractography this network of connections between cortical regions assumes an important role in cognition . At the level of cerebral lobes, this study demonstrated that swm has region - specific roles in normal cognition . In healthy persons, tractography of swm can be used to characterize in more detail the relationship between regional cortical connectivity and specific cognitive processes . In ad, tractography of temporal swm may represent a novel biomarker of neuronal injury that may aid in diagnosis and disease monitoring . All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the helsinki declaration of 1975, as revised in 2000.
A deficincia transversa da maxila uma m ocluso com alta prevalncia em todas as faixas etrias, da dentio decdua permanente . Se no for corrigida, pode agravar - se com o passar do tempo, prejudicando o crescimento e desenvolvimento facial . Alm dos prejuzos oclusais, essa deficincia pode trazer problemas respiratrios tambm severos, devido consequente constrio da cavidade nasal . Em pacientes em crescimento, a sua resoluo relativamente simples, por meio da expanso rpida convencional da maxila . Porm, os pacientes j maduros geralmente so encaminhados para um procedimento mais invasivo, a expanso rpida de maxila assistida cirurgicamente (sarpe). Mais recentemente, pesquisadores tm demonstrado que possvel executar a expanso palatal esqueltica em pacientes adultos sem auxlio de osteotomias, mas sim com auxlio de mini - implantes . O objetivo do presente artigo demonstrar e discutir uma das tcnicas disponveis de marpe, desenvolvida por won moon e colaboradores, na university of california, los angeles (ucla). A tcnica encontra - se detalhadamente descrita, com as etapas laboratoriais e clnicas que devem ser seguidas para sua correta execuo . Para descrev - la, apresentado o caso clnico de uma paciente adulta, detalhando toda a sequncia do tratamento e os resultados obtidos . A tcnica apresentada pode ser uma alternativa no invasiva sarpe na resoluo da deficincia transversa de maxila, podendo ser empregada na maioria dos pacientes com crescimento facial finalizado . A paciente apresentada demonstrou benefcios significativos nos aspectos oclusal e respiratrio, sem a necessidade de interveno cirrgica . The prevalence of transverse maxillary deficiency, which affects an important number of patients seeking orthodontic care, may reach 23.3% within the primary dentition population . This type of malocclusion usually develops during facial growth and development and, if left untreated, will probably affect the permanent dentition, since the chances of spontaneous correction are low . Some of the most prevalent factors on its multifactorial etiology are myofunctional disorders of the stomatognathic system, usually associated with deleterious habits such as thumb sucking . In these cases, the tongue may be in an abnormally lower position, which leaves room for the antagonist muscles (buccinators) to apply dominant forces and consequently constrict the maxillary arch . Intramembranous maxillary bone formation may be affected by and depends on surrounding muscles activity and individual breathing pattern along development . At the same time typical cases are those of patients with class iii malocclusion with mandibular prognathism, in which p561 t polymorphism in the ghr candidate gene, responsible for growth hormone receptors, for instance, determines the excessive growth on condylar cartilage . As a result, maxillary and mandibular posterior teeth may present with increased overjet as the mandible is protruded (fig 1). Figure 1lateral radiograph and coronal cbct slice of a patient with true mandibular prognathism and excessive vertical growth; images show bilateral skeletal posterior crossbite due to mandibular anterior position and lower tongue posture . If not properly managed within appropriate time, maxillary transverse deficiency, associated or not with posterior crossbite, may result in several problems for the patient: different degrees of occlusal disharmony; changes in tongue posture; damage to periodontal structures, such as local bone loss and gingival recession; functional shift of the mandible due to incorrect buccolingual tipping of posterior teeth; asymmetric mandibular position in growing patients; joint disorders and muscle function disturbances; lack of space in the arch for adequate dental alignment . The most serious consequence of maxillary transverse deficiency, however, might be the consequent narrowing of the nasal cavity, which increases nasal air resistance (fig 2) and may be an etiological factor of obstructive sleep apnea syndrome (osas). Figure 2coronal slice shows maxillary transverse deficiency and, consequently, nasal cavity narrowing in adult mouth - breather with moderate osas (ahi = 15.9). Are also noticeable the high - arched palate, low tongue position and anatomic disorders of nasal cavity (turbinate hypertrophy and septal deviation). For the treatment of this condition, according to orthodontic consensus, patients should undergo rapid palatal expansion (rpe) immediately, while still growing . This procedure has been used for over a century in orthodontics, and its positive effects have been widely described and documented . The earlier the treatment is delivered, the better the prognosis and the outcomes, increasing chances of morphological and functional correction and bringing about proper facial development . During primary and mixed dentition and the first years of permanent dentition a recent review of the literature showed that it is a stable procedure in the short and long term, regardless of the type of expander used . Patient growth leads to progressive calcification and interdigitation of craniofacial sutures, including the midpalatal suture, and rpe becomes more difficult as facial growth approaches its completion because of increased mechanical resistance of these structures . The amount of undesired orthodontic movement (buccal tipping of anchor teeth) and its side effects are proportional to patient age and skeletal maturation . Therefore, adolescents tend to have greater tooth inclination and buccal bone dehiscence and, therefore, less orthopedic expansion than children . Although retrospective case series have demonstrated the success of tooth - borne expansion in this age group, no well - designed clinical trials have determined its success rate . . A higher rate of side effects, such as a reduction in alveolar bone thickness and height, bone dehiscence and gingival recession, may be expected as a result of important mechanical forces delivered at the teeth and its supporting structures . Therefore, surgically - assisted rapid palatal expansion (sarpe) is often indicated to these patients . This procedure increases expansion predictability and success, and reduces its side effects . One of the available sarpe techniques consists of a lefort i osteotomy associated with surgical rupture of the midpalatal suture, which decrease the mechanical resistance to the lateral forces that will be applied by hyrax expanders, usually anchored to the first molars and first premolars . However, despite its benefits, sarpe increases biological and financial costs of the treatment . The surgery requires hospitalization and general anesthesia, which might scare patients away from surgical - orthodontic treatment for good . In face of that, some authors have investigated the use of orthodontic microimplants as auxiliary anchorage devices to optimize the application of mechanical forces to circummaxillary sutures, thus avoiding the otherwise indispensable osteotomies . This system, which has been called microimplant - assisted rapid palatal expansion (marpe), applies forces to the microimplants, and not to the teeth or periodontium . Different appliance designs and techniques have been described in the literature, and each leads to specific associated outcomes . A recent clinical study using one of them found an 86.96% success rate in young adult patients (mean age = 20.9 2.9 years), with stable results after 30 months of follow - up . The objective of the present study was to describe one of the techniques available for rapid palatal expansion of non - growing patients, maxillary skeletal expansion (mse), developed and improved along several years by dr . Won moon and colleagues at the university of california - los angeles (ucla). For didactic reasons, this article has been divided into the following sections: introduction; laboratory and clinical procedures, demonstrating the step - by - step manufacture and appliance delivery; case report, to illustrate some of the technique applications; discussion; and conclusion . The laboratory manufacture of the mse appliance is similar to that of a conventional hyrax expander . The steps below should be followed: first visit: thorough explanation of procedures to the patient, clarifying all details and technical limitations and reasserting that failure may occur; placement of separator elastics on the permanent maxillary first molars . Second visit: removal of separators, prophylaxis and band placement on first molars; conventional alginate transfer impression; regular plaster pouring; separators elastics placed again on molars; orthodontic accessories (tubes and brackets) may be soldered to the bands at this stage . Laboratory procedures (fig 3): selection of 8, 10 or 12 mm mse, according to palate width (details below); bending wires to reach the bands, following palate curvature, at a separation of at least 2 mm along all their extension; wire soldering to the bands, followed by finishing and polishing; reverse traction hooks may be soldered to the buccal aspect of bands at this stage . Figure 3laboratory procedures: midline (palatal raphe) and limit between soft and hard palate (clinically determined) traced using lead pencil on model; selection of mse with greatest expansion capacity (8, 10 or 12 mm) that can be placed flush to palatal mucosa; appliance wire segments bended to outline palate curvature, holding at least a 2 mm gap from the mucosa; expander should be centralized to palatal raphe and placed at the most posterior position possible, slightly before limit between soft and hard palate; soldering of wire segments onto the bands, followed by polishing; posterior view shows that expander is flush to palatal mucosa, but should not touch it . Third visit (fig 4): removal of separators, prophylaxis and expander proof; application of topical anesthetics to the palate; appliance cementing, checking the vertical position in relation to palate; local infiltrative anesthesia; self - drilling microimplant placement using appropriate digital key (biomaterials korea, seoul, south korea); immediate expander activation (2 to 3 turns); instructions about hygiene and activation; prescription of analgesic drug of choice for two days (optional); no need for antibiotic coverage if the patient has good general health . Figure 4clinical visit: expander clinical proof, topical anesthesia applied, and expander cemented; after expander is cemented (as shown on plaster model, for teaching purposes), infiltrative anesthesia is applied close to orifices of mi; after region is anesthetized, mi are placed paying special attention to anteroposterior and lateral inclination . Index finger of one hand should hold the digital key, and index and thumb of other hand firmly moves key counterclockwise . During posterior mi placement, patient mouth should be wide open to ensure correct anteroposterior inclination . Follow - up: the patient should be seen more often than in conventional expansion . In some cases, the patient is not able to activate the expander at home due to increased resistance, and the professional support is necessary . At all visits, the distance of the expander from the mucosa should be checked . In case of contact the stability of all mi should be checked regularly using tweezers and, in case any mobility is found, mi should be removed; the treatment may continue, although extra - carefully, even if there is only one mi on each side . Removal: for removal, the same connector used for placement, coupled with the digital key, should be slowly turned counterclockwise . Plaque may accumulate on the mi head, which hinders mi gripping (careful previous cleaning of the site is required). Due to the forces applied, the mi may be removed without anesthesia . Immediately after each mi is removed, a cotton pellet soaked in hydrogen peroxide might be applied to the site to promote asepsis, but no additional care is required . Mi should be discarded after removal, and should never be sterilized or reused . The selected expander should be the one with the greatest expansion capacity that, at the same time, may be kept at an ideal vertical distance from the palatal mucosa . Bicortical anchorage (oral and nasal) is determinant of success and if the expander is too distant from the mucosa (more than 2 mm), microimplants may not reach the nasal cortical bone . Moreover, chances of mi deformation are higher if the force is applied too far from the implant / bone interface (fig 5). The body of the expander should be placed as posterior as possible, close to the junction of hard and soft palate (hard palate mucosa is whiter). The greatest resistance against suture opening is located in the sutures between maxilla and pterygoid plates (fig 6), and forces should be applied more posteriorly to overcome initial resistance and promote parallel opening of the midpalatal suture (fig 7). When forces are applied directly into the center of resistance of the maxilla by means of mi, and not to teeth (as in conventional expansion), the force system is more favorable due to a homogeneous force dissipation, which prevents buccal tipping and produces a more parallel suture opening (fig 8). Figure 5force application too far from bone / microimplant interface, resulting in mi deformation . Figure 6dry skull shows relation between the pterygoid plates of sphenoid bone and maxilla . These structures provide great resistance to lateral forces applied by the expander, and connection between them has to be split apart for real skeletal expansion . Figure 7when expander is placed at a more posterior position, forces concentrate closer to the pterygoid plates, structures that offer great resistance to palatal expansion . Therefore, occurs a parallel opening of the palatine suture anteroposteriorly and vertically, differently from conventional expansion, in which opening takes the form of a " v " (broader in anterior region). Figure 8a) in conventional palatal expansion, forces are applied to teeth, below the center of resistance of the maxilla . This system of forces generates buccal dentoalveolar tipping and an inverted - v opening (coronal view), indicated by the red dotted lines . B) in marpe, forces are applied directly into the maxillary center of resistance by means of the mi, which practically eliminates inclination forces of posterior teeth and promotes more parallel suture opening in a coronal view (indicated by red dotted lines). A small amount of anesthetics (no more than 1/4 of a cartridge) may be applied only once on each side, between the two ipsilateral mi . Anesthetic application local should be carefully chosen, and the needle should always be placed close to the midpalatal suture to avoid contact with the palatine artery . The operator should have extensive knowledge of the position of this artery, which may vary according to palate depth . Whenever possible, a vasoconstrictor combined with the anesthetic should be used to reduce bleeding, which is often absent . Mi should be placed carefully, although the guides (expander holes) facilitates its placement . Mi should be as perpendicular as possible to the palatal bone (each mi parallel to all others) so that the force distribution is effective . Therefore, both the anteroposterior and the lateral inclination should be repeatedly checked during placement . When placing the posterior mi, patient should keep the mouth wide open to avoid changing their anteroposterior inclination (mi tend to distal tipping). Mi can be delivered in most patients without previous bone perforation, using the digital key . If torque is excessively high, bone perforation can be made using a 1 mm diameter drill . A very high - arched and deep palate, typical of chronic mouth - breathers, may hinder the vertical positioning of the mse . For these patients, the anterior or posterior segments of the expander can be trimmed so that the expander can be placed closer to the mucosa (fig 9). This option is acceptable because forces are applied to the mi, leaving to teeth only a supporting purpose during mi placement . Figure 9maxillary occlusal photograph showing removal of anterior wire segments of mse, to improve vertical fit in a very narrow and high - arched palate . Although there are no randomized clinical trials, the following activation protocol is suggested as a reference, based on a sample of over 100 patients seen over 15 years (table 1). In adults, the 8 mm mse has 40 activations (0.2 mm per turn); the 10 mm one, 50 activations; and the 12 mm one, 60 activations . Activations should not reach the limit, because the expander loses rigidity as it approaches the limit and might undergo some deformation . Table 1suggested activation protocol.age groupactivationbeginning of adolescence3 to 4x / weekend of adolescence1x / dayyoung adults2x / dayolder than 25 years2x or + /day a 22-year and 6-month - old female was seen for orthodontic treatment at the orthodontic clinic of the universidade federal do paran, brazil . The patient had not undergone any orthodontic treatment before, but had already made up her mind to avoid maxillary expansion surgery . The smiling photo showed excessive buccal corridor display and easily noticeable transverse maxillary deficiency (fig 10). The mandibular arch had moderate anterior and posterior crowding and left transverse asymmetry due to the posterior crossbite on that side . In the maxillary arch, there was mild crowding and transverse asymmetry (opposite to the mandibular arch) on the left side as well, due to the same crossbite (fig 11). Right molars and canines displayed a class i relationship, bearing normal horizontal and vertical overjet . On the left side, canines had an edge - to - edge relationship (class ii), with posterior crossbite (fig 12). Lateral radiograph showed a good skeletal relationship, as well as good inclination and position of maxillary and mandibular incisors (fig 13). A coronal cbct slice revealed exacerbated inclination of teeth (torque) in posterior crossbite (fig 14). Sagittal slices of the joints revealed that the condyles were not centrally positioned in the fossa, which confirmed the clinically present double - bite (fig 15). Because of her breathing complaints, it was applied the epworth sleepiness scale and quebec sleep questionnaire as screening tools, which revealed a high risk of obstructive sleep apnea syndrome (osas). Therefore, the patient underwent in - home polysomnography (nox medical, reykjavik, iceland), and results revealed an apnea / hypopnea index (ahi) of 7.9, classified as mild apnea syndrome according to the american association of sleep medicine guideline, associated with moderate snoring and isolated episodes of bruxism (fig 16). Figure 11initial occlusal photographs; moderate crowding in mandibular arch due to constriction caused by the maxilla . Note the lingual inclination of left posterior teeth in maxillary arch . Figure 12initial intraoral photographs: good occlusal relationship on the right side and edge - to - edge relationship of left canines (class ii); left superior buccal segment in crossbite . Figure 13lateral radiograph obtained from cbct shows harmonic maxillomandibular skeletal relationship and satisfactory position of maxillary and mandibular incisors: note that mandibular ramus heights are asymmetric . Figure 14coronal cbct slice at the level of maxillary first molars shows excessive palatal inclination of these teeth; tongue is at a low position; measurement indicated maxillary constriction and, consequently, nasal cavity constriction . Figure 15sagittal slice shows incorrect position of condyles into articular fossa, especially on the right side (contralateral to the posterior crossbite). Figure 16baseline in - home polysomnography shows an ahi of 7.9, defined as mild osas; despite that, patient has good oxygen saturation along the night . The first treatment option was non - surgical rapid palatal expansion (marpe) because the patient refused to have sarpe . We thought skeletal expansion was necessary because of the patient s respiratory disorder, reported by the patient herself at first, and later confirmed by the polysomnography . Treatment alternative consisted of fixed orthodontic appliance and microimplants for intrusion and buccal inclination of the left maxillary posterior teeth to compensate the buccolingual inclination of teeth in crossbite area, with possible future side effects on its supporting structures . Treatment started with the placement of a 10 mm maxillary skeletal expander (mse) and three immediate activations (1/4 of a turn, 90 degrees each), followed by two daily activations . By the second week, the patient reported having heard clicks in the region of the palatal suture and, in the following days, appearance of the interincisal diastema (fig 17). There was a discrete opening of the anterior bite due to contact of the buccal cuspid of the left first maxillary molar, which moved in the direction of overlapping the antagonist mandibular molar . Photographs after 34 activations confirmed suture opening and lack of collateral buccal inclination of maxillary molars (fig 18). After 44 activations, at a total of 8.8 mm screw opening, the mse was removed for the placement of another expander, a common practice depending on case severity . At this time, crossbite was still present (fig 19). However, instead of using another mse and continuing with pure skeletal expansion, we decided to place a conventional tooth - borne hyrax expander for two reasons: circummaxillary sutures had already been mobilized, and, therefore, skeletal gains should be preserved; and we would like to ensure buccal inclination of maxillary left posterior teeth to optimize future orthodontic treatment . At this point, the patient had already reported important improvement of sleep quality, with facilitated nose breathing and reduction of rhinitis episodes, frequent in the past . An 8.8 mm expansion at the palatal suture may be classified as substantial, as mean opening in conventional expansion in growing patients is usually around 4 to 5 mm . Figure 17photograph taken after 20 activations (4 mm); interincisal diastema confirms suture opening . Discrete anterior open bite appears due to an overjet reduction of the posterior teeth in crossbite, which generates premature occlusal contacts . Absence of tooth or alveolar bone tipping as left posterior teeth still show palatal inclination . Figure 19photograph taken during mse removal, after 44 activations (8.8 mm); high mechanical resistance bent expander built - in support wires; mi with no deformation when removed are signs of ideal progression of the expansion . On the same day the mse was removed, to eliminate any possibility of relapse, an 11 mm hyrax expander was delivered . The bands were placed onto the first molar, and a palatal wire extended to the first premolar on both sides . We continued the protocol of two daily activations until the desired inclination of the left maxillary posterior teeth was achieved . The increase of interdental diastema was clear during the activation period, confirming the skeletal changes . As unilateral expansion is not feasible, it was necessary to overcorrect the right side until there was buccal crossbite, so that the ideal inclination was achieved in the opposite side (fig 20). Activations continued until the total expansion was 7 mm, when the expander was tied - out (fig 21). Spaces created by the expansion were distributed along maxillary arch . When manipulated to centric occlusion, a substantial transverse increase was evident and the posterior crossbite corrected . Following the expander removal after the recommended retention time a broader maxillary arch allows for the expansion of mandibular arch, which was also constricted . Figure 21occlusal photo taken at hyrax tie - lace; smaller expansion on left side, limited by crossbite occluding forces . Left posterior teeth aligned to canine and second molar, which reduces time of treatment with fixed appliance . Facial photographs after expansion showed decreased buccal corridor display and correction of the lower midline shift at centric occlusion, which confirmed that the mandible was in fact deviated at maximum intercuspation before expansion (fig 22). Post - expansion ct scan confirmed the opening of the palatal suture (fig 23) and also showed a more favorable buccolingual inclination of the left posterior teeth and an increase on the nasal cavity floor (fig 24). However, the greatest treatment benefit was probably the one revealed by post - expansion polysomnography: a reduction of the ahi from 7.9 to 1.5 . It is important to inform that no other concurrent therapy for the breathing issue was delivered and that the patient s body mass index remained the same throughout the treatment . Improved mandibular position and buccal corridors as a result of skeletal and dental expansion promoted by the two expanders . Figure 23cbct slices show homogenous suture opening along anterior and posterior regions and uniform separation of the hemimaxillae . Figure 24coronal slice after expansion shows more favorable buccolingual inclination (torque) of posterior maxillary teeth . Also, nasal cavity floor is 23.2 mm wide, larger than at baseline (15 mm). Wax - bite registration was sent to the radiologic laboratory, but the cbct scan was obtained at maximal intercuspation, suggesting that posterior left crossbite is still present . Figure 25post - expansion in - home polysomnography depicted an ahi of 1.5, a substantial reduction from baseline value of 7.9 . Note anterior overbite improvement, matching of the dental midlines and almost complete crowding dissolution . Figure 27the overjet created on the left cuspids will allow for an increase on the intercanine distance in the mandibular arch, completing its ideal alignment . On the right side, the upper cuspid will also present with some overjet when its torque is corrected . The hyrax expander would be held in place for the next four months to achieve satisfactory suture ossification . After 3 months of inferior fixed appliance therapy, the inclination of posterior teeth was very improved and almost all the crowding was solved, helping to close the anterior bite back to its baseline status . We believe that the expansion made on the mandibular arch to alleviate the crowding has a better relapse prognosis, because the maxillary arch was skeletally expanded with marpe creating overjet for lower intercanine width increase . When the hyrax expander is to be removed, teeth that were overcorrected during expansion on the right side will naturally return to the ideal position, due to muscular activity . After the bones are placed in a favorable transverse position, the case became simple to solve using corrective orthodontic treatment and class ii elastics on the left side . Any other treatment alternative, even using the most sophisticated biomechanics, would be challenged by the lower midline deviation . Even so, the treatment time would be increased and the condyles would remain in an unfavorable position . Another promising application of this technique may be for class ii hyperdivergent patients, who often present with maxillary transverse deficiency . According to buschang et al, the best treatment in these cases is true mandibular counter - clockwise rotation . Using marpe, intrusion of maxillary molars may be executed immediately after maxillary expansion, which promotes counterclockwise rotation of the mandible improving the sagittal relationship . For that purpose, it is enough to remove the bars that hold the expander to the bands after the expansion active retention period . The expander itself will promote transverse retention while it is used for skeletal anchorage to promote posterior maxillary intrusion . In such cases, mi should also be placed in the mandibular arch to avoid compensatory extrusion of the antagonist teeth . Embryologic formation and development of the midpalatal suture are thoroughly described in the literature, particularly in histological studies of human specimens . Medial borders of the hemimaxillae, which grow toward each other until they are mechanically interlocked, progress along the following postnatal development stages: synfibrosis, broad distance between parallel borders; synarthrosis, narrower sinuous course; synostosis, complete interdigitation . However, the age at full suture ossification (synostosis) has not been definitely determined in the literature . Recent histological studies revealed that only the anterior third of the suture was ossified in human beings older than 70 years, although ossification appeared complete on radiographs . In those samples, such studies support the theory that the midpalatal suture may be the only cranial suture that does not achieve full ossification because of the constant mechanical stress that is applied to it . Histological studies have demonstrated that caution should be taken when defining the stage of ossification using imaging exams . Occlusal radiographs or cbct should be requested to confirm marpe success, defined by midpalatal suture opening, because not all cases display an interincisal diastema . However, if the diastema is created, as in the case here reported, suture split and skeletal expansion of the maxilla are evident . It remains unclear why few marpe cases fail, but it is believed that differences in calcification patterns of the midpalatal suture and craniofacial architecture (higher resistance) are contributing factors . As mechanical forces are distributed into the palate by the mi s, the stress on teeth and supporting structures is understated, which might reduce side effects such as gingival recession and buccal bone dehiscence . A clinical study that followed up 69 young adults that underwent marpe did not find any clinically significant side effects . Other studies of conventional palatal expansion in young adults without mi have warned about the risk of side effects . Lin et al recently conducted a direct comparison of marpe and conventional expansion (mean age 18.1 4.4 years) and found that marpe was more orthopedically efficient and had a lower rate of dentoalveolar side effects . This initial data may be suggestive of evidence, which should be further investigated in randomized clinical trials . Moreover, the effect of marpe is basically orthopedic, because forces are applied directly to the bone; therefore, there is no need of overcorrection . In conventional expansion, however, overcorrection is recommended because of the orthodontic effects (buccal tipping), which may often lead to relapse . According to haas, midpalatal suture rupture takes place after the third or fourth complete turn, at a screw opening of about 3 to 4 mm, because of tooth inclination . When using marpe, suture split happens sooner, usually still in the second week of activation, because there is less tooth tipping . A new mse has been recently developed to incorporate some changes that increase efficiency and treatment predictability . Mse new design uses 1.8 mm diameter mi s and has a robust wrench - type activation key . It should be indicated in patients that higher resistance of the circummaxillary sutures is expected . Several marpe techniques, using various designs, are available . Some expanders are supported only by mi (palatal distractors), but most have a hybrid design and are supported by both mi and teeth . A technique developed at yonsei university uses four mi, two of which in the anterior palate, measuring 1.8 mm in diameter, and four teeth for anchorage . It must be kept in mind that different techniques, with differences particularly in mi and expander positioning, have different outcomes . It has been demonstrated that the dimensions of the nasal cavity increase in growing patients as a result of rpe, and that upper airway resistance may be reduced in the short and long terms . Other studies within several medical specialties have gone further and demonstrated that rpe is efficient to treat pediatric patients with osas . Orthodontists should be thoroughly familiar with these longitudinal studies, so that they can give up - to - date information to their patients, as well as to their colleagues in the multidisciplinary team required for the treatment of this syndrome . A recent study found a significant reduction of 56.2% of the ahi of adult patients that underwent sarpe, as well as significant improvements of osas clinical symptoms . In the same line of thought about marpe, we may be looking at an interesting treatment option for patients with osas, which, however, precludes the use of invasive osteotomies . Although the patient reported here had mild apnea, we currently know that osas is progressive, particularly because of the loss of muscle tone and the accumulation of fat in the cervical region as individuals grow older . Our patient is still very young, but, at a more advanced age, the condition might deteriorate . In her current condition, as a result of treatment, we might expect her to be able to control osas in the future with the help of myofunctional therapy to strengthen the oropharyngeal muscles . Marpe efficacy for this purpose remains to be proven, and it should be determined to which groups of patients, with different osas etiologies, this therapy would be most beneficial . However, this treatment may have a high impact on individual quality of life and public health, because moderate / severe osas has an estimated prevalence of 23.4% (95% ci, 20.9 - 26.0) among women and 49.7% (95% ci, 46.6 - 52.8) among men (mean age 57 years), which results in substantial costs for the public and private health care systems . Sufficient evidence has been already gathered to suggest that all orthodontic patients, adult or pediatric, should undergo an evaluation of the risk of osas using validated questionnaires, as a form of screening . Studies in sleep medicine have increasingly highlighted the importance of the orthodontist in the early diagnosis of this syndrome, because the oral cavity has several signs that potentially indicate an increased risk of this syndrome, such as the mallampati classification . This positioning is even more important in pediatric populations, because the orthodontist is one of the first professionals to carefully assess facial growth and the oral cavity, usually at the age of 6 or 7 years old . If diagnosed and approached correctly at this early age by a multidisciplinary team, serious problems such as cardiologic and metabolic sequelae could be avoided, which osas would probably lead to if undiagnosed . The most frequent complication is the inflammation and hyperplasia of the mucosa around the mi, usually associated with inadequate local hygiene . A significant amount of time should be spent to orientate the patient about hygiene importance, using all the tools to optimize it (dental brush and water jet). In cases where mechanical control is not sufficient, if inflammation affects only one mi, it should be removed, and the treatment may progress normally . Hyperplasia may also occur when there is not enough distance from the expander and/or its wires to the mucosa, usually associated with local pain . In patients with slow bone remodeling, such as those with type ii diabetes, additional care should be taken to avoid buccosinusal communication after mi removal, as bone neoformation takes longer . One of the limitations of this technique is associated with very narrow and high - arched palates, which hinders mse vertical positioning and reduces the success rate of the treatment . To our knowledge, this is the first case report to demonstrate non - surgical resolution of maxillary transverse deficiency associated with osas in an adult patient, evaluated in the short term . Recent evidence suggests that non - surgical palatal expansion, assisted by microimplants, is achievable and predictable in young adults . No concrete evidence has shown that the palatal suture is completely fused at the end of facial growth, which makes this treatment theoretically applicable at any age and phase of life . Microimplant - assisted rpe in adults, in addition to an efficient solution for maxillary transverse deficiency in a substantial number of patients, seems to have an important impact on the reduction of upper airway resistance . The robust skeletal anchorage provided by the palatal expander offers novel mechanical possibilities for the treatment of a wide range of malocclusions.
India, yoga combines specific postures (asanas), breathing techniques (pranayama), meditative techniques (dhyana), chants (mantras), and wisdom teachings (sutras) to encourage health and relaxation . Following the worldwide commercial success of yoga, industry reports an increase in yoga participation since 2005 with over 30 million people practicing yoga for health benefits daily . As the number of yoga practitioners and yoga schools in the west increases, interpretations of the practice of yoga vary with several applications of spiritual, cultural, and therapeutic knowledge . The national institutes of health (us) and national health services (uk) describe yoga as a safe and effective intervention to increase strength, flexibility and balance, and treatment for high blood pressure, heart disease, aches and pains, depression, stress, and potentially asthma . The purpose of this study is to investigate the prevalence of yoga research by subject and type in the context of a western healthcare database . Specifically, as the popularity of yoga practice for health in western populations continues to increase, do publication trends in yoga research indicate a similar surge in interest? A service offered by the national library of medicine (nlm), pubmed contains publication information of over 21 billion articles for biomedical literature with citations that include fields of biomedicine, behavioral sciences, chemical sciences and bioengineering . This database is most commonly used to source relevant articles for health professionals and academics in the western hemisphere, primarily because of its subject focus, abundance of material and free - access to medical abstracts in the english language . The annual statistics of pubmed results for yoga - related research is generated from an automated online tool and key - term search strategy (appendix a). A preliminary analysis indicates the first recorded yoga article in pubmed dates to 1948 authored by e. abegg with an unknown title, while the first full - text yoga article appears to be written for 1964 . Therefore, to ensure a comprehensive and pragmatic search any yoga - related publication from the years 1950 to 2012 is included . Any citation that is not included on the pubmed search tool will be excluded and considered a limitation of the research . A service offered by the national library of medicine (nlm), pubmed contains publication information of over 21 billion articles for biomedical literature with citations that include fields of biomedicine, behavioral sciences, chemical sciences and bioengineering . This database is most commonly used to source relevant articles for health professionals and academics in the western hemisphere, primarily because of its subject focus, abundance of material and free - access to medical abstracts in the english language . The annual statistics of pubmed results for yoga - related research is generated from an automated online tool and key - term search strategy (appendix a). A preliminary analysis indicates the first recorded yoga article in pubmed dates to 1948 authored by e. abegg with an unknown title, while the first full - text yoga article appears to be written for 1964 . Therefore, to ensure a comprehensive and pragmatic search any yoga - related publication from the years 1950 to 2012 is included . Any citation that is not included on the pubmed search tool will be excluded and considered a limitation of the research . The total number of yoga - related articles listed on the pubmed database from 1950 to 2012 is 2,099 . Of these titles, 498 (24%) include yoga trials and 324 (15%) systematic reviews [figure 1]. Yoga titles by type on pubmed two yoga - related articles are published in 1950, with a cumulative total of seven titles until 1960 . In 1961, seven further articles are added with annual additions of two to 13 articles until 1973 . This trend of less than 20 new articles per year continues until 1991 with the exception of 3 years (1973 - 1975), [figure 1]. The number of yoga publications in pubmed more than doubles between 1995 and 2000 from 17 to 39 . The rate of publication following 2000 is sporadic until a surge in 2007 when 114 yoga titles are added to pubmed in one year . A steady increase in yoga research is indicated since 2007 with an increased rate of publication by at least 24 articles per year . From 2010 to 2012, 189, 229 and 274 new yoga - related articles are added per year . Of all yoga - based literature on pubmed the results of the key term search indicate a vast majority (90.3%) of all yoga research is associated with 10 health conditions . The most prevalent health conditions for yoga study are stress / anxiety (343), pain (276), depression (207), cardiovascular disease (200), and blood pressure / hypertension (198). Respiratory conditions, cancer, body weight, hormones, and diabetes also indicate high association with yoga research [figure 2]. Sleep, addiction, prenatal care, infection, and injury appear to have less association with yoga research (cumulative <5%). The total number of yoga - related articles listed on the pubmed database from 1950 to 2012 is 2,099 . Of these titles, 498 (24%) include yoga trials and 324 (15%) systematic reviews [figure 1]. Two yoga - related articles are published in 1950, with a cumulative total of seven titles until 1960 . In 1961, seven further articles are added with annual additions of two to 13 articles until 1973 . This trend of less than 20 new articles per year continues until 1991 with the exception of 3 years (1973 - 1975), [figure 1]. The number of yoga publications in pubmed more than doubles between 1995 and 2000 from 17 to 39 . The rate of publication following 2000 is sporadic until a surge in 2007 when 114 yoga titles are added to pubmed in one year . A steady increase in yoga research is indicated since 2007 with an increased rate of publication by at least 24 articles per year . From 2010 to 2012, 189, 229 and 274 new yoga - related articles are added per year . Of all yoga - based literature on pubmed the results of the key term search indicate a vast majority (90.3%) of all yoga research is associated with 10 health conditions . The most prevalent health conditions for yoga study are stress / anxiety (343), pain (276), depression (207), cardiovascular disease (200), and blood pressure / hypertension (198). Respiratory conditions, cancer, body weight, hormones, and diabetes also indicate high association with yoga research [figure 2]. Sleep, addiction, prenatal care, infection, and injury appear to have less association with yoga research (cumulative <5%). The first recorded yoga article in western medical research dates to 1948, authored by e. abegg with an unknown title . The first full - text article found (1964) explores oxygen consumption during yoga - type breathing patterns . Yoga research in the first 30-years of study includes diverse topics such as metabolism, arterial blood gases, body composition bronchial asthma and hypertension . The body of literature still comprises largely of 10 health conditions, thought the recent 5-year trend indicates a shift in focus . Stress / anxiety, pain, and depression remain the most prevalent, with cancer now exceeding the number of articles of hypertension, respiratory conditions, and cardiovascular disease [figure 3]. In 2012 publications, 53% of all publications relate to stress / anxiety (21%), pain conditions (17%), and depression (15%), while 37 new articles in cancer (12%) indicates an area of emerging interest . 5-year publication trends on pubmed by health condition this bibliometric analysis employs a gross method to observe the annual trends of yoga citations in one medical database . As the results of yoga and health conditions are not investigated, no conclusions in terms of the direction of correlation can be drawn from this study . The first recorded yoga article in western medical research dates to 1948, authored by e. abegg with an unknown title . The first full - text article found (1964) explores oxygen consumption during yoga - type breathing patterns . Yoga research in the first 30-years of study includes diverse topics such as metabolism, arterial blood gases, body composition bronchial asthma and hypertension . The body of literature still comprises largely of 10 health conditions, thought the recent 5-year trend indicates a shift in focus . Stress / anxiety, pain, and depression remain the most prevalent, with cancer now exceeding the number of articles of hypertension, respiratory conditions, and cardiovascular disease [figure 3]. In 2012 publications, 53% of all publications relate to stress / anxiety (21%), pain conditions (17%), and depression (15%), while 37 new articles in cancer (12%) indicates an area of emerging interest . This bibliometric analysis employs a gross method to observe the annual trends of yoga citations in one medical database . As the results of yoga and health conditions are not investigated, no conclusions in terms of the direction of correlation can be drawn from this study . A surge of yoga's popularity in western culture in 2005 is matched with academic interest in 2007 . There are now over 2,000 yoga titles on the database, with a rate of more than 200 additions per year . Systematic reviews and yoga trials are increasing, indicating a potential increase in quality of evidence . Three conditions show consistently high correlations with yoga research: stress / anxiety, pain, and depression . A significant rise in the number of cancer publications suggests an area of emerging research . Investigation into the quality and direction of these findings would assist in understanding the potential impact of yoga on several predominant health conditions.
Interleukin (il)-6 is a cytokine featuring redundancy and pleiotropic activity . It was successfully cloned in 1996 as a b - cell differentiation factor, which promotes b - cell differentiation into antibody - producing cells . Subsequent in vitro studies and analysis of il-6 transgenic mice have shown that il-6 acts not only on b cells but also on t cells, hepatocytes, hematopoietic progenitor cells, and various other cells [24]. One of the important functions of il-6 is the differentiation of cd4 nave t cells into effector cells . Il-6 in the presence of tgf- promotes nave t - cell differentiation into th17 cells, while il-6 inhibits tgf--induced regulatory t - cell (treg) differentiation, causing imbalance between th17 and treg, which is a primary pathogenic factor in several autoimmune diseases . Il-6 transmits its signal through its binding to transmembrane receptors or the soluble il-6 receptor (il-6r) [7, 8]. After binding of il-6 to il-6r, the resultant il-6/il-6r complex associates with gp130 and induces homodimerization of gp130, which triggers signal transduction system . The pathological significance of il-6 for diseases was first demonstrated in a case of cardiac myxoma . The culture fluid obtained from the myxoma tissues of a patient who presented with fever, arthritis with positivity for antinuclear factor, increased c - reactive protein (crp) levels and hypergammaglobulinemia and was diagnosed with undifferentiated connective tissue disease, contained a large quantity of il-6, which suggested that il-6 might contribute pathologically to chronic inflammation and autoimmunity . Subsequent studies have shown that dysregulation of il-6 production is implicated in the pathogenesis of castleman's disease, rheumatoid arthritis (ra), and various other autoimmune, inflammatory, and malignant diseases [24]. Because of the biological activities of il-6 and its pathological role in diseases, it was anticipated that il-6 blockage would constitute a novel treatment strategy for autoimmune and inflammatory diseases [4, 1315]. To this end, tocilizumab was developed, which is a humanized anti - il-6r monoclonal antibody (ab) of the igg1 class that was generated by grafting the complementarity determining regions of a mouse anti - human il-6r ab onto human igg1 . Ra is a chronic, progressive inflammatory disease of the joints and surrounding tissues accompanied by intense pain, irreversible joint destruction, and systemic complications such as fatigue, anemia, and fever . At the local level, inflammatory cells invade the otherwise relatively acellular synovium leading to neovascularization, synoviocyte hyperplasia, and formation of pannus tissue, which in turn causes destruction of cartilage, erosion of the adjacent bone, and, ultimately, loss of function of the affected joint . The biological activities of il-6 such as proinflammatory activity, augmentation of synovial fibroblast proliferation, osteoclast differentiation, matrix metalloproteinase (mmp), and vascular endothelial growth factor (vegf) production, as well as lymphocyte differentiation and its elevation in both serum and synovial fluids of patients with ra [1722] indicate that il-6 is one of the key cytokines involved in the development of ra . It has been demonstrated in animal model of ra, that are type ii collagen - induced arthritis (cia), and antigen - induced arthritis, il-6 performs a major role in the development and progression of joint destruction, while il-6 deficiency generated by gene knockout or il-6 blockade by means of anti - il-6r ab reduces the incidence and severity of arthritis in these models [2328]. In the cia model, immunization with type ii collagen predominantly increased the frequency of th17 cells and treatment of mice with anti - il-6r ab during priming markedly suppressed the induction of th17 cells and arthritis development, while treatment with anti - il-6r ab on day 14 failed to suppress both th17 differentiation and arthritis . Similarly, in a glucose-6-phosphate - isomerase- (gpi-)induced arthritis model, administration of anti - il-6r ab on day 0 or 3 suppressed th17 differentiation and protected against arthritis induction, while injection of anti - il-6r ab on day 14, at the peak of arthritis, did not bring about any improvement in arthritis . Arthritis of anti - type ii collagen antibody - induced arthritis (caia) is another arthritis model, but, in this model, the priming phase of t cell dependent antibody generation is skipped . Although il-6 is also elevated in this model, caia was profoundly suppressed in tnf mice but not in il-6 mice, indicating that tnf may play a more significant role in the development of caia than il-6 . These observations suggest that in the priming phase il-6 is a required factor for the activation of t cell response and production of antibodies specific for joint components and that in the effector phase tnf is the main generator of arthritis . We found that tocilizumab was not effective for clinical improvement in the condition of two patients with psoriatic arthritis, for whose development immune activation does not appear to be required . The clinical antiarthritic effect of tocilizumab is slower than that of tnf inhibitors, which may be due to the different pathological roles of il-6 and tnf in the development of ra (figure 1). As shown in table 1, seven phase iii clinical trials of tocilizumab subsequent to phase i and ii studies demonstrated its efficacy either as monotherapy or in combination with disease - modifying antirheumatic drugs (dmards) for adult patients with moderate to severe ra [3440]. A cochrane database systematic review concluded that tocilizumab - treated patients taking concomitant methotrexate were four times more likely to achieve american college of rheumatology (acr) 50 improvement (absolute%, 38.8% versus 9.6%) and 11 times more likely to achieve disease activity score (das) remission (30.5% versus 2.7%) than patients taking a placebo . Furthermore, the samurai and lithe studies proved that radiological damage of joints was significantly inhibited by the treatment . The findings of the radiate trial showed that, among ra patients who had previously discontinued tnf inhibitors 50% achieved acr20, 28.8% acr50, and 12.4% acr70 responses . The acr improvement and das remission criteria include an acute - phase reactant component, so that there was concern that the effect of tocilizumab evaluated with these criteria might be overestimated . However, it was found that, even when criteria such as the simplified disease activity index (sdai) and clinical disease activity index (cdai) were used, remission rates for patients treated with tocilizumab were in the same range as those for patients treated with tnf inhibitors [42, 43]. On the basis of the excellent results obtained for the efficacy of tocilizumab, it was approved in april 2008 for the treatment of ra in japan . The recommended posology of tocilizumab (proprietary name, actemra) is 8 mg / kg, every 4 weeks . Subsequently, the european medicines agency approved tocilizumab (proprietary name, roactemra) for ra in january 2009 at a recommended dose of 8 mg / kg . In the united states, it was approved for ra in january 2010, but the recommended starting dose is 4 mg / kg administered once every 4 weeks followed by an increase to 8 mg / kg depending on clinical response . While the dosage differs among countries, tocilizumab has now been approved for the treatment of ra in more than 90 countries worldwide . In addition to clinical trials, the efficacy of tocilizumab was reconfirmed in actual medical practice . The finding by the three recent studies, the german phase iiib real - life study (tamara study) [44, 45], the danish nationwide cohorts of ra patients (danbio registry) study, and the multicenter retrospective real - life study (reaction study) [47, 48] are shown in table 2 . In the tamara study, 286 patients were registered for an analysis of the effectiveness and safety [44, 45], 41.6% of whom had previously been treated with tnf inhibitors . Acr50 and acr70 response rates at week 24 were 50.7% and 33.9%, respectively, while 47.6% of the patients achieved das remission and 54.9% the european league against rheumatism (eular) good response . Remission rates with the new acr / eular boolean - based criteria for clinical studies were 15.0% after 12 weeks and 20.3% after 24 weeks, and cdai and sdai remission rates were 24.1% and 25.2%, respectively . For the danbio registry in denmark, the disease activity decreased at all - time points, with remission rates for tocilizumab treatment of 39% after 24 weeks and 58% after 48 weeks . These response rates were comparable to those found for patients switching to their second tnf inhibitors and to the response rates previously observed in phase iii clinical trials . In japan, 229 patients were registered in the reaction study for an analysis of the effectiveness of tocilizumab [47, 48]. Clinical remission at week 52 was observed in 43.7% of the patients, radiographic non - progression in 62.8%, and functional remission in 26.4% . The retention rates at 24 and 52 weeks were 79.5% and 71.1%, respectively, and were the same for those with or without previous anti - tnf treatment . These results indeed show the efficacy of tocilizumab for the treatment of ra in actual medical practice . The safety and tolerability profiles of tocilizumab monotherapy for japanese ra patients obtained from six initial trials and five long - term extensions have been published . For these studies, 601 patients with a total exposure to tocilizumab of 2,188 patient - years (pt - yr) were enrolled . The incidence of adverse events (aes), including abnormal laboratory test findings, was calculated as 465/100 pt - yr, with infections being the most common serious aes (6.2/100 pt - yr). Of the patients treated more than 5 years, 59.7% met the das28 remission criteria at 5 years, which demonstrates the excellent tolerability and high efficacy of tocilizumab . In addition, a systemic literature review to assess the risk of aes for ra patients treated with tocilizumab reported that pooled odds ratios (ors) indicated statistical significance for an increased risk of aes for patients treated with 8 mg / kg of tocilizumab plus methotrexate compared with controls (or = 1.53; 95%ci = 1.261.86), as well as a heightened risk of infection (or = 1.30; 95%ci = 1.071.58). The results of an interim analysis of a postmarketing surveillance of all patients treated with tocilizumab in japan were recently reported . This analysis comprised 3,881 patients who received 8 mg / kg of tocilizumab every 4 weeks, and was observed for 28 weeks . Occurrence of a total of 3,004 aes in 1,641 patients (167/100 pt - yr) and 490 serious aes in 361 patients (27/100 pt - yr) was reported . The most frequent ae and serious ae were infection at 31/100 pt - yr and 9/100 pt - yr, respectively, with the majority of infections being pneumonia and cellulitis . . Abnormalities in laboratory test findings, such as increases in lipid and liver function parameters were common, and total and serious aes associated with laboratory test abnormalities were 35/100 pt - yr and 2/100 pt - yr, respectively . The increased lipid level resulting from tocilizumab administration is perhaps mediated by its effecting on lipoprotein receptor expression, since it was recently shown that overproduction of il-6 reduces blood lipid levels via upregulation of very - low - density lipoprotein receptors . In contrast, we and others observed that hba1c levels and insulin sensitivity improved as a result of tocilizumab treatment [53, 54]. While white blood cell and neutrophil counts usually decreased just after tocilizumab injection, this was not related to the incidence of infection . Twenty - five patients died for a standardized mortality ratio of 1.66, which was similar to the results reported for a japanese cohort study of ra . The results of this analysis thus demonstrated that tocilizumab is acceptable in the actual clinical setting . Seven cases of gastrointestinal (gi) perforation in six patients were reported in this postmarketing surveillance . In the worldwide roche clinical trials, 26 (0.65%) cases of gi perforation were found among patients with ra treated with tocilizumab for a rate of 1.9/1,000 pt - yr and most cases appeared to be complications of diverticulitis . This rate is intermediate between the rates of gi perforations of 3.9/1,000 pt - yr for corticosteroids and 1.3/1,000 pt - yr for anti - tnf agents reported in the united health care database . The reactivation of tuberculosis is a major concern during anti - tnf treatment, but there is no medical consensus regarding the effect of il-6 blockade on tuberculosis . Examined the effects of il-6 and tnf blockade on the development of tuberculosis infection in mice and observed that there was less tuberculosis infection for anti - il-6r ab than for anti - tnf ab . In addition, we showed that tuberculosis antigens - induced interferon (ifn)- production was suppressed by the addition of tnf inhibitors (infliximab and etanercept) but not of tocilizumab . Although it seems likely that the incidence of reactivation of tuberculosis is lower during tocilizumab treatment than that during anti - tnf treatment, further detailed studies will be needed to clarify this point . These include anti - tnf blockers (infliximab, etanercept, adalimumab, golimumab, and certolizumab), an il-1 antagonist (anakinra), a b - cell depletor (rituximab), an il-6 receptor inhibitor (tocilizumab), and a t - cell activation blocker (abatacept). These biological modifiers target different molecules and b cells, leading to different clinical effects and causing different adverse effects . Since no head - to - head comparative studies have been made of the efficacy of these various agents, it has not yet been determined which of these biologics should be selected for a given patient . Currently, one of the anti - tnf drugs is chosen as a first - line biologic, but between 14 and 38% of patients show no or little response to anti - tnf treatment, with as many as 40% of patients discontinuing these drugs within a year and 50% within 2 years . The findings of the radiate trial showed that ra patients who had previously discontinued tnf inhibitors, mainly due to their inefficacy, achieved acr20/50/70 responses of 50%, 28.8%, and 12.4%, respectively, when tocilizumab was administered at 8 mg / kg every four weeks . At present, tocilizumab is likely to be prescribed as a second - line biologic therapy but will have to overcome significant competition from established anti - tnf therapies . It is anticipated that tocilizumab will be selected as a first - line biologic for moderately to severely active ra patients with certain complications . Aa amyloidosis is a serious complication of ra, and amyloid fibril deposition causes progressive deterioration in various organs [59, 60]. Since the gene activation of serum amyloid a, a precursor protein of amyloid a fibril, depends primarily on il-6 [61, 62], tocilizumab administration was found to promptly reduce serum concentrations of saa, just as in the case of crp . Three case reports showed the clinical ameliorative effect of tocilizumab on gastrointestinal symptoms due to intestinal amyloidosis [6365], and amyloid a fibril deposits were found to have disappeared in two cases after three injections of tocilizumab [63, 65]. This suggests that tocilizumab may be suitable as a first - line drug for ra patients who are complicated with or are at high risk of developing aa amyloidosis . Remission induction is the current goal for ra, and with the development of biological modifiers, a growing number of ra patients has been able to achieve this goal . The long - term efficacy after cessation of tocilizumab followed by das28 remission was demonstrated in the dream (drug - free remission after cessation of actemra monotherapy) study . The continuous rate of tocilizumab - free efficacy was 35.1% at 24 weeks and 13.4% at 52 weeks . Serum levels of il-6 and mmp-3 are useful markers for identifying patients who may be able to discontinue tocilizumab without risk of recurrence . In addition, the restore study (retreatment efficacy and safety to tocilizumab in patients with rheumatoid arthritis at recurrence) demonstrated that retreatment of all relapsed patients with tocilizumab resulted in re - remission . Systemic lupus erythematosus (sle) is a multisystem autoimmune disorder with a broad spectrum of clinical presentations of unknown etiology that mainly affects young women . The pathogenesis of sle remains unclear, but the concept of apoptosis goes some way towards explaining how the immune system may recognize mainly intracellular antigens . Defects in the clearance of apoptotic cells have been recognized in sle patients, leading to aberrant uptake by macrophages, which then present intracellular antigens to t and b cells, thus driving the autoimmune process . Cytokine dysregulation is pervasive, and its expression profiles may serve as a marker of disease activity and severity . Recent findings have highlighted type i interferon pathway or th17 cell activation in the pathogenesis of sle . Levels of crp have been shown to rise in acute illness but not in sle flares, indicating that il-6, a major regulator of crp production, has a minor role in sle development . However, recent findings suggest that crp dysregulation also plays a part in the pathogenesis of sle and sle may well be a potential target for il-6 blockade . Urinary excretion and renal expression of il-6 was elevated in sle patients with active proliferating lupus nephritis [76, 7881], as were il-6 levels in the cerebrospinal fluid of sle patients with central nervous system involvement . Compared to healthy controls, sle patients had significantly more il-6 secreting peripheral blood mononuclear cells [83, 84]. Lymphoblastoid cells isolated from sle patients produced higher levels of il-6 and blocking of il-6 inhibited anti - double - stranded dna (dsdna) ab production in vitro [85, 86], indicating that il-6 is involved in autoantibody production . In murine sle models, age - associated increases in serum il-6, soluble il-6r, and abnormal expression of il-6r have been detected in mrl / lpr mice [8789]. In old nzb / w mice, anti - il-6 ab reduced and exogenous il-6 increased production of igg dsdna ab by b cells [90, 91]. Furthermore, il-6 administration exacerbated glomerulonephritis [92, 93], while il-6 blockade by means of anti - il-6r or anti - il-6 ab prevented the onset and progression of the disease [94, 95]. Mice with epidermal loss of junb reportedly developed an sle phenotype linked to increased epidermal il-6 secretion, and facial skin biopsies of sle patients displayed low levels of junb protein expression, high il-6, and activated stat3 levels within lupus lesions . These findings led to an open - label phase i dosage - escalation study of tocilizumab (2 mg / kg, 4 mg / kg or 8 mg / kg, every 2 weeks for 12 weeks) with an enrollment of 16 sle patients with mild - to - moderate disease activity . Significant improvement in the modified safety of estrogens in lupus erythematosus national assessment version of the systemic lupus erythematosus disease activity index score was observed in 8 of the 15 evaluable patients, accompanied by a median reduction in anti - dsdna ab levels of 47% . The percentage of cd38cd19igd plasma cells in the peripheral blood, which was higher for sle patients than for normal controls (mean 5.3% versus 1.2%), was significantly reduced to 3.1% at 6 weeks . Systemic sclerosis (ssc) is a connective tissue disease, characterized by fibrosis of the skin and internal organs, vasculopathy, and immune abnormalities . Il-6 in the serum of ssc patients was reportedly elevated and the level correlated with the skin severity score [100104]. Moreover, the culture supernatants of peripheral blood mononuclear cells and skin tissues from ssc patients contained higher concentrations of il-6 than those from controls [105109]. In vitro studies demonstrated that il-6 may contribute to fibrosis by inducing collagen production and induce -smooth muscle actin (-sma) expression by dermal fibroblasts, leading to their differentiation into myofibroblasts . On the other hand, anti - il-6 ab suppressed procollagen type 1 production in fibroblasts derived from ssc patients in vitro . Ssc serum mediated largely by il-6 was found to induce endothelial cell activation and apoptosis in endothelial cell - neutrophil cocultures . Il-6 is also associated with humoral and cellular immunological abnormalities in ssc [98, 99]. Il-6 is thus thought to play a significant role in producing the characteristics of ssc . Moreover, in a ssc model mouse, induced by immunization with topoisomerase i and complete freund's adjuvant, loss of il-6 expression could ameliorate skin and lung fibrosis . We also examined the clinical effect of tocilizumab on two diffuse ssc patients who had been resistant to conventional treatment regimens . Six months after the treatment, both patients showed softening of the skin with reductions of 50.7% and 55.7% for the total z - score determined with the vesmeter, a novel device for measuring the physical properties of the skin, and of 51.9% and 23% for the modified rodnan total skin score . Histological examination showed thinning of the collagen fiber bundles and reduction of the number of -sma positive cells in the dermis . Since there are few therapeutic drugs for ssc at the present time, these improvements suggest that tocilizumab appears to be a promising biologic for the treatment of ssc . The inflammatory myopathies encompass a group of heterogenous muscle diseases which share the common clinical features of slowly progressive symmetrical muscle weakness, decreased muscle endurance, and fatigue . They include polymyositis (pm), dermatomyositis, and inclusion body myositis, but are generally considered to be distinct diseases with different pathophysiological mechanisms . Muscles produce il-6, and il-6 has been also shown to play a regulatory role in muscle wasting . Among these inflammatory myopathies, pm appears to be another suitable target disease for tocilizumab . Excessive il-6 expression has been found in the sera and infiltrating mononuclear cells in the muscles of pm patients [121123]. Infiltrating cytotoxic t cells are thought to be involved in muscle fiber damage, and il-6 functions as a helper factor in the induction of cytotoxic t cells . Moreover, in a model of myosin - induced experimental myositis it was shown that control mice developed clinically manifest muscle damage, whereas il-6-deficient mice showed no clinical or histological signs of muscle damage . In another model of pm, known as c - protein - induced myositis, intraperitoneal administration of anti - il-6r ab suppressed the severity of myositis preventatively as well as therapeutically . We tested the efficacy of tocilizumab in two pm patients who had been refractory to corticosteroids and immunosuppressive drugs . Creatine phosphokinase levels of both patients normalized and mr images showed the disappearance of high - intensity zones in the thigh muscles . These findings suggest that tocilizumab may also be effective as a novel drug for refractory pm . Dermatomyositis is a complement - mediated microangiopathy associated with destruction of capillaries, hypoperfusion, and inflammatory stress on the perifascicular regions, so that the pathology is different from that of pm . Production of il-6 and type i interferon signature genes was recently proposed as a biomarker for disease activity in childhood dermatomyositis, which thus may be another disorder suitable for tocilizumab targeting . The pathological consequence of such inflammation is destruction of the vessel wall, which is histologically detected as fibrinoid necrosis . Takayasu's arteritis (ta) and giant cell arteritis (gca) belong to an entity designated vasculitis syndrome, and involve both large and medium - sized arteries [129, 130]. The pathogenesis of ta and gca remains unclear, but it is clear that il-6 is involved in their development [129133]. Tocilizumab treatment for a 20-year - old woman with refractory active ta improved the clinical manifestations and abnormal laboratory findings, and subsequent studies reported that tocilizumab treatment induced a rapid remission in 2 patients with ta and 5 patients with gca . Surprisingly, two of the patients with gca went into remission without concomitant use of corticosteroids . Moreover, tocilizumab was also shown to be effective as rescue treatment for three gca patients for whom the prednisone dose could not be tapered to less than 30 mg / day . Positron emission tomography / ct scans revealed that in two patients generalized large - vessel vasculitis was detected during the active phase, which completely resolved upon a 6-month course of tocilizumab therapy . These reports strongly imply that il-6 inhibition may serve as an innovative strategy for the treatment of both ta and gca . However, several studies have suggested that gca patients with a lesser inflammatory response without an increase in il-6 expression were at a higher risk of developing ischemic manifestations than were other patients, since the angiogenic activity of il-6 offers protection against ischemia in such gca patients . These findings indicate that further clinical studies are required to evaluate the efficacy and safety of tocilizumab for gca and ta . It is worthy of note that il-6 has been also implicated in the development of other types of vasculitis syndrome such as polyarteritis nodosa (pan) and antineutrophil - cytoplasmic - antibody- (anca) associated vasculitis [139142]. However, so far there have been no reports about off - label use of tocilizumab for pan or anca - associated vasculitis . On the basis of excellent results of the efficacy of tocilizumab for castleman's disease [143, 144] and systemic juvenile idiopathic arthritis [145147], it has been approved and used as the first - line biologic in japan . Pilot studies and case reports with off - label use of tocilizumab also indicate the potential indications of this biologic for various other organ - specific autoimmune and chronic inflammatory diseases . These include relapsing polychondritis, acquired hemophilia a, autoimmune hemolytic anemia, adult - onset still's disease [151165], crohn's disease, bechet's disease with posterior uveitis, polymyalgia rheumatica [135, 168], remitting seronegative, symmetrical synovitis with pitting edema, spondyloarthritides [170175], graft - versus - host disease [176, 177], tnf - receptor - associated periodic syndrome, and pulmonary arterial hypertension complicated with castleman's disease or mixed connective tissue disease [179181]. Further clinical trials are essential, however, to evaluate the efficacy and safety of tocilizumab for these diseases . Acute il-6 synthesis provides a warning signal and protects the host from environmental stress, while its prolonged production causes the onset and progression of various autoimmune diseases . Several clinical trials have verified the efficacy and safety of tocilizumab for ra, systemic juvenile idiopathic arthritis and castleman's disease, resulting in approval of this innovative biologic for the treatment of these diseases . Case reports of off - label use or pilot studies have also raised the possibility that tocilizumab could become the biological drug of choice for other systemic autoimmune diseases including sle, systemic sclerosis, polymyositis and large vessel vasculitis . At present, the mechanisms through which tocilizumab exerts its clinical ameliorative effects on phenotypically different autoimmune diseases are not completely understood . Il-6 blockade may suppress autoantibody production or correct the imbalance of autoantigen - specific th17 and/or th1 versus treg . Thus, clarification of the mechanisms as well as further clinical trials to evaluate the efficacy and safety of tocilizumab for these diseases are important issues.
Femoral and humeral bones of balb / c mouse embryos were removed aseptically on 18.5 dpc, and they were microdissected into esz and growth plate (egp) under a stereomicroscope (zeiss stemi 2000-c, carl zeiss microimaging). Each region was minced and gently digested with 2 mg / ml of collagenase (wako pure chemical) at 37 c for 2 h. they were cultured in growth medium composed of iscove's modified dulbecco's medium (invitrogen) supplemented with 15% fetal bovine serum, and subjected immediately to retroviral infection . Fli1 was introduced into the pmys - ires - gfp vector . The full length ews the per cell normalization method (percellome method) was applied to esz and egp samples . A 10 l aliquot of each lysate was treated with dnase - free rnase a (nippon gene inc ., japan) for 30 min at 37 c, followed by proteinase k (roche diagnostics gmbh . Usa) was added to each well, shaken for 10 s four times and then incubated for 2 min at 30 c . Dna concentration was measured using a 96 well fluorescence plate reader with excitation at 485 nm and emission at 538 nm . Usa) was used as standard . As reported previously, the grade - dosed spike cocktails (gscs) made of the bacillus subtilis rnas corresponding to the sequences in the affymetrix genechip arrays (affx - thrx-3_at, affx - lysx-3_at, affx - phex-3_at, affx - dapx-3_at, and affx - trpnx-3_at) were prepared, and gscs were added to the sample homogenates in proportion to their dna concentrations . The genechip mouse genome 430 2.0 array (affymetrix) was hybridized with the crna generated from esz and egp cells, and murine ewing's sarcoma tissue (table 1). After staining with streptavidin phycoerythrin conjugates, arrays were scanned using an affymetrix genechip scanner 3000 and analyzed using affymetrix genechip command console software (agcc, affymetrix) and genespring gx 11.0.2 (agilent technologies) as described previously . The expression data for esz and egp cells were converted to percellome data, i.e., absolute copy numbers of mrna per one cell, by the homemade software scal4 (spike calculation version 4). Response linearity of the five gsc spikes and the location of spike probe sets in the histogram of all probe sets (fig . N = 3), all the pairs were plotted to a scatter graph as red (expression above detection level) or green dots (below detection level) with the data of five yellow spike probe sets (fig . If any samples did not draw a symmetric scatter plot with yellow dot on the diagonal line, the sample were rejected for evaluation, and they were subjected to additional analyses . Homemade software named rsort (roughness sort) was used . This program sorts the probe sets as upward or downward peaks in a 3d isobologram (fig . 2). To avoid biologically nonsense probe sets such as ones with expression below the detection level, the data were visually checked for their 3d isobologram shape . Femoral and humeral bones of balb / c mouse embryos were removed aseptically on 18.5 dpc, and they were microdissected into esz and growth plate (egp) under a stereomicroscope (zeiss stemi 2000-c, carl zeiss microimaging). Each region was minced and gently digested with 2 mg / ml of collagenase (wako pure chemical) at 37 c for 2 h. they were cultured in growth medium composed of iscove's modified dulbecco's medium (invitrogen) supplemented with 15% fetal bovine serum, and subjected immediately to retroviral infection . Fli1 was introduced into the pmys - ires - gfp vector . The full length ews the per cell normalization method (percellome method) was applied to esz and egp samples . Briefly a 10 l aliquot of each lysate was treated with dnase - free rnase a (nippon gene inc ., japan) for 30 min at 37 c, followed by proteinase k (roche diagnostics gmbh . Usa) was added to each well, shaken for 10 s four times and then incubated for 2 min at 30 c . Dna concentration was measured using a 96 well fluorescence plate reader with excitation at 485 nm and emission at 538 nm . Usa) was used as standard . As reported previously, the grade - dosed spike cocktails (gscs) made of the bacillus subtilis rnas corresponding to the sequences in the affymetrix genechip arrays (affx - thrx-3_at, affx - lysx-3_at, affx - phex-3_at, affx - dapx-3_at, and affx - trpnx-3_at) were prepared, and gscs were added to the sample homogenates in proportion to their dna concentrations . The genechip mouse genome 430 2.0 array (affymetrix) was hybridized with the crna generated from esz and egp cells, and murine ewing's sarcoma tissue (table 1). After staining with streptavidin phycoerythrin conjugates, arrays were scanned using an affymetrix genechip scanner 3000 and analyzed using affymetrix genechip command console software (agcc, affymetrix) and genespring gx 11.0.2 (agilent technologies) as described previously . The expression data for esz and egp cells were converted to percellome data, i.e., absolute copy numbers of mrna per one cell, by the homemade software scal4 (spike calculation version 4). Response linearity of the five gsc spikes and the location of spike probe sets in the histogram of all probe sets (fig . N = 3), all the pairs were plotted to a scatter graph as red (expression above detection level) or green dots (below detection level) with the data of five yellow spike probe sets (fig . If any samples did not draw a symmetric scatter plot with yellow dot on the diagonal line, the sample were rejected for evaluation, and they were subjected to additional analyses . Homemade software named rsort (roughness sort) was used . This program sorts the probe sets as upward or downward peaks in a 3d isobologram (fig . 2). To avoid biologically nonsense probe sets such as ones with expression below the detection level, we describe a unique dataset of mouse embryonic cartilage with or without the ewing's sarcoma fusion oncogene, ews fli1 . The dataset was used in the study published recently and was informative to understand the tumorigenic mechanisms of ewing's sarcoma.
Aspirin - exacerbated respiratory disease (aerd) refers to the development of bronchoconstriction in asthmatics following the ingestion of aspirin or other nonsteroidal anti - inflammatory drugs . It is defined by a clinical syndrome associated with moderate - to - severe asthma and eosinophil inflammation in the upper and lower airways, resulting in chronic rhinosinusitis and asthma . Additionally, the airways of aerd show epithelial disruption, cytokine production, and the upregulation of inflammatory molecules . The prevalence of aspirin hypersensitivity in the general population ranges from 0.6 to 2.5% and is higher in asthmatics . Metabolites involved are prostaglandins (pgs), leukotrienes (lts), and thromboxane (tbx). Inhibition of coxs by acetyl salicylic acid (asa) in the respiratory tract alters arachidonic acid metabolism, leading to a reduction in pge2 . The lipoxygenase (lox) pathway produces the leukotrienes lta4, ltb4, and ltc4 as metabolites . 15-lipoxygenase (15-lo) is one of the lox family members and catalyses the conversion of arachidonic acid to 15-hydroxyperoxyeicosatetranoic acid (15-hpete). 15-hydroxyeicosatetranoic (15-hete), a more stable derivative of 15-hpete, is another important product, which acts as an anti - inflammatory mediator and functional antagonist of lts . Further products of 15-hpete include eoxins (exs) exa4 and 15-hete can be conjugated with glutathione, leading to the formation of exc4, exd4, and exe4 . Aerd has also been correlated with increased cyslt receptors: cysltr1 and cysltr2 [79]. The third cyslt receptor, the g protein - coupled receptor 17 (gpr17), is located at an intermediate phylogenetic position between two distinct receptor families: the purinergic receptor (p2y) and cyslt receptor for extracellular nucleotides and cyslts, respectively, . Overexpression of cysltr1 was detected in the nasal mucosa of patients with aerd, compared with aspirin - tolerant asthma (ata). Considering the pathogenic mechanism of aerd, various genetic markers have been suggested in various ethnic groups and are summarized in this paper . Based on evidence showing a close association of leukotrienes and aerd, initial research was performed on the association between ltc4s 444a> c promoter polymorphism and aerd . In the population investigated (polish), the c allele was identified as a risk factor; however, this finding was not replicated in japanese, american, or korean populations [1215]. Snps of 5-lipoxygenase; alox5 at 1708 g> a, 21c> t, 270 g> a, and 1728 g> a and alox5 activating protein (alox5ap, 218a> g) were studied in a korean population where it was discovered that the haplotype alox5 ht1 [g - c - g - a] was significantly higher in aerd than in ata, suggesting a possible contribution of alox5 in aerd . We identified three snps (634c> t, 475a> c, and 336a> g) in the promoter region of cysltr1, and mutant variants of these snps were associated with the aerd phenotype . The mutant variants showed higher promoter activity, suggesting that these polymorphisms may modulate cysltr1 expression increasing aerd susceptibility . In the case of cysltr2, the frequencies of minor alleles for 819 t> g, 2078c> t, and 2534a> g were significantly higher in the aerd group when compared with ata . Decreased production of prostaglandin e2 (pge2) by nasal epithelial cells of aerd has been observed . Pge2 production in airway smooth muscle cells has been shown to downregulate cox2 mrna expression . Two snps of tbxa2r, 4684 t> c, and + 795 t> c, were shown to be associated with the phenotype of aerd in a korean population [22, 23]. The prostaglandin e2 receptor subtype 2 gene (ptger2) was associated with the risk of aerd by decreasing the level of transcription, resulting in a reduction of the pge2 braking mechanism of inflammation and involvement in the molecular mechanism underlying aerd in the japanese population . A further report in the korean population showed that prostaglandin e2 receptor subtype 3 (ptger3) may be an important genetic factor for aspirin intolerance in korean asthmatics . The human leukocyte antigen (hla) allele dpb1 * 0301 was identified as a strong marker for aerd, because patients with this allele showed typical characteristics of aerd including a decreased forced expiratory volume in 1 s (fev1) and increased prevalence of rhinosinusitis with nasal polyps, as previously noted in a polish population . Increased numbers of eosinophils and mast cells have been observed in the bronchial mucosa of aerd [28, 29]. Recent studies demonstrated that the chemoattractant receptor molecule expressed in th2 cells, the crth2 466 t> c polymorphism, could increase serum and cellular eotaxin-2 production by lowering crth2 expression, leading to eosinophilic infiltration in aerd patients . A further study indicated that the chemokine cc motif receptor (ccr3) may be related to eosinophil migration . The ccr3 520 t> c was significantly associated with aerd patients where mrna expression was also significantly increased after asa provocation . Il-13 polymorphisms at 1510a> c and 1055c> t are associated with the development of rhinosinusitis in aerd patients . Il-13 arg110gln may be associated with an increased eosinophil count and eotaxin-1 level, leading to an increase in eosinophilic inflammation in the upper and lower airways of patients with aerd (table 1). Viral respiratory infections have been suggested to contribute to allergic sensitization, leading to the development of asthma and in subjects with established asthma; they are known to exacerbate allergic disease . Aspirin hypersensitivity is diminished in some aerd patients during acyclovir treatment of herpes simplex infection . Moreover, elevated levels of igg4, derived from chronic antigenic stimulation of viral origin, have been noted in aerd patients . A further study investigating the exacerbation of aerd with airway infection of respiratory syncytial virus was reported . Recently, a study indicated that the polymorphisms in the toll - like receptor 3 (tlr3) gene, tlr3 299698 g> t and 293391 g> tlr3 recognizes dsrna, activates nuclear factors, and increases interferon - gamma, which is a signal to other cells and increases antiviral defenses . As functional deterioration of tlr3 can predispose individuals to increased susceptibility to viral infections, the detection of tlr3 polymorphisms may be informative for risk assessment in aerd susceptibility . The suggested mechanism is that specific cytotoxic lymphocytes are produced in response to viral infection . Activity of these lymphocytes is suppressed by pge2, which is produced by pulmonary alveolar macrophages . If pge2 levels are decreased, cytotoxic reactions are preceded by cox inhibitors and cytotoxic lymphocyte - mediated attacks lead to the destruction of virus affected cells in the respiratory tract . Reactive oxygen species, toxic metabolites, and mediators released then precipitate asthma attacks . The ubiquitin - proteasome pathway - related gene (ube3c) has been recently studied in a korean population and indicated that rs3802122 and rs6979947 is associated with aerd . A further study indicated that the kinesin family number 3a (kif3a) gene and its polymorphism might have an effect on aerd, because rs3756775 revealed a significant association with the percentage decline in fev1 after aspirin provocation . Recently, the genome - wide methylation profile of nasal polyps showed that genes involved in lymphocyte proliferation, cell proliferation, leukocyte activation, cytokine biosynthesis, immune responses, inflammation, and immunoglobulin binding were hypomethylated . In the arachidonic pathways, pgds, alox5ap, and ltb4r the calcium channel voltage - dependent gamma subunit 6 (cacng6) gene encodes a protein that stabilizes the calcium channel . Cacng6 has been studied in aerd, which revealed that rs192808c> t may be associated with the risk of aerd in a korean population . Genome - wide association studies (gwas) have recently emerged as a technology that can predict genetic variations across the genome associated with human diseases and clinical responses to drug treatment . Candidate gene approaches have been used for most of the genetic association studies of aerd . Gwas suggested that the nonsynonymous cep68 rs 7572857 g> a variant, replacing glycine with serine, showed a higher decline in fev1 due to aspirin provocation than other variants and could be a susceptible gene for aerd . Gene - gene interactions have also been proposed in the pathogenesis of aerd, and a few studies indicated that the genetic effects of cyslts and ltc4s 444a> c synthesis increased the lower level of fev1 after lysine asa inhalation . Tbxa2r 795 t> c polymorphism was associated with hla dpb1 * 0301 in aerd patients compared with ata . Recently, a synergistic effect between the tgf - beta1 - 509c / t and il-10 - 1082a / g polymorphisms on the phenotype of aerd was noted when stratified by the presence of rhinosinusitis . Moreover, kim et al . Reported a significant epistatic effect with a four - locus genetic interaction in the susceptibility to aspirin intolerance in asthmatic patients . This model includes four snps: b2adr 46a> g, ccr3 520 t> g, cysltr1 634c> t, and fcer1b 109 t> c . Aerd often produces a moderate - to - severe phenotype; however, diagnosis in these patients is challenging despite the availability of various techniques . A hypothesis has been put forward, mostly focused on the overproduction of cyslts and arachidonic acid pathways . Most of the genetic studies have been performed using techniques such as gwas and the candidate gene approach . However, replication studies in different ethnic groups will be essential to validate the reported data and apply this knowledge in clinical practice . Future areas of investigation should focus on identification of biomarkers for early diagnosis with various diagnostic techniques . These genetic studies will be able to extend our understanding about the molecular genetic mechanism of aerd and to find a genetic marker for predicting drug responses or hypersensitivity reactions . Furthermore, this will be helpful for the determination of new diagnostic tools and therapeutic interventions.
Radical prostatectomy (rp) is the standard of care among the treatment options for patients with clinically localized prostate cancer, especially in men with a life expectancy> 10 years . A recent rp series reported the positive surgical margin (psm) rate to range from 11% to 38% . Psm is defined by most investigators as an extension of the tumor to the inked cut surface of the resected specimen . A psm is more likely to occur in cases with extracapsular extension, if the prostate is not resected widely enough . A psm can also occur in cases of organ - confined disease if resection is performed too close to the prostate, which is often referred to as a capsular incision . A psm suggests incomplete local resection, poor cancer control, and suboptimal patient outcome . In addition, the number and extent of the psm have been shown to be risk factors for biochemical recurrence (bcr) after rp . Laparoscopic radical prostatectomy (lrp), and more recently robot - assisted laparoscopic radical prostatectomy (ralrp), have been accepted as alternatives to open surgical methods at many institutions . These methods have shown comparable oncological and functional outcomes in recent studies, including for psm status . When evaluating the efficacy of rp for treating localized prostate cancer and also for considering possible adjuvant therapy, psm status is an important factor, regardless of approach . Previous data have demonstrated adverse oncologic outcomes associated with psm in patients undergoing rp, although the location of the psm and its effect on bcr - free survival of rp have rarely been examined in patients who have undergone four different types of rp . In this study, we analyzed the location of the psm and its association with the bcr rate in patients who had undergone four different types of rp in a single center . We retrospectively analyzed the medical records of 1,880 patients who underwent rp between september 1995 and december 2011 by five surgeons at samsung medical center, with approval from the institutional review board (2014 - 06 - 049). Among a total of 1,880 patients, 633 patients underwent radical perineal prostatectomy (rpp), 309 patients underwent radical retroperitoneal prostatectomy (rrp), 164 patients underwent lrp, and 774 patients underwent ralrp . The presence of a psm, age, preoperative prostate - specific antigen (psa), prostate size, follow - up length, biopsy gleason score (gs), pathologic gs, clinical stage, pathologic stage, presence of nerve - sparing, and presence of bcr were recorded retrospectively . Locations of the psm were recorded in the four groups as apex, anterior, posterolateral, and base . All rp specimens were coated with ink, sectioned at 3- to 4-mm intervals, analyzed by the same pathology department, and processed by using the stanford technique . The surgical margin was considered positive when the tumor was found at the inked surface . Postoperative follow - up visits were typically scheduled at 3-month intervals for 1 year, biannually for the second and third years, and yearly thereafter . Bcr was defined as two consecutive psa measurements 0.2 ng / ml . Rpp was performed as previously described by harris . Ralrp was performed by the transperitoneal antegrade approach with the use of the da vinci robot system (intuitive surgical inc ., the choice of surgical approach was made by the patient's physician on the basis of the patient's preference after a discussion of benefits, risks, alternatives, and also the special conditions of each patient . In each of the surgical groups, unilateral or bilateral nerve preservation was considered and performed if clinically indicated by preoperative erectile function, age, and oncological parameters . The baseline characteristics of the patients were analyzed with percentages for categorical factors or with the mean and standard deviation for continuous factors . Categorical factors were compared by using the chi - square test and fisher exact test, and continuous factors were compared by using the kruskal - wallis test . The estimated risk of bcr according to site of psm was determined by using kaplan - meier survival analysis and was compared by use of log - rank tests . In all of the tests, 9.4 (sas institute inc ., cary, nc, usa) and r ver . 3.0.3 (r foundation for statistical computing, vienna, austria; http://www.r-project.org/). The total number of patients was 1,880, with 633 (33.7%) undergoing rpp, 309 (16.4%) undergoing rrp, 164 (8.7%) undergoing lrp, and 774 (41.2%) undergoing ralrp . There were no significant differences in mean age, psa level, or prostate size according to operative group (p=0.487, p=0.084, and p=0.389, respectively). The biopsy gs distribution and the clinical stage did not differ significantly by surgical type (p=0.841 and p=0.136, respectively). The pathologic gs distribution and the pathologic stage also did not show a significant difference by surgical type (p=0.783 and p=0.133, respectively). A psm was found in a total of 336 cases (17.9%). Of these, there were 122 cases (18.4%) of rpp, 67 cases (21.7%) of rrp, 28 cases (17.1%) of lrp, and 119 cases (15.4%) of ralrp . The location of the psm was the apex in 136 cases (7.2%), anterior in 67 cases (3.5%), posterolateral in 139 cases (7.3%), and base in 95 cases (5.0%), and did not differ significantly by surgical type (p=0.536, p=0.557, p=0.062, and p=0.109, respectively) (table 2). In all patients, the median follow - up was 48.2 months (interquartile range [iqr], 33.4 - 64.7 months). The median follow - up of each operative group was 67.7 months (iqr, 40.7 - 90.9 months) for rpp, 45.9 months (iqr, 32.4 - 64.4 months) for rrp, 41.4 months (iqr, 27.5 - 55.8 months) for lrp, and 43.5 months (iqr, 32.4 - 55.5 months) for ralrp (p<0.001). The total number of cases of bcr was 390 (20.7%), with 181 cases (28.5%) in the rpp group, 81 (26.2%) in the rrp group, 24 (14.6%) in the lrp group, and 104 (13.4%) in the ralrp group . Cases with a psm showed a higher bcr rate than did cases with a negative surgical margin (p=0.04). In the psm group, the bcr rate did not differ significantly according to the location of the psm in all rp groups combined (p=0.469). The bcr rate according to the location of the psm also did not show a significant difference in the kaplan - meier survival analysis for the four types of surgery (p=0.694, p=0.301, p=0.445, and p=0.309 for rpp, rrp, lrp, and ralrp, respectively) (fig . Yossepowitch et al . Demonstrated that a psm in rp specimens is an adverse outcome following rp . Pathologic tumor margin status seems to be comparable between open, laparoscopic, and robotic series overall . However, the location of the psm and its effect on bcr - free survival of rp has rarely been examined among patients according to four different types of rp in a single center . Therefore, we explored the location of the psm and its association with the bcr rate between four different types of rp . We found that the location of the psm seemed to be unrelated to the type of rp . In addition, the bcr rate did not differ significantly according to the location of the psm in each type of rp . In one retrospective study, there was no significant difference in the incidence of a psm between the rpp (22%) and rrp specimens (16%), and each had a 4% incidence of capsular incision . Moreover, no significant difference was found in the time to psa failure between patients who had undergone rpp with complete excision of the seminal vesicles and those who had undergone rrp . Rpp showed proven long - term cancer control of rrp with rapid convalescence and low morbidity . In a retrospective comparison of bcr, no significant difference was found between the ralrp group and a contemporary series of rrp performed at a single center after control for clinical and pathologic features . In most series of lrp and ralrp . Showed that regardless of surgical approach, the most common site of a psm is the prostatic apex and that insufficient removal of prostatic tissue at the apex for optimizing urethral length and avoiding incontinence can result in psms, even with tumors that do not violate the capsule pathologically (i.e., stage pt2). In addition, brown et al . And khan and partin demonstrated that comparison of surgical margin status between high - volume centers with operations performed by experienced surgeons shows a definitive advantage in achieving negative surgical margins for one surgical approach over the other . These results differ from the results of our study, most likely due to differences in patient selection . Patient selection is the primary factor that determines the psm rate in a given series . The experience of surgeons and the method and detail of pathologic analysis also seems influential . Although this study was retrospective and was performed at a single institution, this enabled a standardized review of all pathology specimens of the four different types of rp and strengthened our study . This study was limited by the difference in follow - up length according to type of surgery . The bcr rate of lrp or ralrp was relatively low compared with the bcr rate for rpp or rrp . The median follow - up and bcr rate according to type of surgery showed statistically significant differences (p<0.001 and p<0.001, respectively) (table 1). Therefore, the tendency of a higher bcr rate of rpp or rrp may come from the relatively shorter follow - up of lrp or ralrp . Oncologic outcomes with 1,000 consecutive lrps performed over a 4-year period with a median follow - up period of 12 months were reported by guillonneau et al . . The rates were 92% for pt2a, 88% for pt2b, 77% for pt3a, and 44% for pt3b by pathologic stage . The actuarial, 3-year, bcr - free survival was 94.5% overall, 98.2% for pt2, and 78.7% for pt3 disease . These two studies of lrp reported better bcr - free survival than did our study (89.7%). This difference in bcr - free survival may be due to the shorter mean follow - up length of those studies compared with our study . Meanwhile, with regard to ralrp, badani et al . Reported a large series of 2,766 consecutive ralrps with a mean follow - up period of 22 months . Their overall actuarial 5-year, bcr - free survival was 84% overall, 84% for pt2, and 66% for pt3 patients, which was a worse bcr - free survival than in our study (90.3%). This difference may be due to the shorter mean follow - up length of our study compared with the study of badani et al . . Because of the relatively long time period included in this study, the learning curve with surgery or progression of surgical technique may have had an effect . Especially, ralrp is a relatively newer method, so the learning curve may have influenced oncologic outcome . Each method of rp was performed by more than one surgeon, except rpp and lrp . Despite this limitation, considering the lack of cases when performing four types of rp in a single center, this study offers a valuable comparison of the different types of rp . In summary, the location of the psm does not seem to be related to the type of rp . The bcr rate according to the location of the psm in each type of rp also showed no significant difference . In the end, a long - term, prospective and randomized trial with a sufficiently large number of cases from established and experienced centers of excellence is required to compare the results of lrp or ralrp with the results of rpp or rrp . In this study, we presented the oncologic outcomes in a large contemporary cohort of patients undergoing four different types of rp . The bcr rate also showed no significant difference according to the location of the psm in each type of rp.
Breast cancer is the most common malignancy in women and number one cause of death in women between the ages of 45 and 55 in the united states . Although metastatic disease, considered incurable, is rarely seen at the time of initial diagnosis, approximately 20% of women with operable breast cancer eventually relapse, with about 70% of the relapses as distant metastases [25]. Approximately 80% of women with metastatic breast cancer have skeletal metastases, which are often the result of bone marrow infiltration of malignant cells with subsequent progression and invasion of the skeletal cortex [69]. The most common complications of skeletal metastases are pathologic fractures, spinal cord compression as the result of vertebral compression fracture or extension of the tumor beyond the epidural space, and hypercalcemia [79]. Furthermore, skeletal metastases sometimes require surgery or radiation therapy to treat pain or an impending fracture . Bone marrow metastases result in the invasion and destruction of the bone tissue matrix by tumor cells . Although bone marrow infiltration by metastases is commonly present among breast cancer patients, total bone marrow infiltration resulting in profound pancytopenia is extremely rare . A 62 year - old female presented with increased fatigue that was interfering with her activities of daily living . Her breast exam was notable for a fixed, 1 cm left axillary lymph node . Her white blood cell (wbc) count was 3.2 k/l, hemoglobin (hgb) was 6.8 g / dl, and platelet count was 3 k/l . Multiple imaging studies, including computed tomography (ct) of the chest, abdomen and pelvis, as well as a bone scintography were completed as part of the initial diagnostic work up . The bone scintography scan showed diffuse skeletal metastatic disease involving multiple vertebrae and the pelvis . A mammogram had also been performed and showed an irregular spiculated nodule in the upper inner left breast . The patient subsequently underwent a core needle biopsy of an enlarged left axillary lymph node . The biopsy revealed a metastatic lobular carcinoma strongly positive for both estrogen and progesterone receptors (er and pr) and negative for her2 and e - cadherin by immunohistochemical staining . To further evaluate the patient s profound pancytopenia, which required frequent transfusion of packed red blood cells (prbc) and platelets, the patient underwent a bone marrow biopsy . The pathology showed that the metastatic carcinoma had entirely replaced the bone marrow (figure 1). The tumor cells were positive by immunohistochemical staining for cytokeratin ae1/ae3, er and pr, but were negative for her2, consistent with metastatic breast cancer . After much discussion of the risks and benefits of therapy in view of profound pancytopenia, the patient initiated systemic therapy with doxorubicin administered as a continuous three day infusion (20 mg / m / day) on a 21 day cycle in the inpatient setting . A continuous infusion of doxorubicin was selected based on small, prior studies suggesting that acute and chronic toxicities, including bone marrow suppression, may be lessened when doxorubicin is administered as a continuous versus bolus infusion [1113]. The doxorubicin dose was reduced by 50% due to increased transaminases that normalized with subsequent cycles of therapy . The second cycle was given at full dose, but was complicated by febrile neutropenia requiring hospitalization . After 3 cycles of therapy, continuous doxorubicin was switched to monthly liposomal doxorubicin (40 mg / m) for ease of administration in the outpatient setting since the patient s platelets had sufficiently recovered . As shown in figure 2, approximately four months after commencing treatment, her wbc rose to 5.4 k/l, hgb increased to 11.6 g / dl and platelet count improved to 131 k/l . Approximately thirteen months after beginning the treatment, the patient experienced a near full recovery from her pancytopenia with no evidence of anemia or thrombocytopenia . Her laboratory tests showed wbc of 4.8 k/, rbc of 4.00 m/l, hgb of 12.8 g / dl and platelet count of 160 k/l at that time, indicating a remarkable response to treatment (figure 2). After four cycles of liposomal doxorubicin, the patient was transitioned to endocrine therapy with an aromatase inhibitor, letrozole . Her laboratory profile remained stable 43 months after initiation of continuous doxorubicin . In her last 12 months of life, her disease progressed in the liver, bone, orbit and brain . She was treated with radiation to the orbital metastasis and brain metastases, but declined further systemic chemotherapy . In her last month of life, her platelets dropped to 15 with normal white blood count and hemoglobin levels . She ultimately died from complications of metastatic breast cancer 44 months after her initial diagnosis . . However, bone marrow failure as the herald of this disease is not typically seen . Very limited data exists as to the safest and most efficacious manner to treat patients with profound pancytopenia due to metastatic solid tumor involvement . In this case, the patient s thrombocytopenia was particularly worrisome, requiring daily platelet transfusions . There was also concern that cytotoxic chemotherapy would exacerbate the patient s thrombocytopenia and increase bleeding risk . The patient s dramatic response to chemotherapy with full platelet recovery is also highly unusual . For our patient, continuous doxorubicin successfully unpacked the bone marrow despite a low baseline platelet level, and without increasing the need for more frequent platelet transfusion or risk of catastrophic bleeding . Given the rarity of this presentation, it is currently unknown if the majority of similar patients experience near full recovery of hematopoietic function after initiation of appropriate systemic treatment for metastatic disease . Pancytopenia is not a common presentation in patients with metastatic breast cancer . In several studies, pancytopenia was noted as the consequence of adjuvant chemotherapy with alkylating agents and topoisomerase ii inhibitors rather than metastatic disease . Some have suggested that the use of growth factor support such as filgrastim or peg - filgrastim may also contribute to the risk of developing pancytopenia related to myelodysplastic syndrome or acute myeloid leukemia, although that association has not been completely established . Saito et al . Reported on a metastatic breast cancer patient with pancytopenia who had evidence of concurrent therapy - related acute myeloid leukemia and bone marrow metastasis . In our case, this has rarely been described in the literature and includes both patients with both positive and negative outcomes from therapy . Several reports highlight patients who have responded to systemic therapy including low dose capecitabine, endocrine therapy and trastuzumab monotherapy [2832]. . Reported on a case of a patient with bone marrow metastasis from breast cancer . The patient developed pancytopenia with disseminated intravascular coagulation (dic) and was given weekly paclitaxel therapy with blood transfusion and g - csf injections . Therapy was ineffective and the patient died of gastrointestinal hemorrhage due to complications of pancytopenia that were likely caused by dic and metastatic disease . Our patient experienced similar bone marrow metastasis and pancytopenia, but was able to achieve sustained disease control through doxorubicin chemotherapy followed by aromatase inhibitor therapy, without bleeding complications . In addition, laboratory tests at the time of diagnosis did not show any evidence of dic in our case . In addition to continuous doxorubicin, our patient received zoledronic acid as a part of management of osseous metastasis from breast cancer . Many studies have focused on the prevention of breast cancer due to subclinical bone marrow metastasis caused by dormant disseminated tumor cells (dtcs) in the bone marrow [1921]. Although dtcs appear to be associated with a higher risk of distant recurrence, they are not known to cause abnormalities in peripheral blood laboratory tests . Solomayer et al . Demonstrated the impact of zoledronic acid on dtcs in primary breast cancer patients . The authors found that dtc - positive patients treated with zoledronic acid were more likely to become dtc - negative following a 12 month treatment interval and concluded that treatment with zoledronic acid improved the elimination of dtcs . The role of bisphosphonates in preventing skeletal metastases in patients with high risk early stage breast cancer is controversial [20,2327]. The contribution of zoledronic acid in bone marrow recovery in this instance is not known . Breast cancer has a tendency to metastasize to the bone marrow, but does not commonly cause bone marrow failure . The case presented describes a patient who developed complete bone marrow infiltration by estrogen receptor positive metastatic breast cancer that led to transfusion dependent pancytopenia . The patient received treatment with continuous doxorubicin and zoledronic acid followed by endocrine therapy, which resulted in complete recovery of normal bone marrow function for 3 years . Drug therapy is a consideration in patients with bone marrow infiltration with good performance status.
The partograph is a very useful graphical record of the course of labor that yields optimum results when employed in labor management by obstetric caregivers (ocgs). As an obstetric tool, evidence abounds that the acquisition of knowledge of its use and ensuring proper application of that knowledge would culminate in a remarkable reduction in the incidence and outcomes of prolonged and obstructed labor, which are reported to be associated with 8%10% of maternal deaths.1,2 aside from the contributions of traditional birth attendants (tbas), who also give primary obstetric care, this service is rendered in nigeria and most developing countries by general duty doctors, nurses, and midwives, and community health workers of diverse training, including the community health extension workers (chews). Having knowledge and making use of this simple tool by these ocgs, will be an important step for designing appropriate intervention strategies that would encompass: training, retraining, and continuous professional educational programs to further empower them in safe motherhood practices . This study aimed to evaluate the ocgs knowledge of partograph use, assess the extent of its use, determine the factors that impede its usage, and unravel the relationship, if any, between years of experience and partograph use among the respondents in this research . The results of this study, in addition to encouraging the design of continuing professional education programs for all ocgs, will enhance the formulation of policies that will improve maternal and child health care delivery and identify areas for development and sustenance of improved midwifery practice that will lead to job satisfaction and delivering of high - quality care by all ocgs in general hospital, calabar, nigeria . This study would also form the basis for further research and will contribute to knowledge on intervention programs for maternal and child health in nigeria and other similar sub - saharan african countries whose roadmap to achieving the millennium development goal 5(mdg 5) in 2015 is still a difficult task ahead . The long - term objective and impact that this study would achieve shall be the continuous and effective use of the partograph in labor monitoring in general hospital, calabar . Prolonged and obstructed labor accounts for 8%10% of maternal deaths1,2 and mechanical obstruction in the second stage is a possible complication in about 1%2% of labors.3 the world health organization (who) estimated annual global obstructed labor - related maternal mortality at 50,000.4 this does not include cases of prolonged labor, which leads to life - long morbidities to both mother and child . One significant and unfortunate complication of both prolonged and obstructed labor is vesicovaginal fistula (vvf). The united nations population fund estimates that there are about 2 million women living with vvf, most of them in sub - saharan africa.5 obstructed labor is therefore a significant cause of maternal morbidity and mortality in nigeria and many other resource - poor areas of the world . The use of the partograph would engender gross reduction in the number of these deaths since abnormal markers in the progress of labor would be identified earlier.1,2,6 additionally, it has been reported that partograph use in obstructed labor management enhances maternal and neonatal well - being.6 this simple tool for the management of labor was formally introduced in the general hospital, calabar on november 15, 2012 . This coincided with the period that a high - profile patient was lost to prolonged and obstructed labor in the study facility . In an earlier study, the enormity of maternal wastage in labor, especially in rural areas of cross river state (crs), was reported.7 although the partograph has found use in many centers, inconsistency in utilization remains a problem in these centers . This was the driving force in the yenogoa study8 and also for the current study in calabar, another capital city in the same geo - political zone of the country . A review of literature shows that the southwestern geopolitical zone of nigeria has witnessed remarkable research on partograph use . This is equally true of the southeastern geo - political zone.913 ultimately, maternal health indices are more favorable in those two zones than the one studied . This unhealthy trend is also evident in the northern geo - political zones of the country.14 the current study is a pioneering research in the crs and the second in the south - south geo - political zone of nigeria . The adoption of the partograph by the who was borne out of the safe motherhood conference in nairobi, kenya in 1987, which set out to address the worrisome statistics of maternal and infant mortalities worldwide . When used effectively, the partograph will prevent prolonged or obstructed labor, which accounts for about 8%10% of maternal deaths.1,2 its use also aids continuity of care and helps the early recognition of abnormalities of labor.1,2 philpott indicated that the partograph serves as an early warning system and assists in early decision in the transfer, augmentation, and termination of labor.6 it is also important to update standards and protocol for service delivery, management and supervision, monitoring, and evaluation of the quality of services, along with feedback from the client and health providers . The partograph is being widely used in a number of countries, especially in developing countries . In nigeria, it is mostly used in teaching hospitals and rarely in general and cottage hospitals . The type of partograph in use involves a uniform spread of both the who type and plain composite partograph.15 the use of the partograph is hindered by poor knowledge,911,15 lack of the charts in the labor wards,10,11 shortage of health care personnel,10 time - consuming tasks for the low numbers of staff,16 and poor appreciation of its advantages in preventing obstructed labor.11 a notable fact in developing countries is that knowledge of the use of the partograph for labor management is very low among nurses, midwives, and doctors working in the primary and secondary health care levels and private health care centers when compared to tertiary level care.9,10,12 additionally, the general inability of the peripheral hospitals to produce benchmarks on the use of this chart in labor,10 poor managerial support regarding the procurement of necessary supplies,17,18and lack of motivation of the health workers,16constitute major obstacles in the use of the partograph . The adoption of the partograph by the who was borne out of the safe motherhood conference in nairobi, kenya in 1987, which set out to address the worrisome statistics of maternal and infant mortalities worldwide . When used effectively, the partograph will prevent prolonged or obstructed labor, which accounts for about 8%10% of maternal deaths.1,2 its use also aids continuity of care and helps the early recognition of abnormalities of labor.1,2 philpott indicated that the partograph serves as an early warning system and assists in early decision in the transfer, augmentation, and termination of labor.6 it is also important to update standards and protocol for service delivery, management and supervision, monitoring, and evaluation of the quality of services, along with feedback from the client and health providers . The partograph is being widely used in a number of countries, especially in developing countries . In nigeria, it is mostly used in teaching hospitals and rarely in general and cottage hospitals . The type of partograph in use involves a uniform spread of both the who type and plain composite partograph.15 the use of the partograph is hindered by poor knowledge,911,15 lack of the charts in the labor wards,10,11 shortage of health care personnel,10 time - consuming tasks for the low numbers of staff,16 and poor appreciation of its advantages in preventing obstructed labor.11 a notable fact in developing countries is that knowledge of the use of the partograph for labor management is very low among nurses, midwives, and doctors working in the primary and secondary health care levels and private health care centers when compared to tertiary level care.9,10,12 additionally, the general inability of the peripheral hospitals to produce benchmarks on the use of this chart in labor,10 poor managerial support regarding the procurement of necessary supplies,17,18and lack of motivation of the health workers,16constitute major obstacles in the use of the partograph . General hospital, calabar is a 100-bed secondary health care facility located in calabar, the capital city of crs . The hospital was completed and commissioned on november 7, 1991 . The hospital records showed that there were 2,370 deliveries and 588 cesarean sections (cs) between january 2010 and december 2012 (3-year period). The indications for cs were: prolonged labor (25.3%), previous cs (27.4%), postdate (11.2%), fetal distress (7.3%), obstructed labor (9%), preeclampsia (6.6%), cephalopelvic disproportion (3.1%), placenta previa (2.9%), multiple pregnancy (2.6%), malpresentation (2.2%), bad obstetric history (1.9%), and failed induction (1%). There were 14 maternal deaths recorded within this period, giving a maternal mortality ratio of 590 deaths per 100,000 live births . The direct causes of maternal deaths were: ruptured ectopic pregnancy, septic abortion, eclampsia, and prolonged obstructed labor . The commonest indirect cause was hiv disease and pulmonary tuberculosis . In response to the high maternal and under - five morbidity and mortality rates in crs, the government through the ministry of health introduced a free treatment program in the year 2012 to cover all pregnant women and children less than 5 years of age . The study included all the 180 ocgs working in general hospital, calabar who consented to participate in the study . The pilot and reliability testing of this questionnaire was conducted in a previous study.8 a four - part questionnaire was used to study the knowledge and utilization of the partograph by all non - physician ocgs working in general hospital, calabar . A similar study was done among midwives in the federal medical center and the university teaching hospital in bayelsa state, also in the south - south geo - political zone of nigeria and the findings have been published.8 purposive sampling techniques were used, ie, all practicing and designated ocgs in the study facility were recruited into the study . Ocgs knowledge scores on the use of the partograph were assessed with 24 composite questions . If an ocg scored less than 12 (<50%), this indicated paucity of knowledge, while scores ranging from 12 and above (> 50%) showed a proper knowledge on the use of the tool (partograph). This study obtained ethical approval from the research ethical committee of the crs ministry of health, calabar . The data entry and analysis was performed using spss for windows (v19.0; ibm corporation, armonk, ny, usa). Descriptive statistics included: frequency, and means and standard deviations to summarize variables, while inferential statistics (chi - square) were used to test the significance of association between two categorical variables . General hospital, calabar is a 100-bed secondary health care facility located in calabar, the capital city of crs . The hospital was completed and commissioned on november 7, 1991 . The hospital records showed that there were 2,370 deliveries and 588 cesarean sections (cs) between january 2010 and december 2012 (3-year period). The indications for cs were: prolonged labor (25.3%), previous cs (27.4%), postdate (11.2%), fetal distress (7.3%), obstructed labor (9%), preeclampsia (6.6%), cephalopelvic disproportion (3.1%), placenta previa (2.9%), multiple pregnancy (2.6%), malpresentation (2.2%), bad obstetric history (1.9%), and failed induction (1%). There were 14 maternal deaths recorded within this period, giving a maternal mortality ratio of 590 deaths per 100,000 live births . The direct causes of maternal deaths were: ruptured ectopic pregnancy, septic abortion, eclampsia, and prolonged obstructed labor . The commonest indirect cause was hiv disease and pulmonary tuberculosis . In response to the high maternal and under - five morbidity and mortality rates in crs, the government through the ministry of health introduced a free treatment program in the year 2012 to cover all pregnant women and children less than 5 years of age . The study included all the 180 ocgs working in general hospital, calabar who consented to participate in the study . A semi - structured, self - administered questionnaire was used . The pilot and reliability testing of this questionnaire a four - part questionnaire was used to study the knowledge and utilization of the partograph by all non - physician ocgs working in general hospital, calabar . A similar study was done among midwives in the federal medical center and the university teaching hospital in bayelsa state, also in the south - south geo - political zone of nigeria and the findings have been published.8 purposive sampling techniques were used, ie, all practicing and designated ocgs in the study facility were recruited into the study . Ocgs knowledge scores on the use of the partograph were assessed with 24 composite questions . If an ocg scored less than 12 (<50%), this indicated paucity of knowledge, while scores ranging from 12 and above (> 50%) showed a proper knowledge on the use of the tool (partograph). This study obtained ethical approval from the research ethical committee of the crs ministry of health, calabar . The data entry and analysis was performed using spss for windows (v19.0; ibm corporation, armonk, ny, usa). Descriptive statistics included: frequency, and means and standard deviations to summarize variables, while inferential statistics (chi - square) were used to test the significance of association between two categorical variables . One hundred and eighty questionnaires were administered to all non - physician ocgs in general hospital, calabar . Out of these, 130 properly completed questionnaires were analyzed . The non - physician respondents were: nurse / midwife 102 (78%), chew eight (6.2%), nurse aid eight (6.2%), and others, eg, junior chew (jchew) 12 (9.2%). Most of the respondents (66, 50.8%) had practiced for over 20 years (table 1). Seventy percent of the respondents indicated that only two ocgs are placed on duty in a shift most of the time in their maternity wards . On the use of the partograph, only 86 (66.2%) of the respondents had used the instrument before and only 47 (36.2%) of them were very confident in using the instrument . Only sixty percent of the respondents had training on the use of partographs in midwifery school . Sixty - six point two percent, 74.6%, 46.2%, and 73.1% agreed that the partograph can be used to reduce maternal morbidity, maternal mortality, child morbidity, and newborn mortality, respectively . Overall, this study shows that 70.8% (92) of the respondents were aware and had good general knowledge of the partograph . However, detailed assessment of their knowledge of the component parts of the partograph and monitoring during normal labor showed poor knowledge, whereas about 86 (66%) did not know the location of the action line and 96 (73.8%) had no idea of the normal labor graph / plotting on a partograph, using the alert and action lines as yardsticks . Many of the respondents (43.8%) did not know the number of uterine contractions in every 10 minutes, 51.5% did not know the minimum duration of a strong uterine contraction, and 50.8% did not know the minimum time required to assess uterine contractions during normal labor . Knowledge of ocgs on the assessment of labor with the partograph (table 2) showed that most of the respondents indicated that the partograph can be used for detecting prolonged labor (73.1%), obstructed labor (64.6%), poor progress of labor (80.8%), inefficient uterine contraction (68.5%), fetal distress (68.5%), identifying abnormal fetal heart rate (66.9%), identifying satisfactory progress of labor (68.5%), detecting need for augmentation of labor (65.4%), and detecting need for cs (63.8%).the respondents were not too sure whether the partograph could be used in identifying dehydration in the mother; 52.3% indicated yes while 47.7% did not . Factors affecting utilization of the partograph in labor monitoring (figure 1) were: little or no knowledge (85.4%), nonavailability of the partograph (70%), shortage of staff (61.5%), and the fact that it is time - consuming (30%). The availability of the partograph among ocgs showed that only 78 (60%) agreed that the instrument was available in the labor ward while 52 (40%) disagreed . Also, 65 (50%) admitted that partographs were employed in the management of labor in the facility whereas 65 (50%) disagreed . Among the 65 (50%) who agreed to the use of the partograph for labor monitoring in the study facility, 50 (76.9%) said they use it routinely, while nine (13.8%) and six (9.2%) said they use it rarely and occasionally, respectively . The relationship between knowledge of partograph, years of experience, partograph availability, and utilization is represented in table 3 . Knowledge of partograph (=12.05, p=0.0001) and partograph availability (=56.5, p=0.0001) had a significant relationship with its utilization . The relationship between years of experience (=0.0, p=1.000) and partograph utilization was not statistically significant . Table 4 showed that nurses / midwives (=7.44, p=0.006) were more knowledgeable on the use of partograph when compared to chews, nurse aids, and jchews . Also, previous training (=9.43, p=0.002) was significantly related to knowledge of partograph . Sex (fisher s exact test: p=1.0), years of practice (=2.84, p=0.09), and years of experience (fisher s exact test: p=0.75) showed no statistically significant association . The present study is focused on ocgs in general hospital, calabar, an urban secondary hospital in crs . It is a first step toward a larger study that will cover all primary and secondary health facilities . Nigeria has a very high maternal mortality ratio19,20 and since nurses and midwives and health professionals form the bulk of skilled birth attendants, it is important that these workers be knowledgeable in partographic labor monitoring . The majority of the respondents (70.8%) were well aware and had good general knowledge of the partograph . For instance, almost two - thirds (66%) of the study respondents could not locate the action line, 73.8% could not locate the alert line, and 51.5% did not know about the minimum duration of strong uterine contraction . The finding in this study is comparable with studies done in enugu in nigeria11 and addis ababa in ethiopia,21 which also showed lack of depth of knowledge . This finding shows that ocgs knowledge on the partograph is far below expectation, and therefore many of them will not be maximally utilized in public health hospitals and primary health care centers . This therefore informs the need for urgent steps to improve the partographic knowledge of ocgs through continuous training programs in order to increase its use . This study also revealed that knowledge of the partograph and its availability had a significant relationship with its utilization . This finding is similar to a study conducted in ogun state, southwestern nigeria, among ocgs in peripheral maternity centers, which showed low levels of utilization due to poor knowledge of the partograph.10 also, analysis drawn from recent studies within the maternity units of the two tertiary health care delivery centers in yenagoa, bayelsa state, nigeria, indicated a statistically significant relationship between the availability and use of this tool in labor.8 prolonged and obstructed labor contribute significantly to maternal morbidity and mortality in nigeria and many other resource - limited areas of the world; therefore, early detection with the use of this simple, cost - effective, and affordable tool could reduce maternal morbidity and mortality and improve neonatal outcomes.1,2,6 a significant relationship was also observed between shortage of staff and partograph utilization . According to oladapo et al,10 the lack of health care staff is strongly implicated as a factor affecting utilization of the partograph as a labor - monitoring instrument . Other researchers have opined that some midwives consider the time spent in completing the partograph to be unduly wasted as they do not appreciate its contribution in helping the woman in labor.16 by implication, when the number of staff on shift duty is favorable, the higher would be the propensity of them completing the partograph during labor monitoring . The few providers on duty are overwhelmed with work such that little or no attention is paid to some important and routine professional details . The present free health care provided to pregnant women and children under the age of 5 years may further compound this issue . Furthermore, it was also observed in this study that previous training had a positive correlation with knowledge of the partograph . Even though many of the participants lacked detailed knowledge of the partograph, the nurses / midwives who had received better formal training were more knowledgeable in the use of the partograph than the chew, nurse aid / orderlies, and jchew . Fawole et al13 confirmed the significance of formal training as a solution for increasing knowledge of the partograph . Unfortunately, the use of the partograph in the primary and secondary health care delivery facilities is still an insurmountable problem . This situation also holds true in private maternity centers, which are perpetually facing a shortage of trained staff.10,17 evidence has shown that knowledge of partograph use for labor management is very low among nurses, midwives, and generalist doctors working in the primary and secondary health care levels and private health care centers when compared to tertiary level care.9,10,13 this brings to the fore the need to introduce hands - on training for all factions of ocgs who practice at the secondary and primary levels of care and encourage them to be engaged in continued professional educational development . There was no statistically significant relationship between sex, years of practice, and years of experience with partograph knowledge in this study . This observation is in accordance with that of engida et al in ethiopia.21 contrary to this observation, opiah et al8 in their study in yenagoa, bayelsa state in the niger delta region of nigeria, reported that there was a significant relationship between years of experience of midwives and the use of the partograph . The disparity in their study could be accounted for by differences in setting, since theirs was conducted among midwives at the tertiary level and at an academic medical center . Midwives in tertiary institutions have more regular training, including regular updates compared to their colleagues at secondary health care facilities . Obviously, the findings from these studies imply that all ocgs should get periodic on - job refresher training on the use of this important tool . The federal and state ministries of health should consider the enforcement of the use of this life - saving tool in hospitals and primary care centers to stem the tide of maternal morbidity and mortality and fetal wastage during labor . The timing of this study coincided with the period that the hospital lost a high - profile patient from ruptured uterus due to prolonged and obstructed labor . Before referral to the general hospital, the index patient was managed in a peripheral health facility where partographs are not used for labor monitoring . Additionally, in the general hospital where this unfortunate maternal death occurred, the use of the partograph was not a mandatory hospital policy . This officially prompted the adoption and use of the partograph by ocgs in general hospital, calabar with effect from november 2012 . This development could have influenced the outcome of this study since the partograph was introduced 2 months before the study was conducted . This study was not conducted with an epidemiological representative sample; rather, it was confined to ocgs based at the general hospital, calabar . A comparative analysis of primary, secondary, and tertiary institutions would have been ideal . But nevertheless, the findings from this study may be a starting point to begin to address the inadequacy in midwifery practice within this study area . These may occur from the tendency of some respondents to give responses they consider as socially desirable or use such defense mechanisms as denial . The timing of this study coincided with the period that the hospital lost a high - profile patient from ruptured uterus due to prolonged and obstructed labor . Before referral to the general hospital, the index patient was managed in a peripheral health facility where partographs are not used for labor monitoring . Additionally, in the general hospital where this unfortunate maternal death occurred, the use of the partograph was not a mandatory hospital policy . This officially prompted the adoption and use of the partograph by ocgs in general hospital, calabar with effect from november 2012 . This development could have influenced the outcome of this study since the partograph was introduced 2 months before the study was conducted . This study was not conducted with an epidemiological representative sample; rather, it was confined to ocgs based at the general hospital, calabar . A comparative analysis of primary, secondary, and tertiary institutions would have been ideal . But nevertheless, the findings from this study may be a starting point to begin to address the inadequacy in midwifery practice within this study area . These may occur from the tendency of some respondents to give responses they consider as socially desirable or use such defense mechanisms as denial . This study implies a lack of detailed knowledge of the partograph by ocgs, nonavailability of the partograph, and poor staff numbers as underlying factors working against optimal utilization of this invaluable labor - monitoring tool in the study facility . The authors therefore recommend that all ocgs should have intensive training on partographic labor monitoring . The knowledge and inclination to use this instrument should be reinforced through periodic continuous medical education by way of unit presentations, seminars, and workshops . These cost - effective labor - monitoring charts should be made available by the hospital management for use at all times in the labor room in line with who recommendations and safe motherhood initiative . The hospital should develop policies and make them mandatory for the partograph to be used for all patients admitted into the labor ward . It is hoped that this study will form the basis for further research and also provoke commitment in the developing world regarding the need to introduce this tested tool in intervention programs for issues concerning reproductive health.
Providing care for patients at risk for suicide is best accomplished collaboratively with cooperation of professionals across health care disciplines and settings . Given that all health care professionals may encounter patients at risk for suicide in their clinical work, collaborative suicide risk management is relevant to a variety of providers and health care settings . Consistent with this approach, simon and gutheil penned the phrase, never worry alone,1 to emphasize the importance of a multidisciplinary approach to suicide . Therapeutic risk management of the suicidal patient (trmsp)2 is an approach for working with patients at risk for suicide that was devised by clinicians and researchers at the rocky mountain mental illness research, education and clinical center in denver, co, usa based on clinical legal concepts described by simon and shuman.3 therapeutic risk management ensures that the role and competence of the clinician is aligned with legal concerns surrounding suicide risk in psychiatric practice.3 the trmsp model involves a three - tiered approach to managing a patient at risk for suicide, which includes the use of objective measures in risk assessment, acute and chronic risk stratification, and safety planning . This model, which has been fully articulated elsewhere,2,46 was originally described for use by individual practitioners in psychiatry . Systems of care may strategically deploy multidisciplinary assets to apply the trmsp model in a manner that enhances patient treatment and safety, is medicolegally sound, and optimizes limited and valuable clinical resources . Readers are referred to the original publications describing each component for detailed descriptions and theoretical underpinnings . The national institute of health defines multidisciplinary care as an approach to health care that brings individual disciplines together to address a common problem.7 one well - documented benefit of a multidisciplinary approach to health care is that it ensures that all bio - psycho - social - cultural aspects of care are provided.8 while a system of care approach is not a new concept to health care,911 it has not been explicitly applied to trmsp . As suicide risk has bio - psycho - social - cultural components,12 systems of care can utilize a multidisciplinary approach to provide comprehensive assessment and management of individuals at risk for suicide . All providers involved should share a commitment to prioritize suicide risk assessment, an appreciation for each discipline s contributions to patient care, and an awareness of the interdependency of practice.13 in addition to providing comprehensive care, multidisciplinary approaches offer an opportunity to ensure care is delivered in ways that optimize patient safety and minimize liability for both the system of care and the individual providers comprising it . The trmsp approach permits systems of care to share responsibility for the treatment of patients at risk for suicide and allows providers to incorporate the totality of available clinical data . This in turn diffuses risk management of high - risk patients, thereby mitigating anxiety at the individual provider level, minimizing defensive practices born of anxiety, and facilitating optimal clinical decision - making . Despite its importance, there exists a paucity of both research and clinical guidelines to multidisciplinary suicide risk assessment and management . In a seminal text of suicidology, maris et al wrote, while suicidologists give lip service to the multidisciplinary study of suicide, in actual fact most of us have very narrow and specialized domain assumption usually those related to our professional training and subdisciplinary paradigms.14 that said, the department of health and human services at the national institute of mental health recommends collaborative care as a best practice for reducing suicidality.15 they describe a model in which a nurse, social worker, or other appropriately trained staff obtains information about suicide risk via screening, which then facilitates an initial treatment plan and follow - up care . This can be followed by consultation with a mental health professional such as a psychiatrist who serves an advisory role to primary care teams . The strength of this stepped - care approach is in its maximization of the effectiveness of collaborative care, with the aim of cost - effectiveness in mind by starting with low - intensity interventions that may progress to more intensive interventions . The value in adopting the trmsp approach is not only to combat the propensity to worry alone, as simon and gutheil noted,1 but also to utilize the various strengths that multidisciplinary team members bring, since not every provider will be able (due to training and/or time constraints) to carry out all the components of trmsp . Ultimately, it is our belief that providers in the settings of primary care, the emergency department (ed), and mental health outpatient can offer a unique perspective that will collectively result in a clearer picture of suicide risk, which can subsequently facilitate a better risk management plan . Figure 1 displays the interconnectedness of the system of care approach to trmsp as an alternative to each discipline operating as a silo . The majority of mental health care in the united states is provided in outpatient primary care settings by non - psychiatrist physicians and nurse practitioners.16,17 additionally, between 75% and 90% of individuals who die by suicide had contact with a primary care provider in 90 days preceding their death.18,19 physician education related to screening for mental health issues has been identified as a primary method to prevent suicide,20 yet licensing and accrediting bodies have not recognized specific tools or procedures for assessment or management of suicide risk in primary care settings.21 similarly, there is little formal education regarding suicide risk assessment in nursing programs both in the united states or internationally,22,23 with evidence suggesting that neither primary care physicians24,25 nor nurses26 are well prepared to assess and treat a person who is at risk for suicide . Eds are often used as an important safety net for persons at risk for suicide in the community, often bridging the gap between outpatient services and inpatient settings . There are approximately 3.7 million ed visits in the united states each year for suicides or suicide attempts, a number that comprises nearly 7% of all ed visits.27 eds typically employ physicians, nurses, and social workers . Such providers report some confidence in screening for suicide risk.28 the purpose of a screening is to evaluate the possible presence of suicide risk to determine if a person needs assessment . Screening for suicide risk involves asking specific questions designed to determine whether a more detailed evaluation is needed . However, once screening for suicide risk has occurred, demands on provider time and limited resources are frequently a barrier to more thorough suicide risk assessment and management in a high - volume ed setting.28 the assessment process, involving defining the scope of a problem and recommending treatment interventions to address the problem, remains highly variable across practice settings.29,30 in addition to professionals working in traditional medical settings, mental health systems could benefit from a concrete roadmap delineating a clinically and medicolegally sound process for suicide risk assessment and management . Training in suicide risk assessment is a core competency requirement in psychiatric residency and for other providers employed in mental health settings . In the united states, there is a recent push for state legislation to require qualified professionals in primary care, ed, and mental health settings to complete training in suicide risk assessment, treatment, and management.31 however, psychiatrists consistently report the need for increased training on the care of patients who are suicidal.3234 furthermore, while multidisciplinary mental health practitioners (eg, social workers, psychologists, case managers, counselors, psychiatrists, and psychiatric nurses) are often trained in suicide prevention and intervention techniques, many have not been provided with the specific training and/or experience to adequately apply such knowledge to clinical practice . This can become particularly challenging in acute crises wherein the anxiety attendant in working with a suicidal patient and making difficult clinical calls as well as fear regarding potential medicolegal consequences stemming from poor outcomes may interfere with optimal skill deployment . Individual providers have their own beliefs, theoretical orientations, and limited resources related to suicide risk assessments, many of which do not optimally align with the complex needs of the patient at risk for suicide.35 in some cases, the electronic health record in use at a particular setting dictates the suicide screening and assessment tools to be used . Yet systems of care can strive to correct for idiosyncratic individual practices or the limited suicide risk tools available at a practice location . A multidisciplinary approach aims to offer sound clinical care and cogent risk management practices by drawing on resources from a network of providers . The national institute of health defines multidisciplinary care as an approach to health care that brings individual disciplines together to address a common problem.7 one well - documented benefit of a multidisciplinary approach to health care is that it ensures that all bio - psycho - social - cultural aspects of care are provided.8 while a system of care approach is not a new concept to health care,911 it has not been explicitly applied to trmsp . As suicide risk has bio - psycho - social - cultural components,12 systems of care can utilize a multidisciplinary approach to provide comprehensive assessment and management of individuals at risk for suicide . All providers involved should share a commitment to prioritize suicide risk assessment, an appreciation for each discipline s contributions to patient care, and an awareness of the interdependency of practice.13 in addition to providing comprehensive care, multidisciplinary approaches offer an opportunity to ensure care is delivered in ways that optimize patient safety and minimize liability for both the system of care and the individual providers comprising it . The trmsp approach permits systems of care to share responsibility for the treatment of patients at risk for suicide and allows providers to incorporate the totality of available clinical data . This in turn diffuses risk management of high - risk patients, thereby mitigating anxiety at the individual provider level, minimizing defensive practices born of anxiety, and facilitating optimal clinical decision - making . Despite its importance, there exists a paucity of both research and clinical guidelines to multidisciplinary suicide risk assessment and management . In a seminal text of suicidology, maris et al wrote, while suicidologists give lip service to the multidisciplinary study of suicide, in actual fact most of us have very narrow and specialized domain assumption usually those related to our professional training and subdisciplinary paradigms.14 that said, the department of health and human services at the national institute of mental health recommends collaborative care as a best practice for reducing suicidality.15 they describe a model in which a nurse, social worker, or other appropriately trained staff obtains information about suicide risk via screening, which then facilitates an initial treatment plan and follow - up care . This can be followed by consultation with a mental health professional such as a psychiatrist who serves an advisory role to primary care teams . The strength of this stepped - care approach is in its maximization of the effectiveness of collaborative care, with the aim of cost - effectiveness in mind by starting with low - intensity interventions that may progress to more intensive interventions . The value in adopting the trmsp approach is not only to combat the propensity to worry alone, as simon and gutheil noted,1 but also to utilize the various strengths that multidisciplinary team members bring, since not every provider will be able (due to training and/or time constraints) to carry out all the components of trmsp . Ultimately, it is our belief that providers in the settings of primary care, the emergency department (ed), and mental health outpatient can offer a unique perspective that will collectively result in a clearer picture of suicide risk, which can subsequently facilitate a better risk management plan . Figure 1 displays the interconnectedness of the system of care approach to trmsp as an alternative to each discipline operating as a silo . The majority of mental health care in the united states is provided in outpatient primary care settings by non - psychiatrist physicians and nurse practitioners.16,17 additionally, between 75% and 90% of individuals who die by suicide had contact with a primary care provider in 90 days preceding their death.18,19 physician education related to screening for mental health issues has been identified as a primary method to prevent suicide,20 yet licensing and accrediting bodies have not recognized specific tools or procedures for assessment or management of suicide risk in primary care settings.21 similarly, there is little formal education regarding suicide risk assessment in nursing programs both in the united states or internationally,22,23 with evidence suggesting that neither primary care physicians24,25 nor nurses26 are well prepared to assess and treat a person who is at risk for suicide . Eds are often used as an important safety net for persons at risk for suicide in the community, often bridging the gap between outpatient services and inpatient settings . There are approximately 3.7 million ed visits in the united states each year for suicides or suicide attempts, a number that comprises nearly 7% of all ed visits.27 eds typically employ physicians, nurses, and social workers . Such providers report some confidence in screening for suicide risk.28 the purpose of a screening is to evaluate the possible presence of suicide risk to determine if a person needs assessment . Screening for suicide risk involves asking specific questions designed to determine whether a more detailed evaluation is needed . However, once screening for suicide risk has occurred, demands on provider time and limited resources are frequently a barrier to more thorough suicide risk assessment and management in a high - volume ed setting.28 the assessment process, involving defining the scope of a problem and recommending treatment interventions to address the problem, remains highly variable across practice settings.29,30 in addition to professionals working in traditional medical settings, mental health systems could benefit from a concrete roadmap delineating a clinically and medicolegally sound process for suicide risk assessment and management . Training in suicide risk assessment is a core competency requirement in psychiatric residency and for other providers employed in mental health settings . In the united states, there is a recent push for state legislation to require qualified professionals in primary care, ed, and mental health settings to complete training in suicide risk assessment, treatment, and management.31 however, psychiatrists consistently report the need for increased training on the care of patients who are suicidal.3234 furthermore, while multidisciplinary mental health practitioners (eg, social workers, psychologists, case managers, counselors, psychiatrists, and psychiatric nurses) are often trained in suicide prevention and intervention techniques, many have not been provided with the specific training and/or experience to adequately apply such knowledge to clinical practice . This can become particularly challenging in acute crises wherein the anxiety attendant in working with a suicidal patient and making difficult clinical calls as well as fear regarding potential medicolegal consequences stemming from poor outcomes may interfere with optimal skill deployment . Individual providers have their own beliefs, theoretical orientations, and limited resources related to suicide risk assessments, many of which do not optimally align with the complex needs of the patient at risk for suicide.35 in some cases, the electronic health record in use at a particular setting dictates the suicide screening and assessment tools to be used . Yet systems of care can strive to correct for idiosyncratic individual practices or the limited suicide risk tools available at a practice location . A multidisciplinary approach aims to offer sound clinical care and cogent risk management practices by drawing on resources from a network of providers . All patients entering into a system of care should be screened for mental illness and suicide risk . One strategy for risk assessment involves the use of formal self - report measures.36 providers across disciplines often experience disdain for the use of structured instruments, favoring their own personalized clinical interview.37 however, vital information regarding suicide risk may be missed during the course of an unstructured clinical interview . This potential may be minimized when reliable and valid structured tools or self - report measures are used as supplements to the clinical interview . Examples of suicide - specific structured instruments include the columbia suicide severity rating scale (c - ssrs),38 beck scale for suicide ideation (bss),39 the beck hopelessness scale (bhs),40 and the reasons for living inventory (rfl).41 the use of structured instruments offers several potential advantages to a system of care . First, standardized use of suicide risk assessment measures helps establish consistent documentation, communication, tracking, and standards of care across treatment settings and disciplines, facilitating informed care, regardless of how familiar a provider may be with a patient . There are very few restrictions by discipline associated with the administration of the c - ssrs, the bss, the bhs, or the rfl; each is relatively easy and takes little time to administer . Furthermore, use of these measures will help standardize statements regarding risk, as providers may have widely different experiences and knowledge of suicide risk assessment . Also, the use of structured instruments in primary care or ed settings may allow for providers who are less comfortable directly asking about suicidal thoughts or behaviors to begin a conversation with a patient . Finally, the use of structured instruments can improve documentation, all while saving costly higher level provider time . For instance, intake personnel might administer structured instruments or self - report measures, the results of which would then be available for review by higher - level clinicians for incorporation into their formulations regarding the level of risk . Such a process might simultaneously yield more robust and nuanced risk assessments, while minimizing the chance of omitting important inquiries . One disadvantage to using structured instruments in a system of care is the need for caution among practitioners who rely solely on numerical results generated by structured instruments to determine suicide risk . While the c - ssrs is an especially rich tool that offers descriptive detail about ideation, attempts, and behaviors that can be used qualitatively, this is less the case with the numerical scores generated by the bss, the bhs, or the rfl . Interpreting a number out of context is too simplistic a response for a highly complex, volatile situation . Therefore, it is important for providers using structured instruments to receive training in assessment and develop a keen understanding of how to interpret results in the context of other qualitative, subjective, and historical information about a patient . For example, an ed social worker with some assessment background who administers the bss might identify suicidal ideation and risk at baseline levels based upon similar responses to the same instrument during mental health sessions in the preceding weeks . However, for this patient, comparison with a baseline score might reveal significant elevation in suicide risk . When combined with clinical risk assessment, the incorporation of suicide - specific structured instruments offers a nuanced approach to suicide risk assessment, with risk assessment as a process as opposed to an event . Importantly, structured assessment tools must be met with great clinical understanding within a system of care to avoid unnecessary and costly admissions, while also minimizing the chance that an acute crisis requiring hospitalization goes missed . From the medicolegal perspective, these instruments can and should populate the medical record, but must be balanced with individualized narratives of the patient s situation . Therapeutic risk assessment of the suicidal patient requires moving beyond a one - dimensional stratification of suicide risk that has traditionally been predicated upon terms such as low, moderate, or high.2,42 such one - dimensional formulations (and documentation) fail to capture the dynamic nature of suicidal ideation . Poor communication between providers spanning different treatment settings is likely to result, and may create untenable medicolegal risk in the unfortunate event of a patient s death by suicide or suicide attempt . For example, an outpatient psychiatrist might designate his or her patient to be at low risk for suicide, thereby justifying the appropriateness of continued outpatient care and the lack of requirement for admission . But what if that patient actually carries numerous risk factors for suicide and frequently becomes acutely suicidal in the face of psychosocial stressors? Might that low risk designation mislead another provider especially a non - mental health clinician who subsequently encounters the patient during crisis? Such a provider might underappreciate the patient s actual risk for suicide . A two - dimensional designation of risk, addressing both acute and chronic risk, can circumvent clinical documentation dilemmas and enhance communications across providers and settings . In this example, the psychiatrist should designate the patient as of low acute risk and high chronic risk, thereby offering a more nuanced and accurate depiction of suicide risk that better communicates the risk and safety needs to other providers . The high chronic designation reflects the patient s tendency to become acutely suicidal in the face of distressing life circumstances, the occurrence of which is difficult to predict . The high chronic designation more effectively communicates this circumstance to other evaluators, indicating a need for routine suicide risk assessment and substantial potential for future self - directed violent behaviors . Ideally, the language used to describe risk should be uniform so that providers across disciplines and treatment settings may accurately discern the meaning of one another s risk formulations . Hence, a multidisciplinary approach to trmsp requires a standardized nomenclature that facilitates consistent documentation and communication between providers and creates a cohesive medical record . Table 1 provides suggested criteria to guide each level of stratification in this two - dimensional scheme, based directly on the work of wortzel et al.5 the third component of the trmsp model involves the safety planning intervention (spi). Historically, many providers have been taught to use no - suicide contracts with their patients.43,44 these contracts are meant to formalize and document an agreement between a provider and a patient that the patient has agreed not to harm himself or herself . However, there is no empirical support for the effectiveness of no - suicide contracts,45,46 and the document offers no legal protection from malpractice claims.47 in a system of care spanning providers from various disciplines, the presence of such a contract might inadvertently diminish the collective level of vigilance required to maintain the ongoing risk assessment process that optimizes safety for high - risk individuals . The spi is an alternative approach to no - suicide contracts developed by stanley and brown that is rooted in empirically supported treatments and suicide prevention theory.48 the spi has been identified as the best practice by the suicide prevention resource center / american foundation for suicide prevention.49 it is a collaborative tool, mutually developed by both patient and provider, and is based upon the most up - to - date content obtained from a patient during risk assessment . The safety plan consists of six steps derived from the safety plan treatment manual to reduce suicide risk.50 table 2 outlines the rationale and instructions for each step . Just as suicide risk assessment is an ongoing process, so is the spi; a safety plan should be a living document that is regularly updated to reflect changes in the individual patient s circumstances.6 safety plans are the property of the patient, but should be visible to all providers within the network of care so that they can be reinforced across treatment settings and enacted in the event of a suicidal crisis . Utilizing the same spi at each point of contact within the system of care may allow a cohesive and collaborative approach to caring for these patients . Such shared implementation and reinforcement of the spi may help systems of care achieve trmsp in a manner that is, at present, seldom realized . All patients entering into a system of care should be screened for mental illness and suicide risk . One strategy for risk assessment involves the use of formal self - report measures.36 providers across disciplines often experience disdain for the use of structured instruments, favoring their own personalized clinical interview.37 however, vital information regarding suicide risk may be missed during the course of an unstructured clinical interview . This potential may be minimized when reliable and valid structured tools or self - report measures are used as supplements to the clinical interview . Examples of suicide - specific structured instruments include the columbia suicide severity rating scale (c - ssrs),38 beck scale for suicide ideation (bss),39 the beck hopelessness scale (bhs),40 and the reasons for living inventory (rfl).41 the use of structured instruments offers several potential advantages to a system of care . First, standardized use of suicide risk assessment measures helps establish consistent documentation, communication, tracking, and standards of care across treatment settings and disciplines, facilitating informed care, regardless of how familiar a provider may be with a patient . There are very few restrictions by discipline associated with the administration of the c - ssrs, the bss, the bhs, or the rfl; each is relatively easy and takes little time to administer . Furthermore, use of these measures will help standardize statements regarding risk, as providers may have widely different experiences and knowledge of suicide risk assessment . Also, the use of structured instruments in primary care or ed settings may allow for providers who are less comfortable directly asking about suicidal thoughts or behaviors to begin a conversation with a patient . Finally, the use of structured instruments can improve documentation, all while saving costly higher level provider time . For instance, intake personnel might administer structured instruments or self - report measures, the results of which would then be available for review by higher - level clinicians for incorporation into their formulations regarding the level of risk . Such a process might simultaneously yield more robust and nuanced risk assessments, while minimizing the chance of omitting important inquiries . One disadvantage to using structured instruments in a system of care is the need for caution among practitioners who rely solely on numerical results generated by structured instruments to determine suicide risk . While the c - ssrs is an especially rich tool that offers descriptive detail about ideation, attempts, and behaviors that can be used qualitatively, this is less the case with the numerical scores generated by the bss, the bhs, or the rfl . Interpreting a number out of context is too simplistic a response for a highly complex, volatile situation . Therefore, it is important for providers using structured instruments to receive training in assessment and develop a keen understanding of how to interpret results in the context of other qualitative, subjective, and historical information about a patient . For example, an ed social worker with some assessment background who administers the bss might identify suicidal ideation and risk at baseline levels based upon similar responses to the same instrument during mental health sessions in the preceding weeks . However, for this patient, comparison with a baseline score might reveal significant elevation in suicide risk . When combined with clinical risk assessment, the incorporation of suicide - specific structured instruments offers a nuanced approach to suicide risk assessment, with risk assessment as a process as opposed to an event . Importantly, structured assessment tools must be met with great clinical understanding within a system of care to avoid unnecessary and costly admissions, while also minimizing the chance that an acute crisis requiring hospitalization goes missed . From the medicolegal perspective, these instruments can and should populate the medical record, but must be balanced with individualized narratives of the patient s situation . Therapeutic risk assessment of the suicidal patient requires moving beyond a one - dimensional stratification of suicide risk that has traditionally been predicated upon terms such as low, moderate, or high.2,42 such one - dimensional formulations (and documentation) fail to capture the dynamic nature of suicidal ideation . Poor communication between providers spanning different treatment settings is likely to result, and may create untenable medicolegal risk in the unfortunate event of a patient s death by suicide or suicide attempt . For example, an outpatient psychiatrist might designate his or her patient to be at low risk for suicide, thereby justifying the appropriateness of continued outpatient care and the lack of requirement for admission . But what if that patient actually carries numerous risk factors for suicide and frequently becomes acutely suicidal in the face of psychosocial stressors? Might that low risk designation mislead another provider especially a non - mental health clinician who subsequently encounters the patient during crisis? . A two - dimensional designation of risk, addressing both acute and chronic risk, can circumvent clinical documentation dilemmas and enhance communications across providers and settings . In this example, the psychiatrist should designate the patient as of low acute risk and high chronic risk, thereby offering a more nuanced and accurate depiction of suicide risk that better communicates the risk and safety needs to other providers . The high chronic designation reflects the patient s tendency to become acutely suicidal in the face of distressing life circumstances, the occurrence of which is difficult to predict . The high chronic designation more effectively communicates this circumstance to other evaluators, indicating a need for routine suicide risk assessment and substantial potential for future self - directed violent behaviors . Ideally, the language used to describe risk should be uniform so that providers across disciplines and treatment settings may accurately discern the meaning of one another s risk formulations . Hence, a multidisciplinary approach to trmsp requires a standardized nomenclature that facilitates consistent documentation and communication between providers and creates a cohesive medical record . Table 1 provides suggested criteria to guide each level of stratification in this two - dimensional scheme, based directly on the work of wortzel et al.5 the third component of the trmsp model involves the safety planning intervention (spi). Historically, many providers have been taught to use no - suicide contracts with their patients.43,44 these contracts are meant to formalize and document an agreement between a provider and a patient that the patient has agreed not to harm himself or herself . However, there is no empirical support for the effectiveness of no - suicide contracts,45,46 and the document offers no legal protection from malpractice claims.47 in a system of care spanning providers from various disciplines, the presence of such a contract might inadvertently diminish the collective level of vigilance required to maintain the ongoing risk assessment process that optimizes safety for high - risk individuals . The spi is an alternative approach to no - suicide contracts developed by stanley and brown that is rooted in empirically supported treatments and suicide prevention theory.48 the spi has been identified as the best practice by the suicide prevention resource center / american foundation for suicide prevention.49 it is a collaborative tool, mutually developed by both patient and provider, and is based upon the most up - to - date content obtained from a patient during risk assessment . The safety plan consists of six steps derived from the safety plan treatment manual to reduce suicide risk.50 table 2 outlines the rationale and instructions for each step . Just as suicide risk assessment is an ongoing process, so is the spi; a safety plan should be a living document that is regularly updated to reflect changes in the individual patient s circumstances.6 safety plans are the property of the patient, but should be visible to all providers within the network of care so that they can be reinforced across treatment settings and enacted in the event of a suicidal crisis . Utilizing the same spi at each point of contact within the system of care may allow a cohesive and collaborative approach to caring for these patients . Such shared implementation and reinforcement of the spi may help systems of care achieve trmsp in a manner that is, at present, seldom realized . The multidisciplinary systems approach addresses economic realities of health care systems, enabling components of trmsp to be shifted toward less costly provider time by expanding their use beyond psychiatry . While each member of the multidisciplinary team may have specific roles in the assessment / treatment process, the ability of team members to overlap and reinforce trmsp tenants across the system of care enhances safety and hopefully improves outcomes . Finally, a multidisciplinary systems approach to trmsp (documented in the medical record wherein collaboration is apparent) should yield cohesive care, offering emotional and medicolegal comfort to providers and systems of care in the unfortunate event of a poor outcome . Perhaps, the lack of progress reducing morbidity and mortality from suicide stems in part from an ongoing tendency to pass the baton of responsibility as quickly as possible as suicidal individuals move across treatment settings . Multidisciplinary trmsp affords the opportunity to reinvent this process as one whereby providers spanning professions and settings collectively and collaboratively share responsibility within the patient s system of care . It is neither in patients nor providers best interest to approach the management of suicidal patients as one that should be passed to a different silo within the system . Instead, we need a system that encourages cooperative engagement by providers across disciplines and treatment settings, sharing clinical information, responsibility, and medicolegal risk . In creating the multidisciplinary approach to trmsp for systems of care, the goal will be to surround suicidal patients with a network of providers who cooperatively optimize care and mitigate risk as shown in figure 1 . A multidisciplinary approach to trmsp for systems of care will potentially yield better medical care and enhance patient safety and more sound medicolegal practices . Patients, individual providers, and the health care system all stand to benefit from this collective process . The model offered herein may serve as a foundation for implementing a collaborative process between and across disciplines and treatment settings constituting a network of care.
A 56-year - old gentleman previously known to have rheumatic mitral stenosis, presented with a recent increase of dyspnea (new york heart association class iii), now associated with orthopnea and paroxysmal nocturnal dyspnea . On examination, he had rapid atrial fibrillation with a pulse rate of 130/min . After control of the pulse rate with digoxin and beta blockers, echocardiography revealed a mitral valve area of 1.1 cm by pressure half - time method, a mean diastolic pressure gradient of 18 mm hg across the mitral valve, a total mitral valve score of 9/16, and no mitral regurgitation . Moderate calcification of the mitral valve was noted; however, since no commissural calcium was found, percutaneous mitral valvuloplasty was planned . Trans - esophageal echocardiography showed no thrombi in the left atrial appendage, a mitral valve annulus diameter of 38 mm, and a thin inter - atrial septum (2 mm). The procedure was performed through a right femoral vein puncture, employing the multi - track technique, using 2 balloons (20 and 18 mm in diameter). Following a smooth trans - septal puncture, the mitral valve was crossed with a judkins right catheter, and 2 wires were secured in the left ventricular apex with a double coil clearly seen in both . The 2 balloons were advanced along the wires and inflated; yet, surprisingly, no clear waist was seen (figure 1). Skeptic about the balloon position, the operator decided to redo the inflation more proximally, wherein a clear waist was seen that ultimately yielded to balloon inflation . Suddenly, blood pressure collapsed to 80/60 mm hg, with a slow though still irregular bedside echocardiography confirmed the presence of moderate pericardial effusion so that pericardiocentesis was immediately performed (figure 2). Roughly, 250 ml of frank red oxygenated blood were drained out of the pericardial sac . In the mean time, heparin was counteracted by protamine sulphate administration, and dopamine infusion started at a rate of 10 g / kg / min . Blood pressure returned back to 100/60 mm hg, and the pulse rate rose to 100 bpm . The pigtail catheter in the pericardial sac stopped to drain any more blood; however, once again, blood pressure started to drop progressively down to 60/40 mm hg, and the pulse rate surged now to 110 bpm . Amazingly, bedside echocardiography unveiled an echo - dense mass posterior to the left ventricle . Unfortunately, however, during transfer to the operating room, the patient was arrested in asystole . Resuscitation started straight away and continued for 15 min, well inside the operating room . Following median thoracotomy, the surgeon was confronted with a large blood clot filling the posterior pericardial sac . The clot was removed at once; the heart restarted to beat at 70 bpm and blood pressure was restored to 100/70 mm hg . A 1.5 cm tear was discovered in the left ventricular apex, which was sutured with teflon sutures . Eventually, the patient was hemodynamically stable but was discharged to the intensive care unit in deep coma, wherein he was supported with mechanical ventilation . His consciousness level improved the next day, so that his glasgow coma score was 6; the day after, it reached 9 . Thereafter, he became fully conscious, and was disconnected off mechanical ventilation; however, he suffered some memory deficit, and mild motor dysphasia . Three days later, he regained an almost normal neurological state and was discharged from the intensive care unit . Post - procedural echocardiography revealed a mitral valve area of 1.9 cm, with no mitral regurgitation, and confirmed the absence of further accumulation in the pericardium . Over the past two decades, percutaneous mitral valvuloplasty has emerged as the procedure of choice in the procedure is associated with mortality rates that range from 0 to 3%, chiefly due to cardiac tamponade, severe mitral regurgitation, or deterioration of the patient's general condition . The incidence of hemopericardium following percutaneous mitral valvuloplasty is reported at 1 - 3%, being related to either trans - septal puncture or left ventricular perforation with guide wires or balloons . The site of perforation is crucial for both the immediate and long - term outcome . Frequently self - limited perforation of the right atrial appendage often responds well to pericardiocentesis, and spontaneous closure is the rule . On the other hand, left ventricular apical perforations can be swiftly fatal due to the high left ventricular pressure, especially if induced by the rigid balloon catheters rather than the guide wires, and therefore frequently need emergency surgical repair . Pick up cases which need life - saving immediate pericardiocentesis, aided by reversal of anticoagulation by protamine sulphate, even before hemodynamic deterioration . Bedside echocardiography is the standard of care for prompt diagnosis, and should be an indispensable workhorse in any cath lab that performs percutaneous mitral valvuloplasty procedures . During pericardiocentesis, one should always ensure a proper position of the pigtail catheter inside the pericardial sac, guided by fluoroscopy and bedside echocardiography . However, if the clinical condition deteriorates despite the absence of effluent from the pericardium, emergency surgical exploration would be mandatory . Moreover, it is widely acknowledged that blood in the pericardial sac does not clot due to the defibrination effect by cardiac motion . In the current case, however, rapid accumulation of a large amount of blood over a brief period of time might have been the cause of blood clotting in the posterior pericardial sac, further augmented by protamine sulphate injection . It would be wise to inflate the balloons extremely cautiously, so that if no waist is seen, inflation should be immediately halted and the balloon catheters pulled back to a more proximal position, in order to avoid injury of the left ventricular apex.
Celem badania bya ocena w codziennej praktyce stratyfikacji ryzyka zakrzepowo - zatorowego w niezastawkowym migotaniu przedsionkw . Spord 81 kolejnych pacjentw dotychczas nieleczonych przeciwzakrzepowo, po uwzgldnieniu kryteriw wykluczajcych, ostatecznie badaniem objto 68 . Pacjentw analizowano w zalenoci od ryzyka wystpienia powika zatorowych: chads2 2 (grupa i) vs chads2 <2 i cha2ds2-vasc 2 (grupa ii) oraz pci . Doustne leczenie przeciwzakrzepowe prowadzono warfaryn, po uwzgldnieniu preferencji pacjenta . U 26 pacjentw (43%), w tym 15 kobiet (57%), kwalifikacja ta bya moliwa jedynie przy zastosowaniu skali cha2ds2-vasc . W porwnaniu z grup chads2 2 chorzy ci byli modsi (72 10 vs 63 10 lat, p = 0,0002), rzadziej obcieni nadcinieniem ttniczym (p = 0,03), a ryzyko krwawie byo u nich nisze (has - bled 1,23 0,65 vs 0,81 0,49, p = 0,03). W porwnaniu z mczyznami u kobiet czciej w wywiadzie wystpowa udar niedokrwienny mzgu (2 vs 18%, p = 0,03), rzadziej choroba wiecowa (58 vs 25%, p = 0,005). W trakcie 18-miesicznej obserwacji leczenia warfaryn incydenty krwawienia wystpiy u 9 chorych (13%), w tym 6 kobiet (67%). Zastosowanie skal cha2ds2-vasc i has - bled pozwala w atwy i precyzyjny sposb zidentyfikowa pacjentw prawdziwie niskiego wydaje si, e kobiety s obarczone wyszym ryzykiem krwawie, pomimo rzadszego stosowania lekw przeciwpytkowych . Anticoagulation therapy with warfarin is highly effective at reducing thromboembolic events (te), but is associated with a higher risk of major bleeding compared to new oral anticoagulants [antithrombotic drugs xa (rivaroxaban, apixaban) and iia inhibitors (dabigatran)] [1, 2]. They appear to have a favorable safety profile, particularly with the decreased risk for intracranial bleeding . However, the clinical decision on antithrombotic management should be based on the balance between an individual's thromboembolic and hemorrhagic risk . The most commonly used scheme to determine this risk of te in patients with nonvalvular atrial fibrillation (af) was the chads2 score . A limitation of this scale was a large part of the eligibility of patients as intermediate risk and skipping other potential risk factors for te, such as myocardial infarction, complex aortic plaque, peripheral artery disease, female sex and age 65 - 74 . The additional risk factors have been expressed in the cha2ds2-vasc score . Finally in current guidelines, the cha2ds2-vasc score is crucial to decide whether or not to anticoagulate . Anticoagulant therapy carries a risk of bleeding, and major bleeding such as intracranial bleeds can be catastrophic . The has - bled score is a simple and user - friendly tool for the assessment of bleeding risk before starting anticoagulation . A has - bled score of 3 points or more indicates increased one - year bleed risk on anticoagulation sufficient to justify caution or more regular review . That risk includes intracranial bleeding, bleeding requiring hospitalization, one with a hemoglobin drop> 2 real life risk stratification scores in patients with nonvalvular sustained af who have not yet received anticoagulation . From 81 consecutive patients with the inclusion criterion as sustained nonvalvular af, who had not been treated with anticoagulants, finally 68 were enrolled in the study . Patients with unstable arterial hypertension (> 160/110 mm hg), alcohol abuse, a serious incident of bleeding requiring blood transfusion, bleeding disorder, liver disease (alt> 3 times the upper reference level), and those who did not agree to further contact were excluded from the study (n = 13) (fig . Recruitment patients for the study we collected the following data: demographic (age, sex), clinical (concomitant diseases, medications, a history of thromboembolic and bleeding events), and basic laboratory (morphology, electrolytes, gfr, glucose, lipids, liver enzymes, bilirubin, c - reactive protein). A follow - up was performed to ascertain outcomes: clinical events (stroke / tia, peripheral embolism, bleeding), change in antithrombotic therapy (discontinuation, duration on therapy); medical history update; and inr records in relation to therapeutic range . Major bleeding was defined as an intracranial hemorrhage, a decrease in blood hemoglobin level of more than 5.0 g / dl, the need for a transfusion of two or more units of blood, the need for corrective surgery, or any combination of these events . Minor bleeding was defined as a subcutaneous ecchymosis or hematoma, gastrointestinal bleeding, urinary bleeding or bloody sputum . Hypertension, diabetes, and lipid disorders were diagnosed according to the latest recommendations, as well as the implementation of antiplatelet therapy . For each patient chads2 and the chads2 score is based on a point system in which 2 points are assigned for a history of stroke or tia and 1 point each is assigned for age 75 years, a history of hypertension, diabetes, or recent cardiac failure . The cha2ds2-vasc scale gives two points for age of 75 years, and in addition to factors of the chads2 score, one point each for age 65 - 74 years, vascular disease, and female sex (table i). The bleeding risk of our patients was assessed by the has - bled score [assigning one point each for the presence of hypertension, abnormal renal / liver function, stroke, bleeding history or predisposition (e.g. Bleeding diathesis, anemia), labile inr (international normalized ratio), elderly (> 65 years), and drugs / alcohol concomitantly]. Risk factors for stroke and thrombo - embolism in non - valvular af according to chads2 and cha2ds2-vasc score initially, the risk of te was calculated on the basis of the chads2 score . Next, in patients with chads2 <2 the risk of te was re - estimated by the cha2ds2-vasc score . Warfarin was indicated in patients at high risk for te (chads2 or cha2ds2-vasc 2), after consideration of the patient's decision . Study groups were analyzed related to scores: chads2 2 (group i, n = 35) vs. chads2 <2 and cha2ds2-vasc score 2 (group ii, n = 26) and gender . The local bioethics committee approved the study and informed consent was obtained from all participants . Continuous variables are presented as mean values sd or medians with interquartile ranges (iqr) in case of their non - normal distribution . An independent t - test (when the distribution in both groups was normal) or the mann - whitney u test (in the absence of normal distribution of measured variables) was used to evaluate differences in the continuous factors between the groups . Independent test was used to investigate the relationship between scores (chads2, cha2ds2-vasc) and qualitative variables . Continuous variables are presented as mean values sd or medians with interquartile ranges (iqr) in case of their non - normal distribution . An independent t - test (when the distribution in both groups was normal) or the mann - whitney u test (in the absence of normal distribution of measured variables) was used to evaluate differences in the continuous factors between the groups . Independent test was used to investigate the relationship between scores (chads2, cha2ds2-vasc) and qualitative variables . The baseline characteristics of patients are summarized in table ii . The studied population consisted mainly of older patients, 21 patients (31%) were 75 years old and 18 (26%) were aged 65 - 74 . Nineteen patients (27%) had experienced myocardial infarction, 10 (15%) stroke, 13 subjects (19%) had diabetes mellitus, and 5 (7%) peripheral artery disease . The most frequent was hypertension (73%), followed by dyslipidemia (72%) and coronary artery disease (42%). Patients had preserved left ventricular systolic function (mean 53 12%, median 56%) and no symptoms of heart failure . Characteristics of the studied patients non - normal distribution bmi body mass index; wbc white blood cells; rbc red blood cells; hgb hemoglobin; hct hematocrit; plt platelets; na sodium; k potassium; cl chlorine; gfr glomerular filtration rate; tch total cholesterol; ldl low density lipoproteins; hdl high density lipoproteins; tg triglycerides; alt alanine aminotransferase; ast aspartate aminotransferase; crp c reactive protein; sd standard deviation; q25 first quartile; q75 third quartile almost half of patients were women (47%). Compared to men, women had significantly more often a history of ischemic stroke (2 vs. 18%, p = 0.03), but less coronary heart disease (58 vs. 25%, p = 0.005). Women had higher levels of hdl (1.47 vs. 1.21 mg / dl, p = 0.003), and lower hemoglobin (13.5 vs. 14.2 g / dl, p = 0.05) and hematocrit (40.4 vs. 43.4%, p = 0.02) than men . In analysis according to gender, the studied population did not differ in age, body mass index (bmi), daily doses of warfarin, scale has - bled, high - sensitive c - reactive protein (hscrp), and kidney and liver function (table iii). Characteristics according to gender and scales chads2 2 (group i), and chads2 <2 and cha2ds2-vasc 2 (group ii) the results are presented as% and mean value sd non - normal distribution bmi body mass index; gfr glomerular filtration rate; tch total cholesterol; ldl low density lipoproteins; hdl high density lipoproteins; tg triglycerides; alt alanine aminotransferase; ast aspartate aminotransferase; crp c reactive protein; cad coronary artery disease; sd standard deviation distribution of chads2 and cha2ds2-vasc scores is shown in fig . Taking into account the mentioned two scales 61 patients (90%) were at high risk for thromboembolic complications . In 26 patients (43%) with one point on chads2, qualifications for vitamin k antagonist (vka) therapy was only possible by using cha2ds2-vasc . In this group were 15 women (57%). Distribution of patients according to chads2 and cha2ds2-vasc risk stratification seven patients (10%), all men, did not require anticoagulation (cha2ds2-vasc = 0). In the studied population concomitant aspirin and vka was used by 42% and dual antiplatelet therapy by 16% of patients . During the 18 2.35 months of follow - up the median daily dose of warfarin was 5 mg (range 1.25 - 8 mg) with therapeutically optimal inr . The total study group was at low risk of bleeding, with a median has - bled score of 1 (98% with has - bled 2 [low risk]). Among hemorrhage events, there was no major bleeding . Minor bleeding was observed in 9 (13%) patients, including 6 women (67%): nose (5 cases), the urinary tract (3) and gastrointestinal tract (1). Three men with bleeding received one of the antiplatelet drugs . During the 18 months of follow - up no incident of thromboembolic complication was recorded . Patients of group ii compared to group i were younger (p = 0.0002), with prevalence of women (58 vs. 43%, p = 0.25), and less frequently burdened with arterial hypertension (p = 0.03). In this group there were higher glomerular filtration rate (p = 0.008), total cholesterol level (p = 0.01) and ldl values (p = 0.02). Moreover, group ii patients had a lower value of has - bled (p = 0.03). The main finding of our study is that the cha2ds2-vasc score identified 26 subjects more at high risk of thromboembolic complications in comparison to chads2, which was a large part of all requiring vka (43%). According to chads2, these patients were qualified to moderate risk level with chads2 = 1 . The crucial factors of high risk of te in group ii were age, female gender and vascular disease, particularly myocardial infarction . Similar results were observed in a danish national registry of 73,538 non - anticoagulated patients with nonvalvular af . As in our study, a large percentage (46%) of respondents were women . They proved that cha2ds2-vasc performed better than chads2 for categorization into risk groups for stroke and for identification of patients at truly low risk and that not all risk factors were associated with an equal risk of te . A particularly high risk was associated with age 65, gender and diabetes mellitus . Opposite to our patients, the danish population was older (31 vs. 54% 75 years), less frequently burdened with arterial hypertension (73 vs. 40%) and vascular disease (42 vs. 17%). . Found that women with af are at higher risk for ischemic stroke than men . However, in a recently published study mikkelsen et al . Revealed that female gender is associated with an increased risk of stroke for af only in patients aged> 75 years (hr 1.2, 95% ci: 1.12 - 1.28). Antithrombotic therapy to prevent thromboembolism is recommended for all patients with af, except those who are at low risk or with contraindications . Atrial fibrillation patients classified as low - risk using chads2 score = 0 have stroke rates of 1.5% per year, so they are not truly low - risk. The evidence shows that cha2ds2-vasc score reclassifies 26% of patients with a chads2 score of 1 to a low risk of stroke . Truly low - risk patients with af, who do not need any antithrombotic therapy [5, 12]. In our study 10% of patients had cha2ds2-vasc = 0 . Those with a cha2ds2-vasc score of 2 (the age - adjusted rate of stroke per year 2.2%) are at high risk of te and should be managed with oral anticoagulation, whether with warfarin or novel oral anticoagulants that overcome the limitations or disadvantages . For those with a cha2ds2-vasc score of 1 (the age - adjusted rate of stroke per year 1.3%), oral anticoagulation therapy with vka or direct thrombin inhibitor, or oral factor xa inhibitor is considered, based upon an assessment of the risk of bleeding complications and patient preferences . The decision to anticoagulate is based not only on thromboembolic risk but also on the risk of bleeding, and the european guidelines on atrial fibrillation incorporate a bleeding prediction scheme has - bled to help with this decision making [4, 5]. It is recognized that several risk factors predisposing to bleeding are also risk factors for stroke, and although some schemas (e.g. Chads2) have modest value for predicting stroke, they are very good at predicting a patient's risk of bleeding . The type and severity of comorbid disorders are the most important risk factors for anticoagulant - related bleeding . Cardiovascular disease, liver dysfunction, and severe renal impairment are associated with increased risk of bleeding [14, 15]. We observed only minor bleeding events, mostly from the upper respiratory (44%) and urinary tract (33%). Absence of major bleeding in our study may result from preserved renal function, especially in our younger patients (group ii). In a dutch center the most prevalent bleeding events were from the gastrointestinal tract, even 40% of all events, as major bleeding requiring a blood transfusion . Opposite to our patients, this population was older (mean age 66 vs. 73 years), less burdened with arterial hypertension (73 vs. 47%) and vascular diseases (42 vs. 11%). Similar to our study, patients received aspirin with vka (42 vs. 39%), but less dual antiplatelet therapy (16 vs. 3.5%). Despite the low risk of bleeding (median has - bled = 1) in our population, there were several incidents of minor bleeding (13% compared to 9% in the other studies) [17, 18]. Possibly that can be explained by concomitant use of aspirin and antiplatelet drugs . However, in such cases in the literature there was observed more frequently major bleeding . Based on our results, it seems that some caution and regular review of the patient is needed following the initiation of antithrombotic therapy not only in the high - risk bleeding group (has - bled> 3) but also in patients with low or intermediate risk (has - bled 2), particularly with concomitant use of antiplatelet drugs . Close control of inr in the therapeutic range can reduce the number of bleeding complications, as confirmed in other studies . Current practice guidelines for stroke prophylaxis recommend warfarin for patients at high risk for stroke including those over 75 years of age or younger patients with additional risk factors . Although these recommendations are strongly supported by the clinical trial evidence, studies show that many patients are not receiving appropriate vka therapy . The approach may be changed with a new generation of anticoagulant drugs, such as dabigatran and rivaroxaban . They offer an interesting alternative to warfarin, because blood tests for monitoring inr are not required and there is lower bleeding risk while offering similar results in terms of efficacy . Their main disadvantage is the low experience of their use and no specific antidote for an overdose . The challenge is defining patients who would best benefit from thromboprophylaxis, and showing how to deliver it in the most effective and safe way . Due to the risk of bleeding complications, there is often suboptimal implementation of prevention among patients with the risk of te . An individual approach to each patient, including risk stratification with the available scores, helps reduce the number of complications in anticoagulation . The main limitation of the study is the small study group and therefore a multivariate analysis was not performed . Moreover, we analyzed data from a single academic center, and thus the strength of the evidence cannot be compared to that obtained in randomized studies . Despite these constraints, the results are consistent with other studies . The reclassifying approach with the implications of cha2ds2-vasc and has - bled schemata provides some improvement over the chads2 score, with the identification of real low and high risk patients . In the era of antiplatelet therapy it seems that women present higher risk of bleeding, although less frequent use of antiplatelet therapy.
There have been many reports with respect to vaginoplasty, including those involving skin grafting, the intestinal tract and skin flaps, since this surgery was first carried out by dupuytren [18]. However, these methods involved forming the lumen of the vagina, with no reports to date on vaginoplasty including the vaginal vestibule reconstruction . We report on a new method for performing vaginoplasty, including the vaginal vestibule reconstruction with respect to a case of congenital absence of a vagina . Twenty - one - year - old female had been undergoing treatment with respect to primary amenorrhea from the age of 16 . At 20 years old, she underwent a laparoscopic gonadectomy with a diagnosis of testicular feminisation syndrome . Findings: although there were no problems with the shape of the labia majora, labia minora, and clitoris, no pubic hair was observed . The vagina measured 3 cm and there was a coecum, while the vaginal vestibule narrowed on the anal side (figure 1). Findings before the operation no pubic hair was observed and the vagina measured 3 cm . Transvaginal ultrasonography: the uterus was absent and the distance from the tip of the vaginal caecum to the retroperitoneum was 2.7 cm (figure 2). Findings of transvaginal ultrasonography the uterus was absent and the distance from the tip of the vaginal caecum to the retroperitoneum was 2.7 cm . Surgical findings: the posterior labial arteries of both sides were confirmed by doppler prior to surgery and vaginoplasty using a bilateral pudendal thigh flap (ptf) was planned . First, in order to ensure space for inserting the skin flap, an obstetrics and gynaecology specialist made a transverse incision at the tip where the vaginal caecum was observed, then exfoliated the connective tissue between the bladder and the rectum to create space . Next, the procedure was taken over by a plastic surgeon to design the ptf . It was designed such that the donor scar matched the contour line of the inner proximal region of the thigh within a range in which sufficient reefing is possible without including the skin of the outer labia . Moreover, the bilateral skin flaps were extended towards the anal side, thereby reconstructing the vaginal vestibule using these parts (figure 3). The bilateral ptfs were elevated under the fascia (figure 4), a tunnel was made underneath the skin of the outer labia, and subsequently, these skin flaps were moved to the centre (figure 5). Design of ptf the bilateral skin flaps were extended towards the anal side, thereby reconstructing the vaginal vestibule using these parts . Next, the skin flaps were sutured together from the distal part of the skin flap in the shape of a lumen, thereby creating an intravaginal cavity (figure 6). This was then inserted into the space between the bladder and the rectum that was made prior to lifting the skin flap, and the remaining vaginal mucous membrane in the urethra was sutured together with the skin flap . Moreover, in order to make the vaginal vestibule, a vertical incision was made in the narrowed vaginal vestibule of the anal side along with the remaining vaginal mucous membrane and subsequently, part of the anal side of the skin flap was inserted (figure 7(a)). The donor site was temporarily reefed along the contour line of the inner proximal region of the thigh (figure 7(b)). Creating an intravaginal cavity . Post operation (a) a vertical incision was made in the narrowed vaginal vestibule of the anal side and part of the anal side of ptf was inserted . (b) the donor site was temporarily reefed along the contour line of the inner proximal region of the thigh . Postoperative prognosis: the skin flap was completely engrafted and the insertion of the silicon prosthesis inside the vagina was commenced for preventing stenosis of the intravaginal cavity two weeks following surgery . Subsequently, the prosthesis was inserted for three months and the length of the vagina was maintained at 8 cm when examined at two years and four months following surgery . No stenosis of the vaginal vestibule or opening of the vagina was observed (figure 8(a)) and a substantially satisfactory vulva shape was thereby acquired (figure 8(b)). Vaginoplasty with respect to the absence of a vagina is classified into nonsurgical method and a surgical method . Regarding the nonsurgical method, the frank method has been reported, which uses a prosthesis for continuously extending the vaginal vestibule mucous membrane and making an intravaginal cavity . However, this method is accompanied by pain and the lumen of the vagina is often found to be insufficient, so the surgical method is currently carried out in most cases . Regarding the surgical method, the mcindoe method has been reported, wherein the connective tissue between the bladder and the rectum is exfoliated to create space and skin grafting is carried out in the inner wall of the space . Although this method is only slightly invasive, in many cases, stenosis is caused due to postoperative scar contracture, thereby making it difficult to maintain an intravaginal cavity of sufficient width and length . With methods that use the intestinal tract such as the ruge method, which uses a sigmoid colon, the intravaginal cavity is the intestinal tract mucous membrane, thus making it less prone to cause stenosis . However, it has some problems in that the surgery becomes highly invasive due to a laparotomy and an odour as well as contamination on the underwear due to the secretion of intestinal tract fluids . In the method using skin flaps, many reports exist including the gracilis myocutaneous flap, ptf, labia minora flap and so on . The shape of the lumen may be maintained regardless of being configured from skin tissues of the intravaginal cavity, and it is a method that is less invasive compared to those using the intestinal tract . Currently, ptf using skin flaps are commonly carried out with respect to vaginoplasty, allowing stable blood circulation and non - exposed donors . Ptf has a problem that dysfunction may occur during sexual intercourse due to pubic hair in the vicinity of the vulva . However, no pubic hair was congenitally observed in the present case, so vaginoplasty by ptf was thus selected . Ptf is also referred to as a singapore flap and it was first reported in 1989 by wee et al . For use with respect to vaginoplasty . Ptf is a fascial skin flap which uses the posterior labial arteryas the feeding vessel . Carried out an anatomical study with respect to ptf and reported that the posterior labial artery dominates the skin from the perineum to the proximal part of the thigh in the vicinity of the femoral triangle for nourishment, with the range of sampling skin flap potentially being safely harvested, and thus having a size of 15 6 cm in adults . Moreover, they mention that ptf becomes the sensory skin flap because the posterior labial nerve branching from the perineal nerve is the dominant nerve of ptf . However, there are several problems associated with the original method reported by wee et al . First, the vagina is an organ shaped with a lumen structure, so it is designed to have a cylindrical shape in the original method . Accordingly, a disordered natural skin line of the inner proximal region of the thigh and deformation of the perineum may be caused due to unreasonable reefing when closing the donor following sampling of the skin flap . Second, the skin suture line of the opening of the vagina may become round in shape, causing scar contracture to the entire circumference and leading to stenosis of the opening of the vagina . Third, in the original method, wherein reconstruction of the intravaginal cavity was the main purpose, reconstruction of the vaginal vestibule was not carried out . It is believed that these facts may cause some dysfunction during sexual intercourse in addition to cosmetic problems of the perineum . Accordingly, when designing the skin flap, the following three ingenuities were contrived: (1) when the outer labia skin is included in the skin flap, the shape of the outer labia becomes greatly distorted, so the skin flap outside the outer labia (mainly above the thigh) was redesigned . (2) the width of the wound border of the skin flap was made to be a width allowing sufficient reefing by drawing the wound border of the femoral part, and was designed such that the suture wound matches the natural skin line of the inner proximal region of the thigh . (3) it was designed such that the skin suture line of the opening of the vagina does not become round by extending the skin flap to the anal side, and such that the vaginal vestibule may be created at that site . Due to these procedures, the shape of the outer labia was maintained and a cosmetically satisfying result was thus achieved by making the suture scar along the natural skin line of the inner proximal region of the thigh . Moreover, due to the part of the skin flap on the anal side becoming sandwiched in the opening of the vagina, it was possible to prevent scar contracture (stenosis in the opening of the vagina) over the entire circumference . Regarding the vaginal vestibule, the part in between the bilateral labia minora is anatomically defined as the vaginal vestibule . The vaginal vestibule has a function of reacting to the glans of the penis during sexual intercourse and leading it to the vagina . Accordingly, when there is no vaginal vestibule due to vaginoplasty, the penis cannot be smoothly inserted during sexual intercourse, thereby causing functional disabilities (figure 9(a)). It is believed that reconstructing the vaginal vestibule with a part of the skin flap in this study was very important to achieve smooth sexual intercourse (figure 9(b)). Although the patient has not experienced sexual intercourse to date following the surgery, sexual intercourse is cosmetically and functionally possible, and it is believed that our techniques for performing vaginoplasty is therefore a useful method . In this study, vaginoplasty including the vaginal vestibule was carried out with respect to vaginal aplasia cases with a narrowing of the vaginal vestibule using ptf . In this case, the vagina was reconstructed using a skin flap while the vaginal vestibule of the anal side was simultaneously reconstructed . Taking into consideration the fact that the vaginal vestibule plays a major role in sexual intercourse, it is believed that this new method is therefore useful and satisfactory in terms of functional reconstruction . The authors declare that there is no conflict of interest regarding the publication of this paper.
The ability to generate maximal forces is critical to many sporting, rehabilitative, exercise, physical therapy, and physical testing settings and procedures . In all such settings, researchers, and practitioners however, what has not been clear is the influence these instructions may have on participants attentional focus and subsequent performance . Utilizing the vertical jump - and - reach task to assess maximal force production from whole - body coordination, wulf and colleagues have attempted to address the potential influences of attentional focusing instructions on maximum force production . In the initial study, 2007) directed attention either onto the rungs (of the vertec measurement device) being reached for (external focus) or onto the fingers reaching for this rung (internal focus). In experiment 1, an external focus resulted in significantly greater jump - and - reach height when compared to an internal focus and under control (no attentional focusing instruction provided) conditions in a within subject design . Given that within air movement patterns (e.g., differences in the reaching movement, not actual jump height) may have caused such an effect, in experiment 2, center - of - mass (com) displacement was also measured . Participants greater jump height using external instructions was associated with greater com displacement compared to when internally focused instructions were utilized . This study therefore presented some of the first direct evidence that verbal external attentional focus manipulation benefits maximal force production as increased jump height was associated with increased force production . Supporting other research, an internal focus was shown to not differ from the no - instruction condition, suggesting that in force production tasks attention could be drawn internally without specific direction . Replicating this jump - and - reach benefits of an external focus wulf and dufek (2009) and wulf et al . (2010) also assessed a number of kinematic and neuromuscular variables (e.g., lower - extremity joint moments). In these studies, internally focused instructions (focusing on the fingers reaching for the rung of the apparatus) resulted in lower jump - and - reach height, com and jump impulse displacement in a within - subjects design compared to externally focused instructions (focusing onto the rung being reached for). Wulf and dufek also demonstrated that lower - extremity joint moments of the ankle, knee, and hip joints were significantly larger when externally focused instructions were provided compared to internal focus instructions, indicating benefited jump kinematic coordination . Whilst, wulf, dufek, lozano, and pettigrew attempted to explain the benefits in terms of activity and coordination patterns among associated muscles (tibialis anterior, biceps femoris, vastus lateralis, rectus femoris, lateral gastrocnemius), no differences in the pre - take - off muscle onset suggested that attentional focus did not influence coordination among muscle groups . The increased jump - and - reach height associated with externally focused instructions was achieved with lower emg activity when compared to the internal instructions, suggesting that coordination within muscles is benefited through an external focus . Collectively, these studies (wulf et al ., 2007, 2010; wulf and dufek, 2009) demonstrated that external attentional focusing instructions onto the movement outcome (object reached for) resulted in greater jump - and - reach heights compared to internally focused instructions (hand reaching with). This increased jump height was achieved through greater force production, which itself was the result of improved lower - extremity joint movements (wulf and dufek, 2009) and enhanced neuromuscular coordination (wulf et al ., 2010). Note that the instructions only manipulated attention around the reaching movement (e.g., targeted rung, or reaching finger). In balance studies utilizing supra - postural tasks (e.g., standing balance whilst touching and keeping a flexible curtain still, or dynamic balance whilst holding a bar level) where attention is manipulated internally and externally toward those tasks, whole - body performance is influenced (e.g., mcnevin and wulf, 2002; wulf et al ., 2003). Similarly, attentional focusing instructions have been shown to influence activity in muscles that are not specifically the focus of the attentional instructions (e.g., vance et al ., 2004; such influences have important implications for manipulating attentional focus in other force generating movements that require whole - body coordination toward a single output . (2010) demonstrated that instructing attentional focus externally enhances standing long - jump performance compared to internally focused instructions . External instructions in this case directed attention toward jumping as far past the start line as possible whilst internally focused instructions emphasized extending your knees as rapidly as possible . Although this study did not address kinematic or force production directly, the results add to those already discussed in highlighting the influence of different instructions on force production requiring whole - body coordination . However, of further interest is the effect of attentional focusing instructions upon force production that requires the manipulation of a specific object . Such tasks reflect many occupation, sporting and exercise tasks requirements, and are reviewed next . (2004) was to compare muscular activity (integrated electromyography, iemg) between different attentional conditions . In two experiments, experienced exercisers lifted a weighted barbell (50% of their maximal force production) using a biceps curl movement in a standing position . Attentional focusing instructions emphasized either the movement of the curl bar (external focus) or the arm and muscular movements (internal focus). In these experiments, iemg of both the agonist (biceps) and antagonist (triceps) was significantly lower with externally compared to internally focused instructions . Additionally, in the first experiment, externally focused instructions were associated with faster movement times compared to internal instruction . The second experiment controlled average movement speed using a metronome . As the weight lifted in each condition was the same, these findings led vance et al . To suggest potential benefits of externally focused instructions when the goal of a task is force production due to the more efficient muscular activation patterns observed . Specifically, focusing on the object that the force is being exerted upon may result in more effective performance than would focusing upon the body movements that produce the action research in our labs (marchant et al ., 2008) further replicated this muscular activity finding with further control over the movement timing . In that study, experienced exercisers completed elbow flexions at 60s on a isokinetic dynamometer under internal and external instructional conditions . Again, externally focused instructions were associated with lower biceps emg when compared to internal and control instructions . Following up these findings, we (marchant et al ., 2009) attempted to directly address the proposal that externally focused instructions could benefit maximal force production . Using an isokinetic dynamometer, experienced exercisers completed elbow flexions at 60s with the aim of producing maximal force during the full range of each movement . Instructions focused on the movement of the bar of the crank arm (external) or the movements of their arms and muscles (internal) during each lift . Significantly greater net joint torque (peak and integrated elbow flexor torque) and lower levels of muscular activity of the biceps (peak and integrated emg) were observed when attention was directed externally compared with internally . As such, maximal voluntary force production was benefited from an external focus onto the object force is being exerted against as proposed by vance et al . Focusing internally has resulted in elevated muscular activity that has not transferred to the movement output . However, no measurement of the antagonist muscle in this study means that discussion of intra - muscular coordination is not possible . Efficient force production requires effective recruitment and coordination of fibers within and between agonist and antagonist muscles (e.g., wulf et al ., 2007). As we will see later, evidence suggests that attentional focus influences antagonist activation during force production . Despite the benefits to force production, participants in marchant et al . 's study expressed a preference for internal over external instructions and rated the latter as harder to follow . However, no manipulation check in this study means that information on instruction use was lacking . Those studies presented so far provide evidence that when instructions direct attention externally, maximal forces can be produced more effectively . However, one could argue that such tasks do not represent the subtleties sometimes required in force production settings . Sporting, exercise, and rehabilitative tasks often require the ability to repeatedly and accurately produce sub - maximal force levels . In a recent series of studies, keith lohse assessed the role of attentional focusing instructions on participants ability participants using external instructions (focusing on pushing against the force platform during an isometric plantar flexion task) were more accurate in producing the force and reduced movement preparation time compared to internal instructions (focusing on the calf muscles) when attempting to generate 25% of their maximum force for 4 s, after previously training without attentional focusing instructions (liao and masters, 2001, experiment 1). In experiment 2, the benefits of externally focused instructions were greater for a lower target (25% maximum force) compared to a higher target (50% maximum force) force . Therefore, the benefits of an external focus of attention may increase with increasingly precise force production requirements (liao and masters, 2001). Externally focused training instructions facilitated retention and transfer testing performance when no instructions were provided, during which participants self - reported attentional focus was clearly linked to the instructions they trained with . After training without attentional instruction, participants attempted to generate 30% of the maximum force for 4 s whilst using either internally (focusing on the calf muscles, specifically the soleus muscle) or externally (pushing against force platform) focused instructions . Supporting the findings of liao and masters (2001), internally focused instructions resulted in greater error in producing this target force . In addition, internally focused instruction increased cocontraction of the agonist (soleus) and antagonist (tibialis anterior), and increased recruitment of the antagonist (indicative of poor intra- and inter - muscular coordination (lohse et al ., 2010b). This latter finding supports research demonstrating increased muscular activity during internally focused instructed movement execution in sporting settings (e.g., zachry et al ., 2005). Related research supports the assertion that attentional focusing instructions influence the production of specifically targeted sub - maximal forces . 2007), examined tongue and hand strengths in relation to attentional focusing instructions to assess their potential utility in speech therapy . Healthy undergraduate students produced rapid pressure bursts (40 bursts of 20% of their maximal strength, at 5 s intervals) of either hand or tongue during impulse force control tasks (suggested to result in relatively fatigue resistant muscular activation). Externally focused instructions (onto pressure exerted on an air - filled rubber bulb held in the mouth or hand) resulted in greater accuracy and less variability in the production of the target submaximal force level in both force control tasks when compared to internal instructions (onto exerting force with the hand / tongue). In summary, 2007) is supportive of the work presented so far in that verbal instructions emphasizing internally referenced information (e.g., muscles and movements associated with force production) result in reduced muscular efficiency compared to externally focused instructions (e.g., the object force is being exerted against) during force production . Verbal instructions emphasizing attentional focus onto a force - plate (liao and masters, 2001; lohse et al ., 2010b), or a air filled pressure sensitive bulb (freedman et al ., 2007 however in these studies, the benefits have been demonstrated in relation to subtle and targeted sub - maximal rather than maximal force production tasks . Clearly these studies represent a limited body of evidence so far, and further work is required on a variety of tasks . Within those studies described, testing protocols required participants to repeatedly generate targeted sub - maximal forces that are relatively resistant to fatigue effects . Of interest next is how the beneficial effects of externally focused instructions on force production and movement efficiency translate to prolonged force generation or repetitive execution of forceful movements that are vulnerable to fatigue effects . As well as assessing the influence of attentional focusing instructions on maximal and accurate force production, researchers have recently attempted to ascertain their influence on the maintenance of force generation in muscular endurance tasks . Wulf and lewthwaite (2010) proposed that an external focus should be associated with more effective maintenance of sub - maximal force production, whereas an internal focus would limit muscular endurance through inefficient movement and muscular activation patterns . Only a limited number of studies have used the internal and external focus conceptualization discussed so far in relation to prolonged sub maximal exertion type tasks . However, a large body of research in the sport and exercise psychology literature does address the impact of attentional focus on endurance . It is not the scope of this review to assess this large body of research (for a discussion, see lind et al ., 2009), which has primarily been concerned with the attentional focusing strategies of association (focus on bodily sensations) and dissociation (actively blocking out painful physiological responses related to task effort). What will be discussed here are those studies that have recently attempted to assess the influence of attentional focusing instructions on prolonged submaximal force production ., in press) recently demonstrated the impact of attentional focusing instructions on trained individuals muscular endurance . Using three typical exercise movements (bench press and squat), experienced exercisers used internally (e.g., focusing on the movements of the limbs involved in the exercise) and externally (e.g., focusing on the movement of and exerting force against the bar being lifted) focused instructions whilst executing continuous repetitions of standardized weights to failure . In the first exercise participants completed a modified version of the ymca bench press test (with males lifting 40 kg and females lifting 20 kg to failure) on a smith machine (ends of barbell attached to free running bearings on two vertical bars allowing for vertical movement only). Externally focused instructions resulted in significantly more repetitions executed before failure than under the internal instruction condition, but not the control condition . In the second exercise, participants completed more repetitions on a free bench press (barbell is free from restriction, executed on a standard bench and rack) at 75% of their one repetition maximum (1rm, maximum weight an individual is able to lift in a single repetition of an exercise). A significantly greater number of repetitions were completed using externally focused instructions when compared to both internal and control instructions . Similar findings were observed in the final exercise, where external instructions resulted in greater repetitions to failure when compared to both internal and control instructions when participants executed free squat (free barbell held behind the neck and across the upper back, lifted with the legs) lifts at 75% of their 1rm . These three progressively more complex weight lifting movements demonstrated increasing sensitivity to the impact of attentional focusing instruction . With movement restricted to the vertical plane in the smith machine bench press, the benefits of an external focus were significant but minimal . The benefits increased when executing movements on the free bench press and during the more complex free squat exercise (in terms of musculature involved and motor unit coordination). These findings demonstrate the influence of subtly different instructional emphasis on this trained population's ability to maintain force production before failure, an effect that increases as movement complexity develops . However, data on the movement kinematics and neuromuscular variables is lacking, and further discussion is limited . Such data would benefit an understanding of how movement form deteriorated during these lifts to failure . For example, research demonstrates that good form deteriorates with fatigue (e.g., duffey and challis, 2007), and methods of reducing this would benefit training outcomes and safety . Similarly, movement time was neither recorded nor controlled . Given that vance et al . (2004) observed faster movement execution times when an external focus was employed, control, or measurement of movement speed would benefit future research . (in press) have assessed how attentional focusing instructions influence intermediate swimmers performance of a prolonged performance task (16 m front crawl). In a novel approach to the instructional manipulation, and one reflecting the complex coordinative nature of swimming, in their first experiment instructions not only directed attention internally and externally, but also toward either the arm and leg components of the stroke . When using internal instructions, participants focused on pulling your hands back (arm stroke) or pushing the instep down (leg kick). In the external condition, participants were instructed to focus on pushing the water back (arm stroke) or pushing the water down (leg kick). All participants completed internal and external instruction trials, but these were specific to the stroke component group that they participated in (e.g., arm pull or leg kick group). Swimming times were faster when using external instructions, regardless of the stroke component emphasized . Experiment 2 only emphasized attentional instructions of the arm pull, and also demonstrated faster swimming times for an external focus . As such, performance benefited from attention being directed toward a force - related outcome (pushing the water down or back) compared with internally focused instructions onto movement mechanics . The novel approach to the instructional manipulation of an external attentional focus in this study generates further discussion . When manipulating an external focus in tasks such as swimming where clear outcomes are limited, freudenheim and this is in line with wulf's (2007) suggestion that instructions which emphasize pushing the water back during swimming may hold external properties (see p. 65). With reference to swimming breaststroke, wulf also suggests that instructions should emphasize the production of a triangle with the arms (see p. 62). Researchers should foster links and discussion with coaching practitioners to highlight potentially useful sources of external emphasis possibly drawing upon analogies (e.g., liao and masters, 2001). Despite such findings, key limitations are also worth considering . Information concerning arm stoke parameters (length and frequency) and the quality of leg movements (e.g., number / type of cycles), would have allowed discussion of whether velocity was affected by changes in stroke length or stroke frequency . The prolonged nature of the sprint task does differ from previous research and provides useful insight, but it requires maximal power production rather than specifically muscular endurance . In summary, there is limited research addressing the maintenance of force production using instructionally manipulated attentional focuses . However, initial evidence suggests that an external focus provides some protection against the development of fatigue so that performance can be maintained when compared to internal focused instructions . Research has emphasized an external focus onto the object or substance which force is being exerted onto (a barbell or the water through which one is swimming) in comparison to internal focus onto the limbs or muscles utilized in the movement . Information on the direct mechanisms of these effects is lacking, particularly in the form of movement kinematics, kinetics, and physiological parameters . Given the improved performance, it can be suggested that an external focus promotes movement efficiency and coordination, but research is required to directly assess this . Researchers in physical performance and therapy settings have long been interested in how to influence force production, and verbal instruction and encouragement are regularly utilized in testing protocols . Despite the full details of instructions not being consistently reported in much force production research, some researchers have assessed the influences of different types of instructions albeit not from an attentional perspective . (2003) assessed the influence of instruction on the control of force during landing movements . Instructions emphasizing increasing knee flexion induced lower landing forces than those emphasizing earlier recruitment of the hamstring during landing and under no - instructions (see also prapavessis and mcnair, 1999; mcnair et al ., 2000), suggesting a protective benefit during control of landing forces if attention is directed to form . Although both instructions are internally referenced in terms of the present discussion, they differ in terms of joint - movement or muscular emphasis . Participants were unable to selectively recruit their hamstring when instructed to do so and inadvertently altered their quadriceps muscle activation to the extent that it was less protective during landing . A finding similar to that of lohse a direct comparison is difficult, but these findings do support the previous work highlighting harmful effects of focusing internally onto muscular activation whilst controlling landing forces . These landing tasks are without an obvious movement outcome for external manipulation, but the emphasis of good movement form (rather than mechanics) appears to offer the best alternative at present . Whilst castaneda and gray (2007) and mcnevin et al . (2003) suggest that different external attentional focuses have different effects, in these cases, different internally referenced instructions have different effects . (2000) demonstrated decreased landing forces when attention was directed externally toward the sound of landing . But these benefits were only observed against a control and an imagery condition, and were not different from those gained from the instruction condition . (2000) concluded that instruction guiding safe jump landing should direct attention to the sound of participants landing in addition to concise lower limb kinematic instruction . Whether such mixed attentional focus instruction can be effective is unclear, but research should identify relevant externally focused instructions that could be employed in such force control settings . Other research addressing attentional manipulations and prolonged movement execution have not fully addressed the conceptualization of attentional focus presented so far . (2009) demonstrated that enhanced running economy values (lower oxygen consumption, but not blood lactate or heart rate) were related to the adoption of an external focus onto the surroundings when compared to focusing on breathing or running movements during a 30-min run at 75% vo2max . Participants indicated that the external condition was the easiest, and that the strategies were used during the majority but not all of the runs . Concluded that running (and associated breathing patterns) was at its most economic when the automatic control processes involved in its execution were not interfered with through conscious control . However, rather than an externally distracting comparison, an effective task - relevant external instructional set is required . Research identifying such information could potentially test wulf's (2007) proposal that focusing upon stride length may be a useful external reference (p. 64 however, contrary to this clingman and hilliard (1990) demonstrated that focusing on cadence was superior to focusing on stride length and a dissociating condition in improving race - walkers performance . Research assessing the impact of different external instructions is warranted for tasks requiring prolonged force production . As already stated, different types or distances of external focus may have differential influences on force production, and this may be an important variable in endurance settings . In reviewing these examples of forceful movement execution research it is clear that the identification and examination of different types of externally focused instruction is necessary for further analysis of tasks such as these . It is also apparent that more cross - disciplinary collaboration is required to disseminate the attentional focusing instruction research findings from movement science settings to strength and conditioning research and testing settings . Given the clear interest by a number of researchers in the influence of instruction on force production, such collaboration may provide a fruitful and insightful opportunity . Overall, it is clear that the emphasis of instructions provided has an impact on performance in the force production tasks discussed . It also appears quite likely that in each of the examples provided, the attention focus emphasized in the instructions provided is a key mechanism in the observed outcomes . The available research findings presented in this review indicate that an external focus allows the motor system to self - organize; efficiently coordinating and directing forces needed for accurate, maximal and sustained force production . The observed associated mechanisms include improved limb coordination (e.g., wulf and dufek, 2009) and intra- and inter - muscular coordination (e.g., marchant et al ., 2009; lohse et al ., 2010b; wulf et al ., 2010). Together, these findings present the developing understanding of different attentional focusing instructions influence on force production outcomes and mechanisms of performance . It appears so far that vance et al.s (2004) proposal that an external focus onto the object through which force is being exerted will be beneficial is correct . Clearly, the research here is at an early stage, and a number of issues require further consideration . Given the nature of the force production tasks employed, different instructional approaches have been utilized (see table 1). Studies have generally provided instruction prior to performance, but in some cases (often to control for encouragement) reminders and prompts are provided during tasks (e.g., marchant et al ., in press; freedman et al ., 2007). Similarly, the nature of the target - force production tasks employed by liao and masters (2001) required the provision of internally and externally focused feedback throughout trials . Research is required to assess the impact of such during task instruction and feedback, and given that encouragement during force production tasks has been shown to influence performance (e.g., bickers, 1993; campenella et al ., 2000) some degree of control over this is required if the instructed attentional focus is to be consistent . Related to this, limited evidence on the use and experiences of the instructions provided was available . Over prolonged force production tasks, the issue of maintaining attentional focus becomes important in terms of general concordance and attentional capacity . Whilst motor control researchers suggest a propensity to focus attention internally (primarily onto explicit awareness of skill execution) when under pressure and that this is a critical mechanisms in choking under pressure (baumeister, 1984; lewis and linder, 1997; beilock and carr, 2001), researchers assessing exertional experiences and tolerances note that attention is drawn internally (primarily onto physiological feedback) under increasing workloads . The ability to shift between attentional focuses depends upon the intensity of exertion (see tenenbaum and hutchinson, 2007). For example, hutchinson and tenenbaum (2007) have demonstrated that at high intensity and prolonged duration workloads, attentional focus shifts internally and becomes less flexible as physiological sensations become increasingly salient . Whereas at lower and moderate levels of exertion, the individual can voluntarily shift attentional focus . This suggests a limit to the influence of instructional manipulations of attentional focus during force production tasks as workload increases . Can providing externally attentional focusing instruction and feedback facilitate efficient movements and force production during these difficult ranges of workload, or is an internal focus at these stages unavoidable? The experiences of effort, force production and exertion are not adequately addressed in this body of research to date . For example, the feed - forward hypothesis proposes a relationship between neuromuscular activation and perceived exertion (e.g., cafarelli, 1982; hasson et al ., 1989; pincivero and gear, 2000). As such, another limitation of an internal focus that it also increases perceptions of effort in line with associated increases in muscular activity? How instruction interacts with perceptions of effort in these settings is important information, and may be useful for guiding future instructional approaches . Similarly, no research has addressed how the effects of attentional focusing instructions in force production settings are moderated by participant characteristics such as expertise and experience, self - efficacy, or attentional control . Worth further consideration for the development of externally focused instructions, de graaf et al . (2004) suggest that muscle force awareness is explicit knowledge of the muscular force produced during voluntary movement and is distinct from movement outcome awareness . In their study, de graaf et al . Demonstrate that whilst kinematic awareness is easily accessible, muscular force awareness is harder to perceive (both in terms of perceived demands and associated cortical activation). Given this difficulty, what implications are there for effective instruction of attentional focus during force production tasks? Reviewing the instructions used to date, for externally focused instructions some studies emphasize force production, others emphasize movement outcomes, and some mix both external components . Likewise, when manipulating an internal focus, some studies emphasize movement kinematics whilst some emphases muscular activation, and some attempt both (see table 1). Other methodological consideration include sound measurement of the mechanisms associated with performance changes as recent work has not provided a clear picture of how different attentional instructions have changed movement execution . For example, no data on lifting kinematics or swimming stroke components is provided in marchant et al . (in press) to explain the benefits of an external focus . Longitudinal research assessing the implications of the present findings on physiological adaptation processes resulting from training with different attentional strategies will also benefit this area . Ives and shelley (2003) indicate an appropriate attentional focus is a key variable in developing specific physiological training adaptations, without which adaptations are limited . Evidence of the long term impact of attentional focusing instructions and feedback is currently lacking . Finally, in addition to muscular activation and biomechanical mechanisms, recent evidence demonstrates influences of attentional focusing instructions at a motor - relevant cortical level . Greater activation of the primary somatosensory, motor, and insular cortices were observed when externally compared to internally focused instructions were utilized during the acquisition of a simple motor skill (key press sequence; zentgraf et al ., 2009). Whilst an external focus enhanced task - relevant tactile information processing necessary for the effective execution of environmental - outcome movements, internally focused instructions disrupted the efficient neural flow between sensory and motor areas . That attentional focusing instructions should have a similar influence during force production task seems plausible considering that evidence points to central neural commands being vital in such tasks (e.g., gandevia, 2001) and that an inadequate activation of motoneurons required for effective force production is associated with suboptimal descending drive from the motor cortex (taylor et al ., 2000). For example, loss of force through fatigue occurs due to suboptimal output from the motor cortex . This is evident in the observed increases in force evoked through activation of the motor cortex by transcranial magnetic stimulation (tms; e.g., gandevia et al ., 1996; taylor et al ., 2000 gandevia (2001) recommended better instruction and feedback to minimize the restrictive impact of these supraspinal variables in testing and training settings, and we have seen here that the influence of instructions depends upon their emphasized direction of attentional focus . As such, attentional focusing instructions may be a practically accessible way of researchers and practitioners maximizing participants output . Research is required to address the impact of attentional focusing instructions on different brain areas in line with observed force production, muscular activation, and biomechanical parameters . Specific research on force production and attentional focusing instructions to date is relatively limited, but adds to the body of evidence demonstrating beneficial effects of externally compared to internally focused instructions on movement skill performance and acquisition . The evidence suggests that an external focus onto the object through which or toward which force is being exerted is beneficial to force production, compared to an internal focus toward the movements and muscles associated with the force production . Specifically, individuals can produce greater forces, more accurate targeted forces, or maintain force production for longer when instructed to focus externally . However, firm conclusions cannot be reached until further research addresses a number of key issues (e.g., mechanisms of effects, moderating variables, instructional consistency). The findings do support wulf's (2007) proposal that an external focus of attention should promote the body's natural propensity to conserve energy through coherence between the outcome and the sensory consequences of that action (p. 121). Underpinning the force production benefits discussed, research highlights movement efficiency as a key mechanism being promoted through an external focus of attention (e.g., muscular activity and limb coordination). However, research is required to substantiate these findings further, incorporating a greater appreciation of the variables associated with force production . The maximal and accurate production and maintenance of forceful movements requires a complex interaction between cognitive, psychological, and physiological variables . Of these many variables, attentional focus has been differentially defined and utilized in both research and practice . For a fuller understanding of the role of task - specific attentional focusing instructions on force production in the future, researchers should consider how those instructions interact with individual and task - specific characteristics . Finally, practitioners and researchers involved in testing or training individuals in force production settings should be aware of the influence even subtle differences in the instruction and feedback they provide can have . Researchers in particular should provide details of the instructions and feedback they have used in force production and testing protocols . This would allow for better comparisons between studies and findings . Furthermore, control for instructional content within force production research protocols should be a priority . Any differences in the attentional focus emphasized in instructions and feedback provided may induce unplanned differences between and within conditions, participants, and trials . In terms of consistency in facilitating performance 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.
Elderly individuals are at high risk to develop chronic airway inflammatory illnesses such as asthma and chronic obstructive pulmonary disease (copd) [13]. Among all airway illnesses, a recent survey estimates that asthma affects 48% of the american population over 65 years of age and numbers are expected to increase in the upcoming years . Most deaths caused by asthma also occur in this age group [2, 3]. Acute respiratory viral infections such as influenza are often the trigger for asthma attacks and therefore considered the primary culprits in the pathogenesis of asthma in the elderly [4, 5]. There is a scarcity of information about the influence of chronic or latent respiratory infections, which could induce prolonged airway inflammation . Chlamydophila pneumoniae (cpn) is an obligate intracellular bacterial pathogen that causes chronic, persistent, and often asymptomatic infections . It is also reported to be implicated in exacerbation of asthma, pharyngitis, and bronchitis . Community - acquired pneumonia (cap) remains a significant cause of morbidity and death worldwide and a significant threat to the elderly despite the availability of effective antibiotics . Cap due to atypical pathogens, cpn, and mycoplasma pneumoniae, as single or copathogens, can cause severe diseases in older patients, resulting in hospitalization . Studies from the literature suggest that mycoplasma and chlamydophila are responsible for 912% and 613% of cap, respectively . Reports also indicate that the prevalence of antibodies specific to cpn increases significantly in the elderly . Furthermore, aged mice infected with cpn displayed an increased severity of infection compared to young animals . The age - associate alterations in the function of immune system that may play a role in the increased incidence and severity of cpn infections in the elderly are not well understood . Dendritic cells (dcs) are present below the epithelial cells lining the airway and are amongst the primary responders to airway infections . Dcs present at the airways sense and capture pathogens through a variety of pathogen recognition receptors (prrs). Subsequently dcs become activated by upregulating the expression of costimulatory and antigen - presenting molecules as well as secreting proinflammatory cytokines . During activation, the activation molecules and cytokines secreted by dcs have a major influence on t cell responses [13, 14]. We and others have reported that advancing age significantly impacts dc functions and alters their response to infections . Dcs from aged donors exhibit enhanced inflammatory responses at the basal level and display increased reactivity to self - antigens [16, 17]. In contrast to self - antigens, the response of dcs from elderly to pathogens is often compromised . For example, dcs from aged subjects are severely impaired in their capacity to produce interferon- (ifn-) and interferon- (ifn-) after activation with influenza virus [18, 19]. Recent in vivo studies in mice have demonstrated that cpn can infect dcs and alter their function to induce a proinflammatory state . Furthermore, cpn may also utilize dcs for dissemination outside the lung [20, 21]. Age - associated alterations in the response of dcs to cpn may therefore play a major role in the increase in cpn infections in the elderly . The objective of the present study was to compare the response to cpn of dcs obtained from both young and elderly subjects . The age of young donors was between 20 and 35 years and that of elderly was between 65 and 90 years . This study was approved by the institutional review board of the university of california (irvine, ca, usa). Peripheral blood mononuclear cells (pbmcs) were separated by ficoll - hypaque density gradient centrifugation . Monocytes were purified from the pbmcs by positive selection with anti - cd14 microbeads (stemcell sep, vancouver, bc, canada). The purity of the isolated cd14 monocytes was> 90%, as determined by flow cytometry . For the induction of dc differentiation, purified cd14 monocytes were cultured in a humidified atmosphere of 5% co2 at 37c in rpmi 1640 supplemented with 10% fbs, 1 mm glutamine, 100 u / ml penicillin, 100 g / ml streptomycin, 50 ng / ml human rgm - csf (peprotech, rocky hill, nj, usa), and 10 ng / ml human ril-4 (peprotech). Half of the medium was replaced every 2 days and dcs (cd14hla - drcd11c cells) were collected after 6 days . The purity of the dcs was> 95% as determined by the expression of cd14, cd11c, and hla - dr . C. pneumoniae cm-1 (atcc, manassas, va, usa) was propagated in hep-2 cells and stored in 2-sucrose phosphate glutamate buffer at 80c . Immature dcs collected on day 6 were stimulated with uv inactivated cpn with an moi equivalent to 0.01 cpn: 1 dc or 0.1 cpn: 1 dc or 1cpn: 1 dc . Control and cpn - stimulated dcs from both age groups of subjects were stained for the surface expression of cd80, cd86, cd83, and hla - dr using directly conjugated abs (bd pharmingen, san diego, ca, usa). A total of 10,000 cd14cd11c cells per condition were acquired using a facscalibur (bd pharmingen). Cytokines, ifn-, il-6, tnf-, il-10, il-1, cxcl-8, cxcl-10, and il-12p40 in the supernatants were measured by flow cytomix (bd pharmingen) as per the manufacturer's protocol . Ifn- was measured using an elisa (pbl biomedicals, piscataway, nj, usa). Subsequently, 1 10 dcs were cultured with 1 10 magnetic bead purified (stemcell, vancouver, bc, canada), nave allogeneic t cells from young individuals . For nave t cell isolation, first the total t cells were isolated by negative selection; subsequently, cd45ro beads were used to remove the memory cells . Dcs from aged and young subjects for each experiment were cultured with allogeneic t cells from one young subject for comparison . After 7 days of incubation, the supernatant was collected and the secretion of ifn-, il-10, il-21 (ebiosciences, san diego, ca, usa), ifn- (bd pharmingen), and il-17 (ebiosciences) was assessed using elisa . Stimulated dcs were cultured with purified, cfse - labeled allogenic t cells from young donors at a ratio of 1: 10 in 96-well plates . The purity of t cells recovered from dc t cell cultured after 7 days was 8591%, as determined by flow cytometry . Cells were then collected and stained for cd4 and cd8 markers . By measuring the dilution of cfse proliferation index (total number of divisions divided by the number of cells that went into division) was calculated using the proliferation platform of flowjo . Cdcs were enriched from the pbmcs of aged and young subjects by negative selection using magnetic beads based kit from stemcell separation . Cdcs (5 10/ml) were stimulated with uv inactivated cpn with a moi equivalent to 1 dc: 1 cpn for 24 h. supernatants collected were assayed for various cytokines as described above for dcs . Data were analyzed and figures were generated using graphpad prism 5.00 software (graphpad software, san diego, usa). Significant differences between groups were determined by mann - whitney test at 90% confidence interval (p value <0.05 was considered significant). Therefore, we investigated whether the upregulation of dc activation markers in response to cpn was altered in dcs from aged subjects as compared to dcs from young subjects . The control population included 24 individuals in the age range of 2032 with an average age of 26 . The geriatric population consisted of 24 individuals in the age range of 6593 with an average age of 78 . Dcs from aged and young individuals were stimulated with uv inactivated cpn at a ratio of 1: 1 for 24 h. this concentration of cpn was found to be optimal (supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/436438). Stimulation with the bacteria resulted in substantial activation of dcs from both aged and young subjects (figure 1). The expression of activation markers was comparable on dcs from aged and young subjects (figure 1(a)). Average of mean fluorescent intensity (mfi) of all subjects suggested that dcs from aged subjects displayed enhanced expression of cd80 and cd83 but the difference was not significant (figures 1(b) and 1(d)). The mfi of expression of cd86 and hla - dr on the other hand was comparable in dcs obtained from both age groups (figures 1(a), 1(b), and 1(e)). This data suggest that infection with cpn leads to slight though insignificant increase in activation and maturation of dcs from aged as compared to dcs from young subjects . After stimulation with cpn for 24 h, supernatants were collected and assayed with flow cytomix and/or elisa to quantify cytokine secretion . Stimulation of dcs with cpn induced the production of increased levels of proinflammatory cytokines (il-6, tnf-, il-1, il-12, ifn-, and ifn-), chemokines (cxcl-8, cxcl-10), and anti - inflammatory cytokine (il-10) over unstimulated dcs in both age groups (figure 2). However, the level of the secreted cytokines and chemokines was substantially different between the two populations . Stimulation with cpn resulted in significantly enhanced secretion of cxcl-10 (p = 0.007, figure 2(a)) and tnf- (p = 0.01, figure 2(b)) in dcs from aged subjects as compared to dcs from young subjects . Dc from aged donors also secreted significantly higher levels of il-12 relative to dcs from young donors (p = 0.01, figure 2(c)). In contrast, secretion of anti - inflammatory cytokine, il-10 was significantly reduced in cpn - stimulated dcs from aged subjects compared to dcs from young subjects (p = 0.02, figure 2(d)). Interestingly this high secretion of il-10 by dcs from aged subjects was also observed when dcs from aged and young subjects were incubated with a lower number of cpn, that is, 0.1 cpn and 1 cpn (data not shown), implying that exposure to cpn, even at lower concentrations, induces an immunosuppressive response rather than an inflammatory response in dcs from young subjects . However, in dcs derived from aged individuals, this response is reversed and is more of an inflammatory response . Previous studies have reported a substantial decline in the production of innate interferons, ifn- and ifn-, by dcs from aged donors in response to respiratory viral infections [18, 19, 23]. Therefore, we investigated whether this deficiency in ifn production extends to respiratory bacterial infections . Similar to our results with viral infections, incubation of dcs from aged subjects with cpn resulted in significantly reduced levels of ifn- (p = 0.02, figure 2(e)) and ifn- (p = 0.015, figure 2(f)) relative to dcs from young subjects . The secretion of all other cytokines, il-6, cxcl-8, and il-1 (figures 2(g)2(i)), was comparable in cpn - stimulated dcs from both groups . In summary, these pieces of data demonstrate that advancing age enhances the capacity of cpn - stimulated dcs from aged to secrete proinflammatory and th1 promoting cytokines and chemokines which is accompanied by reduced secretion of anti - inflammatory and protective cytokines . In the aged subjects subgroup analysis was performed for osteoarthritis, hypertension, and subjects taking vitamins and antioxidants as these subgroups had sufficient subject numbers . However, we did not observe any significant difference in any of the cytokine levels between the two groups (p> 0.5). Based on these subgroup analyses, we feel fairly confident that the comparisons between the young control and aged subject populations are yielding valid results across a general geriatric group . Experiments described above were performed with dcs derived from monocytes in vitro in the presence of rgm - csf and ril-4 . To rule out any possible artifacts in the response of dcs due to in vitro derivation, we investigated the response of directly purified cdcs from the blood of aged and young subjects to cpn . Similar to monocyte derived dcs, stimulation with cpn resulted in significantly enhanced secretion of tnf- (p = 0.002, figure 3(a)) and cxcl-10 (p = 0.04, figure 3(b)) by cdcs from aged subjects relative to cdcs from young subjects . The secretion of il-6 was also significantly higher (p = 0.018, figure 3(c)) in cdcs from aged subjects compared to their young counterparts . In contrast, cdcs from aged donors displayed significant impairment in the production of il-10 (p = 0.001, figure 3(d)) and ifn- (p = 0.008, figure 3(e)) relative to cdcs from young donors . The levels of il-12, il-1, cxcl-8, and ifn- were below the detection limits of our assay . These pieces of data suggest that both in vitro derived dcs and cdcs from the blood of aged subjects display similar responses to cpn stimulation . The rest of the experiments were therefore performed with in vitro derived dcs . In order to determine whether the t cell priming capacities of cpn - stimulated dcs are also altered in the elderly, we investigated the capacity of dcs from aged and young subjects to induce the proliferation of cd4 t and cd8 t cells utilizing an allogeneic dc - t cell coculture system described previously . Cpn - activated and nonactivated dcs were cultured with nave, purified, cfse - labeled t cells from young, healthy individuals (to rule out t cell abnormalities) as the t cells from the elderly have been reported to have numerous defects [24, 25]. Six days later, t cells were assayed for proliferation by measuring the dilution of cfse dye by flow cytometry . Culture of cd4 t cells with cpn - stimulated dcs from aged subjects induced approximately 58% proliferation in cd4 t cells relative to unstimulated dcs which induced 47% proliferation (figure 4(a), p = 0.002). Cpn - stimulated dcs from young subjects also induced the significant level of proliferation of cd4 t cells (45%) over unstimulated dcs (38%) (figure 4(a), p = 0.039). Therefore, the increase in cd4 t cell proliferation was 4% greater than that in coculture of dcs from aged and t cells (figure 4(a)). Proliferation of cd8 t cells followed a similar pattern with significantly increased cd8 t cell proliferation being observed after culture with cpn - stimulated dcs from aged individuals (65%) (figure 4(b), p = 0.002) relative to unstimulated dcs (52%). In contrast, cpn - stimulated dcs from young subjects failed to enhance significantly increased cd8 t cells proliferation (49%) relative to unstimulated dcs (54%) (figure 4(b), p = 0.14). No proliferation was observed in both cd4 and cd8 t cells cultured alone without dcs . Altogether, these pieces of data suggest that dcs from aged subjects induce a more vigorous t cell response against cpn infection compared to dcs from young subjects . Next, we determined the nature of cytokines secreted by t cells primed with cpn - stimulated dcs from aged and young subjects . Given the distinct profile of cytokines secreted by dcs from aged and young donors, we expected differences in the polarization of t helper (th) cell responses towards th1, th2, treg, th17, or tfh . Indeed as is evident from figure 5(a), culture of cpn - stimulated dcs from aged subjects induced significantly higher level (p = 0.03) of ifn- secretion from t cells as compared to their young counterparts . In contrast to ifn-, cpn - stimulated dcs from aged donors induced significantly reduced levels (p = 0.026, figure 5(b)) of il-10 production by t cells compared to young dc t cell coculture, further confirming that cpn - stimulated dcs from aged subjects display enhanced level of activation compared to dcs from young subjects . Secretion of il-17 by t cells was comparable between aged and young subjects (figure 5(c)). We also determined il-21 secretion since il-12 secretion by dcs can induce il-21 in addition to ifn-. Cpn - stimulated dcs from aged or young donors did not induce significant levels of il-21 and the data was comparable in the two groups (figure 5(d)). No ifn-, il-10, il-17, and il-21 were detected in t cells cultured without dcs . Dcs cultured alone without t cells for six days also did not secrete any of these cytokines (data not shown). The elderly population is reported to be at higher risk to develop respiratory illness such as copd, bronchitis, and asthma [2, 3]. Several studies document the importance of persistent cpn infections in the pathogenesis of these diseases [7, 26]. Dcs are shown to be crucial, not only for sensitization to inhaled antigens, but also for establishing inflammation in the lung . In vivo studies have demonstrated that cpn can infect dcs and utilize them for dissemination from the lungs to other organs . Persistent cpn infection also induces a proinflammatory state in dcs [20, 21]. We and others have demonstrated that advancing age results in significant changes in the function of dcs . Dcs from aged subjects are impaired in their capacity to control inflammation due to reduced production of anti - inflammatory cytokines, for example, il-10 . Here we report that age - associated alterations in dc functions enhance their response to cpn . These alterations induce an increased inflammatory response in the elderly as compared to young subjects . Our results with monocyte - derived dcs are in keeping with previous studies which have reported that cpn infected dcs induce proinflammatory th1/th17 response via the production of il-12, il-6, and il-1 [28, 29]. These studies all utilized viable cpn while our studies have been performed with uv inactivated cpn . The differences in the response between viable and uv inactivated bacteria are not apparent at the level of cytokine secretion by dcs since dcs can respond to bacterial components we did not observe differences in phenotype between dcs from aged and young subjects upon cpn stimulation . This is in agreement with previous reports where also phenotype of dcs from aged and young subjects after tlr stimulation was comparable [15, 30] but the secretion of inflammatory cytokines was elevated . The differences in cytokine production by dcs are not always associated with differences in phenotype . In this study, also dcs from aged displayed an enhanced proinflammatory response upon cpn stimulation as is apparent from the increase in tnf-, cxcl-10, and il-12 and reduction in il-10 secretion (figures 2(a)2(d)). We chose to assay cxcl-10 due to its important role as a key chemokine for the recruitment of th1 lymphocytes into tissue and also because of the association of cxcl-10 with inflammatory and allergic diseases . Furthermore, the infection of mice with cpn via the intranasal route also induced cxcl10 in the airways and enhanced inflammation . Increased cxcl-10 secretion along with increased il-12 by dcs from aged subjects will therefore enhance the th1 inflammation in the airways of the elderly . In addition, increased tnf- secretion by the dcs from aged subjects also favors th1 responses . However, tnf secretion in response to cpn also inhibits the growth of the bacteria in dcs and regulates inflammation via induction of indoleamine 2,3-dioxygenase (ido). The induction of ido in dcs is a mechanism to prevent replication or persistence of cpn infection but since the present study was performed using uv inactivated cpn, we did not analyze the expression of ido . Regulation of inflammation is impaired in dcs from aged subjects since il-10 production in response to cpn is not elevated to counter the production of increased proinflammatory cytokines . This is in agreement with our previous observations which also demonstrated that stimulation of dcs from aged subjects with lithium chloride, which is well established il-10 inducer in dcs, failed to do so in dcs from aged subjects suggesting that dcs from aged are inherently defective in the production of il-10 . The increase in inflammatory response is not due to altered tlr expression as we have previously reported that the expression of tlrs is comparable between aged and young dcs . This has been further confirmed with our gene array data on aged and young dcs . The gene array data also did not show any difference in the expression of tslpr, il-33r, or icosl on aged dcs as compared to young dcs . However, we did observe alterations in the nfb signaling pathway which is similar to our previously reported findings that dcs from aged subjects display an enhanced basal level of nfb activation, a major pathway involved in proinflammatory cytokine production from dcs . This enhanced nfb activation in dcs from aged subjects may be responsible for the increased secretion of inflammatory cytokines by aged dcs in response to tlrs and pathogens [22, 30, 36]. Interestingly, we also observed similar enhanced secretion of tnf-, cxcl-10, and il-6 and decreased il-10 secretion from cpn - stimulated cdcs purified from the blood of the elderly (figures 3(a)3(d)) suggesting that increased inflammatory response to cpn is a feature of dcs from the elderly and is not due to the inflammatory nature of monocyte derived dcs . In addition to il-10, cpn - stimulated monocyte derived dcs from aged subjects were also deficient in production of innate interferons, ifn- and ifn-, which serve as a primary host defense mechanism against infections (figures 2(e) and 2(f)). Cpn - stimulated cdcs from the blood of elderly also displayed a similar defect in the production ifn- (figure 3(e)). Ifn- is known to play an important role in the defense against respiratory tract infections, such as influenza a virus, respiratory syncytial virus, and sars coronavirus; however, its role in cpn infection has not been investigated . Our results suggest that ifn- may be an important protective cytokine in response to cpn . Previous reports from our laboratory have also demonstrated age - related impaired secretion of ifn- and ifn- by plasmacytoid dendritic cells and monocyte derived dcs during influenza infection . This suggests that impairment in ifn secretion is an intrinsic age - related alteration and not associated with a specific type of infection . Previous studies have reported the induction of ifn- and il-17 producing cd4 t cells by cpn - stimulated dcs . We also observed the secretion of both of these cytokines though the t cells stimulated with dcs from aged displayed enhanced proliferation and secreted increased levels of ifn- and decreased il-10 compared to t cells stimulated with dcs from young subjects (figures 4(a), 5(a), and 5(b)). The cytokines secreted by dcs in response to an infection dictate the proliferation and cytokine secretion of the t cells . The secretion of il-12 and cxcl-10 by dcs induces ifn- production in t cells and therefore increased production of these mediators by dcs from aged results in increased ifn- production while enhanced il-12 and tnf by dcs from aged may be responsible for increased proliferation of t cells . In addition, decreased il-10 production by dcs from aged may further help increase proliferation and ifn- by t cells [13, 40]. A recent murine study has demonstrated that depletion of il-10 producing t regulatory cells, which control inflammation and exacerbate airway inflammation and sensitization associated with cpn infection . Cd8 t cells are also reported to be necessary for clearing cpn infection in mice as depletion of cd8 t cells results in increased bacterial burden and infection kinetics . Moreover, cd8 t cells also produce ifn- in response to chlamydial infection which complements the ifn- produced by cd4 t cells [43, 44]. Higher uncontrolled production of ifn- by both cd4 and cd8 t cells in aged subjects in response to cpn may therefore lead to aggravation of the inflammatory response in the airway of the aged population . Altogether, our data suggest that dcs from aged are more activated and secrete higher level of inflammatory mediators in response to cpn but are still unable to produce a crucial cytokine such as ifn- involved in protection against respiratory infections . Increased inflammatory response of dcs from aged subjects to cpn induces an enhanced th1 response in t cells which aggravates inflammation.
A rapid development of medical sciences in all fields of laboratory diagnostics entails a need to improve the analytical process, which now must meet increasingly strict requirements of scientific societies that set forth new standards in diagnostics and treatment . The most recent guidelines recommended by esc, acc, aha, and whf concerning biomarkers of myocardial necrosis also apply to the work of clinical laboratories . Both good analytical quality and the time of these 2 closely connected elements allow for obtaining a reliable result of the analysis as quickly as possible, which makes it possible for a physician to promptly perform a medical intervention . It is particularly important in acute coronary syndrome (acs), especially with persistent st - segment elevation, which might indicate coronary artery occlusion . In urgent analyses this period of time is called turnaround time (tat), measured from the moment the analysis is ordered to when a result is obtained . This period of time consists of: taking the material (blood), most often from peripheral venous vessels; transporting it to a laboratory; clotting and centrifugation (if it is required by the methodology of the assay); the analytical procedure itself; and authorization and presenting the result to a physician . Esc, acc, aha, and whf guidelines state that tat should not be longer than 60 min for assays of cardiac biomarkers: cardiac isoforms of troponins t and i (ctnt and ctni), mbmass isoenzyme of creatine kinase (ck - mbmass), and myoglobin (myo). However, it is quite difficult to achieve this tat for assays of laboratory parameters, which just include assays of concentrations of cardiac biomarkers, with use of immunochemical methods . These assays, unlike traditional chemical assays, have a relatively long pre - analytical phase (longer clotting time so as to obtain serum) and a longer analytical procedure (usually lasting 2040 min). A way of determining troponin concentration with the high sensitivity method (hstn) has been a standard method in western european countries for more than a year . Thanks to this test, new possibilities have open up for cardiologists, especially in the treatment of acs in patients with no persistent st- segment elevation and in patients with coronary disease, as a predictive test . Methodological modification for tests used in determining cardiac biomarkers has been in use for more than 1 year now . It reduced the time of the analytical procedure from 18 min (the standard version of the test) to 9 min (the short turn - around time version of the test). We should answer the question: is it possible to determine the markers of acute coronary syndrome easily, promptly, and accurately and receive reliable results? Having analyzed these observations, we decided to determine and compare the analytical quality of the tests in the standard and stat versions for determining serum level: cardiac isoform of troponin t with high sensitivity method (tnths), ckmbmass, and myoglobin, as well as to verify whether the tnths stat test meets requirements for use in patients with acs . These requirements are the following: coefficient of variation <10% at the concentration level close to diagnostic tnths, equal to the 99 percentile of reference population; and tat <60 min for tnths assays, with the use of the standard and stat versions . The study was performed with the use of roche assay kits (tnths stat catalogue no . 0509278190, ckmbmass stat catalogue no . 11731432122, and myoglobin stat catalogue no . 11820788122) as well as reference material for the following reagents (tnths catalogue no . 5092744190; ckmbmass catalogue no . 11821598322, and myoglobin catalogue no . All assays were performed with a cobas e411 hitachi immunochemical analyzer, with the use of calibrators and control materials produced by roche . According to statistical metrology, we evaluated analytical quality by using comparative analysis of exactness (precision and accuracy) of measurement methods of all assay kits, as well as analyzing the correlation of the obtained results using 2 tests: the standard and stat versions . In the first module of the studies, we used standardized materials made by roche the precicontrol cardiac ii and precicontrol troponin . We determined lege artis concentration levels of the analyzed parameters on 2 levels: diagnostic low and pathological high (table 1) in test - to - test repeatability . In the second module of the studies, we evaluated the precision of a series of 21 assays of concentration level of troponin t at a level close to diagnostic level (99 percentile of reference population) in native serum in within - test repeatability . The sample of serum was taken from randomly selected patients from the interventional cardiology unit . While performing quantitative definition, we used a relative measurement of imprecision (i%), which, according to metrological terminology, is coefficient of variation (cv) and contains information about random error (standard deviation, sd) and mean value of measurements (x). The evaluation of accuracy was performed with regard to reference values of concentration levels (xr) in the control material . The difference between these values and mean values obtained from the series of measurements, presenting systematic error, was expressed as bias deviation (b%). Using experimentally determined quantitative information on precision and accuracy, we evaluated exactness of measurement methods and expressed it as the total error value (te%). Then the result was compared with the expected quality of measurements acceptable total error (tea%) obtained from the centre of quality control for laboratory diagnostics in d, as well as with biological variation database and quality specifications, which ricos et al . Obtained (table 1). In the third module of the study, to evaluate the correlation of results, we used 80 samples of native serum of physiological and pathological concentration levels comprising maximum linearity of particular measurement methods . The results were taken from standard assays of cardiac biomarkers concentration levels in patients in the interventional cardiology unit of the military medical academy veterans central hospital . The concentration levels were: 0.003 g / l to 10.160 g / l for troponin t, 1.66 g / l to 377.40 g / l for ckmbmass, and 21 g / l to 1225 g / l for myoglobin . We compared the accuracy of determining the stat series of the analyzed assay kits and standard assay kits as reference assays, assuming that the correlation is strong for correlation coefficient r> 0.95 . In the fourth module of the study, we made some observations of tat with the use of the standard and stat versions . The observations were made in a series of unselected studies in the patients from the admission room of the military medical academy veterans central hospital, in whom all the 3 biomarkers were determined . In the first study, we analyzed tat for assays with the use of the standard version (analysis time: 18 min, n=60); in the second study a similar procedure was used with the use of stat tests (analysis time: 9 min, n=57). Tat started when the sample of the study, ordered to be performed by the physician, was entered into the laboratory information system and finished when it was authorized and made available in the hospital information system . The obtained variables were analyzed with the fisher test (evaluating the divergence of qualitative distribution and random distribution) or mann - whitney test (evaluating medians of continuous variables). Statistical calculations were carried out with microsoft excel calculation sheet and medcalc 11 (medcalc software bvba) statistical program . The exactness analysis of reagent kits made in the first module did not indicate a significant decrease in the measurement quality of stat version reagents in comparison to the standard versions, despite numerical differences in the precision and accuracy . Most significantly, still acceptable precision differences were observed with regard to reagents used in determining troponin t. they were particularly visible in the reference material concentration corresponding to diagnostic concentrations . This is because the method has high sensitivity and the values in the analyzed concentration range are low and remain close to the limit of analytical sensibility . The te% of the stat method with regard to tnths on both the controlled levels is almost 2-fold higher (15.6% and 7%) in comparison to the comparative method (7.1% and 4.8%). Yet, it still remains 2-fold lower than the maximum acceptable by the centre of quality control for laboratory diagnostics in lodz (20%) and almost 3-fold lower than the one connected with biological variability troponin concentration in human serum according to biological variation database and quality specifications bvds (28%). The analysis of a series of 21 samples of tnths concentration level in native serum (module 2) showed a comparable precision of the 2 measurement methods . Measurement imprecision was characterized with almost identical coefficient of variation (cv=3.07% for both of the 2 measurement methods tnths and tnths stat (table 2). It also confirmed that the particular analytical method meets precision requirements at cv <10% for troponin, diagnostic in the clinical aspect (99 percentile of healthy population). The analysis of the results of assays in the patients serum (module 3) confirmed a close correlation between all the parameters (figure 1), especially when the reference material appears to show various values of reference concentrations of the same analyzed parameter referring to the corresponding measurement methods, which also occurred in this study . We also analyzed real tat in a series of unselected studies on the patients (module 4). In total, we analyzed 117 samples to simultaneously determine the 3 analyzed biomarkers: troponin t, ck - mbmass, and myoglobin . In the first stage of the observations, when the analysis was performed with the use of standard reagents, the mean tat obtained for 60 samples was 6513 min . In the second stage of the experiment, performed with the use of reagents having a shortened methodology time, the mean tat obtained for 57 samples was 5112 min (p<0.001) (figure 2). Thanks to the application of a new reagent kit the stat version it was possible to meet the criteria for obtaining tat <60 min to determine troponin concentration . Thus, the percentage of results meeting the criteria for obtaining tat<60 min increased from 40% to 75% (p=0.000008, fisher s exact test). Bearing in mind in the criteria set forth by esc, acc, aha, whf, we asked a question in the introduction: is it possible to determine markers of acute coronary syndrome easily, promptly, and accurately and receive reliable results? The analytical quality of the tested cardiac biomarkers entirely meets all criteria applicable in poland, both those referring to biological diversity and more restrictive ones recommended by the centre of quality control for laboratory diagnostics in lodz . In the new stat kits, the analyzed methods do not significantly differ in terms of the analytical quality of the obtained results of the assays from corresponding standard methods; the evaluated precision of the methods meets all the criteria of the analytical quality . The correlation of results obtained with the use of the standard and stat methods is very high; therefore, the results are analytically reliable and follow adopted standards . The procedure of performing laboratory determination is equally easy with the use of the stat method as well as the standard method . The combination of the quality and speed of the analytical method helps to make a more clear decision either rule out myocardial necrosis or diagnose it and shorten the time the patient will have to spend in a hospital rescue unit or admission room . Hospitals, less than 25% of laboratories that made ctnt / ctni and ckmbmass determinations obtained tat <60 min . The hospital that managed to obtain the shortest tat median for troponin did so within 50 min and needed 48 min for ckmbmass . In more than 90% of the results, tat exceeded 90 min . When the number of patients staying in the hospital rescue unit, or those who are only waiting to be accepted (because the hospital is currently overcrowded), doubles, tat for troponin assays increases by 12 and 33 min, respectively . With regard to the situation in poland and elsewhere, the information might be meaningful in the case of large multi - profile and clinical hospitals . Population studies conducted in finland confirmed mean tat for determining troponin at 69 min, on condition that the laboratory phase of the whole diagnostic procedure, which starts upon admitting the patient to the admission room, takes approximately half the time . This closely corresponds to the laboratory experiment analyzed in this study, because shorter time of the analytical method directly results in reducing the laboratory component of tat . What is interesting in our method is that reducing the analytical time to 9 min resulted in reducing tat by up to 14 min . It should be pointed out that neither the staff in the admission room nor in the laboratory knew about our experiment . We suspect a psychological component could play a role corresponding to laboratory phase of analyses, because laboratory diagnosticians were aware of the type of test kits they work with . However, we suggest more improvement might also be introduced in pre - laboratory procedures . The application of new stat methods led to a decrease in tat by up to 14 min, which was then shorter than 60 min . It should be also emphasized that the reduction of time of the analytical procedure not only does not worsen the analytical quality of assays but also contributes to higher efficiency of the medical laboratory . The reduction of tat for cardiac biomarkers plays a crucial role in strategic decision - making in hospital rescue units, admission rooms, and intensive cardiac supervision units . We assume that determining troponin concentration levels with the high - sensitivity method has already become, or if not, will soon become, common . Clinical specialists should thus take advantage of results obtained thanks to the high - sensitivity method rather than the ones obtained with the use of traditional methods, as the first gives more possibilities, but its interpretation is more difficult . The diagnostic specificity was reduced at the expense of a large increase in diagnostic sensitivity for diagnosing a myocardial infarction (mi) in the first hours following its onset . Difficulty in proper interpretation of troponin assays appeared shortly after they were introduced in 2010 . Until now the saying was very accurate: when troponin was a lousy assay it was a great test, but now that it s becoming a great assay, it s getting to be a lousy test . While interpreting the result of highly sensitive troponins assays, a physician should follow 10 strict clinical recommendations . The first 2 refer to need for close cooperation with the laboratory conducting the analyses . This will facilitate understanding all analytical problems, which might considerably affect the interpretation of results . It is even recommended to avoid using the terms troponin - positive and troponin - negative . Jaffe suggests that detectable levels will soon become the norm and that it should be differentiated from because highly sensitive troponins brought a decrease in diagnostic sensitivity of mi, the delta cardiac troponin values strategy appears to be adequate in clinical routine . We would like to highlight the issues that physicians should be more aware of when cooperating with their laboratories, as those aspects (like the real imprecision of tests) are crucial in diagnostic and monitoring procedures, especially when we consider short periods of test repetition . This is especially true since information obtained from reagent sheets cannot be transferred directly to laboratory practice without checking and adjusting it to the real conditions in which the laboratory works (e.g., different analyzers, different procedures and laboratory equipment) all these factors influence the quality of the results) becoming familiar with analytical procedures and potential errors arising out of the procedures will definitely make it easier to interpret results of assays, especially in troponin and ckmbmass concentrations, close to cut - off values i.e., within the 99 percentile of the reference population . The new generation of stat cardiac markers has high analytical quality, guaranteeing high analytical and clinical reliability of results . Application of myocardium necrosis biomarkers in the stat version contributes to a significant decrease in tat and allows for obtaining a good result of an analysis recommended tat <60 min.
A dental implant consists of components, which transfer chewing forces to the jaw bone . In recent years, the effects of loading on implants and surrounding bone have been widely investigated to design dental implant systems . Biomechanical, mechanical, chemical and biological aspects of dental implants are required to be considered to increase the success rate of dental implants . The most important factors that affect dental implant - bone interface include the type and direction of forces, quantity and quality of the supporting bone and materials of dental implant and prosthesis . Dental implants and prostheses are attached using different types of luting agents, which are commonly used to increase retention and to improve the marginal seal of prosthesis . To investigate the biomechanical factors, stress is the consequence of masticatory load on the prosthesis . In a study by de jager et al . Investigated a simple model that imitated the contraction behavior of luting agents to evaluate the finite element model merit in predicating the contraction stress . They compared the experimental contraction stress by finite element method (fem) analysis and demonstrated that it is a reliable method to predict the actual contraction stress in dental restorations when the luting agent thickness is uniform . Evaluated the stress distribution of a three - unit zirconia based implant - supported fixed dental prosthesis (fdp), using the 3d - fem with different load conditions . Furthermore, they found the highest von mises stress in the cervical area of the frameworks and abutment . Investigated the effect of luting agent types and thickness on the stress distribution within all - ceramic crowns using the fem . The results of their study showed that luting agent thickness does not have a significant effect on stress distribution of the core or veneer . However, the loading conditions and elastic modulus of luting agents play a vital role in stress distribution . Different luting agents have various properties such as modulus of elasticity, compressive and tensile strengths, toughness and poisson ratios . The fem is an efficient method to evaluate the effects of luting agents on the stress distribution . The aim of this study was to evaluate the effect of type of luting agent on stress distribution of the bone surrounding implants in a three - unit fdp using fem analysis . The null hypothesis was that the type of luting agent does not have any effect on stress distribution pattern of a three - unit implant - supported fdp . A 3d fe model of three - unit implant - supported fdp replacing the maxillary first molar with maxillary second premolar and second molar as the abutments was designed based on wheeler's dental anatomy . The geometric mesh of the modeled fixed dental prosthesis the maxillary second premolar and maxillary second molar were supported by two standard - plus screw - shaped implants (4.1 diameter, 043.152s for premolar and 4.8 diameter, 043.252s for second molar, straumann ag, waldenburg, switzerland) with regular neck solid abutments (048.541, straumann ag) with 5.5 height and 6 tapered tightened on the implants . A porcelain veneer with 1 mm thickness and a base - metal core from minimum 0.5 mm to maximum 1.5 mm thickness were established for porcelain fused to metal framework . Although the thickness of luting agent does not have an important effect on stress values, the luting agent thickness was considered 25 m . The fdp model was designed using catia v5 r18 software (dassault system, suresnes cedex, france) based on wheeler's anatomical teeth dimension . Mesh design and fem calculations were carried out using abaqus / cae 6.6 version (hibbitt, karlsson and sorensen inc ., the whole model was created with c3d4 elements (4-node linear tetrahedron). In total, the model was consisted of 465108 nodes and 86296 elements [figure 1]. To simulate the model during mastication movements, three different loads were considered in oblique, vertical and horizontal directions . On the functional cusps of the fdp, 400 n oblique, 200 n vertical and 57 n horizontal loads were applied [figure 2]. To simulate an oblique loading condition, a 400 n load was applied with = 120 according to the horizontal plane to the palatal cusps of each prosthetic unit . Each load case was carried out separately and applied on 8 equal points of each unit . The directions and magnitudes of three load conditions to apply the boundary condition, all nodes in the y z plane at the end of the x - axis in both directions were fixed; no translation was allowed in any direction [figure 2]. All materials were assumed to be linear elastic, homogeneous, time independent and isotropic . The material properties of the fdp unit and different types of luting agents are listed in tables 1 and 2 respectively . A 3d fe model of three - unit implant - supported fdp replacing the maxillary first molar with maxillary second premolar and second molar as the abutments was designed based on wheeler's dental anatomy . The maxillary second premolar and maxillary second molar were supported by two standard - plus screw - shaped implants (4.1 diameter, 043.152s for premolar and 4.8 diameter, 043.252s for second molar, straumann ag, waldenburg, switzerland) with regular neck solid abutments (048.541, straumann ag) with 5.5 height and 6 tapered tightened on the implants . A porcelain veneer with 1 mm thickness and a base - metal core from minimum 0.5 mm to maximum 1.5 mm thickness were established for porcelain fused to metal framework . Although the thickness of luting agent does not have an important effect on stress values, the luting agent thickness was considered 25 m . The fdp model was designed using catia v5 r18 software (dassault system, suresnes cedex, france) based on wheeler's anatomical teeth dimension . Mesh design and fem calculations were carried out using abaqus / cae 6.6 version (hibbitt, karlsson and sorensen inc ., providence, rhode island, usa) the whole model was created with c3d4 elements (4-node linear tetrahedron). In total, the model was consisted of 465108 nodes and 86296 elements [figure 1]. To simulate the model during mastication movements, three different loads were considered in oblique, vertical and horizontal directions . On the functional cusps of the fdp, 400 n oblique, 200 n vertical and 57 n horizontal loads were applied [figure 2]. To simulate an oblique loading condition, a 400 n load was applied with = 120 according to the horizontal plane to the palatal cusps of each prosthetic unit . Each load case was carried out separately and applied on 8 equal points of each unit . To apply the boundary condition, all nodes in the y z plane at the end of the x - axis in both directions were fixed; no translation was allowed in any direction [figure 2]. All materials were assumed to be linear elastic, homogeneous, time independent and isotropic . The material properties of the fdp unit and different types of luting agents are listed in tables 1 and 2 respectively . The stress levels were calculated using von mises stress value which is an appropriate criterion for stress evaluation of ductile materials . Contours of stress distribution on the cortical bone corresponding to three different loads are shown in figure 3 . The maximum von mises stress values were localized in the palatal side of second premolar supporting bone; particularly the area of cortical bone which has interaction with the implant . The maximum value was 48 mpa in all cases [figure 3] and the minimum von mises stress values occurred in the area far from the implants . To compare the results of simulation of the model with different types of luting agents, contours of the cross sectional view of the cortical bone as shown in these figures, there is a little difference between contours of stress distribution . Von mises stress values (mpa) and distribution patterns on the cortical bone when the different loads were applied comparison of applying different types of luting agent materials to cortical bone stress distribution (horizontal load) comparison of applying different types of luting agent materials to cortical bone stress distribution (oblique load) comparison of applying different types of luting agent materials to cortical bone stress distribution (vertical load) figure 7 shows the stress distribution in connectors region under horizontal, vertical and oblique load conditions . The horizontal load condition generated almost the same stress pattern along the connector while the maximum stresses were in the top and bottom of the connector due to stress concentration [figure 7a]. Under vertical load condition [figure 7b], shearing load behavior appeared in the bottom of the connectors which was much more than von mises stress in the horizontal load condition . The oblique load condition is a superposition of horizontal and vertical load conditions, which resulted in the maximum stress in the bottom of the connector [figure 7c]. Stress distribution in connector regions (a) horizontal, (b) oblique, (c) vertical figure 8 shows the stress distribution in implant and abutment regions . Stress distribution in implant and abutment regions (a) horizontal, (b) oblique, (c) vertical the stress levels were calculated using von mises stress value which is an appropriate criterion for stress evaluation of ductile materials . Contours of stress distribution on the cortical bone corresponding to three different loads are shown in figure 3 . The maximum von mises stress values were localized in the palatal side of second premolar supporting bone; particularly the area of cortical bone which has interaction with the implant . The maximum value was 48 mpa in all cases [figure 3] and the minimum von mises stress values occurred in the area far from the implants . To compare the results of simulation of the model with different types of luting agents, contours of the cross sectional view of the cortical bone as shown in these figures, there is a little difference between contours of stress distribution . Von mises stress values (mpa) and distribution patterns on the cortical bone when the different loads were applied comparison of applying different types of luting agent materials to cortical bone stress distribution (horizontal load) comparison of applying different types of luting agent materials to cortical bone stress distribution (oblique load) comparison of applying different types of luting agent materials to cortical bone stress distribution (vertical load) figure 7 shows the stress distribution in connectors region under horizontal, vertical and oblique load conditions . The horizontal load condition generated almost the same stress pattern along the connector while the maximum stresses were in the top and bottom of the connector due to stress concentration [figure 7a]. Under vertical load condition [figure 7b], shearing load behavior appeared in the bottom of the connectors which was much more than von mises stress in the horizontal load condition . The oblique load condition is a superposition of horizontal and vertical load conditions, which resulted in the maximum stress in the bottom of the connector [figure 7c]. Stress distribution in connector regions (a) horizontal, (b) oblique, (c) vertical stress distribution in implant and abutment regions (a) horizontal, (b) oblique, (c) vertical the failure is defined by the criteria which depend on stress distribution and material property . The fem can employ structures of various shapes with many elements defined with specific young's modulus and possion's ratio values . The 3d fe model was designed of three - unit implant - supported fdp to determine the influence of different types of luting agents on stress distribution pattern of the unit . The periodontal ligament is absent in implant - supported fdp, hence the stress occurs as a result of functional forces, which are directly transmitted to the supporting bone . A study by bozkaya et al . Showed that occlusal loads more than 1000 n will overload the compact bone and change its geometric shape . Different types of loading were applied to the framework; the maximum mastication load cases were considered as 400 n oblique, 200 n vertical and 57 n horizontal . As shown in the results, the maximum stress values in surrounding bone, connectors, implants and abutments occurred in the oblique load . The applied oblique load has the maximum value compared with the vertical and horizontal load cases . In addition, the oblique load contains vertical and horizontal components, which can yield horizontal and vertical load effects . Adhesive cements are commonly used to enhance the retention, marginal adaptation and fracture resistance of the restored teeth . Moreover, these types of luting agents are different in terms of chemical and physical properties . For example, zinc phosphate luting agent has the highest modulus of elasticity (13.5 gpa), which protects the supra - structure material of prosthesis from destructive occlusal forces . Furthermore, polycarboxylate luting agent has lower compressive (55 - 85 mpa) and higher tensile (8 - 12 mpa) strength than zinc phosphate agent, that result in more deformation which is not suitable for high force concentration in occlusal area . Demonstrated that permanent luting agents like zinc phosphate agent generate uniaxial retention forces from 2.5 to 4.7 times greater than provisional luting agents such as zinc oxide eugenol . Nejatidanesh et al . In their study have reported a significant difference between the mean retention values of different luting agents . Moreover, the results of their study showed that resin luting agents had the highest retention . On the other hand, resin modified glass ionomer, zinc phosphate, zinc polycarboxylate and panavia f2 the result of the present study showed that the maximum von mises stress of the cortical bone was at palatal side of the second premolar . Evaluated the influence of various occlusal materials on the stress transferred to implant - supported prostheses and supporting bone using the fem . The results of their study showed that von mises stress increased in the coronal one - third and two - third of the lingual surface of the cortical bone . The modulus of elasticity of cortical bone is higher than spongy bone and for this reason, cortical bone is stronger and more resistant to deformation . Hence, higher stress values can be seen in cortical bone compared to spongy bone . The results of this study showed that there were no significant changes in the cortical bone, implant and abutment stress distribution pattern for different luting agent materials . Similar results were observed by comparing panavia f and variolink ii resin composite luting agents . One may conclude, the luting agent plays a role in transferring the load to cortical bone, implant and abutment, but different types of luting agents may not affect the pattern of transferring load to the cortical bone . However, different types of luting agents might slightly change the direction of the load transferred to the bone due to different displacement field of luting agents under the same mastication load . Due to the palatal direction of oblique force on functional cusps, maximum von mises stress values were observed in the palatal area between the cervical region of the implant and supporting bone . Several fem studies have been carried out on fdp's connector that evaluated height, type and design of connectors . In the present study, the effect of type of luting agent on stress distribution in the fdp's connectors was evaluated and the maximum stresses in the fdp were in the top and bottom of the connector region that was due to stress concentration in the sharp edges . It has been reported that regardless of types of connectors material, it is the weakest part of the fdp and also connectors are more likely to fail . In the implant part for all load cases, the stresses were concentrated in the neck of implant due to the rigid connection between implant and bone which was similar to oruc et al . Study . In the present study, all luting agents were not observed and only well - known luting agents were evaluated . In addition, due to high calculation cost for simulation of whole jaw bone, the model of jaw bone was simplified . The achieved results using some assumptions regarding material properties in each layer of the fe model were compared qualitatively with each other in the current study . Therefore, stress distribution patterns may have been different depending on the material properties assigned to each layer of the fe model and the model used in the experiments . Thus, as many in vitro studies, it is difficult to extrapolate the results of this study directly to the clinical situation and the inherent limitations in this study should be considered . Within the limitations of this study, the following conclusions were drawn: the stress distribution depends on the loading conditions.the highest stress value was observed at oblique load condition.the maximum von mises stress was in the palatal side between the cervical region of the implant and supporting bone.the type of luting agents did not affect stress distribution and stress values at the bone surrounding implant.the maximum tensile stress and fracture risk occurs in the connector regions . The maximum von mises stress was in the palatal side between the cervical region of the implant and supporting bone . The type of luting agents did not affect stress distribution and stress values at the bone surrounding implant.
Hain - munk syndrome (hms) (omim #245010) is an extremely rare form of autosomal recessive disorder, which along with papillon - leferve syndrome (pls) (omim #245,000) is classified as type iv palmoplantar keratoderma (ppk). Ppks are a heterogeneous group of disorders, which are characterized by abnormal thickening of the palms and sole and are classified into focal ppk, diffuse ppk, punctuate ppk and palmoplantar ectodermal dysplasia based on clinical sites of lesions, associated lesions and histopathologic findings . Also called as cochin jewish disorder, hms was first reported by haim and munk in 1965 among members of a small jewish community from cochin in india . Beyond its occurrence in the jewish members of cochin, hms has not been reported in other population . However, pls has been found to have a world - wide prevalence with incidence of 1 - 4 cases per million of the general population . The clinical symptoms of hms includes congenital palmoplantar keratosis, severe early onset periodontitis, aggressive periodontitis (agp) accompanied by tooth loss, recurrent pyogenic cutaneous infections, atrophic nail deformities, acroosteolysis, arachnodactyly, radiographic deformity of the fingers and pes planus . Though the palmoplantar hyperkeratotic lesions and aggressive periodontal condition are also seen in pls, the presence of nail deformities, arachnodactyly, acroosteolysis and pes planus distinguishes hms from pls . Cathepsin c (ctsc) is a lysosomal cysteine proteinase coding gene, which encodes for ctsc protein that plays a major role in the activation of granule serine proteases, granzymes a and b, tryptase and chymase, and cathepsin g and elastase from immune cells . High levels ctsc expression has been observed in polymorphonuclear leukocytes, alveolar macrophages, plantar, palmar and gingival epithelium . Hence disruption in the expression or function of ctsc caused by mutations may be expected to produce clinical manifestations . Indeed, genetic analysis of ctsc gene in hms subjects has identified mutations in exon 1 (c. 145c t), exon 4 (c. 587 t c) and exon 6 (c. 2127a g). Since mutation within ctsc has also been reported in pls condition, both hms and pls are described as allelic variants of ctsc . Studies so far have reported the occurrence of this mutation in hms and pls subjects with consanguineous parentage in a majority of cases . However, more recently pls has been reported in subjects born to non - consanguineous parents as well . In the present study, we have investigated the genetic status of ctsc gene in a patient with clinical symptoms of hms to: (1) confirm that the subject indeed had a mutation in ctsc gene, which is indicative of hms and (2) to find out the mutant region with the ctsc gene in the subject, especially since neither the subject nor her parents were from the cochin jewish background, but belonged to the south indian dravidian race . A 23-year - old subject who reported to the institute for evaluation of oral condition was provisionally diagnosed with hms associated with agp . After obtaining informed consent from the subject and accompanying parent, a total volume of 0.2 ml of the whole blood sample was processed for dna extraction with genelute blood genomic dna kit (cat #na2000, sigma - aldrich, st . 100 ng of the extracted genomic dna was amplified with independent set of primers flanking each of exon 1 - 7 [table 1]. An universal amplification program was used to amplify the exons as follows: after an initial denaturation at 94c for 2 min, the exons were amplified for 35 cycles with denaturation at 95c for 45 s, annealing at 55c for 45 s and extension at 72c for 1 min, with a ramp of 1/s, which was followed by a final extension at 72c for 5 min . The amplified regions of each of the exon was run in a 1.2% agarose gel and eluted with genelute dna gel elution kit (sigma aldrich, cat #na1111). A 10 ng aliquot of the eluted pcr amplicons was subsequently subjected to direct sequencing with either forward or reverse pcr primers to identify for the presence of mutations . A 23-year - old subject who reported to the institute for evaluation of oral condition was provisionally diagnosed with hms associated with agp . After obtaining informed consent from the subject and accompanying parent, a total volume of 0.2 ml of the whole blood sample was processed for dna extraction with genelute blood genomic dna kit (cat #na2000, sigma - aldrich, st . 100 ng of the extracted genomic dna was amplified with independent set of primers flanking each of exon 1 - 7 [table 1]. An universal amplification program was used to amplify the exons as follows: after an initial denaturation at 94c for 2 min, the exons were amplified for 35 cycles with denaturation at 95c for 45 s, annealing at 55c for 45 s and extension at 72c for 1 min, with a ramp of 1/s, which was followed by a final extension at 72c for 5 min . The amplified regions of each of the exon was run in a 1.2% agarose gel and eluted with genelute dna gel elution kit (sigma aldrich, cat #na1111). A 10 ng aliquot of the eluted pcr amplicons was subsequently subjected to direct sequencing with either forward or reverse pcr primers to identify for the presence of mutations . A 23-year - old south indian female from a consanguineous family reported with complaints of edentulism and recurrent skin infections that had aggravated since the age of 5 years . Clinical investigation revealed loss of all her primary teeth between the age of 3 and 4 years and premature shedding of permanent teeth since the age of 15 years . Intraoral examination showed severe agp, which was associated with a complete edentulous maxilla, partially edentulous mandible with grade iii mobility of mandibular first and second molars [figure 1a and b]. Orthopantomograph confirmed the clinical observation of agp, which showed a complete edentulous maxilla, partially edentulous mandible associated with generalized loss of alveolar bone [figure 1c]. Hyperkeratotic psoriasiform lesions on an erythematous background were present on the dorsal surface of the hands and feet [figure 1d], which were confirmed by biopsy (data not shown). Besides, nail abnormalities in the form of onychogryphosis were also present, but was clinically pronounced only in the fingers of upper limb [figure 1e]. Radiograph of the wrist and palm region did not exhibit arachnodactyly [figure 1f]. Laboratory investigations for complete blood count, erythrocytic sedimentation rate, serum electrolytes, serum alkaline phosphatase, liver function tests and total bilirubin were found to be normal (data not shown). (a) edentulous maxilla; (b) partially dentulous mandible with aggressive periodontitis; (c) orthopantomogram of maxilla and mandible; (d) healed psoriasiform skin lesion; (e) onychogryphosis of fingers of upper limb; (f) radiograph of wrist and palm the longest isoform of ctsc consists of seven exons, of which mutation in exons 1, 4 and 6 have been reported to be associated with hms . In order to examine the genetic status of ctsc gene in the hms subject, the genomic dna isolated from peripheral blood sample was amplified with intronic primers that flanked each of the seven exons from 50 bp to 100 bp away from splice acceptor and donor sites and subjected to direct sequencing . Sequence analysis identified a single heterozygous point mutation within exon 7 of ctsc that caused substitution of the codon at position 453 from ata to gta with a resultant change in the encoded amino acid from isoleucine to valine (ile453val) [figure 2]. Further analysis of the intronic regions that were co - amplified along with each of the seven exon identified single nucleotide polymorphisms (snps) within intron 2 at position g. 88068052c> a [figure 3a] and intron 5 at position g. 88033661 t> c [figure 3b] in homozygous condition . The wild type base, a (indicated by a green arrow) and the mutant base, g (indicated by a black arrow) occur in heterozygous condition in the hain - munk syndrome subject, but not in the subject's father chromatogram of genotypes of the single nucleotide polymorphisms (snps) in intron 2 and intron 5 . (a) snp in intron 2 at position g.88068052c> a (indicated by a black downward arrow); (b) snp in intron 5 at position g.88033661 t> c (indicated by a black downward arrow). Note that both snps occurred in homozygous condition in the hain - munk syndrome patient and the father to investigate the pattern of inheritance of the exon 7 mutation and the intron 2 and 5 snps, the genomic copy of the subject's father, who is the lone surviving parent was analyzed by direct sequencing . Sequence comparison identified no mutation within the exon 7, which clearly indicated that the mutant allele was not inherited from the subject's father . The homozygous occurrence of intron 2 and 5 snps in both the subject and that of her father confirmed consanguineous parentage . A 23-year - old south indian female from a consanguineous family reported with complaints of edentulism and recurrent skin infections that had aggravated since the age of 5 years . Clinical investigation revealed loss of all her primary teeth between the age of 3 and 4 years and premature shedding of permanent teeth since the age of 15 years . Intraoral examination showed severe agp, which was associated with a complete edentulous maxilla, partially edentulous mandible with grade iii mobility of mandibular first and second molars [figure 1a and b]. Orthopantomograph confirmed the clinical observation of agp, which showed a complete edentulous maxilla, partially edentulous mandible associated with generalized loss of alveolar bone [figure 1c]. Hyperkeratotic psoriasiform lesions on an erythematous background were present on the dorsal surface of the hands and feet [figure 1d], which were confirmed by biopsy (data not shown). Besides, nail abnormalities in the form of onychogryphosis were also present, but was clinically pronounced only in the fingers of upper limb [figure 1e]. Radiograph of the wrist and palm region did not exhibit arachnodactyly [figure 1f]. Laboratory investigations for complete blood count, erythrocytic sedimentation rate, serum electrolytes, serum alkaline phosphatase, liver function tests and total bilirubin were found to be normal (data not shown). (a) edentulous maxilla; (b) partially dentulous mandible with aggressive periodontitis; (c) orthopantomogram of maxilla and mandible; (d) healed psoriasiform skin lesion; (e) onychogryphosis of fingers of upper limb; (f) radiograph of wrist and palm the longest isoform of ctsc consists of seven exons, of which mutation in exons 1, 4 and 6 have been reported to be associated with hms . In order to examine the genetic status of ctsc gene in the hms subject, the genomic dna isolated from peripheral blood sample was amplified with intronic primers that flanked each of the seven exons from 50 bp to 100 bp away from splice acceptor and donor sites and subjected to direct sequencing . Sequence analysis identified a single heterozygous point mutation within exon 7 of ctsc that caused substitution of the codon at position 453 from ata to gta with a resultant change in the encoded amino acid from isoleucine to valine (ile453val) [figure 2]. Further analysis of the intronic regions that were co - amplified along with each of the seven exon identified single nucleotide polymorphisms (snps) within intron 2 at position g. 88068052c> a [figure 3a] and intron 5 at position g. 88033661 t> c [figure 3b] in homozygous condition . The wild type base, a (indicated by a green arrow) and the mutant base, g (indicated by a black arrow) occur in heterozygous condition in the hain - munk syndrome subject, but not in the subject's father chromatogram of genotypes of the single nucleotide polymorphisms (snps) in intron 2 and intron 5 . (a) snp in intron 2 at position g.88068052c> a (indicated by a black downward arrow); (b) snp in intron 5 at position g.88033661 t> c (indicated by a black downward arrow). Note that both snps occurred in homozygous condition in the hain - munk syndrome patient and the father to investigate the pattern of inheritance of the exon 7 mutation and the intron 2 and 5 snps, the genomic copy of the subject's father, who is the lone surviving parent was analyzed by direct sequencing . Sequence comparison identified no mutation within the exon 7, which clearly indicated that the mutant allele was not inherited from the subject's father . The homozygous occurrence of intron 2 and 5 snps in both the subject and that of her father confirmed consanguineous parentage . In the present study, we have reported a case of hms, which was diagnosed based on the clinical observation of early onset destructive periodontitis, recurrent pyogenic skin infections, nail deformities and hyperkeratotic lesions on the dorsal surface of the limbs . Though the periodontal pathology and skin infections are also observed in pls, the condition was diagnosed as hms based on the specific occurrence of nail deformities and severity of hyperkeratotic skin lesions . The subject, however, did not present other cardinal features of hms like the palmoplantar keratosis, pes planus, arachnodactyly, acroosteolysis and radiographic deformity of fingers and hence distinguished the present condition from that of other reported cases . Since genetic mutations in the ctsc gene have been reported to occur in hms subjects, we sought to investigate the status of ctsc gene mutation in the above subject by direct sequencing of all seven coding exons of ctsc along with at least 50 bp of corresponding intronic regions . Sequence analysis revealed a single heterozygous point mutation in exon 7, which resulted in the substitution of encoded amino acid, isoleucine by valine in the mutant allele (ile453val). Though the mutation event involved substitution between two nonpolar aliphatic (hydrophobic) amino acids, the ile453val mutation has earlier been shown to cause a reduction in the protease activity of ctsc protein . The ile453val mutation, however, was not identified in the subject's father . Wherein the subjects were found to be carriers of mutations in either exon 1 or 6 of ctsc in homozygous condition when their respective parents carried the same mutation in heterozygous condition . Besides neither the parents nor sibs of the affected subjects in the above studies, who were heterozygous for the mutant and wild type alleles were known to manifest any of the clinically identifiable hms or other ppk conditions . Hence, whether the occurrence of ile453val ctsc mutation in heterozygous condition suffixes the development of hms symptoms or it requires compound mutations with involvement of other genes becomes essential to be investigated . It is very likely that the hms subject of the present study harbors compound mutations in other genes, which in the presence of heterozygous ctsc ile453val mutation may have caused the manifestation of the hms symptoms . This is likely because, ctsc ile453val mutation has also been reported in non - syndromic agp, where in the only known clinical manifestation appears to be early tooth loss . It may be noted that the hms subject of the present study carried a few of hms symptoms in addition to agp with tooth loss . Analysis of the associated intronic regions of the seven exons of ctsc in the hms subject and the lone surviving parent also identified snps within intron 2 (g. 88068052c> a) and intron 5 (g. 88033661 t> c). According to the snp database of national center for biological information, the snp within intron 2 identified in the present study has earlier been documented as rs217076 while the snp within intron 5 is a novel finding . Both snps nevertheless occurred in homozygous condition in the hms subject and the subject's father . These findings strongly suggest that the parents of the affected subject were in fact consanguineous . Since hms has been reported in the probands of consanguineous parents, it is possible that the ile453val mutant allele might have been inherited from the mother . However, from discussions with the hms subject's father, it was found that the mother had not exhibited any of the clinical symptoms of hms, including the agp condition, which is the most obvious and easily identifiable even by non - clinicians . This leads to the suggestion that the subject's mother may not have had the ile453val mutation while it occurred in the hms subject as a sporadic event . Such subject specific sporadic (non - inherited) mutation has been described in exon 2 (c. 203 t g) of ctsc gene in pls subjects alone, where in their respective parents were found to carry wild type base (c. 203t / t). In the present scenario with available clinical and genetic data, the reported condition may be regarded as a non - familial and milder form of hms as, (1) the subject carried the clinical features of hms that distinguished it from pls, (2) the surviving father is disease free while the subject's deceased mother was not known to have carried any of hms symptoms, (3) most of other symptoms of hms as has been elaborated by hart et al . Were not observable in the subject and (4) the iie453val mutation occurred in heterozygous condition in the subject . However, it remains to be determined whether the co - occurrence of skin lesions and nail deformities alone with agp is sufficient to consider the condition as hms . This may be addressed by further clinical and genetic investigations on hms subjects with defined lineage and generation of consanguineous parents as the severity of symptoms is likely to vary among the hms subjects born to different generations of consanguineous parents due to the cumulative effect of detrimental mutations . Taken together, the findings of the present study provides clinically significant data as the ile453val mutation in exon 7, rs3888798 in intron 2 and a novel snp in intron 5 have been observed in a compound manner in ctsc gene for the first time in a hms subject . Hence the findings may be used as a lead to further studies on hms to clinically describe and classify the occurrence of the symptoms in combination with genetic status of ctsc gene into mild, moderate and severe forms of hms.
Throat swabs and urine sediments were obtained from patients with serologically confirmed cases of measles . Clinical specimens were inoculated onto b95a cells (24), and the cells were observed for cytopathic effect (cpe). Inoculated cells were blind - passaged up to three times before being discarded . Total cellular rna was extracted from infected b95a cells or directly from clinical specimens, if virus isolation was not successful, by the guanidinium acid - phenol method (25). Cdna corresponding to the 565 nucleotides coding for the cooh - terminus of the nucleoprotein (n) and the full - length open reading frame for the hemagglutinin (h) gene were synthesized by using avian myeloblastosis virus reverse transcriptase and amplified by polymerase chain reaction (pcr), as described previously (2,3). Sequences of the pcr products were derived by automated sequencing with the bigdye terminator chemistry according to the manufacturer s protocol (perkin elmer - applied biosystems, foster city, ca), and reaction products were analyzed on an automatic sequencer (abi 373, abi 3100, perkin elmer - applied biosystems). Sequence data were analyzed by using version 10.1 of the genetics computer group sequence analysis software package (genetic computer group, madison, wi) (26). Genotypes were assigned on the basis of phylogenetic analyses performed by using paup version 4.0 (27). The epidemiologic information was gathered through case investigation by state and local health departments and reported to the centers for disease control and prevention through the national notifiable diseases surveillance system . For the years 19972001, the number of reported measles cases in the united states has been at record low levels . A total of 138 cases were reported in 1997, 100 cases / year in both 1998 and 1999, 86 cases in 2000 (4), and 108 cases for 2001 (provisional data). Most cases were imported from other countries or spread from imported cases . During this 5-year period, specimens for viral isolation were obtained from 31 (64%) of 48 outbreaks, and at least one virus was isolated in tissue culture from 19 of the 31 outbreaks from which specimens were submitted (table 1). In addition, reverse transcriptase (rt)-pcr was successfully used to detect measles rna in clinical specimens from two outbreaks when attempts to isolate virus failed (table 1). Overall, genetic information was obtained from 21 (44%) measles outbreaks, while specimens from 10 outbreaks failed to yield a viral isolate or positive pcr signal . The inability to isolate mev or detect measles rna was due to failure to collect specimens within 5 days after the onset of rash or to improper storage . In addition to the outbreaks, 176 sporadic cases and 28 chains of transmission with 2 cases were reported during this period . Twenty - eight measles viruses were isolated from sporadic cases and two - case chains (table 1); six additional specimens from sporadic cases were positive for measles rna by rt - pcr . Mvi indicates that sequence was obtained from a viral isolate; mvs indicate sequence was obtained directly from the specimen . For sporadic cases, viruses representing 11 of the 20 genotypes recognized by who in 2001 (20) were isolated in the united states in 19972001 (tables 1 and 2). Among the 13 chains of transmission for which an imported source of mev was not detected by classical epidemiology, number indicates the number of times a genotype was associated with either an outbreak or a case . Thirteen (24%) of the measles sequences detected from chains of transmission in the united states in 19972001 belonged to genotype d6, which has previously been associated with importation from european countries (2,3). Within the last 6 years, viruses in genotype d6 have been isolated in brazil, uruguay, argentina, united kingdom, spain, germany, russia, poland, and luxembourg (9,10,16,28). During 19972001, genotype d6 viruses were imported into the united states from italy, greece, ukraine, croatia, cyprus, and the united kingdom . During 1997, viruses from genotype d6 were imported into the united states from the large measles outbreak that occurred in so paulo, brazil, and spread to other south american countries (2933). The viruses that were isolated from cases imported from brazil to minnesota (mn33 - 97) and pennsylvania (pa28 - 97) during 1997 had sequences identical to those obtained from measles viruses isolated during the outbreak in brazil . Measles virus wa31 - 97 was isolated at the same time as mn33 - 97 and pa28 - 97 from a case in washington with an unknown source of infection . The sequence of wa31 - 97 was identical to those of the two viruses imported from brazil, suggesting that this virus may also have been imported from brazil (figure). Genetic relationship between measles viruses isolated in the united states in 19972001 and the reference strains established by the world health organization (who) (20). Phylogenetic tree was based on the nucleotide sequences coding for the cooh - terminus of the nucleoprotein . Reference strains as established by who are shown in bold and designated by their genotype name . Twelve (22%) of the 55 sequences obtained from viral isolates or directly from clinical specimens were placed in genotype d5 (tables 1 and 2). Genotype d5, along with d3, is one of the genotypes known to have endemic circulation in japan (11). Epidemiologic investigations indicated that japan was the source of infection for 6 of the 10 sporadic cases and two of the outbreaks . The largest outbreak in the united states during 19972001 occurred in anchorage, alaska, during 1998 . However, since the imported case and the outbreak occurred in the same place and within two generations of transmission, they were likely epidemiologically related . Although no viral specimens were received for the imported case, the sequence of the virus isolated during the outbreak was closely related to the sequences of viruses known to be circulating in japan . Genotype d5 was also detected in specimens from a small outbreak that occurred in new york in july 2001 among a group of japanese students who were visiting a university in new york city . Viruses from genotype d4 were isolated from eight chains of transmission during 19972001; in seven of these chains, a foreign source of infection was identified (tables 1 and 2). Importations from kenya were associated with a 15-case outbreak in virginia in 1999 and a sporadic case in minnesota in 2001 . At this time, no information is available about the genotypes of wild - type measles viruses circulating in kenya, but a genotype d4 virus was imported into the united states from kenya in 1996 (1). A genotype d4 virus imported from ethiopia was responsible for a six - case outbreak in vermont during 2000 . In 2001, a genotype d4 virus was isolated from a small outbreak in massachusetts, which was traced to a student from pakistan . During 1999 genotype d4 is known to be circulating in pakistan, southern africa, india, and ethiopia (17,21,34). A genotype d4 virus (ca1 - 00) this finding was unusual because genotype d4 viruses have never been detected in japan despite extensive virologic surveillance . Previous studies had shown that wild - type measles viruses isolated in the people s republic of china were members of a distinct genotype designated h1 (14). More recent information indicated that measles viruses circulating in both china and vietnam were members of clade h but were sufficiently divergent from each other to be considered two separate genotypes, h1 and h2, respectively (35). Analysis of viruses imported from both china and vietnam showed that these new genotype designations will be useful in epidemiologic surveillance . In 1997 and 1998, two viruses from genotype h1 were isolated from cases imported from china, and one virus in genotype h2 was isolated from a case imported from vietnam . Two other viruses from genotype h2, ak16 - 00 and mn36 - 01, were isolated from sporadic cases that occurred in alaska during 2000 and minnesota during 2001, respectively ., genotype h1 viruses were isolated from an outbreak in washington (wa2 - 01) and four sporadic cases in florida (fl26 - 00), washington (wa9 - 01), illinois (il5 - 01), and minnesota (mn9 - 01). Concurrently, the republic of korea was experiencing a measles epidemic that began in 2000 . Korea was identified as the source of the washington outbreak and the sporadic case in illinois . The sequences from the washington outbreak and the sporadic case in illinois were identical to those of genotype h1 viruses isolated in korea (36). The person diagnosed with measles in florida in 2000 had traveled to los angeles and las vegas shortly before onset of illness and probably was infected by a genotype h1 virus (fl26 - 00) while in transit . The sequence of fl26 - 00 was identical to that of the two viruses imported from korea, suggesting that the source of this case may have also been the korean outbreak (figure). The sequence of the virus (wa9 - 01) from the sporadic case in washington, which was imported from china, was very closely related to the sequences of the korean viruses . This finding suggests that the genotype h1 viruses circulating in both china and korea are closely related (figure). The sequence of the virus identified in the minnesota case (mn9 - 01) with an unknown source was identical to that of the washington case imported from china (wa9 - 01). Viruses from genotype c2 were isolated from two imported cases in the united states . Detecting a genotype c2 virus (ca2 - 97) in association with an importation from germany was not unusual since genotype c2 viruses have frequently been detected in europe (21), but the isolation of a c2 virus (in16 - 98) from a patient returning from zimbabwe was unexpected . No viral isolates from zimbabwe have been characterized, and genotype c2 viruses have not been detected in any countries in southern africa . In imported cases, the source of infection is usually assumed to be the country in which the person was traveling during the incubation period . However, this patient may have been infected while in transit from africa to the united states via europe . In fact, in this case, rash onset was 14 days after completion of travel, suggesting that the infection occurred near the end of the trip . Measles viruses in genotype d2 are known to circulate in both south africa and zambia (13,17). A genotype d2 virus was obtained from a single case imported into the united states from dublin, ireland, in 2000 (ny11 - 00). Although genotype d2 viruses are probably not endemic in ireland, genotype d2 viruses were isolated during an outbreak that occurred in an immigrant community in dublin that had low vaccination coverage . The sequence of ny11 - 00 was identical to the sequence of a virus isolated during the irish outbreak (37). Viruses from genotype d3, the genotype associated with the resurgence of measles in the united states in 19891991, were detected from small outbreaks in california and maryland in december 2000 and january 2001, respectively . In both outbreaks, the only other genotype d3 virus detected in the united states after 1993 was also imported from the philippines to california in 1996 (3). Six of the sequences obtained from cases in 1999 and 2001 (tx28 - 99, wa12 - 99, il50 - 99, ca38 - 01/1, az35 - 01, ca38 - 01/2) were closely related to the recently recognized genotypes d7 and d8 (figure). A retrospective study showed that viruses from genotype d7 were isolated in australia as early as 1985 (5). The sequences of il50 - 99, ca38 - 01/1, az35 - 01, and ca38 - 01/2 were most closely related to the sequences from the australian genotype d7 viruses . Interestingly, a virus isolated in canada in 2000 from a case with a travel history to mexico had a sequence nearly identical to that of il50 - 99 (a who reference strain for genotype d7), which was imported into the united states from sweden (38). In 2001, genotype d7 viruses (ca38 - 01/1, ca38 - 01/2, az35 - 01) were associated with two small outbreaks in california and a sporadic case in arizona . Genotype d7 viruses were isolated from cases that were imported into el salvador from europe (39) as well as from outbreaks in germany (40), suggesting that d7 may be another endemic european genotype . The two viral isolates from genotype d8, tx28 - 99 and wa12 - 99, had identical h gene sequences, although the sources of importation were different . The sequences of these viruses were most closely related to that of a virus isolated in the united kingdom in 1994 (10), which has been designated the reference strain for genotype d8 . Viruses in genotype d8 have been detected in ethiopia and nepal in 1998 and 1999, respectively (34,41). While no viral isolate was obtained, measles rna was detected in some of the clinical samples . Sequences of the pcr product showed that a virus in genotype g2 was responsible for the outbreak . Viruses in genotype g2 are known to be circulating in indonesia and malaysia and were associated with importation of virus from indonesia to the netherlands (22,23). However, virologic surveillance has not been established in most areas of asia, and genotype g2 viruses may be circulating in countries other than malaysia and indonesia . This study demonstrates the utility of virologic surveillance, especially for countries in the elimination phase of measles control . Sequence data obtained from 55 viral isolates or clinical specimens from measles cases in the united states during 19972001 indicated that 11 genotypes of virus were represented . Rather, the diversity of genotypes reflects multiple, imported sources of measles virus . When the source of virus was identified by standard epidemiologic investigation, virologic surveillance helped to confirm the source of the virus and to build a genetic database of viral sequences associated with imported cases from different areas . Virologic surveillance was especially useful for characterizing 13 chains of infection in which the source of infection could not be identified by standard epidemiologic methods . Seven different genotypes were detected in these 13 chains, indicating multiple sources of infection . This finding suggests that these unknown source cases were the result of imported virus and not caused by circulation of an endemic genotype . These results underscore the need to improve the mechanism for obtaining appropriate specimens for viral isolation from all suspected cases, especially outbreak - associated cases . In countries that are in the elimination phase of measles control, obtaining specimens for viral isolation at first contact with all suspected measles cases is important . Viruses isolated during the resurgence of measles in the united states in 19891991 were all in genotype d3, suggesting that d3 viruses had spread throughout the entire country (2,3). Following the resurgence, both standard epidemiologic and virologic surveillance indicated that endemic transmission was interrupted in the united states in 1993 (2,3,42,43). After 1993, only three viruses from genotype d3 were isolated in the united states, and all three were the result of importations from the philippines (3). The pattern of mev genotypes observed in the united states in 19932001 suggests an absence of indigenous transmission of virus since no genotype was consistently isolated . The pattern of viral genotypes reported for the united states after 1993 has been observed in other areas of the world that have good virologic surveillance and have achieved a high level of measles control . In canada, the pattern of viral genotypes detected over the last 10 years has been very similar to the pattern in the united states (38). Likewise, virologic surveillance in victoria, australia, over a 25-year period suggested that repeated importation of multiple genotypes had occurred (5), and a similar pattern has been reported for the united kingdom (10). In contrast, in countries that still have indigenous transmission of measles, only a limited number of genotypes are circulating (14,15). The tremendous reduction of measles cases in the united states after 1991was due in part to the successful measles control and elimination program initiated by the pan american health organization (paho) in the early 1990s . However, during 1997, brazil had a resurgence of measles, with nearly 50,000 cases reported (44,45). Genetically homogeneous viruses in genotype d6 viruses were associated with all the recent measles activity in south america except for two cases imported into el salvador in 2001 . In the last 2 years, paho has reported a record low number of measles cases in the americas and <2,000 cases were reported for the year 2000 (46); most cases occurred in the dominican republic and haiti . Virologic surveillance will play a key role in documenting the elimination of endemic transmission of the genotype d6 viruses in south and central america in the same manner that was used to document the elimination of the genotype d3 viruses in the united states . Efforts are under way to improve laboratory capacity and expand virologic surveillance in the americas . Strengthening virologic surveillance activities will not only contribute to our understanding of the transmission pathways of mev but also increase the sensitivity of measles diagnosis . As the prevalence of disease decreases, the positive predictive value of serologic testing also decreases . Having the laboratory capacity to detect mev or viral rna will be especially helpful for measles surveillance in areas where indigenous transmission has been interrupted and many of the suspected cases are sporadic . The identification of new genotypes indicates that our current understanding of the extent of genetic diversity in measles strains throughout the world is incomplete . Virologic surveillance has not been initiated in many countries, and others are just beginning to collect appropriate samples . Virologic surveillance activities need to be initiated or expanded in countries that are in the outbreak control phase of measles control to obtain an accurate record of the pattern of endemic viral genotypes present in all areas of the world.
Hepatitis b virus (hbv) infection and hepatitis c virus (hcv) infection are the main causes of end - stage liver disease requiring orthotopic liver transplantation (olt). However, the post - olt course is quite different between the two types of hepatitis . The post - olt hepatitis b recurrence rate is> 80% without any prevention, while> 90% of recurrent infections can be controlled with a combination of hepatitis b immunoglobulin (hbig) and nucleos(t)ide analogues (nas). Non - olt chronic hepatitis b patients are treated with antiviral proliferative na agents, with> 90% long - term control with minute side effects . The first commercially available na, lamivudine (lam), produced a rapid and definite short - term antiviral response, but 1520% of the patients experienced annual recurrence of resistant virus and 70% of them did so after 5 years . Fewer than 3% of patients treated with newer nas such as entecavir (etv) or tenofovir (tdf) experience resistant virus; these newer nas are accepted as first - line and long - term treatment [3, 4]. The mechanism for controlling hbv viral recurrence is direct viral replication control by a combination of nas and hbig as passive immunoprophylaxis . As hbig combination therapy has important roles, the b - cell - related adaptive immune response appears to play a role in controlling hbv after olt . However, as hbv - induced hepatitis is characterized by t - cell immune response, both b- and t - cell adaptive immune responses have vital roles . When active immunization of patients with hbv vaccine is performed, hbv - specific and non - hbv - specific immune responses can be obtained . Hcv reinfects> 90% of patients, with more than half of these patients developing chronic hepatitis requiring interferon- (ifn-) based antiviral treatment . Non - olt chronic hepatitis c patients have been treated with ifn - based immune reaction - related treatment . Recently, pegylated ifn (peg - ifn) has been used in combination with ribavirin and, more recently, with the addition of a direct - acting antiviral agent (daa) targeting the hcv nonstructural protein (ns) 3/4a protease . Single peg - ifn resulted in only 30% of patients experiencing sustained viral response (svr), representing undetectable hcv - rna longer than 24 weeks after finishing ifn . This demonstrated> 99% viral eradication, while the peg - ifn and ribavirin combination resulted in 50% svr [10, 11]. Peg - ifn plus ribavirin and telaprevir or simeprevir resulted in> 80% svr for patients with genotype 1, which is the more difficult - to - treat genotype of hcv . All - oral, ifn - free regimens are expected to become commercially available in the near future . Graft reinfection is universal after olt, leading to high - titer hcv viremia, with cirrhosis developing within 5 years of transplantation in approximately 20% of patients and within 10 years in 50% . Thus, hcv infection after olt differs completely from chronic hepatitis c (chc) without transplantation . However, the mechanisms underlying accelerated hcv - induced liver damage after olt are poorly understood . Several factors appear to be involved in the risk of hepatitis c recurrence, particularly those related to viral and immune responses . Immunosuppressive therapy is a likely cause for the severe accelerated course of hcv - related hepatitis after olt [16, 17]. In particular, high - dose steroids, immunosuppressive drug combinations, powerful induction treatments, and acute rejection can worsen patient outcomes . The pathology of hcv - related disease reflects immune reactions to virus - infected hepatocytes . In post - olt settings, the effects of interferon - based treatment are limited to 3050% of patients, with especially poorer results in post - olt patients, and also carry the possibility of inducing mortal chronic rejection that should be avoided . In this review, we summarize the aberrant immune system in hbv- and hcv - related hepatitis, together with the changes in these diseases after olt . After infecting a patient once, hbv persists in the liver for the rest of a person's life, even after the patient achieves a clinically cured condition with seroclearance of hbv envelope antigen (hepatitis b surface antigen, hbsag) and emergence of hbs antibody (hbsab). In controlling viral replication, immune function has been revealed to be important, as immunosuppressive treatment for cancer chemotherapy or organ transplantation can induce viral replication even in hbsag negative with hbsab positive clinically cured patients and organ transplant recipients [21, 22]. Hbv is an enveloped dna virus containing a relaxed circular dna genome, which is converted into a covalently closed circular (ccc) dna that persists in the nucleus of infected cells as minichromosomes . Natural killer (nk) cells work as the innate immune modulator to induce the death of microbial - infected cells with strong cytotoxic activity and the production of high levels of certain cytokines and chemokines in a nonmajor histocompatibility complex- (mhc-) restricted manner distinct from t and b cells . Upon hbv infection, nk cells migrate to the liver, with a decrease in their numbers in the spleen and bone marrow, suggesting the recruitment of nk cells from these organs . As hepatocytes normally express little mhc class i, nk cells may play a more important role in the early defense against hbv infection before the mhc class i expression is upregulated after viral replication in hepatocytes . Antigen - presenting cells (apcs), such as kupffer cells (liver resident macrophages) and dendritic cells, have important roles in intermediating the innate to adaptive immune responses . Kupffer cells or macrophages behave in both an immunostimulatory and immunoregulatory fashion upon hbv exposure . The addition of hbv particles and hbsag induces the production of proinflammatory cytokines interleukin- (il-) 1, il-6, cxcl-8, and tumor necrosis factor (tnf)- by human cd68 macrophage - enriched cells via nf-b (nuclear factor kappa - light - chain - enhancer of activated b cells) activation . However, another study reported no such cytokine production with immunoregulatory cytokine transforming growth factor- (tgf-) production . The immune system activates kupffer cells to eradicate hbv, while hbv evades the kupffer cell - related pathway to reduce the inflammatory pathway and change the environment to be favorable for survival . Pretreatment of nonparenchymal cells, including kupffer cells, with hbsag or hbv virion, abrogates the toll - like receptor- (tlr-) related antiviral response such as ifn-, interferon - stimulated gene (isg), or nf-b . In the liver biopsy specimens of patients with active hepatitis b, kupffer cells have been revealed to possess higher expression of galectin-9, which is an immunoregulatory molecule . Kupffer cells accumulate around injured hepatic loci and produce several cytotoxic and fibrosis progression - related molecules . However, they also have an important function in scavenging apoptotic hepatocytes, which could function as a bait for inflammation, and depletion of kupffer cells could induce worsening of hepatitis [31, 32]. Kupffer cells function as both proinflammatory and anti - inflammatory and profibrotic and antifibrotic cells in their environment . Both the hepatitis state and kupffer cell polarity are needed to understand the immunological pathogenesis in hbv - related hepatitis . Strong hbv - specific cd8 t - cell responses have been shown to correlate with viral and hepatitis control during acute infection . In chronically infected patients, hbv - specific cd8 t - cell responses are weak and occur with low frequency, while patients with low viral load exhibit multispecific strong responses . The epitopes targeted by the cd8 t cells have been deeply analyzed in hla - a and -b restricted epitopes, as these have been believed to have antiviral impact [35, 36]. However, a recently characterized hla - c restricted epitope has also been revealed to have a clinical impact and is an especially frequent allele in patients who live in southeast asia . Several factors have been suggested to explain this phenomenon . In hbv - specific cd8 t cells, proapoptotic protein bcl2-interacting mediator (bim) is upregulated, nave t - cell phenotypes such as cd45ra, cd27, cd28, and ccr7 are highly expressed, and immune regulatory molecules such as programmed death-1 (pd-1), cytotoxic lymphocyte antigen 4 (ctla-4), and t - cell immunoglobulin mucin-3 (tim-3) are also highly expressed [29, 3841]. Several experimental trials that were conducted to block such immunoregulatory exhaustion molecules showed reversal of these immunoregulatory conditions [41, 42]. Similar to the cd8 t cells, cd4 t cells have also been found to exhibit a lower response in the acute phase of infection in patients who developed chronic hepatitis later . The cd4 t - cell response in patients who recovered was found to be more frequent, stronger, and more multispecific than that observed later in patients with chronic hepatitis . The ifn- producing antiviral th1 response against hbv core has been revealed to be stronger in patients with resolved infection even several years after infection . The humoral immune response has been acknowledged as an avenue for understanding the clinical course of acute and chronic hepatitis b . The antibody responds against viral structural antigens such as the core antigen (hbcag) and the envelope antigen (hbsag). Anti - hbcag igm antibody (igm - hbcab) is accepted as the earliest and most diagnostic marker of acute infection . Anti - hbc igg antibody (igg - hbcab) develops during acute infection and remains positive for the duration of the patient's life . Hbsag emerges in serum from the acute phase of infection and remains when the patient exhibits chronic hepatitis b, while in patients who experience an acute self - limiting course hbsag could be cleared . Anti - hbs antibody (hbsab) is a virus - neutralizing antibody recognized as having lower viral and disease activities . The lack of anti - hbs in chronic infection can be attributed to a selective exhaustion of b cells and il-10 secreting immunoregulatory b - cell expansion [45, 46]. Since hcv is not also a cytopathic virus, immune reactions play a central role in the development of chronic hepatitis (figure 1) [47, 48]. The lack of a strong th1-type helper t - cell response and cytotoxic t - cell response against hcv leads to chronic infection with this virus [4951]. High - magnitude, broad, polyfunctional, and sustained t - cell responses correlate with spontaneous recovery [35, 49, 52], but these responses are not correlated with interferon - induced viral clearance . The role of nk cells in chronic hepatitis c is not completely understood . However, as nk cells are the first immunological walls against hcv, much evidence has been uncovered . An nk - cell activating and inhibitory receptor gene polymorphism has been discovered to have roles in the course of hcv infection [54, 55]. As ifn- is the basic treatment for chronic hepatitis c, ifn - producing nk cells have been defined as key immune cells . Nk cells can produce ifn-, ifn-, and tnf- and induce dendritic cell activation and support innate to adaptive immune response bridging . Nk cells can also lyse hcv - infected hepatocytes, t cells, and apcs and modulate immune responses . However, hcv itself has been revealed to have a role in the potential inhibition of nk cell function, resulting in chronic hepatitis . Antigen presenting cells such as kupffer cells, macrophages, or dendritic cells (dcs) behave in both an immunostimulatory and immunoregulatory manner upon hcv exposure, as in hbv . In chronic hepatitis c patients, kupffer cells are increased and activated as the higher expression markers cd163 and cd33 [59, 60]. In vitro analysis has revealed that hcv core and ns3-affected kupffer cells secrete proinflammatory cytokines such as il-1, il-6, and tnf- and also immunosuppressive cytokine il-10 . Proinflammatory cytokine release might explain the induction and persistent inflammation in chronic hepatitis c, while immunosuppressive cytokine release explains the difficulty in the eradication of hcv - infected hepatocytes . The direct effects of hcv on the inflammatory signal in kupffer cells have been revealed to upregulate the immunoregulatory molecule pd - l1 . Probably, hcv interferes with kupffer cell - related antiviral activities but induces strong enough inflammatory cytokines to result in chronic inflammation . The effects of kupffer cells on liver fibrosis progression are similar to those in hbv infection . The kupffer cells accumulate around inflammatory foci and express cytotoxic molecules such as granzyme b, perforin, and reactive oxygen species to induce inflammation and fibrosis . Myeloid dcs (mdcs) produce a large amount of il-12 upon stimulation, while plasmacytoid dcs (pdcs) produce a large amount of ifn- in viral infection . However, several reports have indicated contradictory results that dc function is not impaired in chc patients [6973]. Most of these reports are studies with mdcs; however, pdcs are also reportedly functionally impaired and reduced by increased apoptosis . Since culture conditions and chronic hepatitis conditions in the patient may change the phenotype of immune cells, functional differences in dcs during chronic hcv infection remain contentious . In vitro transfection or the addition of hcv proteins such as core, because of the scarcity of in vitro culture systems for hcv, these experimental results are also contentious . With the recent establishment of infectious cell culture - produced hcv, the role of the humoral immune response in the clearance of hcv is not well understood . After viral clearance, most antibody titers wane despite the persistent t - cell response . The target of the response is placed in and around the envelop proteins e1 and e2 and the hypervariable region near the amino terminus of e2 . Neutralization epitopes have been revealed to be masked by extensive glycosylation and by virions covered with lipid droplets and might not be effectively targeted [78, 79]. In addition, as the rna - dependent rna polymerase of hcv lacks proofreading activity, it is easy for hcv - rna to mutate and escape from the host immune pressures . Although 2030% of infected patients recover from the infection with strong t - cell response memory and possible neutralization antibody b - cell response, they could be reinfected with the virus, indicating difficulties in producing disease - controlling vaccines . Strong hcv - specific cd4 t - cell and cd8 t - cell responses have been shown to be evident in hcv patients with resolved infection, while diminished in patients with chronic hepatitis c . To recognize viral - infected hepatocytes or apcs interferon upregulates mhc class i expression; however, replicating hcv - rna reduces that expression . Interferon is released from nk cells and dcs during an early phase of viral infection and has important roles in eradicating hcv, as this is the key drug for treatment . This hcv interference with mhc expression must be one reason why chc patients show reduced cd8 t - cell responses . Recent attention has focused on regulatory t cells (tregs) and their contribution to chc . Their mechanism of immunosuppression depends on both cell - cell contact and immunosuppressive cytokine secretion . A subpopulation of tregs that express cd18 and also cd49b - expressing type 1 regulatory t (tr1) cells have also attracted attention, because they produce large amounts of immunosuppressive cytokines such as il-10 and tgf-, with which they inhibit type 1 and 2 helper responses . Tregs and tr1 cells may contribute to hcv persistence by suppressing hcv - specific t - cell responses [8789]. Treg frequencies and activities are apparently higher in chc patients than in those who have achieved viral clearance . Recently discovered t - cell regulatory molecules such as pd-1, 2b4, and tim-3 have been revealed to be coexpressed in intrahepatic hcv - specific cd8 t cells, indicating that hcv - induced t - cell functional exhaustion represses viral eradication . Strong innate and adaptive immune responses are responsible for hcv clearance; however, the virus itself affects many sites of the immune system, ameliorating the effective antiviral immune functions . To control nk, kupffer cells, b cells, or t cells a multicenter study in europe in 1993 identified the risk of post - olt hbv recurrence . The risk was low in patients with acute liver failure who were intolerant of hbv . However, the recurrence rate in patients with liver cirrhosis, especially with high serum hbv - dna at olt, was> 80% . As the immune system is repressed with steroids and calcineurin inhibitors, recurrent hepatitis b produces severe hepatitis with a high incidence of mortal liver failure . However, present protocols that use na in combination with long - term hbig have resulted in> 90% control of hbv recurrence . The first trial of long - term hbig combined with the first - generation na lamivudine (lam) was conducted in 1998 . Monthly hbig administration with lam resulted in all patients surviving for 1 year after olt without serum hbv - dna positivity . The historical progression of controlling post - olt hbv recurrence is summarized in table 1 . As patients with positive hbv - dna before olt were more likely to later have hbv recurrence, to maintain anti - hbs antibody titers> 500 iu / l was recommended . If hbv - dna was negative before olt, the anti - hbs antibody titer could be reduced to 100150 iu / l with or without lam . From the standpoint of cost savings, the hbig dose requirement was able to be decreased as clinical data accumulated [9597]. Currently, hbig is administered as required only when anti - hbs antibody titers fall below target levels . Some reports indicate that only a short duration of hbig administration is required and that it can be withdrawn several months after olt . If hbv - dna was negative at the time of olt, hbig could be withdrawn at several months after olt . For acute liver failure patients who had been infected with the virus shortly before hepatitis development, hbig of course, strict monitoring of hbv - dna and hbv surface antigen (hbsag) titers should be continued throughout the patient's life . The mechanism of protection against hbv reactivation by the combination of drugs is not well defined . The cccdna episome is the transcriptional template for hbv messenger rna transcripts that encode viral structural and ns proteins and the pregenomic rna template for reverse transcription and synthesis of the viral genome . Nas inhibit the reverse transcription of pregenomic rna, resulting in a rapid decrease in serum hbv - dna, but cannot eliminate the cccdna reservoir . Hbig contains high - titer antibodies against hbsag, which is the major component of the envelope of the hbv virion . The possible mechanisms through which hbig prevents hbv transmission are that it neutralizes circulating virus by immune complex formation, protects nave hepatocytes against hbv released from extrahepatic sites through blocking the putative hbv receptor, or anti - hbs antibody internalizes into hepatocytes, interacts with hbsag, and inhibits hbsag secretion from cells . To protect against hbv infection of nave hepatocytes might be difficult, since recent studies have revealed that intrahepatic hbv - dna is detectable in> 50% of even well - controlled patients after olt . The hbv virion released from the infected cells could be blocked with anti - hbs antibody . In an in vitro assay, the internalized antibody was seen to induce the accumulation of intracellular viral particles even after the antibody was removed from the cell culture supernatant . Several new nas such as adefovir dipivoxil (adv), entecavir (etv), telbivudine (ldt), and tenofovir (tdf) have become commercially available . Because of the risk of developing resistance, lam is no longer recommended as a first - line treatment for hepatitis b. the currently recommended first - line agents are etv and tdf, which have resulted in a very low emergence of resistance [3, 4]. Such newer nas are very effective when combined with hbig even during short duration, post - olt hbv control [103109]. Because of low resistance and the powerful antiviral response evoked by etv and tdf or a combination of nas, several institutions have developed successful hbig - free protocols if the hbv - dna titer is low enough at the time of olt [103, 110]. As the strong nas are very effective in hbv control, immune cell - related treatments are not administered, although hepatitis b infection is an immune mediated disease . The practice of active immunization of post - olt recipients with hbv vaccine is emerging . For a successful vaccine response, most studies report low response rates, even with doubled concentrations or prolonged injections of vaccines (table 2) [111115]. Patients who had not been hbv carriers (such as adult patients with acute liver failure due to sexual transmission and nonchronic hbv carriers with anti - hbc antibody - positive donor livers) are good candidates for vaccine administration [112, 116121]. Patients with acute hbv infection who undergo olt are often positive for anti - hbs antibody even before olt and have powerful immune responses . Such patients should respond well to vaccination since they have not developed tolerance to hbv, unlike chronic carriers . Since noncarriers respond well to hbv vaccination, even under prednisolone and calcineurin inhibitor usage, immune tolerance is expected to play a large role in this process . In non - olt hbv patients, analysis has revealed that hbsag - positive newborns had higher regulatory t - cell frequencies and dysfunctional cd8 t cells, which represent immune tolerant status . However, another report analyzing the immunological characteristics of hbsag - positive young carriers and aged patients with active hepatitis revealed comparable peripheral t - cell proinflammatory cytokine production capacity and hbv - specific ifn- responses . These findings indicate that tolerant carriers can react with hbv antigens and can show active immunity against hbv vaccination, if regulatory t - cell function diminishes . With good responses to newer nas after olt, hbv - dna decreases even in the liver, and this might recover compressed hbv - specific t cells to react with hbv . Chronic hbv carrier recipients, including patients with positive hbv - dna at olt, do not respond well to hbs - antigen - containing vaccine, with response rates being mostly <30% [114, 115, 119, 124, 125]. The immune status of these patients was evaluated by a mixed lymphocyte reaction (mlr) assay in response to antidonor and anti - third - party allostimulation using an intracellular carboxyfluorescein diacetate succinimidyl ester- (cfse-) labeling technique . Third - party refers to healthy volunteers with the same blood type as the patients . The autologous lymphocytes, the donor lymphocytes, and the third - party lymphocytes were irradiated and used as the stimulator cells, and the recipients' lymphocytes were used as the responder cells in mlr . The investigators minimized immunosuppression until the donor lymphocytes showed no response as autologous lymphocytes, but third - party lymphocytes showed a positive response . The investigators found that vaccination was successful in patients showing a donor - specific mlr hyporesponse, with a well - maintained response to the third - party stimulus . The vaccine was not successful in patients showing hyporesponse to both the donor and the third party . These results provide encouragement that even immune tolerant liver cirrhosis patients can react with hbv vaccines under lower immunosuppressant protocols after olt . Another protocol of repeated vaccine administration resulted in successful immunization in 40% of patients with post - olt liver cirrhosis . The donors to good responders were the spouses of recipients and had high anti - hbs antibody titers before donation . The spouses with high - titer anti - hbs antibodies were probably infected with hbv by the recipients after marriage, resulting in the anti - hbs antibody boost . The adoptive immune transfer of the hbv - specific immune response could be achieved . To successfully transfer immune memory to recipients luo et al . Have shown that a high anti - hbs antibody titer (> 1000 these results suggest that pre - olt hbv vaccination for candidate living donors might facilitate improved post - olt vaccine responses in recipients with liver cirrhosis . Several experimental adjuvant vaccines have also been tried with up to 44.8% success rates [111, 119, 129]. The vaccine response depends on immune tolerance to the virus in both recipients and donors . The liver is the largest immune organ in the abdomen; therefore, it plays a critical role in immune responses . Multiple populations of nonhematopoietic liver cells, including sinusoidal endothelial cells, stellate cells located in the subendothelial space, and liver parenchymal cells, can function as apcs . The viral - specific immune competence of the grafted liver might overcome general immune tolerance to the virus in chronic hbv carriers . As a shortage of donor organs is a universal problem, anti - hbc positive healthy carriers could be candidate donors . With regard to the above vaccination protocols, non - hbv - related patients who received anti - hbc antibody positive donor livers have fared quite favorably . The post - olt incidence of de novo hepatitis b occurring in anti - hbc antibody - positive donors without prophylaxis is high (33100%) [22, 131, 132]. These hbv - nave patients are good candidates for the hbv vaccine because 5080% tend to respond well [112, 120, 121]. Pre - olt vaccination is also possible if patients have sufficient time before undergoing olt . In countries with universal vaccination programs, the recipients might already have anti - hbs antibody and could be boosted with additional vaccination before olt, resulting in 78% of prospective recipients having a high titer of anti - hbs antibody (> 1000 the vaccination responses were observed to be good in recipients with higher anti - hbs titers at the time of olt and lower tacrolimus levels at the time of vaccination . As hcv recurrence is observed in almost all the patients who receive olt, hcv eradication before olt has been tried, although with unsuccessful outcomes [135, 136]. Hcv genotypes 1b and 4 seem to be negative predictive factors for recurrence because of a lower response to pegylated interferon (peg - ifn) and ribavirin combination therapy . The host and donor factors associated with poorer outcomes are female gender, older donor age, steatosis of the graft, and the il-28b single nucleotide polymorphism (snp) [137140]. A human genomewide association study recently uncovered many disease - susceptible genes or drug sensitivity - related genes . In chc patients, the il-28b gene snp was found to be related to spontaneous clearance and susceptibility to treatment with peg - ifn plus ribavirin [141143]. The combination of recipient and donor il-28b genetic polymorphism has been revealed to be important in post - olt hcv treatment outcomes . Recently, direct - acting antivirals such as ns3 protease inhibitors or ns5 polymerase inhibitors or a combination of them have come to represent a new highly effective treatment strategy . The triple combination therapy of peg - ifn, ribavirin, and a protease inhibitor (telaprevir) has been accepted as a highly effective treatment for non - olt chc, producing> 75% sustained virological response (svr). However, as telaprevir inhibits cytochrome p450 3a4 and reduces the metabolism of calcineurin inhibitors, the trough levels of cyclosporine a (cya) increase to 4.6-fold and fk 506 (fk) to 70-fold . The second generation protease inhibitor simeprevir very weakly inhibits cytochrome p450 3a4 and is safer than telaprevir . Triple therapy including simeprevir is safer than triple therapy with telaprevir and is currently recommended . In post - olt settings, although the t - cell response is repressed with calcineurin inhibitors, post - olt chc patients often show severe hepatitis recurrence with high viral load . In post - olt several reports have mentioned that hcv - specific immune responses correlate with post - olt hepatitis c progression [149, 150]. The frequency of hcv - specific il-17-secreting cd4 t cells was shown to be increased in severe inflammation in liver fibrosis patients . The serum cytokine profile of these patients with severe recurrence exhibited higher inflammatory cytokines (il-17, il-1, il-6, il-8, and monocyte chemoattractant protein [mcp]-1), decreased antiviral cytokine ifn-, and increased ifn- reducing cytokine il-10, suggesting the presence of the inflammatory phenotype with repressed antiviral immune response . Moreover, tregs and tr1 cells are overexpressed in patients with severe hepatitis c recurrence compared with patients with no or minor recurrence [86, 153]. These results suggest that tregs and tr1 cells are involved in hcv recurrence after olt . Because the strength of immunosuppressive therapy and the viral load would be changed after olt, the time course of the immune response has important roles . Recently, we have shown that tr1 frequency was repressed in 40 days after olt under the condition of persistently normal alanine aminotransferase (alt), even at 3 years after olt . Tr1, which has a strong il-10 production capacity, may reduce hcv - specific t - cell responses and induce active hepatitis with alt elevation . Monitoring tr1 frequency might be a way to determine which patients would develop active hepatitis . However, hcv - specific cd4 t - cell ifn- production, which was higher in patients with persistently normal alt until 3 years after olt, was found to diminish after 3 years (tsuzaki r. et al . This result indicates that, although the adaptive immune response could control hepatitis, the strength of the response might diminish over time . These results from our experience indicate that ifn - based anti - hcv therapy could be applied for patients with higher tr1 after olt, who might show active hepatitis until 3 years after olt . Whether the tr1 reduction treatment will become the next treatment strategy is not clear, as selective reduction of tr1 might be difficult . However, as calcineurin inhibitors reduce regulatory t cells, minimum usage of calcineurin inhibitors might be the way this can be accomplished now . Innate immune responses have also been identified as hcv targets and could be depressed with respect to their functions . Dendritic cells (dcs) and nk cells are thought to play a central role in the interplay between the innate and adaptive immune responses . Kupffer cells are also involved in post - olt hepatitis c recurrence, as nf-b was highly expressed in patients with post - olt hcv recurrence . However, the specificity for the disease state is not well characterized . In post - olt settings, nk cells have also been deeply investigated with respect to their activities in hcv infection and hepatitis . Nk cells are implicated in various viral infections, including hcv and front - line anticancer immune responses . The hcv - e2 protein has been revealed to bind the nk cd81 receptor and decrease the release of ifn-, resulting in noneffective antiviral responses . Another nk cell receptor, the killer immunoglobulin receptor (kir), which displays an inhibitory function, has been revealed to be correlated with post - olt hepatitis c. the kir - ligand mismatch and recipient kir2l3 haplotype have been shown to correlate with recurrent hepatitis c . Ifn treatment susceptibility of post - olt hcv recurrence has also been shown to be correlated with the nk receptor haplotype kir2ds2 . Intravenous administration of living donor perfusate of nk cells could reduce the hcv - rna increase after olt . As acutely infected hepatitis c patients show self - recovery at a rate of 2030%, a strong nk cell response might control hepatitis c even under immunosuppressive treatment . Adaptive immune response in post - olt hbv remains a problem that should be investigated, as this virus continues to be difficult to be eradicated from the infected liver . However, the anti - hcv treatment protocol is drastically changing because several clinical trials of new daa with> 80% viral eradication might result in these drugs being introduced to the market; therefore, the importance of investigating the immune system in post - olt hcv will probably consolidate to selected refractory patients in the next 10 years . The adaptive immune response in post - olt hepatitis b recurrence is hidden under strong antiviral hbig and na combination treatment . However, the effectiveness of active immunization is dependent upon adaptive immune responses being effective for patients with non - hbv - related disease who have received anti - hbc antibody - positive donor livers and patients with acute liver failure who are not immune tolerant to hbv . Vaccination is not sufficiently effective for patients with liver cirrhosis; nevertheless, the donor immune memory for hbv and the strength of the immunosuppressant drugs have important roles . Adaptive immune responses, especially of the cd4 and cd8 t cells and the treg, have strong effects in post - olt hepatitis c viral recurrence and in recurrent hepatitis activities . The regulatory t cells and tr1 cells affect the clinical course and could be used as prediction markers . As ifn - based treatments have risks after olt, forecasting the patient's course with such markers could be beneficial.
Current structure - based research has used high - resolution mx and nmr - derived structures to guide hypothesis - driven research . This has been effective for well - folded, compact enzymes, and has enabled atomic - level dissection of an enzyme s active site . Nevertheless, estimates suggest that over 50% of eukaryotic proteins contain significant functional unstructured regions (vucetic et al . Macromolecular flexibility is an important aspect of the regulatory mechanisms of biological systems (henzler - wildman and kern 2007; perry et al . Mx, nmr, and electron microscopy (em) are regarded as the most reliable methods for determination of structure; nonetheless, these techniques are limited by macromolecules with functional flexibility and intrinsic disorder (fink 2005). Validation of macromolecular flexibility in solution by small - angle x - ray scattering (saxs) has recently become a central tool in the new area of characterizing multi - state systems within structural biology (bernado et al . Has the potential to address how specific complexes and flexibility drive biological processes (putnam et al . 2007; although saxs has some inherent limitations, there is sufficient information within the one - dimensional scattering profile to distinguish between well - defined conformations and the conformational space occupied by a flexible assembly (fig . 1). The theoretical basis for solution scattering has been the subject of an excellent review (koch et al . Previously, i authored a review providing a general framework for experimental design, data processing, and data interpretation that combined saxs with atomic - resolution structures from crystallography (putnam et al . The purpose of this review is to discuss different tools and methods that have recently been developed for saxs analysis of flexible multidomain assemblies.fig . 1validation of flexibility using saxs curve (a) and rigid - body modeling (b). A experimental saxs profiles (black and blue) for the human dna ligase iii (cotner - gohara et al . 2010) in a match with theoretical profiles calculated for the crystal structure (red) (cotner - gohara et al . 2010) and its dynamic model (green) obtained by bilbomd and mes (pelikan et al . Baseline convergence necessary for assessing flexibility is misleading for the saxs curve with insufficient buffer subtraction (gray). Pair distribution p(r) function calculated for the experimental (black) and the theoretical saxs (red, cyan). Crystal structure, full - length and ensemble models used to calculate theoretical saxs profiles are shown in the panel a (data adapted from cotner - gohara et al . B schematic diagram of typical rigid - body modeling performing building of initial model, conformational sampling, and ensemble analysis validation of flexibility using saxs curve (a) and rigid - body modeling (b). A experimental saxs profiles (black and blue) for the human dna ligase iii (cotner - gohara et al . 2010) in a match with theoretical profiles calculated for the crystal structure (red) (cotner - gohara et al . 2010) and its dynamic model (green) obtained by bilbomd and mes (pelikan et al . 2009). Baseline convergence necessary for assessing flexibility is misleading for the saxs curve with insufficient buffer subtraction (gray). Pair distribution p(r) function calculated for the experimental (black) and the theoretical saxs (red, cyan). Crystal structure, full - length and ensemble models used to calculate theoretical saxs profiles are shown in the panel a (data adapted from cotner - gohara et al . B schematic diagram of typical rigid - body modeling performing building of initial model, conformational sampling, and ensemble analysis debye law as a powerful tool for distinguishing between rigid and flexible particles (rambo and tainer 2011). In particular, it was shown that for comparative saxs experiments, application of the law can distinguish between discrete conformational changes and localized flexibility relevant to molecular recognition (devarakonda et al . 2011; williams et al . . This approach aids insightful analysis of fully and partly flexible macromolecules that is more robust than traditional kratky analysis (porod 1982). Kratky analysis relies on visual inspection of the kratky plot, which can be confounded by a limited observational q range (q <0.2), the presence of high experimental noise, or by non - ideal buffer subtraction (fig . 1a). Intensity measurements at high scattering angles are exponentially more sensitive to the buffer blank subtraction than measurements near the guinier region . Therefore, small errors during the buffer blank subtraction may confound the baseline convergence necessary for assessing flexibility by kratky analysis (fig . Debye law resides within the low - resolution region of the saxs profile, typically q <0.15, that is routinely well measured and not prone to buffer blank subtraction issues . For example, kratky analysis of the saxs data collected for the atp - free and bound forms of mre11rad50 (williams et al . 2011) did not clearly identify flexibility of the atp - free state and rather led to the hypothesis that the particle is switching between two distinct conformational states, similar to pyr1(nishimura et al . However, inspection of the porod plot suggests a fundamentally different mechanism . In the presence of atp, the complex forms a distinct particle with a sharp scattering contrast, as evidenced by the porod plateau (fig . In fact, inspection of the porod debye region demonstrates a loss of the plateau, supporting the hypothesis that mre11rad50 is flexible in the absence of atp . These types of analysis provide qualitative information about conformational states that give credence to modeling the solution state as an ensemble of conformers.fig . 2detecting conformational flexibility . A saxs data for the mre11rad50 complex in both the presence (black), and absence (red) of atp (williams et al . 2011), and an exemplary intrinsically disordered domain rad51 ap1 (blue). Inset comparison of the kratky plots for mre11rad50 complexes does not confidently demonstrate flexibility of the complex in the absence of atp (black and red). However, the kratky plot of rad51 ap1 (blue) is hyperbolic in shape, clearly demonstrating the full unfolded particle . Rigid and flexible states of mre11rad50 are presented with crystal structure of mre11rad50-atps (lim et al . Data for rad51 ap1 were kindly provided by gareth williams at the lawrence berkeley national laboratory detecting conformational flexibility . A saxs data for the mre11rad50 complex in both the presence (black), and absence (red) of atp (williams et al . 2011), and an exemplary intrinsically disordered domain rad51 ap1 (blue). Inset comparison of the kratky plots for mre11rad50 complexes does not confidently demonstrate flexibility of the complex in the absence of atp (black and red). However, the kratky plot of rad51 ap1 (blue) is hyperbolic in shape, clearly demonstrating the full unfolded particle . Rigid and flexible states of mre11rad50 are presented with crystal structure of mre11rad50-atps (lim et al . 2011) and dynamic model of mre11rad50 (williams et al . Data for rad51 ap1 were kindly provided by gareth williams at the lawrence berkeley national laboratory methods of analysis based on the concept of a single conformer cannot provide a complete three - dimensional model of dynamic proteins . Using a single best conformer to represent the ensemble at most provides a model representing an average of the conformations that exist in solution . Best single model of the macromolecular state can still be informative by helping guide a hypothesis regarding the macroscopic conformational state (hammel et al . 2002; iyer et al . 2008; jain et al . 2009; pascal et al . 2004; williams et al . For example, if the crystal structure of a macromolecular assembly is known, a theoretical scattering profile can be calculated from the atomic coordinates . This provides the opportunity to evaluate several user - generated models (fig . 1). If an extended conformer fits saxs data better than a compact crystal structure, then an opening of the assembly in solution may be assumed (nagar et al . Crystal packing forces are a selective pressure on a ensemble that typically promote a single conformer within the crystal lattice . Differences between crystal and solution states often reflect the presence of crystal packing forces (cotner - gohara et al . 2010; datta et al . 2009; duda et al . 2008; nishimura et al . 2009; stoddard et al . 2010) that can be used to gain new insights into a protein's flexibility (nishimura et al . Direct comparisons of different conformational states with model saxs profiles calculated from atomic - resolution structures have been quite successful in identifying and decomposing the relative fractions of conformers of a sample in solution, such as with the archaeal secretion atpase gspe . The mx structure of the hexameric ring revealed a mixture of open and closed states of the individual subunits (yamagata and tainer 2007). In contrast, saxs studies of gspe suggested a much different conformational state in solution . In the presence of the transition state atp analogue, amp - pnp, saxs experiments suggest the enzyme s subunits assume an all - closed state . In the next step of the catalytic cycle, the adp - bound state, saxs experiments suggest gspe exists as a mixture of all - closed and all - open states . The original crystal structure of alternating open closed states in a ring failed to explain the saxs experiments and raises significant questions regarding the proper biological state of the crystallized gspe . 2010); consequently, a structural biology approach solely dependent on mx will be limited in scope . High - quality saxs experiments from advanced instrumentation (hura et al . 2009) lead to more precise data and confident assignment of the conformational state(s) of a given sample . Notwithstanding instrumentation developments, accurate calculation of a saxs profile is essential for the accuracy of solution structure modeling . Several methods are available to calculate saxs profiles from atomic models, and differ in the use of the inter - atomic distances, estimation of excluded volume, treatment of the hydration layer, or background adjustment (grishaev et al . Calculation of an saxs profile from atomic coordinates requires spherical averaging that can be efficiently accomplished by representing a macromolecule in terms of inter - atomic distances (schneidman - duhovny et al . 2006) or by using spherical harmonic reconstructions (grishaev et al . 2010; liu et al . 1995). Explicit calculation of inter - atomic distances with solx software (zuo et al . 2006) requires more intensive computation, but results in good agreement throughout the large q range with experimental scattering profiles (putnam et al . Calculating profiles for anisometric shapes or unfolded regions is also more problematic for spherical harmonic reconstructions (reviewed by putnam et al . 2007) and inaccuracies in fitting can be compensated by over - adjustment of excluded volume or the density of the hydration layer . As the data quality becomes extraordinary good, full atomistic models are required for accurate interpretation of the experimental saxs profiles (fig . 3). In this example of a high - resolution experimental saxs of the cellulase cel5a catalytic domain, explicit calculations using inter - atomic distances of several models demonstrate that the calculation of accurate profiles may detect small unfolded regions (fig . 3). Saxs can detect these unstructured regions only because they affect the overall / globular shape of the protein . However, the example presented clearly shows the kind of information content stored in the saxs profiles or its p(r) functions derived from them (fig . The fact that the full - atomistic model is important to match experimental data has been further shown by analysis of 19 proteins containing a 19-residue his tag (hura et al . 2009). His tags increase dmax, and should be modeled explicitly with available core atomic models.fig . A comparison of the experimental scattering curves of cellulase cel5a catalytic domain (black) with the theoretical curves for cel5a crystal structure missing the c - terminal unfolded region (pdb 1edg) (blue), full - atomistic model (red), and coarse - grain (cg) model (green), shown in panel b. bottom panel the discrepancy between theoretical and experimental profiles is calculated as intensity(experiment)/intensity(model). Please note the large discrepancy for the cg model (= 1.7) and crystal structure (= 1.8) in comparison with the full - atomistic model calculated by foxs (= 1.2). Better profile matches are obtained by calculating explicit atom distances (foxs = 1.2) in comparison with the saxs profile calculated by spherical harmonics using crysol cp(r) functions calculated for saxs profiles shown in a have been calculated by use of the software gnom (svergun 1992). The production and purification of the cellulase cel5a catalytic domain has been described elsewhere (fierobe et al . Saxs experiments were performed at the european synchrotron radiation facility (grenoble, france) on beamline id02 as described by (hammel et al . A comparison of the experimental scattering curves of cellulase cel5a catalytic domain (black) with the theoretical curves for cel5a crystal structure missing the c - terminal unfolded region (pdb 1edg) (blue), full - atomistic model (red), and coarse - grain (cg) model (green), shown in panel b. bottom panel the discrepancy between theoretical and experimental profiles is calculated as intensity(experiment)/intensity(model). Please note the large discrepancy for the cg model (= 1.7) and crystal structure (= 1.8) in comparison with the full - atomistic model calculated by foxs (= 1.2). Better profile matches are obtained by calculating explicit atom distances (foxs = 1.2) in comparison with the saxs profile calculated by spherical harmonics using crysol cp(r) functions calculated for saxs profiles shown in a have been calculated by use of the software gnom (svergun 1992). The production and purification of the cellulase cel5a catalytic domain has been described elsewhere (fierobe et al . Saxs experiments were performed at the european synchrotron radiation facility (grenoble, france) on beamline id02 as described by (hammel et al . 2004a) fitting theoretical models to saxs profiles requires that a measure be established for determining the agreement between two scattering curves . I am not convinced that a best measure of assessing agreement between experimental and theoretical curves has been adequately developed . The values become less informative as the high resolution saxs profiles with low - noise are used to fit atomistic models . For additional assessment of the quality of model - data agreements this residual - ratio clearly displays discrepancies in the important small q region whereas the standard log10-based presentation of log (i) versus q frequently does not (figs . Better quality experimental data promotes the need for increased accuracy and computations of saxs profiles . By using explicit - all atom distances (schneidman - duhovny et al . 2010) and water models to account for the effect of solvent (grishaev et al . 2010) superior fits between experimental high resolution structures and saxs data are obtained (fig . The explicit representation of the molecule is particularly useful for multidomain - flexible assemblies, which frequently adopt highly anisometric shapes the foxs algorithm explicitly computes all inter - atomic distances that include the first solvation layer based on the atomic solvent accessible areas (fig . As foxs is available through a web server, it enables uploading and simultaneous analysis of a collection of atomic coordinate input files against experimental data . In combination with the mes (pelikan et al . 2009) that is also part of the suite, the user is provided with powerful tools to identify the heterogeneity or flexibility of the experimental system . These powerful analytical techniques, together with advanced instrumentation, have been the basis for visualizing minimum conformational changes in human complement c3b (chen et al . A experimental scattering curves for free c3b (black) and in the complex with extracellular fibrinogen - binding protein (efb) from staphylococcus aureus (c3b / efb) (blue) were fit to mes model (red line). Bp(r) functions indicate conformational changes between c3b (black) and c3b / efb (blue), where broadening of p(r) for c3b / efb - c is consistent with reorientation of the cub - ted domain . C comparison of rg for the two predominant mes conformers of either c3b (black) or c3b / efb (blue) as obtained by bilbomd sampling with their maximum dimensions (dmax). Rigid - body modeling - derived c3b conformers are shown in gray with efb highlighted in red . (d, e) superposition of the bilbomd - mes - derived conformers of free c3b (d, magenta and green) and c3b / efb (e, blue / red) with the crystal structure of c3b (gray). (2010) efb - induced conformational changes in human complement c3b as revealed by saxs . A experimental scattering curves for free c3b (black) and in the complex with extracellular fibrinogen - binding protein (efb) from staphylococcus aureus (c3b / efb) (blue) bp(r) functions indicate conformational changes between c3b (black) and c3b / efb (blue), where broadening of p(r) for c3b / efb - c is consistent with reorientation of the cub - ted domain . C comparison of rg for the two predominant mes conformers of either c3b (black) or c3b / efb (blue) as obtained by bilbomd sampling with their maximum dimensions (dmax). Rigid - body modeling - derived c3b conformers are shown in gray with efb highlighted in red . (d, e) superposition of the bilbomd - mes - derived conformers of free c3b (d, magenta and green) and c3b / efb (e, blue / red) with the crystal structure of c3b (gray). Although comparison of model saxs profiles with the experimental data is one of the most straightforward applications of saxs, the uniqueness of arrangements of atomic resolution structures that fit saxs data must also be evaluated . The determination of multidomain or subunit assemblies using rigid - body modeling in conjunction with saxs data involves preparing a large number of possible atomic models and comparing them with experimental data . The models can either be refined directly against experimental data (petoukhov and svergun 2005) or prepared independently using the saxs data as a filter to select the best fit model(s) (boehm et al . 1999; forster et al . 2008). The biggest challenge in trying to model flexible multidomain systems using saxs data is to avoid over - fitting . Most commonly, over - fitting can be detected by visually inspecting the selected models and examining for large unfolded regions or unrealistic inter - domain distances . Successful fits of experimental data derived from aggregated or heterogeneous samples (reviewed by putnam et al . For example, studies of mammalian lipoxygenase illustrate the need for establishing monodispersity of sample in cases where domain flexibility is proposed (dainese et al . The discrepancy between the experimental curve of mammalian lipoxygenase and the profile calculated from the atomic coordinates were interpreted in terms of a very large movement of the n - terminal domain (hammel et al . (2011) found that mammalian lipoxygenase, besides its flexible n - terminal domain, forms a transient dimer that also leads to an elongated saxs signal . Therefore, samples that are suspected of possessing intrinsic flexibility must be carefully characterized to ensure monodispersity before saxs modeling (rambo and tainer 2010b). A number of techniques have been used to generate realistic atomic models that sample conformational space of multi - modular proteins . 2011) based on exploration of the dihedral angles in connection regions (akiyama et al . 2004; curtis et al . 2012), torsion / cartesian simulated annealing (schwieters et al . 2010), and minimal molecular dynamics (minimal md) (boehm et al . 1999; hammel et al . 2005; yang et al . This approach was applied to solution structure determinations of human and chimeric antibodies (reviewed by perkins and bonner 2008). The technique uses a large number of conformers that are built with directed md computations applied only to the inter - domain connections . These models are filtered on the basis of their agreement with properties extracted from experimental saxs curves, for example the radius of gyration, radius of gyration of cross sections, and the overall fit of the theoretical scattering from the model to the experimental data (abe et al . Constrained modeling confirms the experimental data analysis and produces families of best - fit models . Although these molecules are most likely an ensemble with a wide range of conformations, the selected best fit conformers are sufficient to reveal conformational switching or flexibility . The recently developed bilbomd approach uses a similar minimal md strategy and describes the final model as a population - weighted ensemble selected from the entire pool of conformers (pelikan et al . 2009) (figs . 4 and 5).fig . 5solution structure modeling of intramolecular hg transfer between flexibly linked domains of mercuric ion reductase (mera). A comparison of experimental and calculated scattering profiles for full - length mera (mutmera). Experimental saxs data (gray), single best - fit conformation to the experimental scattering profile with = 1.96 (blue line), and combined profile from five contributing conformations identified by mes (red line) with = 1.39 . Superposition of the five models identified by mes with the metallochaperone - like n - terminal domains in a different color weighted by the factors 0.40 (pink), 0.29 (green), 0.16 (cyan), 0.08 (purple), and 0.07 (gray). B experimental saxs data for the disulfide - cross - linked handoff complex (ss mutmera) (gray) and calculated scattering data for the single best - fit conformation = 1.02 (blue line). Residuals iexperiment / imodel are shown as blue dots and as a blue line for smooth residuals . (2011) solution structure modeling of intramolecular hg transfer between flexibly linked domains of mercuric ion reductase (mera). A comparison of experimental and calculated scattering profiles for full - length mera (mutmera). Experimental saxs data (gray), single best - fit conformation to the experimental scattering profile with = 1.96 (blue line), and combined profile from five contributing conformations identified by mes (red line) with = 1.39 . Superposition of the five models identified by mes with the metallochaperone - like n - terminal domains in a different color weighted by the factors 0.40 (pink), 0.29 (green), 0.16 (cyan), 0.08 (purple), and 0.07 (gray). B experimental saxs data for the disulfide - cross - linked handoff complex (ss mutmera) (gray) and calculated scattering data for the single best - fit conformation = 1.02 (blue line). Residuals iexperiment / imodel are shown as blue dots and as a blue line for smooth residuals . (2011) conformational sampling may also be performed with simplified coarse - grain (cg) models, where amino - acid residues are presented as spherical beads centered at corresponding c atom positions (rozycki et al . Although extremely simplified, cg incorporates the main generic features and folding data of the protein under investigation . The cg models are used to not only speed up the production phase of conformational sampling but also to speed up the saxs calculation . However, cg models are coarse representations, and it has been shown that full atomistic models are required for accurate calculation of saxs profiles (grishaev et al . The atomistic representation is essential for accurate representation in solution when the particles deviate from a canonical globular shape (fig . Accurate assignment of the flexible regions is crucial to realistic conformational sampling . In most cases, analysis of high - resolution structures can indicate plausible regions of structural flexibility (chen et al . 2010b) or regions with a high isotropic atomic displacement factor (adf also called the b - factor) (duda et al . 2008; williams et al . . Empirical determination of flexible regions can be achieved by hydrogen deuterium exchange mass spectrometry (hdx) which specifically follows changes in conformational states of proteins . For example, hdx clearly assigned the flexible region in the complement c3b molecule after its activation (hammel et al . 2007b). This hdx experiment guided saxs based rigid - body modeling used to visualize the c3b molecule as a highly dynamic system . Saxs modeling also revealed that c3b flexibility may be effected by an allosteric inhibitor, for example the extracellular fibrinogen - binding protein (efb) from staphylococcus aureus . This is the first reported evidence that the system is controlled by allosteric inhibitors and supports new views in which modulators may stabilize preexisting intrinsic conformations rather than inducing completely new domain arrangements (chen et al ., realistic models may by derived by incorporating additional information about the system in question, for example known distance constraints . Techniques that provide local distance and angle information, for example frster resonance energy transfer (fret) (rochel et al . 2011) and nmr (bertini et al . 2007) may provide useful restriction in inter - domain movement and guide conformational sampling . The rigid body / torsion / cartesian simulated annealing strategy developed by grishaev et al . (grishaev et al . 2005; mittag et al . 2010) integrated both nmr and saxs observations into a unique synergistic method for atomistic modeling . From nmr, residual dipolar coupling (rdc) data were used to orient the symmetrically related protein domains relative to the symmetry axis of the protein core whereas translational, shape, and size information was provided by saxs (schwieters et al . Fret in combination with saxs guided rigid - body modeling to aid elucidation of the structural basis of the role of dna in the spatial organization of nuclear hormone receptors in complex co - activators (rochel et al . Distance restraints may also be generated from simple biochemical techniques, for example site - direct mutagenesis . For example, integrated site - directed mutagenesis and saxs combined with conformational sampling of dna binding sites were used to determine the dna - binding properties of mpnk (bernstein et al . 2009) and reveal the intramolecular metal ion transfer between flexibly - linked domains of mercury ion reductase (johs et al . Although exhaustive conformational sampling significantly increases the number of realistic models to be used for modeling experimental saxs data, a single best - fit conformation may be incapable of explaining the observed saxs profile . The lack of convergence of a single best - fit conformation has been shown to correlate with conformational disorder rather than a limitation of the search space algorithm (pelikan et al . The measured scattering is derived from the population - weighted thermodynamic ensemble, and the interpretation of dynamic systems requires analysis beyond best fit conformations (figs . 4 and 5). In recent years, new saxs modeling techniques have been developed to describe dynamic systems in terms of ensembles of structures (bernado et al . ; yang et al . 2010). Four promising approaches for modeling the ensemble are pushing saxs into an exciting new direction, the ensemble optimization method (eom) (bernado et al . 2007), minimal ensemble search (mes) (pelikan et al . 2009), ensemble refinement of saxs (eros) (rozycki et al . 2011), and basis - set supported by saxs (bss - saxs) (yang et al . 2010). Because of the nearly infinite number of conformations that can be adopted by flexible proteins in silico, obtaining meaningful models requires the development of robust statistical approaches that determine the probability a particular multi - conformational equilibrium will exist (bertini et al . 2010). Again, a common problem with multi - conformational analysis is over - fitting, which occurs when an ensemble model describes noise or aggregation in the experimental system, rather than the desired underlying relationship . Mes avoids over - fitting by asserting the minimum number of states that could be distinguished from saxs data . In addition, to avoid over - fitting the data with the multiple conformations (bernado et al . 2007), a quantitative description of the ensemble also requires the weighting of each conformer's distribution (pelikan et al . For the purpose of avoiding over - fitting of raw data, rozycki et al . Constructed a pseudo free energy scheme to refine the statistical weights attributed to configurations generated by simulation (rozycki et al . These saxs ensemble methods seem enormously successful on the basis of analysis of several key biological systems: identification of the correct subunit positions for full - length ku (hammel et al . 2010b), demonstration of the flexibility in full - length polynucleotide kinase (bernstein et al . 2009), establishment of the configurational space of lys-63 linked tetraubiquitin (datta et al . 2009), elucidation of the flexibility mode in a ubiquitin - pcna complex involved in dna replication and repair (tsutakawa et al . 2011), and describing the partially unfolded state of xrcc4 (hammel et al . The single conformation description of a macromolecule is only a snapshot of a macromolecular ensemble . We have seen that integrative methods that utilize nmr and mx with saxs are proving to be essential for providing a larger description of the macromolecular ensemble . Using saxs data as a source of experimental restraints for modeling macromolecular flexibility is an exciting and relatively underdeveloped discipline . Saxs data can provide important experimental feedback, and can be extended to include dynamic conformational changes characterized by time - resolved experiments . Time - resolved measurements require very high x - ray flux and fast detectors designed for rapid electronic shuttering . Both are now available, and saxs, unlike traditional nmr and fluorescence experiments, is not affected by molecular rotation times, so time - resolved saxs can be performed in an equivalent manner to the traditional static experiments . The development of the approaches for characterizing highly fluctuating conformational equilibria on the basis of traditional static experiments are becoming essential in the description of intrinsic dynamic biomolecular systems (bernado and blackledge 2010). Macromolecular machines with flexible and unstructured regions are now tractable to direct structural investigation (bernado 2010; bernado and svergun 2012). These are some of the reasons why saxs - based solution structure modeling of flexible macromolecular assemblies are gaining popularity and will be used in the future to elucidate the roles of dynamic equilibrium in biological processes (rambo and tainer 2011). A natural complement to the global shape and conformation from saxs deuterium exchange mass spectrometry, which can approach single - residue resolution as shown for the photocycle changes of photoactive yellow proteins (brudler et al . Thus, saxs is well positioned to become an important technique, with new weak - field aligned nmr and fluorescence experiments that can probe samples in the biologically interesting millisecond time frame . With appropriate resources for directed efforts, saxs can provide complementary experimental data on flexibility in macromolecular interactions with widespread effects.
Mycobacterium tuberculosis is one of the most important pathogens in the world (1). The emergence of multi - drug resistant strains (mdr) and co - infection with the hiv virus is considered one of the biggest health problems (2). According to the world health organization reports, in 2013, nine million people developed tb out of which 1.5 million lost their lives due to this disease . The who report in 2014 showed the number of people with tb was on the rise; the report also emphasized that the number of mdr - tb had increased in the past two decades (3). As iran is in close proximity to high - prevalence countries, prevention of the spread of tuberculosis cases particularly mdr - tb is one of the priorities of the country (4). The use of methods that can identify and track tb transmission is helpful in controlling the disease . Planning for tb control needs an identification of the sources of infection and the spread of disease . With the development of molecular epidemiology in recent years, the possibility of studying the epidemiology of infectious diseases has increased significantly . For understanding the path for disease transmission, molecular epidemiology studies are essential in order to prevent the spread of disease (5). Therefore, genotyping techniques are powerful tools for identifying the outbreak, contact tracing, and studying the diversity of strains . Also, increasing knowledge in this area may be considered as an effective way to prevent transmission (6). Many different molecular epidemiology techniques have been proposed for identifying the genetic relationship between different strains of mycobacterium tuberculosis and bovis (68). M. tuberculosis has very conserved genome; as little nucleotide diversity was reported in their genome, this organism is genetically monomorphic (9). Although genome of m. tuberculosis complex is highly conserved in comparison to other bacterial pathogens, some variation does exist (10, 11). Today, there are various methods for genotyping of m. tuberculosis which is called fingerprinting techniques . Genotyping methods can be briefly classified as follows: sequence - based standard methods such as whole genome sequencing.non sequence - based methods: these methods are generally classified into two categories: non - amplified methods (gel- based techniques) such as pulsed field gel electrophoresis (pfge) and restriction fragment length polymorphism (rflp)amplified methods (pcr - based genotyping methods) such as spoligotyping, mycobacterial interspersed repetitive unit - variable number tandem repeat (miru- vntr), random amplified polymorphic dna polymerase chain reaction (rapd - pcr), repetitive element palindromic pcr (rep - pcr). Non sequence - based methods: these methods are generally classified into two categories: non - amplified methods (gel- based techniques) such as pulsed field gel electrophoresis (pfge) and restriction fragment length polymorphism (rflp)amplified methods (pcr - based genotyping methods) such as spoligotyping, mycobacterial interspersed repetitive unit - variable number tandem repeat (miru- vntr), random amplified polymorphic dna polymerase chain reaction (rapd - pcr), repetitive element palindromic pcr (rep - pcr). Non - amplified methods (gel- based techniques) such as pulsed field gel electrophoresis (pfge) and restriction fragment length polymorphism (rflp) amplified methods (pcr - based genotyping methods) such as spoligotyping, mycobacterial interspersed repetitive unit - variable number tandem repeat (miru- vntr), random amplified polymorphic dna polymerase chain reaction (rapd - pcr), repetitive element palindromic pcr (rep - pcr). Often these techniques are based on the repetitive sequences (6). There are two types of repetitive units, interspersed repeats (ir) (direct repeats [dr], insertion sequence - like repeats [is]) and tandem repeats (tr) (variable - number tandem repeats [vntr]) (10, 11). Each method has some advantages and disadvantages (6, 9, 12, 13). The aim of this study was to evaluate the molecular typing methods used in iran . By evaluating discriminatory power of each method and comparing the results, we can assign appropriate weight to each technique and propose a common strategy for future epidemiological studies . Hunter - gaston discrimination index (hgdi) was used to evaluate the discriminatory power for each method (12). We searched several databases such as pubmed, web of science, scopus, iran medex, google scholar, and scientific information database (sid) to identify studies addressing m. tuberculosis molecular epidemiology in iran . Keywords that were selected for this research were: molecular epidemiology, tuberculosis and iran . Moreover, to search for articles that were published in persian, the corresponding persian keywords were used . Having the search conducted, 25 articles in english and 9 persian articles were shortlisted; while others were excluded from the study due to lack of relevance or unavailability . Papers either contained hgdi index or the information necessary to calculate this index; we reviewed the selected articles and used the already existing indexes or calculated the index based on the data extracted from the articles . The indexes were calculated according to the following equation: d=11n(n1)j1snj(nj1). Where n is the total number of strains in the sample population, s is the total number of types described, and nj is the number of strains belonging to the jth type . D can take any figure between 01 while the lowest and largest discriminatory power indexes are represented by 0 and 1, respectively . Assessing and determining the discriminatory power of the molecular epidemiology is important because based on the results of this study, a more powerful tool can be selected for the genotyping research . We searched several databases such as pubmed, web of science, scopus, iran medex, google scholar, and scientific information database (sid) to identify studies addressing m. tuberculosis molecular epidemiology in iran . Keywords that were selected for this research were: molecular epidemiology, tuberculosis and iran . Moreover, to search for articles that were published in persian, the corresponding persian keywords were used . Having the search conducted, 25 articles in english and 9 persian articles were shortlisted; while others were excluded from the study due to lack of relevance or unavailability . Papers either contained hgdi index or the information necessary to calculate this index; we reviewed the selected articles and used the already existing indexes or calculated the index based on the data extracted from the articles . The indexes were calculated according to the following equation: d=11n(n1)j1snj(nj1). Where n is the total number of strains in the sample population, s is the total number of types described, and nj is the number of strains belonging to the jth type . D can take any figure between 01 while the lowest and largest discriminatory power indexes are represented by 0 and 1, respectively . Assessing and determining the discriminatory power of the molecular epidemiology is important because based on the results of this study, a more powerful tool can be selected for the genotyping research . After evaluating all articles, 34 relevant articles (published from 2000 to 2014) were selected for analysis (diagram 1). All 34 articles contained information necessary to calculate hgdi index . As presented in diagram 1, molecular typing techniques frequently used in iran were rflp (is6110, pgrs and dr), miru - vntr, spoligotyping, pfge and rapdpcr . The majority of the methods used in the literature were spoligotyping with 18 studies followed by is6110-rflp and miru - vntr with 9 and 7 cases respectively . Number of methods and articles reviewed in the study number of methods used in the articles reviewed here our search demonstrated that is6110 - rflp had the highest hgid (an average hgid of 0.9897), followed by polymorphic gc - rich repetitive sequences (pgrs) - rflp (average hgid: 0.9904) and miru - vntr (average hgid: 0.9638) and spoligotyping (average hgid: 0.9433) and the lowest discrimination power (average hgdi: 0.6974) was obtained for pfge . In most studies, results are presented in table 1 . Comparative analysis of genotyping methods of mycobacterium tuberculosis published by iranian scientists . Nritld: the national research institute of tuberculosis and lung diseases (tehran, iran) each genotyping method has a different value for this index in different articles depending on various parameters such as number and distribution of samples (14). A minimum of hgdi value more than 0.90 is desired for a test to distinguish among related organisms (12). The analysis of hgdi values for each typing method in this study showed that is6110-rflp had the highest hgdi value of 0.9990 and the lowest hgdi belonged to pfge with an average value of 0.4159 . The analysis of hgdi values for is6110-rflp showed an average of 0.9897 ranging from 0.9990 to 0.9751 . The highest value was achieved by the analysis of isolates obtained from five provinces of iran (15), and the lowest of them was obtained from tehran province (16). The major advantage of this method is the higher discriminatory power it provides but it suffers from the long time required and technical difficulties in conducting . Furthermore, is6110-rflp has low discriminatory power in strains with fewer than 6 copy number of is6110 sequences (13, 17). Also, it has been proven that pgrs fingerprinting is useful for differentiating m. tuberculosis strains with less than six copies of is6110 that cannot be successfully examined by is6110 fingerprinting (1, 18). In the present study, miru - vntr showed high hgdi average value of hgdi = 0.9638 after rflp . In this study, maximum hgdi value for miru - vntr was 0.9966 (19) and the lowest was 0.8126 (20). The discriminatory power of miru - vntr technique depends on the number and type of selected loci, in addition to the number and distribution of samples (21). The highest hgdi value for miru - vntr in iran has been reported by asgharzadeh and colleagues who used a 15-locus miru - vntr typing method (19). Believe that the use of 12 appropriate loci in miru - vntr will provide high discriminatory power (22). (24) who used the 12 loci miru - vntr, obtained hgdi indexes 0.9932 and 0.9869, respectively . In both articles, etr and miru loci have been used . It seems that little difference in the discriminatory power of these two studies was related to sample size (127 samples versus 53 samples). The advantages of miru - vntr are high discriminatory power, high reproducibility (25) and the ability to create a numeric code for each isolate which facilitate its tracing in the database as well as ease and cost - effectiveness . Due to these advantages proposed a 15-locus system as a new standard for routine epidemiological discrimination of m. tuberculosis isolates and a 24-locus system as a high - resolution tool for phylogenetic studies (21). The simultaneous use of two techniques enhances the discriminatory power . In a relevant study, asgharzadeh et al . Showed that the combination of is6110 and miru - vntr had the greater discriminatory power than either method alone (19). In another study, the third most widely used technique in the articles we reviewed was spoligotyping, and the average hgdi value for spoligotyping was 0.9041, the highest hgdi average value was 0.9917 and the lowest was 0.8076 . However, because the results of some papers were incomplete, or they were identified only up to the super family level, the value of hgdi was lower in these articles (marked with * in table 1). Therefore, considering only the articles with full data on the calculations, the average hgdi for this technique will be 0.9433 . As observed, the discriminatory power of spoligotyping was less than the two other techniques . The reason for the lower discriminatory power of this method is that it targets only a single genetic locus, included less than 0.1% of the m. tuberculosis complex genome (26). An important advantage of spoligotyping is its sensitivity which can be performed by 10 fg of chromosomal dna, equivalent to dna from 23 bacterial cells (27), so that the method can be directly performed on clinical samples, without the need for prior culture . Moreover, spoligotyping has proven to be practical for typing on nonviable samples such as paraffin - embedded tissue sections or ziehl - neelsen stained slides (28, 29). Other techniques that we encountered to be used in the articles were rapd - pcr and pfge . The lowest discrimination power was represented by pfge (average hgdi= 0.6974). In this review, the discrimination power of pfge was low because in one manuscript, poyide et al . Used only one restriction enzyme (xba1) and obtained extremely low discrimination power (hgdi value = 0.4159) (30). Although in another study conducted by khosravi et al ., they used two restriction enzymes (dra1 and xba1) and the discrimination power was higher (hgdi value = 0.9790) (31). In general, each typing system has its own advantages and disadvantages, and it is difficult to decide clearly which of them is superior to others . But an ideal molecular typing method must comply with the needs of researchers in terms of performance feasibility as well as analytical standards (26). The performance parameters included technical simplicity or ease of implementation, repeatability, robustness, time and cost - effectiveness . Another special advantage of the method was that it can be standardized and the results would be easily interpretable . The results should simply be comparable with the results of other laboratories as well as global databases . Another attractive advantage of a technique is its capability to be performed directly on clinical samples (26). A general rule is that the higher discriminatory power of the method, the more reliable the results obtained . It has been confirmed that discriminatory power of a molecular marker relates directly to its stability . Half - life of is6110-rflp profile is much shorter than the profile of spoligotyping (about 3 to 8 years in rflp compared to spoligotyping with more than 50 years). The half - life of miru - vntr is slightly longer than rflp profiles and shorter than spoligotyping . The half - life of a desirable molecular marker should be short enough to separate unrelated samples from each other and on the other hand be long enough to be able to find the relationship between epidemiological samples (26). Selecting a genotyping method, in compliance with good discriminatory power, a technique with high discriminatory power with short half - life of genetic pattern (e.g. Rflp and miru - vntr techniques) is more useful to distinguish reactivation from reinfection, while a method with a long half - life of genetic pattern (such as spoligotyping) is more useful for global strain tracking and evolutionary studies . Gold standard for m. tuberculosis genotyping was is6110-rflp, but it needs to change to miru - vntr technique because of its similar discriminatory power in addition to feasibility, time and cost - effectiveness as well as the interpretation of results . More importantly, the pcr - based typing methods require fewer bacteria and can be performed in a shorter period of time . Thus, many researchers have focused on the miru - vntr method as a standard technique . Spoligotyping method has attracted the attention of researchers because of simplicity and cost - effectiveness in addition to the advantage that the results are expressed as positive or negative, according to the presence or absence of the spacers (digital format) (32). Therefore, spoligotyping is recommended to be used as a first - line screening test, followed by techniques with higher discriminatory power such as miru - vntr or is6110-rflp (33). Also, it should be emphasized that the present study was limited to techniques reported in iran . There are other genotyping techniques used by researchers around the world which were not included in this article . According to the present study, combination of miru - vntr with spoligotyping can be recommended for large - scale genotyping in iran . It seems appropriate to consider spoligotyping as the first techniques for screening followed by other techniques with higher discrimination abilities such as miru - vntr or is6110-rflp.
Atrial septal defect (asd) is one of the most common congenital heart diseases in adults . Adult patients with unrepaired asd are at risk of developing major cardiovascular events, especially atrial fibrillation (af). Transcatheter closure has evolved as a standard therapeutic option for the treatment of asd in adult patients . Correction of asd cannot only improve the symptoms and increase exercise tolerance but improve cardiac chamber geometry and hemodynamics . There is an increased incidence of af in adult patients with asd, especially in those over 40 years . Af - related remodeling is believed to underlie the progressive nature of the arrhythmia and contribute to the complexity of long - term management . Up to now, little is known about whether transcatheter closure could reverse cardiac remodeling in asd patients with permanent af . In this retrospective clinical study, we analyzed asd patients admitted in our center with and without af who underwent successful transcatheter closure in an attempt to determine the feasibility of device closure of asd and observe resultant hemodynamic changes in such patients . The clinical medical records of 289 consecutive adult patients older than 40 years who underwent transcatheter asd closure at our center between october 2009 and october 2013 were analyzed retrospectively . The exclusion criteria were, patients with transthoracic echocardiographic maximal asd diameter> 40 mm and rims <5 mm . Of the 289 asd patients, 63 (21.8%) patients with permanent af were selected as the case group and the other 226 (78.2%) patients without permanent af as the control group . When the occluder was deployed across the defect, its position and stability were assessed by fluoroscopy and transthoracic echocardiography . Echocardiography was performed using a sonos ie33 machine (philips medical systems, usa) according to a standard protocol including color doppler data . Measurement was performed by two trained cardiologists independently, and any disagreement was solved by discussion . The right and left ventricular volumes were measured in an apical four - chamber view at end - diastole . The right atrial (ra) volume and left atrial (la) dimension were measured in the same view at end - systole . The left ventricular end - systolic dimension (lvesd), left ventricular end - diastolic dimension (lvedd) and left ventricular ejection fraction were also measured . An appropriate long - term warfarin dose was used in the case group with the international normalized ratio maintained between 2.0 and 2.5 . Aspirin (35 mg / kg per day) was administered for 6 months in the control group . Electrocardiographic monitoring, chest x - ray, and transthoracic echocardiography were performed at 1 and 6 months after the procedure . The cardiac geometric location of the occluder, thrombi, valvular regurgitation and residual shunt were also evaluated by transthoracic echocardiography . Continuous data are presented as median and range if not normally distributed and as mean standard deviation if normally distributed . Nominal data between groups were compared using chi - square or fisher's exact test . The variables of pre and postprocedure were compared using paired, two - sided student's t - test . Continuous data between groups were compared using unpaired, two - sided student's t - test . Data were analyzed using statistical package for social sciences, version 20.0, for mac (spss inc ., chicago, illinois, usa). The clinical medical records of 289 consecutive adult patients older than 40 years who underwent transcatheter asd closure at our center between october 2009 and october 2013 were analyzed retrospectively . The exclusion criteria were, patients with transthoracic echocardiographic maximal asd diameter> 40 mm and rims <5 mm . Of the 289 asd patients, 63 (21.8%) patients with permanent af were selected as the case group and the other 226 (78.2%) patients without permanent af as the control group . The shsma (shanghai shape memory alloy co., ltd ., shanghai, china) asd occluder was used in all patients . When the occluder was deployed across the defect, its position and stability were assessed by fluoroscopy and transthoracic echocardiography . Echocardiography was performed using a sonos ie33 machine (philips medical systems, usa) according to a standard protocol including color doppler data . Measurement was performed by two trained cardiologists independently, and any disagreement was solved by discussion . The right and left ventricular volumes were measured in an apical four - chamber view at end - diastole . The right atrial (ra) volume and left atrial (la) dimension were measured in the same view at end - systole . The left ventricular end - systolic dimension (lvesd), left ventricular end - diastolic dimension (lvedd) and left ventricular ejection fraction were also measured . An appropriate long - term warfarin dose was used in the case group with the international normalized ratio maintained between 2.0 and 2.5 . Aspirin (35 mg / kg per day) was administered for 6 months in the control group . Electrocardiographic monitoring, chest x - ray, and transthoracic echocardiography were performed at 1 and 6 months after the procedure . The cardiac geometric location of the occluder, thrombi, valvular regurgitation and residual shunt were also evaluated by transthoracic echocardiography . Continuous data are presented as median and range if not normally distributed and as mean standard deviation if normally distributed . Nominal data between groups were compared using chi - square or fisher's exact test . The variables of pre and postprocedure were compared using paired, two - sided student's t - test . Continuous data between groups were compared using unpaired, two - sided student's t - test . Data were analyzed using statistical package for social sciences, version 20.0, for mac (spss inc ., chicago, illinois, usa). In the case group, there were 27 (42.9%) males and 36 (57.1%) females with a mean age of 68.3 15.4 years . In the control group, there were 99 (43.8%) males and 127 (56.2%) females with a mean age of 47.4 13.9 years . Patients in the case group were significantly older than those in the control group (p <0.001). Baseline clinical characteristics asd: atrial septal defect; nyha: new york heart association . The cardiac geometry was significantly improved immediately after the procedure and during the follow - up period in both groups . In the case group, the right ventricular (rv) end - diastolic volume decreased from 81.57 31.47 ml preprocedure to 62.92 19.07 ml postprocedure during a median interval of 6 months after closure (p <0.001), and the ra volume decreased from 106.93 54.17 ml preprocedure to 79.07 40.39 ml postprocedure (p <0.001). The la dimension, lvesd, lvedd and left ventricular ejection fraction remained unchanged significantly [table 2]. Echocardiographic variables before and 6 months after asd closure lvesd: left ventricular end - systolic dimensions; lvedd: left ventricular end - diastolic dimensions; lvef: left ventricular ejection fraction; la: left atrial; rv: right ventricular; ra: right atrial; asd: atrial septal defect . Both rv end - diastolic volume and ra volume decreased significantly in the control group during the follow - up period from 76.72 27.56 to 59.39 10.21 ml (p the la dimension, lvesd, lvedd and left ventricular ejection fraction remained unchanged significantly [table 2]. The rv and ra volumes were decreased significantly in both the groups during the 6 months follow - up period after closure . Changes of the rv volume and ra volume in the case group were significantly smaller as compared with those of the control group (20.74 10.68 ml to 24.69 9.46 ml, p = 0.005; 27.53 12.68 ml to 36.63 13.79 ml, p <0.001) [figure 1]. Of the 63 patients with permanent af in the case group, 25 (39.7%), 29 (46.0%), and 9 (14.3%) patients were considered to have new york heart association (nyha) class i, ii and iii, respectively . Of the 226 patients in the control group, 95 (42.0%), 129 (57.1%), and 2 (0.9%) were graded as having nyha class i, ii and iii, respectively . Nyha functional classification was improved in both the groups after the procedure [figure 2]. Changes of the right ventricular (rv) volume and right atrial (ra) volume . The 6 months follow - up period showed that changes of the rv volume and ra volume in the case group were significantly less than those in the control group (* p <0.05; rv: right ventricular; ra: right atrial). Heart function improvement in both the groups . In the case group, 25 (39.7%), 29 (46.0%) and 9 (14.3%) patients were considered to have nyha class i, ii and iii . In the control group, 95 (42.0%), 129 (57.1%) and 2 (0.9%) were graded as having nyha class i, ii and iii . Nyha cardiac function was improved in both the groups after the procedure (nyha: new york heart association). In the case group, there were 27 (42.9%) males and 36 (57.1%) females with a mean age of 68.3 15.4 years . In the control group, there were 99 (43.8%) males and 127 (56.2%) females with a mean age of 47.4 13.9 years . Patients in the case group were significantly older than those in the control group (p <0.001). Baseline clinical characteristics asd: atrial septal defect; nyha: new york heart association . The cardiac geometry was significantly improved immediately after the procedure and during the follow - up period in both groups . In the case group, the right ventricular (rv) end - diastolic volume decreased from 81.57 31.47 ml preprocedure to 62.92 19.07 ml postprocedure during a median interval of 6 months after closure (p <0.001), and the ra volume decreased from 106.93 54.17 ml preprocedure to 79.07 40.39 ml postprocedure (p <0.001). The la dimension, lvesd, lvedd and left ventricular ejection fraction remained unchanged significantly [table 2]. Echocardiographic variables before and 6 months after asd closure lvesd: left ventricular end - systolic dimensions; lvedd: left ventricular end - diastolic dimensions; lvef: left ventricular ejection fraction; la: left atrial; rv: right ventricular; ra: right atrial; asd: atrial septal defect . Both rv end - diastolic volume and ra volume decreased significantly in the control group during the follow - up period from 76.72 27.56 to 59.39 10.21 ml (p <0.001) and from 84.27 32.68 to 43.21 20.48 ml, the la dimension, lvesd, lvedd and left ventricular ejection fraction remained unchanged significantly [table 2]. The rv and ra volumes were decreased significantly in both the groups during the 6 months follow - up period after closure . Changes of the rv volume and ra volume in the case group were significantly smaller as compared with those of the control group (20.74 10.68 ml to 24.69 9.46 ml, p = 0.005; 27.53 12.68 ml to 36.63 13.79 ml, p <0.001) [figure 1]. Of the 63 patients with permanent af in the case group, 25 (39.7%), 29 (46.0%), and 9 (14.3%) patients were considered to have new york heart association (nyha) class i, ii and iii, respectively . Of the 226 patients in the control group, 95 (42.0%), 129 (57.1%), and 2 (0.9%) were graded as having nyha class i, ii and iii, respectively . Nyha functional classification was improved in both the groups after the procedure [figure 2]. Changes of the right ventricular (rv) volume and right atrial (ra) volume . The 6 months follow - up period showed that changes of the rv volume and ra volume in the case group were significantly less than those in the control group (* p <0.05; rv: right ventricular; ra: right atrial). Heart function improvement in both the groups . In the case group, 25 (39.7%), 29 (46.0%) and 9 (14.3%) patients were considered to have nyha class i, ii and iii . In the control group, 95 (42.0%), 129 (57.1%) and 2 (0.9%) were graded as having nyha class i, ii and iii . Nyha cardiac function was improved in both the groups after the procedure (nyha: new york heart association). Our study demonstrated that the transcatheter closure of asd in patients with permanent af is safe and feasible as in the case with patients without af . Transcatheter asd closure can improve the patient's quality of life and cardiac remodeling even in patients with permanent af . In addition, it can improve the symptoms and reverse remodeling of the right - heart in adults of all ages . Studied 650 adult asd patients and concluded that asd in adults is safe and effective, offering excellent long - term outcomes . Patel et al . Also showed that asd closure could decrease the rv size and improve the clinical symptoms significantly with an extremely high success rate . In our study, the nyha cardiac function was improved in both groups after transcatheter closure . Although most adult asd patients with permanent af in our series were older and often had significant comorbidities, our study still brought encouraging outcomes in the cardiac function and structural improvement . Above all, transcatheter asd closure should be recommended as the treatment of choice in patients with and without permanent af . The present study showed that the magnitude of the decrease in the ra and rv size in patients with permanent af was smaller than that in patients without af . This may be partly due to the older age of the patients in the case group . Patients with significant shunting are more likely to develop rv failure and atrial tachycardia, which can lead to significant morbidity and potential mortality . Af is more frequently seen in older patients, and not a few patients may continue to experience fibrillation after asd closure . Rosas et al . Analyzed 200 un - operated patients over 40 years and found that age was a predictor of poor outcomes . Thus, asd closure should be considered as early as possible after confirmation of the diagnosis . However, many adult asd patients in china remain unrepaired even though the transcatheter procedure has been well practiced for nearly two decades . One reason is the unbalanced development of the medical conditions; for instance echocardiography remains unavailable and unaccessible in many central and western regions of china, especially in remote mountainous areas, and therefore asd cannot be detected at an early age in many patients . Although asd was detected in some patients during their youth hood, treatment was often delayed because of the economic reason . Taniguchi et al . Analyzed nine old asd patients with permanent af and found that the nyha cardiac function was improved in all patients . Rv remodeling was also improved significantly in terms of both right and left diameter ratios and plasma b - type natriuretic peptide level . Our study confirmed that both the structure and function of the right - heart were improved significantly in all asd patients with af after occlusion . As a result, it can be concluded that the closure of the defect is still the right thing to do in patients with asd and af . Additionally, as patients with af are at high risk of developing ischemic events, anticoagulation therapy should be considered instead of antiplatelet medication . No device thrombosis or major bleeding complications were observed during the 6 months follow - up period in both the groups, indicating that anticoagulation therapy with warfarin is safety and effective . In addition, given potential thrombi in the la appendage, transesophageal echocardiography should be performed before the procedure . In the present study, transesophageal echocardiography was not taken in all patients with af . However, no ischemic complication occurred during the follow - up period, partly because of the relatively small sample size in the case group . The present study has several limitations, including those inherent to the retrospective design and the relatively short follow - up period . Although thrombus formation was not detected during the follow - up period, long - term follow - up evaluation is mandatory . Evaluation by transesophageal echocardiography may be helpful to examine thrombus formation more closely . In conclusion, our study has demonstrated that the procedure can improve cardiac remodeling and heart function in patients both with and without af, even in patients with permanent af.
The brain is a highly complex organ that consists of various types of neurons and glia . How the complex brain architecture develops from neural stem cells (nscs) is one of the central questions of neuroscience . Much interest has been paid to the mechanisms of nsc maintenance, proliferation, migration, and differentiation into various cell types . A second focus of interest in neurogenesis is the fact that neurogenesis continues throughout life in particular brain regions . The long - held belief that neurons are no longer regenerated once development of the central nervous system (cns) ceases was challenged about 50 years ago . Progress in the several decades since has made adult neurogenesis in the hippocampus and olfactory system widely accepted . The mechanisms underlying adult neurogenesis and its functional significance have been pursued as an important focus of neurogenesis research . Further, the finding that newly generated neurons and glia from nscs can be used in the treatment of degenerative disease and trauma of the cns has attracted the wide interest of neuroscientists and clinicians in stem cell biology . These therapeutic approaches are strongly facilitated by recent advances in es cells and ips cells . In addition, further interest has been shown in the correlation between impaired neuro / gliogenesis and psychiatric diseases, including mood disorders and chronic stress syndromes, along with recent progress in basic research into these diseased states . A closer understanding of the nature of nscs and neuro / gliogenesis is therefore crucial to various aspects of neuroscience, including the development, remodeling, and restoration of brain architecture and function . Stem cell and neuro / gliogenesis biology is also strongly linked to the plastic potential of the cns . Nscs show remarkable plasticity in differentiating into a variety of cell types that play a critical role in the formation of the complex brain architecture . After the brain develops to maturity, its plastic function is generally exhibited through the plasticity of preexisting neurons generated during the developmental and neonatal periods . It is notable, however, that nscs in the mature brain likely confer far stronger plastic potential . In addition, restoring appropriate neuro / gliogenesis in the diseased brain can be regarded as the process of adjusting the brain s plasticity for structural and functional restoration . Thus, stem cell and neuro / gliogenesis biology highlights the remarkable plastic potential of the brain . This research field provides an opportunity to integrate structural plasticity and functional plasticity at the cellular, circuit and behavioral levels and has attracted wide interest from basic scientists, clinicians, and society . In 2015, the japanese association of anatomists and the physiological society of japan held a joint annual meeting in kobe, japan . One symposium, neurogenesis from embryo to adult, attracted a large audience and generated vigorous discussion . In this review article, we summarize the five presentations we made in this symposium, with the goal of providing a comprehensive understanding of the current progress and future directions of this research field . First, itaru imayoshi introduced the role of transcription factors in the self - renewal and differentiation of nscs in the developing neuroepithelium . Dr . Imayoshi revealed the pivotal role of the expression dynamics of basic helix - loop - helix (bhlh) transcription factors, oscillatory or sustained, on the nsc self - renewal and fate decision . Second, tatsunori seki raised the question of how nscs produce granule cells in the embryonic and early postnatal hippocampus and how the early neurogenesis is succeeded by adult neurogenesis . Seki revealed that the first progenitors of granule cells arising in the dentate anlage in the developing hippocampus express glial fibrilar acidic protein (gfap) and that these gfap - expressing progenitors form a proliferative zone in the subpial and hilar zones during the perinatal stage and later in the subgranular zone, where neurogenesis continues throughout life . Third, nobuaki tamamaki introduced the emerging view that adult neurogenesis may also occur in the neocortex . Tamamaki revealed that neuronal progenitors can be induced in the leptomeninges of the neocortex by electrical stimulation and showed their migration and differentiation in the neocortex . Fourth, masahiro yamaguchi introduced how adult - born olfactory neurons are selected for survival or death in the olfactory bulb . Dr . Yamaguchi revealed that the key mechanism of activity - dependent selection is the integration of sensory inputs from the periphery and top - down inputs from higher brain regions . Lastly, yoshitaka hayashi, yoshitaka tatebayashi, and seiji hitoshi introduced the relation between major depressive disorder and oligodendrocyte genesis in the human prefrontal cortex . Hayashi et al . Established a method to quantify oligodendrocytes and their progenitors and revealed the dysfunction of oligodendrocyte development in the diseased prefrontal cortex . Nscs are multipotent and self - renewable cells that give rise to neurons, astrocytes, and oligodendrocytes in the brain . Nscs of the lateral ventricular wall in the forebrain undergo changes in morphology and produce different progeny as brain development proceeds . Nscs begin as neuroepithelial cells, become radial glial cells, and then finally develop many astrocytic characteristics in the adult brain . During neural development, nscs (neuroepithelial cells / radial glial cells) initially undergo symmetric cell division: each nsc divides into two nscs . By repeating symmetric cell division, these cells then undergo asymmetric cell division: each nsc divides into two distinct cell types, one nsc and one immature neuron / neuronal precursor or a basal progenitor cell . Immature neurons or neuronal precursor cells migrate outside the ventricular zone (vz) into the outer layers, where they become mature neurons, whereas basal progenitor cells migrate into the subventricular zone (svz), proliferate further, and give rise to more neurons . By repeating asymmetric cell division, after the production of neurons, nscs finally differentiate into glial cells, but some of them are maintained as nscs in the postnatal and adult brain . Bhlh transcription factors play pivotal roles in the self - renewal of nscs and fate determination of neurons, astrocytes, and oligodendrocytes [813]. Repressor bhlh factors such as hes1 regulate the self - renewal of nscs as downstream effectors of notch signaling, whereas proneural bhlh factors such as ascl1 (also called mash1) promote neuronal differentiation . However, in addition to hes1, some bhlh fate determination factors, such as ascl1 and olig2, have roles in nsc maintenance or proliferation . It is not completely understood how these various and sometimes opposing functions of each bhlh factor in the self - renewal and fate - choice events of nscs are achieved . Showed that transcription of some bhlh genes is differentially regulated in nscs and differentiating cells, prompting imayoshi et al . To analyze the expression dynamics of bhlh factors in further detail . . Indicated that bhlh factors are expressed by nscs in an oscillatory manner and that one of them becomes dominant during the fate choice event (fig . They then proposed that the multipotent state of nscs correlates with the oscillatory expression of several bhlh factors, whereas the differentiated state correlates with the sustained expression of a single bhlh factor [8, 9, 15].fig . 1expression dynamics of bhlh factors in multipotency and cell fate choice . In multipotent nscs, levels of hes1 and ascl1 oscillate with periods of 23 h, while that of olig2 oscillates with a period of 58 h. by contrast, during cell fate choice, one of the bhlh factors is expressed in a sustained manner, while the others are repressed expression dynamics of bhlh factors in multipotency and cell fate choice . In multipotent nscs, levels of hes1 and ascl1 oscillate with periods of 23 h, while that of olig2 oscillates with a period of 58 h. by contrast, during cell fate choice, one of the bhlh factors is expressed in a sustained manner, while the others are repressed to examine the functional significance of these bhlh factor expression dynamics, a novel optogenetic method (photo - activatable gal4/uas system) was developed to artificially manipulate the expression patterns of bhlh factors with blue light illumination . This analysis demonstrated that sustained expression of ascl1 induces neuronal differentiation, whereas oscillatory expression of ascl1 activates cell proliferation of nscs . Thus highlighted the importance of the expression dynamics of bhlh factors in the self - renewal, multipotency, and fate determination of nscs [8, 9, 15] (fig . During the embryonic period, neurons of both the neocortex and hippocampus are initially generated from the vz of the pallium: the neocortex develops from the dorsal and lateral pallium and the hippocampus from the medial pallium . In the neocortex, neurogenesis occurs only during the embryonic and early postnatal stages and ceases by the early postnatal stage, as typically occurs in most brain regions . However, neurons are exceptionally produced in the adult hippocampus, where dentate granule cells arise from gfap - expressing astrocyte - like adult neural progenitor cells, which characteristically differ from embryonic neural progenitor cells [2, 1618]. In an attempt to understand how neurogenesis persists in the hippocampus, seki et al . Explored the neurogenic process of dentate granule cells from the embryonic to late postnatal stages with special reference to gfap - expressing progenitors, namely when these progenitors appear during the embryonic period and how they form the granule cell layer . Analysis using gfap - gfp transgenic mice showed that a distinct gfap - gfp - expressing cell population first appears in the vz of the medial pallium around a ventricular indentation or in the dentate notch at the dorsal edge of the fimbria at e13.514.5 . These are radially oriented cells with apical and/or basal processes that seem to migrate from the vz to the marginal zone, a region below the pia surface . No gfap - gfp expression is found in the dorsal or lateral pallium, which develops into the neocortex . During the perinatal period, gfap - gfp cells increase in number and form a migratory stream in the suprafimbrial region, as well as in the subpial region between the fimbria and dentate gyrus, alternatively called the fimbrio - dentate junction or fimbrio - dentate juncture . Three proliferative zones are formed by gfap - gfp - expressing cells in these regions: the primary dentate matrix in the vz, secondary dentate matrix in the suprafimbrial area and regions below the pia membrane and hippocampal fissure, and the tertiary dentate matrix in the core region of the dentate gyrus, the putative hilus . Gfap - gfp cells in the migratory stream and the developing dentate gyrus are positive for sox2 and ki67, suggesting that they contain proliferative progenitors . They also express neurogenin2 in the vz, tbr2 and neurod in the migratory stream and developing dentate gyrus, and prox1 in the developing dentate gyrus . It is therefore concluded that distinctive gfap - expressing progenitors arising around the dentate notch form germinal regions in the migratory stream and developing dentate gyrus and gradually differentiate into granule neurons . This also indicates that in the dentate gyrus, astrocyte - like neural progenitors continue to generate granule neurons from the beginning of development and throughout life . The site of origin, proliferative activity, and migratory pattern of the gfap - gfp - expressing cells agree well with those suggested by studies with h - thymidine autoradiography, and nestin - gfp . Although previous reports showed the existence of gfap - expressing cells and fibers in the embryonic hippocampus [21, 23, 24], these are thought to belong to the radial type of astrocytes that function as scaffolds for migrating neuroblasts . However, the existence of abundant gfap - gfp - expressing cells with neuronal marker proteins clearly demonstrates that principally gfap - expressing cells and fibers are not derived from radial astrocytes, but rather gfap - expressing migrating spindle - shaped neuronal progenitors and precursors . Interestingly, the dentate gyrus shares some similarities with the cerebellum in terms of their morphogenesis . For example, the hippocampus and cerebellum are derived from neuroepithelium around the edge (cortical hem and rhombic lip) of the dorsal portions of the telencephalon and metencephalon, respectively, both of which are attached to the choroid plexus (fig . 2). Both progenitors are generated in the ventricular zone (primary proliferative zone) and migrate to the subpial region, in which secondary proliferative zones (secondary dentate matrix and external granular layer) are formed . The progenitors in the secondary proliferative zone give rise to small interneurons or granule cells . Production of both neurons in the subpial regions continues into the early postnatal period to form the cerebellar and dentate granule cell layers . These facts suggest that there may be a similar mechanism in early postnatal neurogenesis in the subpial regions of the dentate and cerebellum . Furthermore, the dentate gyrus, unlike the cerebellum, establishes a tertiary proliferative zone (tertiary dentate matrix) in the hilus . During the late postnatal period, the progenitors in this tertiary dentate proliferative zone are gradually confined to the subgranular zone, where neurogenesis persists in adults.fig . 2 a progenitors of granule cells in the developing hippocampus . Gfap - gfp - expressing progenitors (green) arise from the dentate notch (arrowhead) and migrate to the dentate gyrus . Chp choroid plexus, cp cortical plate, egl external granular layer, gcl granule cell layer, sdm secondary dentate matrix, sgz subgranular zone, svz subventricular zone, tdm tertiary dentate matrix, vz ventricular zone (modified from) a progenitors of granule cells in the developing hippocampus . Gfap - gfp - expressing progenitors (green) arise from the dentate notch (arrowhead) and migrate to the dentate gyrus . Chp choroid plexus, cp cortical plate, egl external granular layer, gcl granule cell layer, sdm secondary dentate matrix, sgz subgranular zone, svz subventricular zone, tdm tertiary dentate matrix, vz ventricular zone (modified from) although adult neurogenesis is a common phenomenon in the non - mammalian neocortex, the scale of adult neurogenesis in the neocortex appears to decrease significantly as the phylogeny approaches the human being . In apparent contrast, the limbic area of the telencephalon appears to be a site of continuous generation of neurons . In particular, the medial edge of the mammalian telencephalon, the dentate gyrus, produces granule cells constantly throughout life in humans, while the svz surrounding the lateral ventricle continuously produces gabaergic granule cells for the olfactory bulb . Granule cells produced in the dentate gyrus are integrated into the circuitry of the hippocampus and save new information, whereas granule cells produced in the svz of the lateral ventricle migrate rostrally and are integrated into the olfactory circuitry . In other words, these new neurons are not integrated into the neocortex . However, if a significant number of new neurons is produced in the neocortex and integrated into the neocortical circuit, we cannot deny the possibility that these new neurons ruin the circuits for memory and thought in the human neocortex . Macklis reported that the synchronous apoptotic degeneration of corticothalamic neurons may induce generation of the same number of neurons in the neocortex and may recreate the same circuit as was originally present . In this case, neurogenesis in the neocortex would not disturb the circuit in the neocortex and would be welcomed . Our goal here is to stimulate the interest of many researchers to join the hunt for neuron progenitors in the mouse neocortex . As the first hunter, costa et al . Sought the source of neuron progenitors using a neurosphere assay method and found a novel source of neuron progenitors outside the two major germinative zones (vz and svz), namely the marginal zone of the neocortex . These neuronal progenitors in the marginal zone of the developing cerebral cortex were notably distinct from those in the vz and svz . As the second hunter of the neuron progenitors in the adult neocortex, ohira and colleagues sought nscs and neuronal progenitor cells (npcs) in the adult rat neocortex . The vast majority of neocortical neurons were eliminated and the remnant contained more nscs and npcs . They then observed npcs in neocortical layer 1 of adult rats . On injection of a simple retrovirus to visualize these npcs in layer i, they found that no npcs or related cells were neurons, but rather had a glia - like appearance . Moreover, none of the npcs had axons . As the third hunter of the neuron progenitors in the adult neocortex, nakagomi and colleagues found that neural stem / progenitor cells (nspcs) reside in many regions of the cns, including the svz of the lateral ventricle, subgranular zone of the hippocampal dentate gyrus, cortex, striatum, and spinal cord . In addition, they demonstrated that the leptomeninges (pia mater and arachnoid membrane) also exhibit nspc activity in response to ischemia and reported that the nspcs express nspc markers, such as nestin, and form neurosphere - like cell clusters with self - renewal activity . When the mouse brain was stimulated by electrodes and nestin - immunoreactivity (-ir) in the leptomeninges was examined, neurogenic progenitors were clearly revealed (fig . 3). If the electrical stimulation of the brain was continued, nestin - ir was replaced with gad67-ir or neurod6-ir in granule cells . When the neocortex was continually stimulated for up to 1 or 2 weeks, neuron progenitors in the leptomeninges migrated into the neocortex and projected dendrites into the cortical plate and axons into the white matter . In this case, we consider that the newly generated neocortical neurons would not ruin the circuit in the neocortex.fig . Nestin immunoreactive sites are in green piaprogenitors on the outer surface of the pia mater induced by kindling stress . Adult - born olfactory neurons are generated in the svz and migrate rostrally to the olfactory bulb (ob), the first relay in olfactory information processing . They then differentiate into granule cells (gcs) or periglomerular cells, which act as interneurons in the ob . In rodents, roughly one percent of total ob gcs are newly generated each day, and more than 60% of gcs in the adult ob are estimated to be adult - born gcs [3, 34]. Under normal conditions only half of the new gcs succeed in living longer than 1 month after generation, while the other half are eliminated by apoptosis . Initial excess neurogenesis and subsequent elimination commonly occur in both embryonic and adult neurogenesis . Because proper neuronal selection between life and death is crucial to refining the neuronal circuitry throughout the life of the animals, it is important to understand the mechanism of how adult - born neurons are appropriately selected . First, the selection of adult - born new gcs depends on olfactory sensory experience . Exposure to novel odors increases the survival rate of new gcs, while olfactory sensory deprivation remarkably increases the apoptosis of new gcs [3, 35]. Second, the selection of new gcs depends on brain state . In many cases brain plasticity is influenced by the brain state, most notably by the wake - rest / sleep cycle . In the hippocampus and neocortex, consolidation of spatial memory is considered to occur during the rest / sleep period that follows the spatial learning . On the supposition that the selection of new gcs may occur during the time course of olfactory sensory experience and rest / sleep, yokoyama et al . Examined whether the fate decision of new gcs is associated with feeding behavior . Under food restriction, mice initially showed extensive eating behavior following food availability and then showed grooming, resting, and sleeping, which are typical postprandial (after - meal) behaviors . Interestingly, apoptosis of gcs increased approximately two - fold during the postprandial behaviors, whereas perturbation of these postprandial behaviors remarkably suppressed this increase in gc apoptotic elimination . Further, electroencephalogram analysis showed that postprandial slow - wave sleep (sws) was well correlated with the gc elimination . So, what mechanisms promote gc elimination during postprandial behaviors, particularly during postprandial sws? Neurons in the olfactory cortex (oc) repeatedly generated synchronized spike discharges during sws . Importantly, this synchronized firing of oc neurons generated synchronized top - down synaptic inputs to the ob, which were considered as the candidate signal that promoted gc elimination . In fact, electrical stimulation of the top - down inputs from the oc to the ob promoted gc elimination, and pharmacological blockade of the top - down inputs during the postprandial period suppressed gc elimination . These observations demonstrate that top - down inputs during the postprandial period crucially promote gc elimination . These findings raised the further question of whether enhanced gc elimination occurs only during the postprandial behaviors or during other behavioral states also . Komano - inoue et al . Examined whether fear responses to noxious stimuli induce gc elimination . Mice delivered an electrical foot shock showed startle and fear responses during and immediately after shock delivery . Interestingly, gc elimination was very rapidly enhanced in mice that showed these behavioral responses extensively . Moreover, this enhanced gc elimination after shock delivery was inhibited by the suppression of neuronal activity in the oc . These results show that gc elimination can be promoted during various behaviors and that top - down inputs from the oc to the ob represent the common signal for inducing gc elimination . One intriguing possibility is that behaviors associated with important life events, such as food eating and experience of noxious stimulation, might promote reorganization of the ob neuronal circuit, including the enhanced gc elimination . The oc receives synaptic inputs not only from the ob but also from other nonolfactory brain regions, such as the amygdala and prefrontal cortex . Important life events might recruit wide brain areas for activation and thereby potentiate the activity of the oc and top - down inputs from the oc to the ob to reorganize the ob neuronal circuitry . In conclusion, the life and death of new gcs in the ob is determined by the interplay between bottom and up olfactory sensory inputs from the periphery and top - down inputs from higher cortical regions (fig . Gcs are presumed to receive these inputs at different synapses in different layers of the ob [3, 41]. It is important to understand the manner of integration of these synaptic inputs onto a given gc . It is also important to reveal what kind of information is represented by the top - down inputs . Considering that the top - down inputs reflect the activity of higher cortical regions, they might represent the subjective meaning of the odor stimuli for the animal, including the valence of the odor and odor - evoked emotional and motivational responses . The fate of new gcs thus needs to be understood with regard to the activity of large neuronal networks.fig . 4activity - dependent selection between survival and death for new gcs in the ob . A model of how synaptic inputs integrate and determine the fate of new gcs . During olfactory experience, some newly generated gcs (green) receive odor input (red arrows for right gc) while others do not (left gc). Top - down input from the oc to the ob (blue arrow), which typically occurs during the sleep state, eliminates new gcs that did not receive an odor input (left gc) and leave new gcs that did receive an odor input to survive (right gc). Thus, top - down input from the oc might reflect the activity of various brain regions . This figure was modified from activity - dependent selection between survival and death for new gcs in the ob . A model of how synaptic inputs integrate and determine the fate of new gcs . During olfactory experience, some newly generated gcs (green) receive odor input (red arrows for right gc) while others do not (left gc). Top - down input from the oc to the ob (blue arrow), which typically occurs during the sleep state, eliminates new gcs that did not receive an odor input (left gc) and leave new gcs that did receive an odor input to survive (right gc). Thus, top - down input from the oc might reflect the activity of various brain regions . Major depressive disorder (mdd) is a debilitating mental illness that causes a persistent feeling of sadness, loss of interest, deep despair, and death by suicide . Brain imaging studies of patients with mdd have demonstrated abnormalities in gray matter volume, metabolism, and blood flow in the prefrontal cortex and hippocampus [4244]. Neuropathological studies in these brain regions in mdd have long been of interest in psychiatry . A small number of histological assessments of the prefrontal cortex from patients with mdd revealed abnormalities in the size of neurons and the density of oligodendrocytes [4547]. However, the stereological techniques often used for the quantification of numbers of cells in brain sections pose a serious methodological challenge to neuropathologists . Using flow cytometry, hayashi et al . Established a novel cell - counting method to quantify the number of nuclei of total cells, neurons, and oligodendrocytes in brain regions of interest from postmortem brains (fig . The nuclei were obtained by mechanical homogenization of unfixed frozen brain, because the homogenization process breaks the cell membrane and cytoplasm but leaves the nucleus intact . The nuclei in suspension were stained with 7-aad, neun, and olig2 as markers of dna (total nuclei), neurons, and oligodendrocytes, respectively, making it possible to quantify the number, size, and fluorescence intensity of the cells with a flow cytometer . Furthermore, the fluorescence intensity of olig2 immunostaining is stronger in oligodendrocyte precursor cells (opcs) than in mature oligodendrocytes . The validity and accuracy of this novel cell - counting method have been verified by estimating the number of total and neuronal nuclei from the whole cerebral cortex of rats . This method has been applied to the gray matter of the frontopolar (brodmann area 10) and inferior temporal (brodmann area 20) cortices of postmortem brains from mdd patients and healthy controls . In the present report, hayashi et al . Found a reduction in olig2 oligodendrocyte - lineage cells in the gray matter of the frontopolar cortex from mdd patients compared to that from healthy controls (fig . By measuring the fluorescence intensity, they were able to separate the olig2 cell population into olig2 mature oligodendrocytes and olig2 opcs (fig . Detailed olig2 population analysis showed a significant reduction in the densities of both olig2 and olig2 nuclei in the frontopolar cortex of mdd patients compared to healthy controls (fig . These results suggest that the frontopolar cortex, which is considered to be closely associated with the pathogenesis of mdd, processes the oligodendrocyte abnormality and that patients with mdd have a dysfunction in oligodendrocyte development (such as in proliferation and/or maturation) in childhood and/or adolescence.fig . 5quantification of frontopolar cortex from mdd and healthy control . A negative controls of flow cytometry staining with igg . B a novel cell counting method can separate nuclei from postmortem brain into neurons (neun) and oligodendrocytes (olig2), astrocytes, and microglia (neun / olig2). C the olig2 nuclei were further divided into two populations of strongly and weakly olig2-positive nuclei, which are oligodendrocyte precursor cells and oligodendrocytes, respectively . The density of olig2 (d), olig2 (e), and olig2 (f) nuclei in 1 mg of gray matter from the frontopolar cortex (10 cells / mg). * p <0.05 by unpaired t test . Results of immunostaining with ki-67, a marker of proliferating cells, in the hippocampus (g) and subependymal zone (h) of adult cynomolgus monkeys . A part of this figure was rearranged from quantification of frontopolar cortex from mdd and healthy control . A negative controls of flow cytometry staining with igg . B a novel cell counting method can separate nuclei from postmortem brain into neurons (neun) and oligodendrocytes (olig2), astrocytes, and microglia (neun / olig2). C the olig2 nuclei were further divided into two populations of strongly and weakly olig2-positive nuclei, which are oligodendrocyte precursor cells and oligodendrocytes, respectively . The density of olig2 (d), olig2 (e), and olig2 (f) nuclei in 1 mg of gray matter from the frontopolar cortex (10 cells / mg). * p <0.05 by unpaired t test . Results of immunostaining with ki-67, a marker of proliferating cells, in the hippocampus (g) and subependymal zone (h) of adult cynomolgus monkeys . A part of this figure was rearranged from continuous neurogenesis in the adult brain is observed in the hippocampus and subependymal zone in rodents as well as cynomolgus monkeys (macaca fascicularis) (fig . Volumes of literature report that chronic stress suppresses neurogenesis in rodents and that antidepressants stimulate neurogenesis in the subgranular zone in the hippocampus and the subependymal zone in the lateral ventricles [50, 51]. Although newly generated oligodendrocytes were observed in the adult hippocampus of a non - human primate, oligodendroglial generation from nscs in the adult brain is controversial . Recently, opcs that express ng2 chondroitin sulfate were recognized as generators of newly myelinating oligodendrocytes in the adult brain . The proportion of opcs was reported to constitute about 5% of total cells in rodent brains, which appears comparable to the number of olig2 nuclei in the gray matter of the frontopolar cortex of the human brain (fig . Because myelination in humans continues from adolescence into the 5th or 6th decade of life (see review), adult oligodendrogenesis in the prefrontal cortex and hippocampus may play a significant role in the pathology of mdd [57, 58]. Thus, it is an urgent issue to understand the molecular mechanisms underlying the generation of opcs from nscs, maintenance of opcs, and production and maturation of oligodendrocytes from opcs in the adult primate brain and to clarify the causal relationship between the dysfunction of adult opcs / oligodendrocytes and pathogenesis of mdd . These topics indicate that stem cell and neuro / gliogenesis biology is involved in various aspects of neuroscience . This biology encompasses a wide range of animal ages, from the embryo to adulthood; wide brain regions, from the developing neuroepithelium to higher neocortical regions; and various conditions of the brain, including development, maturation, and disease . Collectively, this research field aims to reveal the structural and functional plasticity of the cns including its genesis, remodeling, and restoration, by incorporating multidisciplinary approaches at the molecular, cellular, circuit, and behavioral levels . The broad scope of stem cell and neuro / gliogenesis study raises questions about inherent similarities and differences in the properties of nscs at different animal ages and in different brain regions, as well as in the process of neuro / gliogenesis in the developing, mature, and diseased brain . It appears important to understand both the intrinsic and extrinsic mechanisms of the nsc fate decision, including cell - to - cell interaction between immature cells, between newly generated cells and preexisting cells, and between new healthy cells and old diseased cells . Further, with regard to functional aspects, how are new neurons and glia incorporated into neural circuits to develop, improve, and restore brain function? To understand this, we need to know more about the constitution of functional neural networks and the interactions of the network with new neurons and glia . We also need to determine how neural activity contributes to the appropriate incorporation of new neurons and glia into functional networks . Recent advances in optogenetic and pharmacogenetic approaches will facilitate these studies . To obtain a comprehensive view, it is crucial to integrate knowledge under different situations and to try to reveal similarities and differences among these situations . The 2015 joint meeting was an ideal opportunity to facilitate the interchange of knowledge among researchers with different backgrounds . We hope that this review will further facilitate this interaction and raise the interest of those who are not directly engaged in this research field . We wish to emphasize that neuro / gliogenesis from nscs is a drastic process involving significant structural and functional changes in the cells and that integration of newly generated neurons and glia into neural networks is accompanied by significant structural and functional changes in the network . Thus, stem cell and neuro / gliogenesis biology provides an ideal platform for the integration of structural and functional plasticity and will contribute to our understanding of the remarkable plastic potential of the cns . We also hope that progress in this field will facilitate the interaction of neurobiologists and clinicians and encourage many people by revealing the tremendous plasticity that is buried in our own brain.
In research and development of herbal formulae, intra - laboratory verification of the effectiveness is an important method to establish evidence based korean medicine . Despite the frequent use of medicinal plants, few scientific studies have been undertaken to determine the safety of traditional medicinal herbs . If the safety of medicines and plant products intended for human consumption is to be determined, systematic toxicological studies must be performed using various experimental models to predict the toxicity and to set the criteria for selecting a safe dose in humans . Through long practice theories and techniques have been developed to eliminate or reduce the toxicity, as well as the drastic actions and side effects, of some drugs . Sulfur is the third most abundant mineral in the body and is essential for life . Sulfur makes up the vital amino acids used to create proteins for cells and tissues and for hormones, enzymes, and antibodies . However, direct administered of sulfur to the human body will generally results in strong toxic side effects . Therefore, super key (processed sulfur), the product remaining after sulfur has been processed to remove the poison, was developed for medicinal use . This study was performed to examine the oral toxicity and the lethal dose of super key . The testing methods were visual observation of the general symptoms, body weight changes and necropsy findings in 8-weeks - old sprague - dawley (sd) rats . The current research trend for oral toxicity testing of extracts is to study acute and subacute toxicity through good laboratory practice (glp) regulations . All the experiments for this research were conducted at medvill, an institution authorized to perform non - clinical studies, under the glp regulations . The super key was prepared in a sterile room at wonkwang university gwangju korean medical hospital . After having been weighed by using an electronic scale and mixed with corn oil, it was prepared at normal potency . The reason sd rats were chosen is that they have been widely used in safety tests in the field of medicine, so the results can be easily compared with many other databases . The mean weights of the rats were 285.3 314.5 g and 200.5 222.9 g, respectively, for the male and the female rats at the time of super key administration . For all animals, a visual inspection was conducted, and all animals were weighed at the beginning of the experiment . During the seven days of acclimatization, the general symptoms of the rats were observed once a day . The temperature of the lab was 19.7 22.6c, and the humidity was 48.1% 75.6% . However, an aberration occurred on july 19, 2014, from 17:30 pm to 18:00 pm when the humidity was 75.6% . The above temporary aberration occurred due to a malfuncion of the air conditioning equipment and the reinstallation of a temperature and humidity sensor . In spite of this, no abnormalities of the general symptoms were observed . Enough food (lab diet 5053) and ultra violet (uv)-filtered water were provided . Animals were selected if their weights and general symptoms were normal . In total, 20 male rats and 20 female rats were selected . The animals were randomly distributed into 4 groups (5 male and 5 female rats per group) as shown in (table 1). In this study, 2,000 mg / kg was set as the high dose, and 1,000 mg / kg and 500 mg / kg were set as the mid and the low doses, respectively . In the control group, a dose of 10 ml / kg of normal saline solution was administered . Super key and normal saline were administered into the mouths of the rats in all groups by using disposable syringes . This study was conducted under the approval of the institutional animal ethics committee of medvill co., ltd . From the 1 day to the 14 day after treatment, the general symptoms were examined once a day . On the day of dosing (day 0), the general symptoms (side effects, revealing time, recovery time, etc . ), as well as the mortality, were examined at 30 minutes and at 1, 2, 4, and 6 hours after oral administration . The weights were measured immediately before treatment and at 1, 3, 7 and 14 days after treatment . After observations had been terminated, necropsies were conducted on the rats after their abdominal aorta and vein had been cut under co2 anesthesia, and the organs of all surviving animals were visually inspected and microscopically examined . The weight data from the experiments were analyzed by using the statistical package for social science (spss) program (spss 16.0). A levene test was conducted to evaluate the homogeneity of the variance and the significance . The one - way analysis of variance (anova) test was conducted when the homogeneity of the variance was recognized (significance level: 0.05 on both sides). Also, the kruskal - wallis test was conducted when heterogeneity of the variance was recognized (significance level: 0.05 on both sides). The weight examinations showed slight decreases in the weights of four female rats (two female rats in the 1,000 mg / kg group and two female rats in the 2,000 mg / kg group). Finally, in both the control and the experimental groups, no meaningful differences in the necropsy findings were noted . Since then, sulfur has been used for various reasons, such as to treat skin diseases, and as an intestinal drug sulfur is a bright yellow crystalline solid or powder . Sulfur is the seventh most abundant mineral in the body, and the body of a human weighing approximately 70 kg contains roughly 140 g of sulfur . Sulfur is acquired through the consumption of food primarily in the form of sulfur - containing amino acids (saas), such as methionine, cysteine, cystine, and taurine, and in its glucosinolate form, which is found in cruciferous vegetables, such as cabbage and cauliflower . In dongui bogam, sulfur is mainly used for the treatments of an abdominal mass and pathogenic qi in the pit of the stomach, stagnation of cold qi, chronic cold syndrome in the back and the kidneys, loss of sensation due to cold wind, and coldness, pain, and loss of power in the legs . It fortifies the sinews and bones, tonifies yang qi, removes balding, malignant furuncles, infantile malnutrition affecting the nose and other conditions in the external genitals, and kills scabies parasites . Sulfur is widely used to detoxify the body, treat scabies [6, 7], heal sores and eliminate itching . Kong et al showed that the treatment of human gastric adenocarcinoma cells (ags) with extracts from young radishes that had a high organic sulfur glucosinolate content inhibited cancer cell growth . In addition, bak et al reported that treating ht-29 human colon cancer cells with kimchi extract made with sulfur treated radishes also inhibited the growth of cancer cells . Choi and kim reported that, when diverse cancer cells were treated with extracts from a hot water extraction from regular ducks or organic - sulfur - fed ducks, a noticeable effect in proliferation inhibition was seen in the cells treated with the extract from organic - sulfur - fed ducks . In previous studies, inorganic sulfur has been demonstrated to inhibit the proliferation of breast cancer cells . The study showed that inorganic sulfur reduced cell proliferation by inhibiting the expression and the activation of epidermal growth factor receptor (egfr) and by increasing the expression of bcl-2-associated x (bax) in estrogen - independent breast cancer (mda - mb-231) human breast cancer cells . Another study investigated the inhibitory effect of sulfur on prostate cancer (pca) in vivo . In that research, prostate tumors were developed by injecting 22 rv1 or du 145 pca cells into sulfur treated and untreated nude mice . The results showed that sulfur inhibited the growth of androgen independent prostate cancer in vivo . Furthermore, the intake of refined inorganic sulfur has been reported to reduce the clinical side effects of radiotherapy in cancer patients . However, the direct administration of sulfur to the human body will generally results in a strong toxic side effect . Therefore, super key, processed sulfur with the poison removed, was developed for medicinal use . Although super key has been used in clinics, safety studies on super key are insufficient . Toxicity tests provide important data and are essential for evaluating the safety of test substances in medications . Doses of 500 mg / kg, 1,000 mg / kg, and 2,000 mg / kg of super key were administered to the experimental groups, and a dose of 10 ml / kg of normal saline solution was administered to the control group . In all four groups, no deaths occurred, and no significant differences in the clinical signs or weights were noted between the control group and the experimental groups . The necropsy results to check for abnormalities in organs showed no significant findings . Animal testing is the most fundamental and basic way to assess the safety of materials to be used for medical purposes . For that reason, the korea food & drug administration of korea has testing protocol guidelines for the study of toxicity, and all experiments should be conducted following glp regulations . The results of our study, which followed that protocol and those regulations, showed that the administration of 2,000 mg / kg of super key did not cause any changes in the weights of sd rats or in the results of necropsy examinations . It also did not result in any mortalities, which indicates that super key is safe to use as a treatment . However, the acute and the chronic side effects of super key need to be studied more in order to assess its oral toxicity, and more hematology and blood chemistry studies are required . The results of this reseach showed that administration of 500 2,000 mg / kg of super key did not cause any changes in the weights of sd rats or in the results of necropsy examinations . Neither did it result in any mortalities, which indicates that the lethal dose of super key is higher than 2,000 mg / kg . The results obtained in this study suggest that administration of super key as treatment may be safe . S.d ., standard deviation; n, number of animals.
It is well established that the presence of neoplasms in the upper tract urothelium necessitates the en bloc removal of the ipsilateral kidney, ureter, and bladder cuff as the standard of care based on the premise that urothelial cancers are caused by a field change or defect . However, due to the morbidity associated with open nephroureterectomies, conservative management may be appropriate for poor surgical candidates . Percutaneous and ureteroscopic techniques have been utilized in the management of urothelial tumors with percutaneous access required to treat larger of upper tract urothelial cancers being used in suitable candidates.various techniques for this approach have been reported including monopolar electrocautery, laser, rollerball, and electrovaporization . The reported recurrence rates have been low, and disease - free survival is comparable to that in patients treated with nephroureterectomy . The use of monopolar electrosurgery however can interfere with cardiac pacemakers resulting in potentially fatal dysrhythmias . We report the case of an upper tract urothelial carcinoma in the renal pelvis resected with bipolar cautery through percutaneous access without deactivation of the cardiac pacemaker . To our knowledge an 82-year - old man who was a lifelong nonsmoker presented with a history of intermittent gross hematuria . His medical comorbidities included non - insulin - dependent diabetes, hypertension, hypercholesterolemia, hypothyroidism, chronic renal insufficiency (creatinine 2.4 mg/ dl), multiple transient ischemic attacks, stroke, sleep apnea, peripheral vascular disease resulting in a below - knee amputation, colon resection for diverticulitis, and a vdd pacemaker (model #640, vitatron, minneapolis, mn) for bradycardia . His preoperative cardiac risk was goldman class ii - iii, and he was at high risk of developing a deep venous thrombosis . The retrograde pyelogram on the right side was normal, but the left retrograde pyelogram demonstrated moderate hydronephrosis in a bifid collecting system and a 2-cm filling defect in the renal pelvis with renal malrotation (figure 1). A ct scan of the abdomen and pelvis revealed a tumor limited to the left renal collecting system with no evidence of metastases . Retrograde pyelogram displaying a renal filling defect in a bifid renal collecting system with a malrotated kidney . Based on a background of renal insufficiency, the options were discussed with the patient and a tumor ablation with the holmium: yag laser and flexible ureteroscopy was carried out . The ureteroscopic procedure was ineffective in completely ablating the tumor at its stalk because the ureteroscope could not fully access the tumor due to the capacious renal pelvis and renal malrotation . This patient was dependent on his vdd pacemaker, thus making monopolar cautery potentially hazardous . Therefore, a percutaneous resection using a bipolar resectoscope was planned under a second general anesthetic without deactivating the pacemaker . With the patient under general anesthesia, percutaneous access to the kidney was obtained, the tract was dilated to 30 f, and an amplatz sheath was inserted . A resectoscope using bipolar cautery (vista, acmi corporation, south - borough, ma) was used to resect the tumor (settings: cut, 6; coagulation, 6) through the amplatz sheath . The operation lasted 30 minutes without any evidence of dysrhythmias or interference with the pacemaker . A 22 f nephrostomy tube was left indwelling until postoperative day 1 when it was removed, and the patient was discharged home without any complications . The patient used a continuous positive airway pressure (cpap) machine postoperatively that helped prevent any postoperative respiratory complications . The patient was seen in follow - up at 3 months with an intravenous pyelogram (ivp) that was free of any filling defects and negative cytology . Serum creatinine increased only slightly from 2.4 mg / dl (preoperatively) to 2.6 mg / dl, postoperatively . At 8 months, a retrograde pyelogram revealed a recurrence of his upper tract tumor that was too large for ureteroscopic management, and percutaneous bipolar resection was once again undertaken . The patient declined upper tract bacille calmette - gurin therapy and laparoscopic nephroureterectomy due to his declining health including a recent cerebral vascular accident . At 6 months, the patient remains free of recurrent or metastatic disease with stable renal function (2.5 mg / dl). Bipolar electrocautery was introduced over 20 years ago as an energy source for endourologic procedures . The potential benefits of bipolar energy include the use of isotonic normal saline irrigation and improved hemostasis . Importantly, it can also be used on patients with permanent cardiac pacemakers and has been described in a case involving transurethral resection of a bladder tumor in a similar pacemaker - dependent patient . Electrocautery - induced cardiac pacemaker failure or malfunction has been well described.since the invention of cardiac pacemakers nearly 50 years ago, electromagnetic interference has been a concern for patients who wear them . Over the years, despite improved technology, permanent cardiac pacemakers remain susceptible to this electromagnetic interference, including electrocautery, during surgical procedures . Traditionally, the use of monopolar electrosurgery is avoided in patients with cardiac pacemakers because the monopolar current passes from the instrument tip through the patient's body towards the ground plate and may adversely affect the pacemaker, resulting in potentially fatal dysrhythmias . Bipolar electro - surgery can reduce the level of interference between electrocautery units and pacemaker electrodes . Bipolar electrosurgery has also been shown to reduce the depth of thermal damage at the site of surgery . In an ex vivo porcine kidney model, wendt - nordahl et al compared the effects of the vista bipolar system to monopolar cautery . The depth of thermal damage was significantly deeper at 300 m measured with the monopolar resectoscope but only reached 160 m with the bipolar device even at the highest output level (level 8). Excessive heat may cause problems with the urothelium of the renal collecting system or penetrate deeply into parenchyma resulting in arteriovenous fistulae . However, in patients with significant medical comorbidities who are not candidates for radical surgery, bipolar resection through a percutaneous access is an alternative . Given the patient's cardiac status, a bipolar resectoscope was used not only to perform tumor resection but also to minimize the risk of electrical interference with the pacemaker . Both ureteroscopy and percutaneous laser ablation would have been tedious due to tumor size and location . Disease recurrence occurred due to multifocality and not due to failure of the initial bipolar procedure . It can be argued that the patient underwent 2 endoscopic procedures while under general anesthesia, whereas a laparoscopic nephroureterectomy would have offered treatment in a single procedure; however, the single procedure would have required more surgical time in one sitting and is much more invasive than 2 short endoscopic procedures . The mortality and morbidity risk would be higher with a nephroureterectomy than 2 shorter endoscopic procedures . While the use of the bipolar electrocautery may be advantageous in the percutaneous resection of upper tract urothelial tumors, potential drawbacks of this technique include limited utility depending on tumor location and access, and dessication of the tissue creating more artifacts for pathological examination . It is advisable to perform a cold biopsy of the tumor for both pathology and margin status before using the bipolar electrocautery . More importantly, this approach is not a replacement for nephroureterectomy but may be an option for nonsurgical candidates or patients who may benefit from nephron - sparing procedures . It may be a palliative procedure in some cases and potentially therapeutic in others . As illustrated in this case, the use of bipolar electrocautery circumvents the potential risks of using electrocautery in patients with cardiac pacemakers . Bipolar electrocautery is a safe and feasible resection modality in percutaneously approached upper tract urothelial neoplasms in patients with cardiac pacemakers.
, cataract surgery has continued to evolve, with smaller incision procedures, development of premium intraocular lenses and laser - assisted surgery.13 in the field of retina, anti - vascular endothelial growth factor (anti - vegf) intravitreal injections have revolutionized treatment of macular edema and choroidal neovascularization.46 the field of glaucoma has experienced a new wave of therapeutic tools, with the development of microinvasive glaucoma surgery such as trabeculectomy ab interno and trabecular meshwork bypass shunts.7,8 in the cornea subspecialty, advances in partial thickness corneal transplant surgeries have transformed the field.9 all of these unique therapies continue to advance ophthalmology and offer hope for improved visual rehabilitation in various conditions . While expanding the array of therapeutic tools, these advances also challenge medical educators with the task of training residents with these new skills and in traditional management approaches . Given these recent advances, many studies have evaluated shifts in practice patterns among practicing ophthalmologists.1013 however, few studies have evaluated shifts in the experience of ophthalmology residents.14 the accreditation council for graduate medical education (acgme) is a nonprofit organization whose mission is to support high - quality resident and fellow education through accreditation of residency training programs . It collects resident case logs annually as part of maintenance of accreditation . In this study, we evaluate trends in resident surgical experience with core ophthalmic procedures from 2009 to 2015 by reviewing the acgme resident case logs . Acgme resident surgical case logs on cataract, cornea, retina, glaucoma, pediatrics, plastics, and trauma procedures published from 2009 to 2015 were analyzed for year - to - year trends in the average number of cases performed as primary surgeon.15 the yale university institutional review board (irb)/ethics committee ruled that irb approval was not required for this study as it did not involve human subjects . From 2009 to 2015, the average number of residency programs per year was 166.3, and the total number of residents was 2,797 for all 6 years . During this 6-year period, the average number of phacoemulsification cases performed by residents as primary surgeon steadily increased from 143.8 to 173.6 . Concurrently, the average number of nonphacoemulsification / extracapsular cataract extraction (ecce) cases decreased from 3.8 to 2.2 . Average number of primary anterior vitrectomy cases also declined from 3 to 1.8 over the same 6-year period (table 1). For cornea, the average number of keratoplasty and other cornea procedures remained relatively stable, ranging from 2.1 to 2.5 and 4 to 4.5 cases, respectively, over the 6-year period (table 1). For retinal procedures, average vitreous concurrently, average laser panretinal photocoagulation (prp) decreased from 59.6 to 45.5 cases and focal lasers decreased from 15.7 to 6.8 cases . Posterior vitrectomy cases remained stable, ranging from 4.8 to 5.4 from 2009 to 2015 (table 1). For glaucoma, from 2009 to 2015, average filtering procedures decreased from 6 to 4.5 cases, while average shunting procedures with glaucoma drainage implants (gdis) increased from 4.5 to 6.7 cases (table 1). Pediatric, plastics, and trauma cases performed by residents as primary surgeon remained relatively stable from 2009 to 2015 . Average number of extraocular muscle surgery cases remained steady, ranging from 24.4 to 26.6 over the 6-year period . Ptosis repair and blepharoplasty / reconstruction cases slightly increased, ranging from 6.1 to 6.7 and 9.1 to 11.2, respectively . Globe rupture repair ranged from 7.2 to 7.6 cases over this 6-year period (table 1). A review of the acgme ophthalmology resident case logs from 2009 to 2015 revealed significant trends in the areas of cataract, retina, and glaucoma procedures, while cornea, pediatric, plastic, and trauma procedure experience remained relatively stable . In the category of cataract surgery, resident primary cases in phacoemulsification steadily increased while ecce cases declined . Since the advent of phacoemulsification, ecce has largely been reserved for mature or hypermature cataracts . The trend in resident experience with ecce is likely a reflection of practicing ophthalmologists shift toward phacoemulsification for standard cataract surgery . This trend suggests that over the next few years, residents may no longer have ecce experience during their training . It raises the question of whether ecce case minimums should be established to equip residents with an alternate method of cataract extraction in cases not suitable for phacoemulsification . This trend suggests that educators may need to provide more didactic education on this technique and highlight important steps during other procedures with related surgical maneuvers . Additionally, since ecce is commonly practiced in other nations, where patients may present with more advanced cataracts, it may be advantageous for us residents to be familiar with this approach, which may prepare them better for experiences in international ophthalmology . The field of retina has undergone tremendous advances over the past decade, with the development of intravitreal anti - vegf therapies . Most notably, primary resident vitreous tap / inject procedures almost tripled over this 6-year period, while laser prp and focal laser experience slowly declined . Although average prp experience demonstrated a declining trend, the average number of cases remained relatively high at 45.5 in the 20142015 academic year . Therefore, prp does not seem to be a skill at risk of becoming lost to future residents . However, the number of focal laser cases was quite low and on a declining trend, which could make it absent from residency curricula in the future . This shift in practice pattern has been demonstrated in american glaucoma society surveys of practicing glaucoma specialists as well as medicare claims data, and has likely influenced trainee surgical experience.1214 the decline in filtering surgery experience is concerning considering it is a core surgical management procedure in glaucoma . Glaucoma educators may need to find a way to maintain trabeculectomy training in the setting of decreasing surgical volume . Construction of partial thickness scleral flaps for gdi tube coverage may be a means of teaching flap creation for trabeculectomy . While corneal surgery has experienced many advances in the area of partial thickness graft surgery, it does not seem to have impacted resident cornea surgery experience yet . Resident cornea procedure average values remained relatively stable . Because cornea procedures were categorized broadly as keratoplasty and other cornea procedures, it is difficult to analyze resident cornea experience in further detail . Botox injection for strabismus correction emerged as a new therapeutic option, but does not appear to have affected resident surgical experience with traditional muscle surgery and is not currently part of the case logs.16 likewise, in plastics, while many new technologies continue to emerge, resident case numbers with ptosis repair, blepharoplasty, and entropion / ectropion repair have remained stable . Lastly, resident trauma experience with globe rupture repair remained relatively stable from 2009 to 2015 . In reviewing the acgme case logs of ophthalmology residents over the past 6 years, significant changes in surgical experience in the area of cataract, retina, and glaucoma were identified . The trends in these subspecialty areas correspond to the development of new techniques or therapies in these fields . These findings highlight a need for educators to be aware of how such shifts can influence ophthalmology residents experience and education . They also suggest that required procedure minimums and resident achievement of these minimums should be periodically reevaluated as new technologies emerge, to help preserve training in core skills as well as remain current with new advances . Finally, educators may need to become innovative in teaching surgical technique of less common procedures.
Cytomegalovirus (cmv) is a common viral infection in renal transplant recipients, affecting more than 50% of recipients of major organ transplants . The most common clinical presentation is a viral syndrome characterized by fever, leucopenia, thrombocytopenia, atypical lymphocytosis and other well - documented manifestations including pneumonia, liver dysfunction and gastrointestinal disease ., we report an unusual case of a renal alograft recipient who developed ulcers over the right axilla, scrotum and penile skin not responding to antibiotics, and the skin biopsies from the axillary and scrotal ulcers revealed several enlarged endothelial cells of the dermal blood vessels containing intranuclear and intracytoplasmic inclusions positive for cmv antigen on immunohistochemistry . A 52-year - old, live related renal allograft recipient, after a 2.5-month post - transplant period, presented with a history of vesicular eruptions over the right axilla, scrotum and penile skin which later developed into ulcers (figure 1). Both the donor and the recipient were cmv immunoglobulin g (igg) antibody positive and cmv igm antibody negative before transplantation . He developed new - onset diabetes in the immediate post - transplant period for which he was on insulin therapy . He had c4d - negative acute graft rejection after 20 days of transplant, which was treated with three doses of methyl prednisolone and graft function become normal . On presentation, he had pyrexia of 38.5c, pulse rate 90/min and bp 130/84 mmhg, and other physical examinations were within normal limits . Laboratory findings revealed a haemoglobin of 10.5 g / dl, total leucocyte count of 17 000/l with 83% neutrophils, platelet count of 310 10/mm, fasting blood glucose of 69 mg / dl, serum creatinine of 1.71 mg / dl and normal liver function tests . His urine examination showed mild proteinuria and two to four leucocytes per high - power field . A chest radiograph was normal . A swab culture from the axillary and scrotal ulcers revealed pseudomonas infection . The patient was treated with intravenous (iv) cefoperazone sulbactam combination antibiotic for 7 days . Subsequently, the patient was treated with another set of antibiotics, cefuroxime and piperacillin tazobactam combination for further 7 days . The skin ulcers and pyrexia did not respond to this therapy either and antibiotic treatment was discontinued . Then skin biopsy was done and samples were sent for microbiology and histology . The total leucocyte count at this time was 12 500/l with 72% neutrophils, and serum creatinine was 1.86 mg / dl . The skin biopsies from the axillary and scrotal ulcers revealed intranuclear and intracytoplasmic inclusions in several enlarged endothelial cells of the dermal blood vessels (figure 2). These inclusions were positivity for cmv antigen (dako) on immunohistochemistry (figure 3). Serology tests yielded cmv igg positivity, and cmv viral load as measured by quantitative real - time polymerase chain reaction was 66 900 copies / ml . The patient was treated with iv ganciclovir 2.5 mg / kg at 12-h intervals for 14 days and then the patient was discharged with advice to continue oral valganciclovir 450 mg daily for 2.5 months as maintenance therapy . He became afebrile with healing and regression of the skin ulcers after 2 weeks of ganciclovir therapy (figure 1). At the time of discharge, the intensity of immunosuppression was reduced during the period of cmv treatment by reducing the dose of cyclosporine . On a follow - up visit 3 months later, the patient was completely asymptomatic with a total leucocyte count of 4800/l and serum creatinine of 1.38 mg / dl . The presence of cmv inclusion bodies, presence of cmv antigen on immunohistochemistry, high cmv dna copies per millilitre and response to anti - cmv therapy indicated cmv as primary causative organism of ulcer in this patient . Photograph of axillary and scrotal ulcer before and after treatment . Microphotograph of biopsy from axillary ulcer showing endothelial cells with intranuclear and intracytoplasmic inclusions (arrow) (h&e, 400). Immunohistochemistry for cmv antigen showing nuclear positivity in the endothelial cells of dermal blood vessels (arrow) (200). In this case study, we report a very rare manifestation of cmv disease, a cutaneous ulcers, in a live related renal transplant recipient . The usual clinical features of active disseminated cmv disease include fever, leucopenia, thrombocytopenia, petechiae, pneumonitis, hepatitis, chorioretinitis, adrenalitis and encephalitis . However, ulcers, mostly around genitalia, perineum, buttocks and thighs, have been described in immunocompromised patients particularly in acquired immunodeficiency syndrome (aids) patients . However, to our knowledge, axillary ulcers have not been described in cmv - associated cutaneous lesions in renal allograft recipients . Recently, the true pathogenetic role of cmv in cutaneous lesions has been the focus of interest . It has been thought that cmv may be simply an innocent bystander during herpes simplex virus infection among non - aids patients . But the demonstration of cmv antigen on immunohistochemistry, cmv inclusion bodies, high titre of cmv dna and overall response to anti - cmv therapy with ganciclovir and valganciclovir suggest the true pathogenetic role of cmv in cutaneous ulcers in renal transplant recipients . One should always consider a differential diagnosis of cmv skin ulcers in a non - healing ulcers in renal transplant recipients . Choi et al . Have also shown a pathogenetic role of cmv in cutaneous lesions, it is not simply a bystander in skin ulcers . Choi et al . Have reported nine cases (five haematological and four organ recipients) of cmv skin lesion in nine non - aids immunocompromised patients; six of them had multiple anogenital ulceration, two had a solitary nodule and one had maculopapular rash . Cmv usually causes intracellular lesion by nature and ulceration of a lesion is not common . Have also reported three cases of cmv skin ulcers in transplant recipients; the first case was associated with extravasated inotrope - induced skin ulcers, second with candida infection and third with pneumonia . Of the three cases, have shown that cmv infection, when detected in the skin, appears to be associated with grave prognosis and reported mortality is 85%, but the fatal cases had either concurrent disseminated cmv infection or mixed cutaneous or systemic infections . When the infection is localized in the skin wounds, the prognosis seems fairly good . The high leucocyte count and growth of pseudomonas from the wound at presentation was misleading concerning the diagnosis of cmv and the patient was treated with antibacterial agents . It is known that cmv is an immunomodulatory virus and predisposes the patients to other associated opportunistic bacterial, viral and fungal infection . Our patient also had associated pseudomonas infection which may be the reason of the high leucocyte count in this patient contradictory to the usual presentation of leucopenia in cmv infection . Recently, roco et al . Reported a case of ureterostomy cytomegalovirus infection presenting as stoma ulceration . The summary of the review of literature of cmv skin ulcers in renal transplant recipients is shown in table 1 . Summary of review of literature of cmv cutaneous ulcers in organ recipients cmv infection is caused by a virus of the family herpesviridae and is usually latent and asymptomatic in those with a normal immune system . Infection by cmv may be frequent and severe in children or adults with congenital or acquired defects of cellular immunity, with underlying malignancy, patients on immunosuppression and aids patients . There are three major patterns of cmv infection in transplant patients: (i) primary infection occurs in seronegative recipient from seropositive donor . (iii) super infection occurs when allograft from a seropositive donor is transplanted into a seropositive recipient . Both our recipient and donor were seropositive and cmv disease appears to be due to either reactivation or super infection of cmv in this patient . We used to give ganciclovir prophylaxis if a seronegative recipient receives a kidney from a seropositive donor and with the use of anti - thymocyte globulin for either induction purpose or for the treatment of rejection . Therapeutic treatment of established cmv disease is primarily with the antiviral agent ganciclovir iv induces remission of cmv disease in renal transplant recipients in all but rare cases that are ganciclovir resistant . Ganciclovir - resistant strains of cmv are generally sensitive to foscarnet which is otherwise avoided because of nephrotoxicity . Valganciclovir is the l - valyl ester prodrug of ganciclovir which provides an excellent oral alternative to ganciclovir for the prevention of cmv in transplant recipients . Although valganciclovir is an established drug for cmv disease treatment, this is the first case report of the treatment of cmv skin ulcers with valganciclovir . In summary, cmv - induced skin ulcers, particularly in axillary regions, have not been previously reported in renal allograft recipients . Cmv - induced skin ulcers should be added to the list of manifestations of cmv disease in renal transplant recipients . Skin biopsy may be of help in the prompt diagnosis and treatment of cmv skin ulcers.
Periodontal disease is a multifactorial infectious disease triggered by the development of dental biofilm, which can harm periodontal tissues . There are several factors which influence periodontitis: personal and social characteristics, behavioral, systemic, genetic and dental factors, and the microbial composition of the dental biofilm [1, 2]. Stress may be defined as the combination of physical and mental responses caused by certain external stimuli (stressors) which allow an individual (human or animal) to overcome certain environmental demands, and the physical and mental weariness caused by the stress process itself . Stress has been related not only to a higher occurrence of periodontal disease but also to its severity . Other social and behavioral factors that could be related to periodontal status are tobacco use, social economic status, nutritional status, psychological aspects, and abusive alcohol intake . Among these factors, alcohol has become relevant because it is one of the few socially accepted psychotropic drugs . This social acceptance has contributed to a rise in alcoholism, which has become a serious public health problem . According to silva furtado amaral et al . The world health organization has reported that the rates of death and functional limitations associated with abusive alcohol intake have surpassed those associated with tobacco smoking . Alcoholism per se imposes a state of stress because, besides its physiological demand, it acts as a chemical aggressor to the body . All the alcoholic drinks consumed by an individual do not remain stored in the body and, before being eliminated, it is first metabolized in the liver, which results in high levels of reactive oxygen species [7, 8]. Several cross - sectional studies have verified an association between the abusive consumption of alcohol and poor periodontal health [911]. Abusive consumption of alcohol has also been associated with the severity of periodontitis . However, many questions on this subject are still not answered and laboratorial research could be helpful to clarify these doubts . Animal model studies may more specifically help elucidate questions related to alcohol intake, stress, and periodontal disease [12, 13]. As a lifestyle factor drinking, and stress seem to contribute to poorer periodontal status . In the present study it was hypothesized that alcohol consumption by stressed animals could be accompanied by an increase in the severity of periodontitis . Therefore, aiming to confirm or reject this hypothesis we evaluated whether forced alcohol intake by stressed animals affects histometrical parameters of periodontal breakdown . Thirty - two animals of the rattus norvegicus species lewis breed (two - month old), weighing average 250 g, were selected for the present study . The animals were divided in groups of four and each group was kept in a cage (polyethylene 16 40 30) [14, 15]. They all received standard rat feed and water or alcohol (ethanol 20%) ad libitum . They were kept in light / dark cycles of 12 hours; the temperature was controlled at 24c and humidity at> 50% . The experiment took place at the university center of vrzea grande univag and it was approved and registered by the ethics in animal research committee (cep / unic-2009 no . Initially, a research assistant randomly divided the animals in four experimental groups: group gal: alcohol + ligature (n = 8);group gasl: alcohol + stress + ligature (n = 8);group gpc: stress + ligature, positive control (n = 8);group gcn: negative control (n = 8). Group gal: alcohol + ligature (n = 8); group gasl: alcohol + stress + ligature (n = 8); group gpc: stress + ligature, positive control (n = 8); group gcn: negative control (n = 8). After the division, the animals in the gal and gasl groups were offered alcohol (ethanol 20%) ad libitum for 60 days [11, 16] while negative control (gcn) received water ad libitum for the same period of time . Because alcohol intake is very limited on the first day, ligatures were inserted only in the second experimental day (gal, gasl, and gpc). The animals received general anesthesia through the intramuscular administration of 0.05 ml xylazine hydrochloride (rompun, bayer . Animal health, so paulo, sp . Brazil) and 0.1 ml ketamine (dopalen, agribrands . Animal health, paulnia, sp, brazil) per 100 grams of body weight . After anesthesia, a sterile silk suture number 4 (ethicon, johnson and johnson, so paulo, sp, brazil) was placed in the gingival crevice of the second right upper molar . The animals in the positive control group were kept in the same environment in the first experimental day, drinking water, but without suffering any kind of intervention . The animals in the negative control group (gnc) did not receive any type of intervention but were kept in their cages in the same environment during all the study . The model of physical stress induction chosen for this study was immobilization done during 60 days, six times a week, in different times of the day as previously described [18, 19]. At room temperature, the animals in the gasl group were placed in polyvinyl chloride tubes compatible to their body sizes . The tubes were closed on both ends with metallic wire, which enabled the animals to breathe while they were immobilized for four consecutive hours . After that process, the jaw segment was decalcified in edta for approximately five weeks (edta was renewed six times); following dehydration in graded alcohol, the samples were embedded in paraffin . The tissue blocks included in paraffin allowed 6 m histological slices that followed the long axis of the teeth in the mesio - distal plane and were stained with hematoxylin and eosin . For the histometric analysis, ten serial sections containing the 1st and 2nd upper molars and the following structures (a) dental pulp, (b) the mesial cementoenamel junction of the 2nd molar, (c) proximal bone crest, and (d) connective attachment had to be observed . Histometry was performed in the mesial aspect from first molars, by the same blinded examiner, through the capturing of images in the microscope and measured in millimeters (software imagelab 2000-diracon bio informtica ltda . Previous to the study, the error was calculated by double measurements of 10% of the specimens with a one - week interval . During the study, paired t - test statistics were run and no differences were observed in the mean values for comparison (p> 0.5). Additionally, pearson's correlation coefficient was obtained between the 2 measurements in each time point and revealed a very high correlation (0.98 and 0.99; p <0.01). The distance between the cementoenamel junction and the bottom of the junctional epithelium (cej - je) in the mesial site of the first molars was defined as the loss of histological attachment . In addition, the distance between the cementoenamel junction and the alveolar bone crest was also analyzed and it was considered as the indicator for bone loss (cej - bc). Analysis of variance (anova) and tukey (p <0.05) tests were applied . There were no statistically significant differences among the body weights of the groups at any of the evaluation periods (anova, p <0.05). Loss of histological attachment (cej - je) and bone loss (cej - bc) (p <0.05) were statistically different in the animals exposed to alcohol (gal) and the ones exposed to alcohol and stress (gasl). The most severe periodontal breakdown was observed among the nonstressed animals exposed to alcohol, followed by the group of stressed animals exposed to alcohol . This last group (gasl) did not differ from the positive control group (gpc). Figures 1 and 2 illustrate results from gasl group at two different experimental times . Finally, the negative control group showed the lowest values of cej - je and cej - bc when compared to the animals in the other groups (table 1). The severity and rate of the progression of periodontitis are influenced by a great variety of determinants and risk factors . Oral hygiene, tobacco smoking, diabetes, age, and the presence of specific microorganisms can be considered as risk factors, since individuals under these conditions seem to be more likely to develop periodontitis . However, the combination of these factors is not enough to explain the difference in the progression rate and severity of the disease . Other systemic conditions such as stress, osteoporosis, obesity, and abusive alcohol drinking have been considered as partially responsible for this variability as well . The effects of alcohol to the body and the functional imbalances associated with its consume have been constantly investigated . Such interest is justified by the wide range of injuries and toxic effects that alcohol drinking causes the organs and tissues, which can reflect in different systemic diseases . Besides, the world health organization estimates that two billion people around the world are alcohol consumers, and that 76 million show some kind of systemic adverse event related to that . In brazil, it is estimated that 68% of the population consumes alcohol and that 11.2% are alcoholics . De souza et al . Demonstrated that alcohol consumption directly framed the progression of bone loss in an induced periodontitis model . On the other hand, instead of a direct action of alcohol over the periodontal tissues, these effects are based on poor oral hygiene, which could better explain a higher occurrence and severity of periodontal disease in people who drink alcohol . In this context, animal studies are important as they may help understand the biological events once they allow the elimination of certain bias by the control of some variables . Therefore, animal studies have observed that alcohol affected the periodontal tissues through different mechanisms . Reduced host responses, due to deficiency and functional changes in neutrophils, as well as changes in the protein metabolism and in the healing of tissues have been previously reported . In the present study alcohol drinking negatively impacted the progression of periodontitis . Similarly de souza et al . Also observed a direct effect of alcohol drinking in the bone loss progression in a ligature - induced periodontitis model . One common question is in which level the alcohol as a water substitute under experimental conditions reflects human alcohol drinking . The answer to that is not simple, especially considering that the metabolism of rats is faster than that of humans . According to the world health organization mild drinkers are men who drink 21 alcohol units (1 unit = 10 grams) per week . For women, for example, a glass of red wine (90 ml) has 12% or 1.7 unit of alcohol . A beer has 5% (1.1 unit) while distilled drinks such as vodka and whisky have 40% or 2.0 alcohol units . Supporting dose dependence, de souza et al . Reported a significantly higher alveolar bone loss in rats receiving 20% ethanol in comparison to rats receiving 10% ethanol or water . . Carried an interesting cross - sectional study to evaluate the effect of the consumption of different alcoholic drinks (wine, beer, and liquor) on the severity of periodontal disease . They observed that the effects over the periodontal tissues did not change depending on the kind of drink taken . These same authors also verified contrary activity of alcohol over some periodontal pathogens such as actinobacillus actinomycetemcomitans and porphyromonas gingivalis . Clinical results suggested that alcohol seems to affect more severely the gums, followed by the periodontal ligament and the alveolar bone and that the effect of alcohol on periodontal disease may depend on the dose, frequency, and time of drinking . . Also demonstrated that individuals that consumed more than 15 grams of alcohol a day had a significant increase in the progression rate of periodontal disease and a higher inflammatory infiltrate, besides a higher number of periodontal pockets when compared to those who did not consume alcohol . Although, dental biofilm acts besides some recognized risk factors such as tobacco use, as previously mentioned, more recently, researchers are considering whether and in what degree lifestyle could affect the periodontium . In this context, stress seems to affect not only periodontal status but also personal lifestyle . Individuals exposed to stressful situations tended to smoke a higher number of cigarettes and become alcohol consumers . Also, a decrease in oral hygiene was observed in a group of stressed individuals . Furthermore, direct tissue damage could be attributed to the stress process . Due to the frequent combined occurrence of the studied factors, this study evaluated the effect of alcohol intake by stressed animals . The fact that the stressed animals exposed to alcohol showed better periodontal conditions than the nonstressed animals was only a partially unexpected result of this study . Yaroslavsky and tejani - butt evaluated the relation between stress and alcohol consumption and observed that rats exposed to chronic stress consumed a greater amount of alcohol . In addition, changes in central dopamine type-2 receptor sites were found indicating an altered dopamine neurotransmission following stress and alcohol exposure . Since stressed animals consumed more alcohol, the authors concluded that it is possible that the consumption of alcohol reverses these alterations related to dopamine, suggesting a self - medicating phenotype . Compared the immunological effect of the discontinuous feeding of a liquid diet containing a moderate amount of ethanol to that of continuous ethanol administration or a control diet . The discontinuous alcohol group received the ethanol diet for 3 days and the control liquid diet for the remaining 4 days of each week (for a total of 4 weeks). The authors concluded that discontinuous drinking of a moderate amount of ethanol can be more harmful for the immune system than a continuous ethanol intake, as the model applied in the present study . Those authors suggested that probably the discontinuous alcohol intake induces a greater stress as indicated by the searched immune indicators . Therefore, future studies must be carried as to elucidate the isolate and combined effects of alcohol and stress over periodontal tissues . Our data suggest that although stress and alcohol may be harmful separately, their association probably triggered different processes . The evaluation of these factors is relevant because the combination of stress and alcohol drinking is a frequent condition in human beings with periodontitis . Although the association did not result as expected, the effects of alcohol drinking confirmed to be an important factor for the development of a periodontal disease . However, nonstressed animals who also consumed alcohol for the same period showed a pattern of periodontal breakdown even more severe . Therefore, the factors of alcohol drinking and stress do not seem to show any synergic effect.
Actinic granuloma is an uncommon granulomatous reaction hypothesized to be an autoimmune response to actinic damage to elastic tissue (o'brien, 1978). It is more commonly seen in women, with a female male ratio ranging from 1.2:1 to 2:1 (gutierrez - gonzalez et al ., 2013, limas, 2004, o'brien, 1975, ragaz and ackerman, 1979). Case studies and observational studies suggest that factors associated with this condition include diabetes mellitus and exposure to sun, radiation, intense light, tanning beds, and high heat (table 1). Actinic granuloma classically presents as an annular plaque, most commonly on sun - exposed areas of the head, neck, and upper extremities, with an atrophic or hypopigmented center and elevated erythematous borders . There is also an ocular variant of actinic granuloma, which has been reported to occur as a yellow to pink plaque on the conjunctiva (konar et al ., 2014, mittal et al ., 2013). We present this case and review of the literature to emphasize the association between actinic granuloma and temporal arteritis, a serious inflammatory condition that could lead to blindness if misdiagnosed . A 62-year - old caucasian male presented during the summer with a 3-month history of asymptomatic papules on the arms, legs, and trunk . The patient was previously treated with a course of methylprednisolone, which resulted in some improvement . Physical examination revealed multiple tan - to - pink infiltrated annular papules on the chest, back, upper arms, and legs . Laboratory studies showed a normal complete blood count, normal lyme and anaplasma titers, and negative hepatitis b and c serologies . Liver function testing showed aspartate aminotransferase (ast) of 57 and alanine aminotransferase (alt) of 79 . Erythrocyte sedimentation rate (esr) was elevated to 104 mm / hr . Histopathologic examination showed an interstitial granulomatous infiltrate in the superficial and mid - dermis composed of histiocytes, lymphocytes, occasional eosinophils, and numerous multinucleated giant cells (fig . 2). Verhoeff - van gieson stain showed fragmented elastin fibers within the cytoplasm of occasional multinucleated giant cells (fig . Periodic acid - schiff stain (pas), acid - fast bacillus (afb), and fite stains were negative . Differential diagnosis based on histopathology included actinic granuloma, interstitial granulomatous dermatitis, interstitial granulomatous drug reaction, and granuloma annulare . Given the presence of numerous multinucleated giant cells, findings on the verhoeff - van gieson stain, and solar elastosis with the time of presentation during summer, the patient was diagnosed with actinic granuloma . Although the patient s headaches were not classic enough to prompt an earlier workup for temporal arteritis, temporal artery biopsy was performed because the patient complained of headaches and had an elevated esr . Biopsy results revealed giant cell arteritis of the right temporal artery, and verhoeff - van gieson stain revealed focal disruption in the temporal artery internal elastic lamina . The patient was started on hydroxychloroquine 200 mg orally bid and prednisone 2.5 mg daily for his temporal arteritis, with partial clearance of his skin lesions . Though his skin lesions drastically improved on this regimen, persistent esr elevation required an increase in prednisone to 40 mg po daily and the addition of methotrexate . The term actinic granuloma was first proposed by john obrien in 1975 after he observed histologic similarities in patients with annular lesions . Because of its clinically similar appearance to granuloma annulare, some critics have questioned whether the condition should be classified as a separate entity or simply as granuloma annulare occurring in sun - damaged skin (ragaz and ackerman, 1979). Others have accepted the distinction but have reclassified the lesions morphologically as annular elastolytic giant cell granuloma (hanke et al ., 1979). Both of these terms, however, are descriptive only, while actinic granuloma offers an etiologic implication (limas, 2004). Histopathologic examination of actinic granuloma shows an inflammatory process limited to the superficial dermis, with usually nonpalisading granulomas, greater frequency of multinucleated giant cells, solar elastosis, and no increase in mucin (al - hoqail et al ., 2002). This is in contrast to the deep, as well as superficial, dermal infiltrate with more commonly palisading granulomas; reduced frequency of multinucleated giant cells; and increased mucin deposition seen in granuloma annulare (al - hoqail et al ., 2002). Four general histologic patterns of actinic granuloma have been described: histiocytic, giant cell, necrobiotic or vascular, and sarcoid (o'brien, 1985). The giant cell pattern is the original pattern, initially consisting of a small granuloma that becomes annular as it advances into elastotic tissue nearby . The necrobiotic variant, also known as the vascular variant, consists of areas of ischemic necrosis in the advancing granulomatous edge . Lastly, the sarcoid variant often demonstrates atrophic stroma, fibrosis, and a persistent inflammatory reaction . Of the four histologic patterns, the giant cell and necrobiotic histologic patterns appear to be more common patterns in females than males (gutierrez - gonzalez et al ., 2013). In general, treatment for actinic granuloma includes topical and intralesional corticosteroids, psoralen ultraviolet a therapy, antimalarials, cyclosporine, methotrexate, and cryotherapy (reisenauer et al ., 2012). Case reports and observational studies have demonstrated variable results with these treatment modalities (table 1). Temporal arteritis, also known as giant cell arteritis (gca), is a form of medium - to - large - vessel vasculitis with the possible complication of vision loss if left untreated (smith and swanson, 2014). Criteria for diagnosis include the presence of three or more of the following: age of 50 years or older, new headache, a clinically abnormal temporal artery, an esr greater than 50 mm / hr, and an abnormal temporal artery biopsy (smith and swanson, 2014). Interestingly, temporal arteritis occurs preferentially in women (smith and swanson, 2014). Giant cell arteritis has been most notably associated with polymyalgia rheumatica (smith and swanson, 2014). Comorbidities in addition to polymyalgia rheumatic in gca include osteoporosis, cardiovascular conditions, diabetes mellitus, hypokalemia, and pseudophakia, all conditions that appear to have a high burden in the populations affected by gca (petri et al ., 2015). Temporal arteritis has also been associated with extreme ear pain (aui - aree et al ., 2010). Other cutaneous conditions and manifestations with which temporal arteritis has had rare associations include psoriatic arthritis, scalp abscess, scalp ulceration with necrosis, butterfly rash of the face, tongue changes, gangrene or ulcers of the leg, purpura and ecchymoses, urticaria, edema, lividity, tender nodules, hyperpigmentation, tortuous arteries, and supravascular pallor (aui - aree et al ., 2010, baum et al ., 1982, corli et al . The association between actinic granuloma and temporal arteritis was first postulated in 1978 by john obrien (see also lau et al ., 1997). A few years later, the same author noted a histologic finding of actinic arteritis in a small vessel within the dermis of a polymyalgia patient (lau et al . The underlying basis of this association is thought to be related to actinic degeneration of elastic tissue not only of the skin, but also of the internal elastic lamina of vessels (gutierrez - gonzalez et al ., 2013, o'brien and regan, 1999). Since then, few reports in the literature have demonstrated the association between actinic granuloma and temporal arteritis (lau et al ., 1997, shoimer and wismer, 2011); interestingly, all cases demonstrating the association have occurred in males, despite the higher prevalence of actinic granuloma in females . Our patient had a unique clinical presentation that differed from previously reported cases of actinic granuloma associated with temporal arteritis . (1997) described two elderly gentlemen who presented with solitary lesions on the forehead, and shoimer and wismer (2011) described an elderly gentleman who presented with erythematous annular confluent plaques in predominantly sun - exposed locations . Our case further supports the possibility of temporal arteritis in a variety of presentations of actinic granuloma . Given the severe consequences of temporal arteritis, most notably blindness, we emphasize the importance of ruling out temporal arteritis in any patient presenting with actinic granuloma and headaches . If an associated temporal arteritis is suspected, systemic corticosteroids should be started to suppress the immune system and prevent blindness.

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