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Several primary and secondary prevention trials have established the beneficial role of implantable cardioverter defibrillator (icd) therapy in preventing sudden cardiac death (scd) [16]. Therefore, the guidelines indicate icd implantation in both survivors of cardiac arrest (secondary prevention) and in patients who are at high risk of developing life - threatening ventricular arrhythmias (primary prevention). Nevertheless, only a minority of patients will experience life - threatening ventricular arrhythmias (va) necessitating icd therapy, this being even more prominent in primary prevention patients [5, 6, 8, 9]. Improved patient selection is important to maintain maximum survival benefit of icd implantation while reducing the number of unnecessary implants . Microvolt t - wave alternans (mtwa) is a promising electrocardiographic risk marker for predicting scd and life - threatening va [1014]. Mtwa is a phenomenon of beat - to - beat variability in the amplitude of the t - wave . One hypothesis suggests that mtwa reflects spatial and temporal heterogeneity or dispersion in the ventricular repolarisation, which could lead to va by means of formation of functional re - entry circuits . Despite conflicting results regarding its predictive value and feasibility [1620], mtwa testing is currently incorporated in the guidelines as a diagnostic tool to improve risk stratification in patients with ischaemic and non - ischaemic severe left ventricular dysfunction [7, 21]. In this study, we aimed to prospectively evaluate the predictive value of mtwa in a real - life population of icd recipients the twente icd cohort study (tics; nl13939.044.06) is a prospective single - centre observational study of icd recipients, designed to evaluate the prognostic value of mtwa and other potential arrhythmic risk factors in predicting mortality and life - threatening va . All consecutive patients between september 2007 and march 2010 who received an icd for primary or secondary prevention of scd according to the esc guidelines were eligible for inclusion in the tics . In the currently presented mtwa substudy, only patients with ischaemic or non - ischaemic left ventricular dysfunction were included . Ischaemic heart disease was defined as left ventricular dysfunction associated with a documented history of myocardial infarction, prior coronary artery bypass surgery, prior percutaneous coronary intervention or significant narrowing of at least one of the major coronary arteries . The choice and programming of the device were left to the discretion of the implanting physician . Standard settings for our centre are ventricular fibrillation (vf) zone> 230 beats per minute (bpm), and ventricular tachycardia (vt) zone> 185 bpm with antitachycardia pacing (atp). Careful skin preparation was performed and high - resolution electrodes were used to minimise noise . In addition to the standard 12-lead ecg, three orthogonal x, y and z leads were recorded as well . Measurements were made with a hearttwave system ii (cambridge heart inc ., bedford, massachusetts, usa) using the spectral analysis method during exercise . After gradually increasing the workload to achieve a constant heart rate, a target heart rate between 100 and 110 bpm was attained and kept stable for 2.5 min . Subsequently, during 1.5 min, a target heart rate between 110 and 120 bpm was maintained . The result of the mtwa test was automatically interpreted by the alternans report classifier within the system and carefully reviewed by trained physicians . Each mtwa report was classified positive, indeterminate or negative using accepted criteria [22, 23]. A test was defined positive if the mtwa voltage was 1.9 v for at least 1 min with an onset heart rate <110 bpm or at rest in any of three orthogonal leads (x, y or z), or in two adjacent precordial leads . If the recording did not prove positive and the heart rate was> 105 bpm for at least one minute, the mtwa test was defined as negative . An mtwa test was considered indeterminate if the test did not meet the criteria for being classified as positive or negative . Based on prior literature about further analysis, patients with indeterminate or positive tests were combined as non - negative mtwa . All patients were followed on a regular basis (every 36 months) in our outpatient icd clinic, either by visits or telemonitoring . The primary endpoint was the combined endpoint of all - cause mortality and appropriate icd shock therapy for vt or vf . Secondary endpoints were all - cause mortality and appropriate icd shock therapy for vt or vf . Continuous variables are presented as mean sd, and categorical data are summarised as frequencies and percentages . Differences in baseline characteristics between mtwa positive and negative patients were analysed using student s t - test or the mann whitney u - test, as appropriate, if continuous, or chi - square or fisher s exact test if categorical . Unless otherwise specified, p - values and confidence intervals (cis) were two - sided and a p - value <0.05 was considered significant . Univariate and multivariate logistic regression analyses were performed to evaluate mtwa as an independent predictor of mortality and/or appropriate shock . All variables were evaluated by univariate analysis as possible predictors, and only those with a significance at or below p = 0.15 were analysed using multivariate logistic regression analysis . The twente icd cohort study (tics; nl13939.044.06) is a prospective single - centre observational study of icd recipients, designed to evaluate the prognostic value of mtwa and other potential arrhythmic risk factors in predicting mortality and life - threatening va . All consecutive patients between september 2007 and march 2010 who received an icd for primary or secondary prevention of scd according to the esc guidelines were eligible for inclusion in the tics . In the currently presented mtwa substudy, only patients with ischaemic or non - ischaemic left ventricular dysfunction were included . Ischaemic heart disease was defined as left ventricular dysfunction associated with a documented history of myocardial infarction, prior coronary artery bypass surgery, prior percutaneous coronary intervention or significant narrowing of at least one of the major coronary arteries . The choice and programming of the device were left to the discretion of the implanting physician . Standard settings for our centre are ventricular fibrillation (vf) zone> 230 beats per minute (bpm), and ventricular tachycardia (vt) zone> 185 bpm with antitachycardia pacing (atp). Careful skin preparation was performed and high - resolution electrodes were used to minimise noise . In addition to the standard 12-lead ecg, three orthogonal x, y and z leads were recorded as well . Measurements were made with a hearttwave system ii (cambridge heart inc ., bedford, massachusetts, usa) using the spectral analysis method during exercise . After gradually increasing the workload to achieve a constant heart rate, a target heart rate between 100 and 110 bpm was attained and kept stable for 2.5 min . Subsequently, during 1.5 min, a target heart rate between 110 and 120 bpm was maintained . The result of the mtwa test was automatically interpreted by the alternans report classifier within the system and carefully reviewed by trained physicians . Each mtwa report was classified positive, indeterminate or negative using accepted criteria [22, 23]. A test was defined positive if the mtwa voltage was 1.9 v for at least 1 min with an onset heart rate <110 bpm or at rest in any of three orthogonal leads (x, y or z), or in two adjacent precordial leads . If the recording did not prove positive and the heart rate was> 105 bpm for at least one minute, the mtwa test was defined as negative . An mtwa test was considered indeterminate if the test did not meet the criteria for being classified as positive or negative . Based on prior literature about further analysis, patients with indeterminate or positive tests were combined as non - negative mtwa . All patients were followed on a regular basis (every 36 months) in our outpatient icd clinic, either by visits or telemonitoring . The primary endpoint was the combined endpoint of all - cause mortality and appropriate icd shock therapy for vt or vf . Secondary endpoints were all - cause mortality and appropriate icd shock therapy for vt or vf . Continuous variables are presented as mean sd, and categorical data are summarised as frequencies and percentages . Differences in baseline characteristics between mtwa positive and negative patients were analysed using student s t - test or the mann whitney u - test, as appropriate, if continuous, or chi - square or fisher s exact test if categorical . Unless otherwise specified, p - values and confidence intervals (cis) were two - sided and a p - value <0.05 was considered significant . Univariate and multivariate logistic regression analyses were performed to evaluate mtwa as an independent predictor of mortality and/or appropriate shock . All variables were evaluated by univariate analysis as possible predictors, and only those with a significance at or below p = 0.15 were analysed using multivariate logistic regression analysis . From september 2007 until march 2010, 503 patients received an icd . Of these patients, 300 no informed consent was obtained . Of these 300 patients, 269 patients with ischaemic or non - ischaemic left ventricular dysfunction were eligible for this mtwa substudy . Mtwa testing was performed in 134 of 269 (49.6%) patients . In 62 of 269 (23.0%) patients mtwa was not performed due to logistic reasons (mainly secondary prevention icd recipients who received their icd during hospitalisation), 7 of 269 (2.6%) patients refused to participate and 66 of 269 (24.5%) patients were not capable of performing exercise - based mtwa testing . In the 134 tested patients (81% male, mean age 62 years, mean left ventricular ejection fraction (lvef) 26.5%), mtwa was positive in 48 (35.8%) patients, negative in 48 (35.8%) patients and indeterminate in 38 (28.3%) patients (due to noise (9 (6.7%) patients), frequent ventricular or atrial ectopy (15 (11.2%) patients), or not achieving the target heart rate (14 (10.4%) patients). The clinical characteristics are presented in table 1 . Among patients with a non - negative test result, there was a trend for association between lower lvef and non - negative mtwa.table 1clinical characteristicsallnegativenon - negativeineligiblenumber of patients200488666general age (years sd)63.0 (10.3)59.8 (11.9)63.0 (9.5)65.8 (9.8) * * gender (male)170 (84.6)33 (68.8)75 (87.2)*62 (93.9) * * lvef (% sd)26.7 (12.2)29.1 (13.1)25.0 (11.2)27.0 (12.8)comorbidities hypertension53 (26.4)12 (25.0)24 (27.9)18 (27.3) dm47 (23.4)11 (22.9)14 (16.3)22 (33.3) copd21 (10.4)2 (4.2)11 (12.8)7 (10.6) cva / tia14 (7.0)3 (6.2)4 (4.7)7 (10.6) af52 (25.9)5 (10.4)9 (10.5)40 (60.6)**indication primary161 (80.5)38 (79.2)74 (86.0)49 (74.2) secondary39 (19.5)10 (20.8)12 (14.0)17 (25.8)aetiology ischaemic135 (67.2)34 (70.8)53 (61.6)48 (74.2) dilated66 (32.8)14 (29.2)44 (38.4)17 (25.8)medication beta - blocker169 (84.1)42 (87.5)70 (81.4)55 (83.3) acei / arb169 (84.1)50 (83.3)75 (87.2)53 (80.3) diuretics153 (76.1)32 (66.7)66 (76.7)55 (83.3) * amiodarone20 (10.0)4 (8.3)6 (7.0)10 (15.2) nyha functional class 353 (26.8)7 (14.6)18 (21.2)29 (45.3) * * qrs duration (ms sd)125 (31)115 (30)125 (28)131 (34)**p <0.05 and * * p <0.01 in comparison with twa negative group lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart association clinical characteristics * p <0.05 and * * p <0.01 in comparison with twa negative group lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart association during a mean follow - up of 38 10 months, 21 patients (15.6%) reached the primary endpoint of death (10 (7.5%) patients) and/or appropriate shock therapy (11 (8.2%) patients). Predictors for the combined endpoint of mortality and appropriate shocks (table 2) were wider qrs duration (p = 0.03) and chronic obstructive pulmonary disease (p = 0.01). No significant relation was found between non - negative mtwa testing and the primary endpoint (p = 0.58, fig . 1).table 2predictors for the combined endpoint of mortality and appropriate therapyunivariate analysishazard ratio p age1.03 (0.991.07)0.16male gender1.13 (0.423.04)0.89lvef0.99 (0.961.03)0.67comorbidities hypertension0.54 (0.181.59)0.26 dm1.25 (0.460.37)0.66 copd3.47 (1.368.84)0.01 cva / tia0.04 (0.0064.08)0.40 af1.51 (0.455.10)0.50prophylactic indication0.55 (0.221.39)0.21ischaemic cardiomyopathy0.74 (0.331.69)0.47nyha functional class 31.22 (0.453.30)0.69qrs duration (ms sd)1.01 (1.001.03)0.03non - negative mtwa1.29 (0.533.13)0.58hazard ratio (95% ci). Hazard ratio for qrs duration per 1 ms increase in duration lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart associationfig . 1survival curves regarding the role of mtwa in predicting a mortality and/or appropriate shock therapy, b mortality and c appropriate shock therapy predictors for the combined endpoint of mortality and appropriate therapy hazard ratio (95% ci). Hazard ratio for qrs duration per 1 ms increase in duration lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart association survival curves regarding the role of mtwa in predicting a mortality and/or appropriate shock therapy, b mortality and c appropriate shock therapy ischaemic left ventricular dysfunction was present in 87 (64.9%) patients, of whom 53 (60.9%) had a non - negative test result . Non - ischaemic left ventricular dysfunction was present in 47 (35.1%) patients, of whom 33 (70.2%) had a non - negative test result . Mtwa was neither predictive for the combined endpoint of death and/or appropriate shock in ischaemic or non - ischaemic lv dysfunction (all p - values> 0.66). Regarding the secondary endpoint mortality, significant associations were found with lower lvef (p = 0.03), chronic obstructive pulmonary disease (p = 0.05), nyha functional class 3 (p = 0.02), non - negative mtwa (p = 0.05, fig . 1) or qrs duration (p = 0.03). After constructing a multivariate model using these parameters, multivariate analysis showed that mtwa was no longer an independent predictor (p = 0.15). In the patients with ischaemic left ventricular dysfunction, 6 (6.9%) patients died, all in the non - negative mtwa group . Using kaplan - meier survival analysis, a significant relation between non - negative mtwa and mortality was found (p = 0.04). Other significant univariate variables were lower lvef and wider qrs duration . After correction using multivariate regression analysis, non - negative mtwa lost its significance . In the non - ischaemic lv dysfunction group, 5 (10.6%) patients died, four in the non - negative group and one in the negative group . No relation was found between mtwa and mortality (p = 0.55). Appropriate shock therapy as alternative secondary endpoint occurred in 11 (8.2%) patients, 5 (5.8%) patients with a non - negative mtwa and 6 (12.5%) patients with a negative mtwa (p = 0.19, fig . 1). In the patients with ischaemic left ventricular dysfunction, 8 (9.2%) patients experienced appropriate shock therapy . Three (37.5%) patients had a non - negative test, and 5 (62.5%) had a negative test (p = 0.14). In the patients with non - ischaemic lv dysfunction, 3 (6.3%) patients experienced appropriate shock therapy . Two events (66%) occurred in patients with a non - negative test result whereas 1 (33%) patient with a negative test result experienced appropriate therapy (ns, p = 0.97). In 66 of 269 (24.5%) patients, mtwa by means of exercise stress testing was technically not feasible because of atrial fibrillation (n = 43), pacemaker - dependency (n = 8), or clinical state (n = 15). P = 0.01), older (p <0.01), more often known with atrial fibrillation (p <0.001), and a lower functional class (p <0.001). The combined primary endpoint of mortality and appropriate shock was reached in 26 (39.4%) patients . This was significantly higher than in the non - negative (rr 2.5 (95% ci 1.3 - 4.7) p <0.01) and negative (rr 3.3 (95% ci 1.4 - 7.6), p <0.01) mtwa group . After multivariate correction for confounders, ineligibility for twa testing remained the only significant predictor of the combined endpoint of mortality and appropriate shock therapy (fig . 1). Regarding the secondary endpoints, compared with non - negative patients, ineligible patients experienced both higher rates of mortality (p = 0.01, fig . 1) and appropriate shocks (p = 0.01, fig . 1). From september 2007 until march 2010, 503 patients received an icd . Of these patients, 300 no informed consent was obtained . Of these 300 patients, 269 patients with ischaemic or non - ischaemic left ventricular dysfunction were eligible for this mtwa substudy . Mtwa testing was performed in 134 of 269 (49.6%) patients . In 62 of 269 (23.0%) patients mtwa was not performed due to logistic reasons (mainly secondary prevention icd recipients who received their icd during hospitalisation), 7 of 269 (2.6%) patients refused to participate and 66 of 269 (24.5%) patients were not capable of performing exercise - based mtwa testing . In the 134 tested patients (81% male, mean age 62 years, mean left ventricular ejection fraction (lvef) 26.5%), mtwa was positive in 48 (35.8%) patients, negative in 48 (35.8%) patients and indeterminate in 38 (28.3%) patients (due to noise (9 (6.7%) patients), frequent ventricular or atrial ectopy (15 (11.2%) patients), or not achieving the target heart rate (14 (10.4%) patients). The clinical characteristics are presented in table 1 . Among patients with a non - negative test result, there was a trend for association between lower lvef and non - negative mtwa.table 1clinical characteristicsallnegativenon - negativeineligiblenumber of patients200488666general age (years sd)63.0 (10.3)59.8 (11.9)63.0 (9.5)65.8 (9.8) * * gender (male)170 (84.6)33 (68.8)75 (87.2)*62 (93.9) * * lvef (% sd)26.7 (12.2)29.1 (13.1)25.0 (11.2)27.0 (12.8)comorbidities hypertension53 (26.4)12 (25.0)24 (27.9)18 (27.3) dm47 (23.4)11 (22.9)14 (16.3)22 (33.3) copd21 (10.4)2 (4.2)11 (12.8)7 (10.6) cva / tia14 (7.0)3 (6.2)4 (4.7)7 (10.6) af52 (25.9)5 (10.4)9 (10.5)40 (60.6)**indication primary161 (80.5)38 (79.2)74 (86.0)49 (74.2) secondary39 (19.5)10 (20.8)12 (14.0)17 (25.8)aetiology ischaemic135 (67.2)34 (70.8)53 (61.6)48 (74.2) dilated66 (32.8)14 (29.2)44 (38.4)17 (25.8)medication beta - blocker169 (84.1)42 (87.5)70 (81.4)55 (83.3) acei / arb169 (84.1)50 (83.3)75 (87.2)53 (80.3) diuretics153 (76.1)32 (66.7)66 (76.7)55 (83.3) * amiodarone20 (10.0)4 (8.3)6 (7.0)10 (15.2) nyha functional class 353 (26.8)7 (14.6)18 (21.2)29 (45.3) * * qrs duration (ms sd)125 (31)115 (30)125 (28)131 (34)**p <0.05 and * * p <0.01 in comparison with twa negative group lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart association clinical characteristics * p <0.05 and * * p <0.01 in comparison with twa negative group lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart association during a mean follow - up of 38 10 months, 21 patients (15.6%) reached the primary endpoint of death (10 (7.5%) patients) and/or appropriate shock therapy (11 (8.2%) patients). Predictors for the combined endpoint of mortality and appropriate shocks (table 2) were wider qrs duration (p = 0.03) and chronic obstructive pulmonary disease (p = 0.01). No significant relation was found between non - negative mtwa testing and the primary endpoint (p = 0.58, fig . 1).table 2predictors for the combined endpoint of mortality and appropriate therapyunivariate analysishazard ratio p age1.03 (0.991.07)0.16male gender1.13 (0.423.04)0.89lvef0.99 (0.961.03)0.67comorbidities hypertension0.54 (0.181.59)0.26 dm1.25 (0.460.37)0.66 copd3.47 (1.368.84)0.01 cva / tia0.04 (0.0064.08)0.40 af1.51 (0.455.10)0.50prophylactic indication0.55 (0.221.39)0.21ischaemic cardiomyopathy0.74 (0.331.69)0.47nyha functional class 31.22 (0.453.30)0.69qrs duration (ms sd)1.01 (1.001.03)0.03non - negative mtwa1.29 (0.533.13)0.58hazard ratio (95% ci). Hazard ratio for age per 1 year increase in age . Hazard ratio for lvef per 1% increase in lvef . Hazard ratio for qrs duration per 1 ms increase in duration lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart associationfig . 1survival curves regarding the role of mtwa in predicting a mortality and/or appropriate shock therapy, b mortality and c appropriate shock therapy predictors for the combined endpoint of mortality and appropriate therapy hazard ratio (95% ci). Hazard ratio for qrs duration per 1 ms increase in duration lvef left ventricular ejection fraction, dm diabetes mellitus, copd chronic obstructive pulmonary disease, cva cerebrovascular accident, tia transient ischaemic attack, af atrial fibrillation, acei angiotensin - converting enzyme inhibitor, arb angiotensin receptor blocker, nyha new york heart association survival curves regarding the role of mtwa in predicting a mortality and/or appropriate shock therapy, b mortality and c appropriate shock therapy ischaemic left ventricular dysfunction was present in 87 (64.9%) patients, of whom 53 (60.9%) had a non - negative test result . Non - ischaemic left ventricular dysfunction was present in 47 (35.1%) patients, of whom 33 (70.2%) had a non - negative test result . Mtwa was neither predictive for the combined endpoint of death and/or appropriate shock in ischaemic or non - ischaemic lv dysfunction (all p - values> 0.66). Regarding the secondary endpoint mortality, significant associations were found with lower lvef (p = 0.03), chronic obstructive pulmonary disease (p = 0.05), nyha functional class 3 (p = 0.02), non - negative mtwa (p = 0.05, fig . 1) or qrs duration (p = 0.03). After constructing a multivariate model using these parameters, multivariate analysis showed that mtwa was no longer an independent predictor (p = 0.15). In the patients with ischaemic left ventricular dysfunction, 6 (6.9%) patients died, all in the non - negative mtwa group . Using kaplan - meier survival analysis, a significant relation between non - negative mtwa and mortality was found (p = 0.04). Other significant univariate variables were lower lvef and wider qrs duration . After correction using multivariate regression analysis, non - negative mtwa lost its significance . In the non - ischaemic lv dysfunction group, 5 (10.6%) patients died, four in the non - negative group and one in the negative group . No relation was found between mtwa and mortality (p = 0.55). Appropriate shock therapy as alternative secondary endpoint occurred in 11 (8.2%) patients, 5 (5.8%) patients with a non - negative mtwa and 6 (12.5%) patients with a negative mtwa (p = 0.19, fig . 1). In the patients with ischaemic left ventricular dysfunction, 8 (9.2%) patients experienced appropriate shock therapy . Three (37.5%) patients had a non - negative test, and 5 (62.5%) had a negative test (p = 0.14). In the patients with non - ischaemic lv dysfunction, 3 (6.3%) patients experienced appropriate shock therapy . Two events (66%) occurred in patients with a non - negative test result whereas 1 (33%) patient with a negative test result experienced appropriate therapy (ns, p = 0.97). In 66 of 269 (24.5%) patients, mtwa by means of exercise stress testing was technically not feasible because of atrial fibrillation (n = 43), pacemaker - dependency (n = 8), or clinical state (n = 15). Ineligible patients were more frequently male (p = 0.01), older (p <0.01), more often known with atrial fibrillation (p <0.001), and a lower functional class (p <0.001). The combined primary endpoint of mortality and appropriate shock this was significantly higher than in the non - negative (rr 2.5 (95% ci 1.3 - 4.7) p <0.01) and negative (rr 3.3 (95% ci 1.4 - 7.6), p <0.01) mtwa group . After multivariate correction for confounders, ineligibility for twa testing remained the only significant predictor of the combined endpoint of mortality and appropriate shock therapy (fig . 1). Regarding the secondary endpoints, compared with non - negative patients, ineligible patients experienced both higher rates of mortality (p = 0.01, fig . 1) and appropriate shocks (p = 0.01, fig . 1). In this study, non - negative mtwa was not associated with mortality and/or appropriate shock therapy . In fact, there was no relation between va requiring icd shock therapy and non - negative mtwa . Furthermore, patients in whom mtwa is not technically feasible experienced highest risk for mortality and appropriate shock therapy . Some studies state that mtwa is a potentially useful predictor of va and mortality in patients with ischaemic or non - ischaemic heart disease [1014]. Other studies reported results which are less promising . The relation with mortality was still significant, but no relation between mtwa and va was found . The results of the mtwa substudy of the scd - heft are in concordance with our study, namely not showing a relation between mtwa and mortality or appropriate icd therapy . The differences in outcome with prior studies which did find significant relations between mtwa and scd [1014] can possibly be explained by the use of appropriate icd therapy as a surrogate endpoint for arrhythmic mortality which is an overestimation, and the use of beta - blockers during exercise testing although this is recommended in the current consensus [16, 17, 21]. Recently, doubt has been raised about the feasibility of mtwa testing in potential icd recipients . In our study, mtwa testing was not technically feasible in 24.5% of included patients . In the literature, if testing in ineligible patients is mandatory, mtwa can be assessed using a protocol based on pacing or pharmacological increase of heart rate . These protocols, however, are not useable in patients with atrial fibrillation or other irregular heart rhythms, but are very useful for patients who cannot reach the target heart rate . Concordance between these different protocols varies between 55 and 98% [2529]. Found no relation with death, sustained ventricular arrhythmia or appropriate icd therapy using an atrial paced protocol . No data are reported on the prognostic value of ventricular pacing - based protocols or pharmacological intervention protocols to increase heart rate . The question remains whether these patients need to be tested by means other than exercise stress testing mtwa protocols since in both our study and the studies by shizuta and jackson, patients in whom exercise - based mtwa testing is not possible experience the highest risk of mortality and va requiring icd therapy, which remains statistically significant after correction for possible confounders [19, 20]. The main limitation of this trial is the number of patients in combination with the low event rate . The main limitation of this trial is the number of patients in combination with the low event rate . In the present study, we did not find a significant relation between non - negative mtwa testing and mortality or appropriate shock therapy.
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Prior to the antivascular endothelial growth factor (anti - vegf) era, age - related macular degeneration (amd) was considered the leading cause of severe visual loss and blindness in the developed world among people over the age of 50 years . Various imaging methods are available for the diagnosis and classification of amd . Until recently, color fundus photography (fp) was the gold standard for grading and staging in amd clinical trials [24]. By permitting visualisation of the choroidal and retinal microcirculation and providing detailed information about the presence of pathological vessels as well as the integrity of the blood retinal barrier, fluorescein angiography (fa) had become a central tool for detecting and classifying cnv as well as cnv activity in eyes with neovascular amd [5, 6]. During the past years, oct has dramatically gained importance for the diagnosis and management of patients with chorioretinal disease by noninvasively providing cross - sectional images of the neurosensory retina and the subretinal space, thus allowing a detailed characterization of structural changes . Thus, oct is increasingly used to determine the presence and activity of cnv and the need for (re-) treatment [79]. New - generation spectral domain oct (sdoct) instruments provide even higher resolution and more dense coverage of the macular area compared with time domain oct . Therefore, sdoct imaging is now widely used for the followup of patients with cnv undergoing anti - vegf therapy . This study aims to compare fp, fa, and sdoct imaging regarding their sensitivity and specificity for detecting amd, cnv, and cnv activity and to analyze whether sdoct may have the potential to replace the other imaging techniques . The european genetic database (eugenda), a database collecting amd patients as well as healthy controls, was retrospectively reviewed, and fp, fa, and sdoct images of 120 eyes of 66 consecutive patients were randomly collected . Eyes with early, intermediate, or late amd as well as control cases were included . Control eyes were required to show no signs for amd, but other chorioretinal diseases including cnv secondary to any other disease but amd was allowed . To be eligible for this study, all images had to be performed on the same day at the university of cologne, germany . Fa images were performed using the spectralis hra system (heidelberg engineering, heidelberg, germany). The standard protocol included 30 stereo images of the transit phase, mid phase, and late phase up to 10 minutes following intravenous injection of fluorescein . Sdoct images were acquired using the spectralis sdoct instrument (heidelberg engineering, heidelberg, germany). Sdoct volume scans (15 20) composing of 37 parallel oct b - scans were used for analysis . For each oct b - scan, 20 images were averaged using the automated real - time (art) function . Images were independently analyzed by reading center graders (tr, nfm, and sl) at the cologne image reading center (circl), which have been trained and certified in image interpretation of amd patients . Discrepancies between graders have been solved by open adjudication . During analysis of one imaging technique, for all images, the presence of amd, cnv, and cnv activity was noted (table 1, figure 1). Amd was defined as the presence of 10 small (63 m), hard drusen and pigmentary changes or at least 1 intermediate (64124 m) or large (125 m) drusen inside the 6 mm etdrs grid . Cnv was considered present on fp, if subretinal or subrpe fibrosis and fibrovascular tissue or fibrin were seen; on sdoct, subretinal hyperreflective material or pigment epithelial detachments (peds) other than single drusen were considered signs for cnv . On fa, cnv lesions were graded according to the modified macular photocoagulation study (mps) grading protocol utilized in the treatment of amd with photodynamic therapy (tap) and verteporfin in photodynamic therapy (vip) studies [11, 12]. Briefly, classic cnv was identified as an area of uniform early hyperfluorescence that showed extensive leakage in the mid and late phases . Occult cnv was classified as areas of stippled hyperfluorescence that appeared in the mid and late phases of the fluorescein angiography . Cnv was graded as present on fa, if classic or occult cnv lesion components or staining scar tissue was detected . Cnv activity was noted, if fluid or hemorrhage was present on fp that was not related to any other retinal vascular disease but cnv, if classic or occult cnv leakage was detected on fa or if diffuse or cystoid intraretinal fluid or subretinal fluid accumulation was seen on sdoct . For each parameter to be evaluated, the following imaging modalities were defined as the gold standard: for presence of amd, fp was used as the gold standard . For the presence of cnv cnv activity was considered present if it was detected on either sdoct or fa (ground truth). Signs for amd were detected on fa in 77 eyes with a sensitivity of 92% (69 out of 75) and a specificity of 82% (in 8 cases, amd was noted on fa but not on color fundus photographs). Disagreement between fa and fp was mainly related to small drusen that have been noted on fa but not on fp, and rpe hyperpigmentation that has been seen on fps but not on fas . On sdoct, amd was considered present in 78 eyes with a sensitivity of 89% (67 out of 75) and a specificity of 76% (in 11 cases, signs for amd were noted on sdoct but not on fp). Disagreement between sdoct and fp could mainly be explained by small or intermediate drusen and rpe changes that have been missed on sdoct and by intermediate drusen that have been noted on sdoct but not on fps . Twenty out of those eyes showed pathologies other than amd, including high myopia, chorioretinitis, retinal vein occlusion, epiretinal membranes, diabetic retinopathy and central serous retinopathy, or idiopathic cnv . Fifty - four out of those cases were diagnosed with amd based on fps . In 14 cases, cnv was seen in the control group (idiopathic or related to high myopia or chorioretinitis). Fp showed a sensitivity of 78% (53 out of 68 eyes) for the detection of cnv, and a specificity of 100% . Sdoct images showed signs for cnv in 64 out of the 68 cases (94%), specificity for detecting cnv was 98% (in one case, cnv was diagnosed based on sdoct but not on fa). In the 64 cases with agreement between fa and sdoct regarding the presence of cnv, a classic cnv lesion component was detected on fa in 25 eyes . Ten out of those 15 cases showed staining scar as a lesion component on fa, and 5 cases demonstrated occult cnv leakage only . Thirty - eight out of the 64 cases showed occult cnv lesion components on fa, with all of those demonstrating a ped on sdoct . In addition, a ped was seen in 22 eyes without occult cnv lesion components on fa, with 18 (82%) out of those cases demonstrating staining scar as a lesion component and 4 (18%) cases showing classic cnv leakage only . Out of the 68 cases with cnv diagnosed based on fa, a total of 60 cases (88%) showed signs for active cnv either on sdoct (53 eyes) or fa (53 eyes), with an agreement between both imaging modalities in 79% out of all 68 cases (46 cases showed active cnv and 8 eyes no signs for cnv activity on both imaging modalities). In 7 cases, fluid was detected on sdoct without evidence for cnv leakage on fa and vice versa . If the ground truth for sdoct and fa was considered the gold standard for cnv activity, sensitivity was 88% for fa, 88% for sdoct, and 38% (23 out of 60) for fp, respectively . Specificity for fp was 98% (in one case, cnv activity was suspected based on fp but not seen on fa or sdoct). Sdoct is increasingly used in clinical trials as well as in clinical practice for the diagnosis and followup of patients with neovascular amd undergoing anti - vegf therapy . As a noninvasive imaging tool, it provides high - resolution cross - sectional images of retinal pathology, allowing to qualitatively and quantitatively analyze various parameters relevant for (re-) treatment decisions . Our study confirms that sdoct is highly sensitive for detecting amd, cnv, and cnv activity; however, it may not yet fully replace the information provided by fa and fp . The presence of characteristic features of amd on fp such as drusen and rpe changes was missed on sdoct in 11% of cases in our study . This may be explained by the sdoct volume scan settings used, as the gap between two parallel oct b - scans was approximately 120 m; thus pathological changes may fall in between two adjacent scans and may be overlooked or may appear smaller than they truly are . On the other hand, amd was diagnosed on sdoct based on the presence of intermediate drusen in 24% of cases that were graded as control cases on fp . On fp, those pathological features have been either interpreted as rpe changes or small drusen, or they have been overlooked due to reduced image clarity . Thus, sdoct may be helpful in identifying drusen and in differentiating drusen from hypopigmentation and thus may improve the quality of image interpretation in eyes with amd compared to fp imaging alone . However, care should be taken not to transfer size definitions for intermediate or large drusen from fps to sdoct, as due to their shape, drusen may appear larger on sdoct compared to fp, or they may appear smaller if they are captured at the border . Further studies are needed to compare drusen sizes between those different imaging modalities before sdoct imaging can be reliably used for staging of early and intermediate amd in clinical trials . Other imaging techniques such as autofluorescence imaging provide additional information concerning drusen and rpe changes and may thus be helpful to identify and classify those features . Based on fa, cnv lesions components are categorized as classic or occult cnv leakage or staining scar tissue that may develop over time and indicate longstanding disease with poor visual function . Reported a sensitivity of only 40% for the detection of new - onset cnv on time - domain oct . The low sensitivity may be explained by the use of time - domain oct, as pathological features may be overlooked more easily compared to sdoct due to the less dense scan pattern, lower image resolution, and higher rate of movement artifacts . Occult cnv on fa is believed to correspond histologically to type 1 cnv, located between the rpe and bruch's membrane . In accordance with this, all eyes with occult cnv on fa demonstrated a ped on sdoct in our study . In contrast, classic cnv lesion components on fa histologically correspond to type 2 cnv, positioned in the subretinal space . Thus, type 2 cnv lesion components are expected to present as hyperreflective material in the subretinal space on oct . This could be confirmed in our study as all cases with classic cnv lesion components on fa demonstrated subretinal hyperreflective material on sdoct . However, in order to correctly interpret oct images, it is crucial to consider that oct scans only represent pseudohistological images, created using information about the reflectivity and axial distribution of various structures . Hence, subretinal hyperreflective material on oct scans may not only represent type 2 cnv, but may include, for example, subretinal hemorrhage, fibrinous material, or photoreceptor debris . This may explain why subretinal hyperreflective material was seen in our study on sdoct in 5 cases demonstrating occult cnv on fa without the presence of a classic cnv leakage or staining scar as lesion components . Additionally, a ped was seen on sdoct in 22 eyes without the presence of occult cnv on fa . In those cases, other cnv lesion components such as staining scar or classic cnv may have covered the cnv membrane located in the sub - rpe space; thus occult cnv leakage was not detectable on fa . This finding indicates that high - resolution cross - sectional images provided by sdoct may add important information regarding subretinal and sub - rpe pathology compared to the two - dimensional en - face view of fa and fp imaging . Agreement between sdoct and fa regarding the activity of cnv lesions in our study was seen in 79% of all 68 cases diagnosed with cnv on fa . Seven eyes demonstrated cnv leakage on fa in the absence of intra- or subretinal fluid on sdoct, and 7 eyes showed signs for cnv activity on sdoct without evidence of cnv leakage on fa . Reported a sensitivity of 90% and specificity of 47% for sdoct to detect cnv activity seen on fa . The disagreement between both imaging modalities may be explained by the fact that fa and sdoct imaging provides different information about retinal pathology . Fa is used to obtain information about the perfusion and the growth of new vessels as well as the integrity of the blood - retinal barrier; thus fluorescein leakage over time can be seen during angiography . This information is missing on oct images; thus oct provides detailed information about pathological changes like, for example, the presence of cystoid spaces; however, it is not possible to detect whether they are caused by fluid accumulation from acute leakage from pathological vessels . Thus, cystoid spaces on sdoct may not necessarily correspond to fluorescein leakage on fa, but may represent structural defects indicating chronic disease (figure 2). Increase or decrease in the amount of fluid seen on oct may thus more reliably indicate cnv activity than the presence of fluid seen at one time point . In addition, care should be taken to not confuse intraretinal cystoid spaces or subretinal fluid with in their paper, the authors state that degenerating photoreceptors may become arranged in a circular or ovoid fashion in chronic diseases affecting the outer retina and rpe . In contrast, cnv activity seen on fa may be missed on sdoct if only intraretinal cystoid spaces and subretinal fluid accumulation are considered to represent cnv activity on sdoct . Recently reported that intraretinal hyperreflective flecks and the inherent reflectivity and boundary definition of subretinal hyperreflective material may indicate active cnv even in the absence of intra- or subretinal fluid accumulation . Additionally, fluid accumulation in the sub - rpe space such as serous components of a ped may indicate cnv activity . Fa imaging at baseline in addition to sdoct is helpful to assess the cnv lesion subtype and the initial severity of cnv leakage; during followup, fa may confirm evidence of cnv activity whenever sdoct interpretation is challenging or inconsistent with retinal function . Sdoct, fa, and fp imaging provide complementary information about pathological changes in chorioretinal diseases . Our study indicates that drusen and rpe changes as signs for amd are best appreciated on fp . Sdoct is highly sensitive to identify cnv and cnv activity; however, it cannot fully replace fa in the management of patients with cnv . Further studies are needed to evaluate which sdoct parameters (e.g., cystoid spaces, diffuse intraretinal fluid, subretinal or sub - rpe fluid, inherent boundary definition of subretinal hyperreflective material, or a change in the amount of fluid) best indicate cnv activity.
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Thus, many recent studies have sought to extract interaction information from biomolecular text using natural language processing technology . However, we have insufficient biomolecular data annotated with linguistic information . In 2005, the icml05 workshop on learning language in logic (lll05) task provided a small training dataset annotated with pos - tags and syntactic relations . Previous studies have insisted that linguistic information was useful for improving the detection of gene interactions . However, the experimental results for the lll05 data gave a reasonable precision but poor recall . To improve recall without sacrificing precision, we propose a three - phase method to detect gene interactions using syntactic relation information, and apply it to a small training dataset lacking domain knowledge . Through experimentation, we show that our proposed method significantly outperforms existing methods, and describe the contribution of each phase to its performance . Section 4 describes the training and test data used for our experiments and presents experimental results that demonstrate that our three - phase method is effective for detecting gene interactions . The task of relation mining in the biomedical domain has been studied extensively in recent years . Current research includes protein - protein interactions [2, 3], subcellular locations, and disease - treatment relationships, and systems based on sequence modeling and pattern- or rule - based extraction best detect protein - protein interactions [2, 6, 7]. Using text mining technology for automatic protein(gene) interactions resulted in high precision, but low recall . Many studies have used linguistic information to improve performance in detecting gene interactions . To improve recall without sacrificing precision, otasek et al . Expanded the diversity of sentence structures recognized by a syntactic parser through additional training, and park et al . Presented a method using bidirectional incremental parsing . Experiments deduced 182 relations out of 492 sentences showing 48% recall and 80% precision . Many linguistic processes have been used to deduce gene interactions, including bidirectional incremental parsing, combinatory categorical grammar (ccg), coordination, apposition, compound noun processing, and positive / negative predicate learning . With these methods, assumed that sentences derived from sets of abstracts contained a significant number of protein names connected by verbs that indicate the type of relationship between them . They restricted the problem domain and imposed several strong assumptions that included prespecified protein names and a limited set of verbs to represent actions . Then, they learned patterns with finite - state automata based on a genetic algorithm . For example, agent1, target3, pattern2 implies that agent1 interacts with target3 via pattern2 . In biomolecular text, the agent or target can be encapsulated in another term based on some conditions, for example, apposition, modifying nouns, and so on . However, the method in cannot deal with a situation in which genes are encapsulated in other terms via syntactic relations . Error analysis revealed that they wrongly detected an agent and its target in a pair of genes, although they correctly detected two genes that interact with each other . The nodes in the dependency trees from which patterns were derived were either a lexical item or a semantic category, such as a gene, protein, agent, or target . They extended the patterns based on eight seed patterns and trained the model using the basic dataset without coreference, as provided by the lll05 challenge organizers . The failure of the system to extract meaningful relations can be traced back to the errors that minipar introduced in the dependency trees . Used gleaner as an inductive logic programming approach and further applied brill tagger, a shallow parser based on conditional random fields, and porter stemmer . They also used much linguistic information, including sentence - structure predicates, the frequencies of words, lexical properties, and semantic knowledge using mesh . Gleaner suffered from not distinguishing between an agent and a target well because no syntactic structure was used . Popelinsky and blatak used brill tagger and wordnet, and katrenko et al . Created a simple ontology specifically for use in the lll05 challenge riedel and klein obtained the best performance on the lll05 challenge task using syntactic chains . Therefore, they generated a set of clauses based on chains of syntactic relations between two genes . The method achieved an f - measure of 52.6% on the dataset without coreferences, demonstrating that using syntactic information from the annotated datasets significantly improved performance . However, recall was only 46.2%, and the system needs to improve recall . For genia and atcr data, they find agents and targets from the syntactic patterns directly connected with interaction verbs with subject or object functions . So, they do not consider the case that agent or target is encapsulated in another term, and indirectly connected with interaction verbs . In addition, there is a limit that they find agents and targets only from the subject and object relations . Combining syntactic dependency information with features based on word sequences could lead to further improvements in performance, as demonstrated by the more recent approaches to relation extraction [1921]. We build on the conclusion of the previous work that linguistic information, especially syntactic information, is an important key for detecting gene interactions . However, we need a more robust method to improve recall without sacrificing precision . Based on syntactic relation information, we propose a three - phase - based method for detecting gene interactions . Mentioned the failure of the system to extract meaningful relations can be traced back to the errors of the applied syntactic analyzer . If we use the annotated lll05 syntactic relation information, we cannot testify the robustness of our system in real time . So, we also experiment the performance of our system based on a real - syntactic analyzer . To objectively compare the performance of our system with that of previous systems, we use lll05 data . In the next section, the first subset does not include coreferences or ellipsis, unlike the second subset . The training set without coreferences consists of 55 sentences, including 106 examples of genic interactions . It contains 70 examples of action, 30 examples of binding and promoter, and 6 examples of regulation . A syntactic relation is important linguistic information for detecting the structure of text . Algorithm 1 shows one example of syntactic relations between two genes in the lll05 data . The syntactic relations provided in lll05 were of the form r, w, w), where r is one of a fixed set of syntactic relations between w and w assigned by the lll parser . The chain of terms is spo0a(agent) protein(n) depend(v) transcription(n) gene(n) spoiig(target). In the chain, node depend(v) is the verb that indicates the interaction between spo0a(agent) and spoiig(target). However, depend(v) has direct syntactic relations with protein(n) and transcription(n), not with spo0a(agent) or spoiig(target). In other words, spo0a(agent) was encapsulated in protein(n) with the relation (mod_att), and spoiig(target) was encapsulated in transcription(n) with the relation (mod_att) and (comp_of). Without any domain knowledge of biomolecular text, we automatically detect gene interactions using syntactic relations annotated in the lll05 data . In the first phase, to improve recall, we detect the relations that encapsulate an agent or target . In the second phase, next, to improve precision, we must determine which of the two genes is the agent and which is the target . To determine the agent and target for two genes, we learn direction rules on the relations from agent to target in the third phase . An agent or target gene is usually encapsulated in another term, and the verb that indicates the interaction between two genes has syntactic relations with two terms that encapsulate the genes . To improve recall for gene interactions first, we find the syntactic chain from an agent to its target . In figure 1, depend(v) is the verb that indicates an interaction between spo0a(agent) and spoiig(target). In this paper, we call the verb that indicates the interaction between an agent and its target an interaction verb . As mentioned above, depend(v) has syntactic relations with protein(n) and transcription(n), but not with spo0a(agent) or spoiig(target). In a syntactic chain from an agent to its target, we call the node preceding an interaction verb a metaagent, and the node following an interaction verb a metatarget . In figure 1, protein(n) is a metaagent, and transcription(n) is a metatarget . We define the syntactic categories connecting an agent(target) and a metaagent(metatarget) syntactic encapsulation categories . In figure 1, mod_att and comp_of are examples of the syntactic encapsulation categories . To detect a metaagent and a metatarget however, in the automatically obtained syntactic chains, we do not know which verb is an interaction verb . To overcome the problem, we extract the syntactic encapsulation categories from the syntactic chains that include only one verb in the training dataset . To detect gene interactions, we must recognize the interaction verbs . In the second phase, we retrieve the interaction verbs that indicate an interaction between two genes . If we consider only the syntactic chains that contain only one verb, the size of the interaction verbs becomes very small . Since the lll05 training dataset is small, we collect all the verbs in the syntactic chains from an agent to its target . According to the first and second phases previous studies made many errors in attempts to recognize which of two genes was the agent or target . Therefore, a new method is required to recognize an agent and its target correctly in a pair of genes . In the third phase, we propose learning the directions of the syntactic relations in the syntactic path from an agent to its target . If we do not permit the reverse direction, the agent and target will not be detected wrongly and thus improve the precision . We learn the direction of a syntactic relation related with an interaction verb . For a syntactic relation, if a syntactic relation is relation(syntactic category, current node, next node), the direction is right, since the next node is written to the right of the current node . If a syntactic relation is relation(syntactic category, next node, current node), the direction is left because the next node is written to the left of the current node . Figure 1 also shows an example of direction information of a syntactic path . Among the directions, we retrieve only the direction information of an interaction verb . The direction information is dependent on the syntactic category of the relation and the lexical word of the current node . In learning, we retrieve a syntactic category (a lexical word) and direction information for an interaction verb, and we make a template lexical word, syntactic category, direction. We construct direction information for all relations concerning interaction verbs in the training data . Based on the direction information, we learn direction rules . Let us explain the direction rule - learning algorithm, which is shown in algorithm 3 . One is a positive rule set obtained by learning the direction from an agent to its target . The other is a negative rule set obtained by learning the direction from a target to its agent in reverse order . Figure 2 shows the reverse syntactic path from a target to its agent of the sentence in figure 1 . The positive and negative rules for the sentence in figure 1 are shown in table 1 . From the positive and negative rule sets, we construct direction rules according to the following subsections . Align means the modification of any conflict in a rule set . For any lexical word a and relation b, if a conflict of two direction rules exists in a rule set, then we remove both rules, and add a modified rule a, b, any. Because the direction information is not trustworthy, we set direction any . The process for aligning a rule set is shown in 1> and 2> of algorithm 3 . After alignment of positive and negative rule sets, we construct direction rules from the two rule sets . The algorithm used to obtain direction rules is shown in 3> of algorithm 3 . Consider every rule a, b, c in the positive rule set, for any lexical word a and relation b, and direction c. in algorithm 3, (3.1) case indicates that direction information c is changed to any . Since the same direction exists in both the positive and negative rule sets, the direction information is not trustworthy . Therefore, we change the direction information into any . In (3.2) case, the direction information c in the positive rule is still used in the obtained direction rule . If c is right, then opposite c means left . Otherwise, if c is left, then opposite c means right . Since the direction in the negative rule set is opposite with that in the positive rule set, the direction information in the template is trustworthy . Case indicates that the negative rule set does not have any rule concerning a and b. the obtained direction rule is same with the original template in the positive rule set . For an interaction verb a, the relations not learned in the training data can appear in the test data . So, we add a default rule a, otherwise, any as described in table 2 . The default rule permits any direction is okay for other relations not appearing in the training data . Because the training data is so small, the default rule can resolve data sparseness problem . We detect agent candidates from the test set using the gene dictionary provided by lll05 . The obtained syntactic encapsulation categories, interaction verbs, and direction rules through three phases are applied to test data according to the following procedure . For each syntactic chain, we repeat the following procedure . If a current node is a gene and syntactic chain contains any interaction verb, then we determine that the current node is a target, and stop the extension of the syntactic chain . Otherwise, if the category of the syntactic relation of the next node candidate is a syntactic encapsulation category, we extend the syntactic chain by adding the next node candidate.otherwise, if the current lexical word is an interaction verb and the direction of the next node candidate is consistent with the direction rules, then we extend the syntactic chain . In the finally obtained syntactic chains, we determine that the first node is an agent and the last node is its target . If a current node is a gene and syntactic chain contains any interaction verb, then we determine that the current node is a target, and stop the extension of the syntactic chain . Otherwise, if the category of the syntactic relation of the next node candidate is a syntactic encapsulation category, we extend the syntactic chain by adding the next node candidate . Otherwise, if the current lexical word is an interaction verb and the direction of the next node candidate is consistent with the direction rules, then we extend the syntactic chain . With more and more biomedical datasets becoming publicly available, there has been some research effort on corpus design issues and usage in biomedical natural language processing [22, 23]. For a reasonable comparison with previous methods as mentioned before, the lll05 training dataset without coreference consists of 55 sentences, including 106 genic interactions, and the test data consist of 144 sentences . Based on the lll05 syntactic tags, the performance of our three - phase method versus that of previous methods . Based on a real - syntactic analyzer, the performance of our three - phase method versus that of previous methods . The change in performance when each phase is removed . In the experiments, we obtained the following five results . Based on the lll05 syntactic tags, the performance of our three - phase method versus that of previous methods . Based on a real - syntactic analyzer, the performance of our three - phase method versus that of previous methods . The change in performance when each phase is removed . Our three - phase detection method for gene interactions achieved an f - measure of 67.2% using lll05-annotated syntactic relations, and 44.0% using a real - syntactic analyzer (see tables 3 and 4).using lll05 syntactic tags, our three - phase method achieved an improvement of 14.6% to 37.6% over previous methods (see table 3).our method significantly outperformed greenwood et al ., which also used minipar (see table 4). When the second or third phase was removed, the precision became significantly worse (see table 5).when the first phase was removed, there were no interaction results . It means the first phase is important for the improvement of recall (see table 5). As shown in table 3, of the systems evaluated, our system performed the best with a precision of 67.9%, recall of 66.6%, and an f - measure of 67.2 percent . Our three - phase detection method for gene interactions achieved an f - measure of 67.2% using lll05-annotated syntactic relations, and 44.0% using a real - syntactic analyzer (see tables 3 and 4). Using lll05 syntactic tags, our three - phase method achieved an improvement of 14.6% to 37.6% over previous methods (see table 3). When the second or third phase was removed, the precision became significantly worse (see table 5). It means the first phase is important for the improvement of recall (see table 5). We will summarize the significance of each phase introduced in section 3 . As shown in table 5, every phase is important for its performance . Without the first phase, if no syntactic relations are considered encapsulation categories, then no pairs of genes are generated . Only this result shows the decrease of recall among three results in table 5 . It demonstrates that the syntactic encapsulation categories contribute to the improvement of recall . Without the second phase, if all the verbs are considered interaction verbs, the precision is very low, which results from the generation of too many wrong syntactic paths . Without the third phase, if we do not consider direction information, then the recall increases and the precision significantly decreases, which also result from the construction of many wrong syntactic paths . The experiments prove that the second and third phases contribute to the improvement of precision, and the first phase to the improvement of recall . We conclude that all three phases are important for detecting gene interactions . To experiment the robustness of our method in real time, we have used minipar, an existing syntactic analyzer . The system based on annotated syntactic relations in lll05 significantly outperforms that using minipar . This is because of the errors in syntactic relations and pos - tags that minipar produced . To improve recall without sacrificing precision, this paper proposes a three - phase method for the automatic detection of gene interactions using syntactic relations . The proposed method does not require domain knowledge . To improve recall, in the first phase, we construct syntactic encapsulation categories of agent and target . In the second phase, we construct interaction verbs that connect pairs of genes that interact with each other . To improve precision, in the third phase, we learn direction information to detect which of the two genes is the agent or target . The experimental results show that our three - phase method performs significantly better than previous methods . Our method achieved a precision of 67.9%, a recall of 66.6%, an f - measure of 67.2% using lll05 syntactic relations . We conclude that our proposed three - phase method is effective for detecting gene interactions . Furthermore, we demonstrated that every phase is important for performance . In the future, we need to expand the size of the training dataset and experiment with a large dataset.
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Ovotesticular disorder of sex development (ot - dsd) is a rare disorder of sexual differentiation characterized by the presence of testicular tissue with distinct seminiferous tubules and ovarian tissue with mature ovarian follicles in the same gonad (ovotestis) or separately in a single individual (1, 2). Ot - dsd it constitutes between 3 and 10% of the total dsd and presents significant diagnostic and management challenges (3, 4). Typically, both mullerian and wolffian duct derivatives are seen, and most affected individuals commonly present with ambiguous external genitalia as neonates or infants . However, the phenotype of the external genitalia may range from normal male to normal female depending on the degree of testicular tissue present . The ovotestis is usually the most common gonad in individuals with ot - dsd, and such gonads are known to be at an increased risk of developing germ cell tumors (5). More than five hundred cases of ot - dsd including familial cases have been reported in medical literature (3, 6, 7). Epidemiologically, the geographical distribution of ot - dsd shows a higher prevalence in the african continent, especially among south african blacks, with the number of published cases being 17 per 100 million people, followed by europe at 15.3 . Age and mode of presentation is often variable and usually comprises hypospadias, unilateral or bilateral cryptorchidism, inguinal hernia, urogenital sinus, gynecomastia at pubertal age or lower abdominal mass in adulthood (4). Internally, the presence of an ovotestis is the most common finding followed by ovaries (4, 9). The chromosomal findings in ot - dsd were first reported in 1959 by hungerford, who demonstrated a 46,xx chromosomal complement in peripheral blood lymphocytes of an individual with this disorder (10). Ot - dsd is a genetically heterogeneous condition with the predominant karyotype being 46,xx, while 46,xx/46,xy chimerism, 46,xy karyotype and x - y translocation are less frequent (4). The objective of this paper is to describe the diverse clinical, cytogenetic and histopathological features of five patients diagnosed with ot - dsd in sri lanka . This is a report of the clinical, cytogenetic and histopathological data of patients with ot - dsd who were referred to the human genetics unit for cytogenetic evaluation between 2005 and 2011 . This facility serves as the main referral center for cytogenetic testing in sri lanka, and the majority of children with dsd in the country undergo karyotype testing at this center . The diagnosis of ot - dsd had been confirmed by histopathological examination of the gonads, which were biopsied either laparoscopically or at exploratory laparotomy . Five milliliters of peripheral blood was obtained from each patient, and chromosomal analysis was performed on routinely cultured lymphocytes after gtg - banding . Karyotyping was done according to guidelines of the international system for human cytogenetic nomenclature (iscn, 2005). At least 30 well - spread and well - banded metaphases were examined in each patient by an experienced cytogeneticist . The age, sex of rearing, mode of presentation, status of the external and internal genitalia and karyotypes of the patients are summarized in table 1 . Their ages ranged from 2 mo to 47 yr, and all were reared as males . In four of the patients (cases 1, 3, 4 and 5), two patients (cases 1 and 3) had perineal hypospadias with bilateral cryptorchidism, while the other two patients (cases 4 and 5), who were siblings, both had male external genitalia with a short, blind - ending vagina and bilateral inguinal lumps . Case 2 was reared as a male but started feminizing and developed female secondary sexual characteristics at puberty with gradual atrophy of both testes . Case 1 underwent surgical correction for perineal hypospadias during early childhood but remained undiagnosed until an exploratory laparotomy performed for an intra - abdominal lump led to the diagnosis of ot - dsd at the age of 47 yr . In addition to the lower abdominal mass, this patient was observed to have gynecomastia . Two patients had bilateral ovotestis: one had an ovotestis on one side and an ovary on the other side, the other had an ovotestis on one side and a testis on the other side; the remaining patient had an ovary and testis separately on either side . Four patients (cases 1, 2, 4 and 5) had a 46,xy chromosome complement, while 46,xx/46,xy chimerism was observed in the remaining patient (case 3). There were no cases of 46,xx karyotype . Table 1 profiles of the five patients with ovotesticular disorder of sex developmentcaseagesex of rearingindication for investigationexternal genitaliainternal genitalia / gonadskaryotype147 yrmaleambiguous genitalia at birth / abdominal mass in adulthoodperineal hypospadias, bilateral cryptorchidismuterus with tubes, left ovotestis and right testis 46,xy223 yrmalefemale secondary sexual characteristics at puberty with gynecomastiamale external genitalia with bilateral atrophic testes uterus with tubes and bilateral streaky ovaries and atrophic testes46,xy32 momaleambiguous genitalia / inguinal lumpperineal hypospadias, bilateral cryptorchidismuterus with tubes, left ovary and right ovotestis46,xx/ 46,xy49 momaleambiguous genitalia / bilateral inguinal lumpsmale external genitalia with blind - ending vaginauterus with tubes and bilateral ovotestis46,xy55 yrmaleambiguous genitalia / bilateral inguinal lumps male external genitalia with blind - ending vaginauterus with tubes and bilateral ovotestis46,xy ot - dsd is a rare disorder of sexual differentiation that is phenotypically and genetically heterogeneous with wide - ranging manifestations . The presence of well - differentiated ovarian and testicular tissue in the same individual, whether as a single tissue - type gonad or an ovotestis, is the hallmark of this condition (9). Most patients with ot - dsd have ambiguous genitalia and are often diagnosed within the first few months to years of life (1, 4, 9). In addition to ambiguous genitalia, patients may present with inguinal hernias, gynecomastia during adolescence or lower abdominal mass in adulthood (4, 11). Four out of 5 patients in this series had ambiguous genitalia diagnosed in the first few months to years of life, and all were reared as phenotypic males . Inguinal hernias and gynecomastia were also present in some patients . A small number of ot - dsd cases are incidentally picked up during laparotomy for unrelated causes . One of the patients in this series had a similar presentation; he presented with a lower abdominal mass in the fourth decade that was later discovered to be an 18-wk - size uterus at laparotomy . A similar case of ot - dsd in a 42 yr old was reported in north india (4). In ot - dsd, the degree of virilization of the external genitalia depends on the capacity of the testicular tissue to secrete testosterone . Previous studies indicate that although approximately 70% of ot - dsd patients are raised as males, less than 10% have normal male external genitalia (1). It is generally believed that the leydig cell function of the dysgenetic testis is inadequate for normal virilization (4), and this is exemplified in this series by case 2, who had bilateral atrophic testes and started feminizing at puberty . Previous reports have also indicated that testicular tissue in ot - dsd patients becomes dysgenetic and that germ cells begin to disappear with increasing age (12). The patients in this study had internal genital organs ranging from streaky ovaries and a uterus to atrophic testes, but the common presenting gonad was the ovotestis . It is known that gonadal tissue may be located at any level along the route of embryonic testicular descent and is frequently associated with an inguinal hernia . A sub - classification of ot - dsd based on the type and location of the gonads has been described (13). According to this classification, ot - dsd is considered to be lateral if a testis is present on one side and an ovary is present on the other side (case 2), unilateral if an ovotestis is present on one side and a testis or ovary is present on the other side (cases 1 and 3) and bilateral if an ovotestis is present on both sides (cases 4 and 5). (9) reported that the ovotestis was the most frequent gonad, accounting for 59% in their series of 16 patients with ot - dsd, and krob et al . (3) also reported that an ovotestis was found in 44.4% of 568 gonads examined . The same observation is reflected in this series, in which 4 out of 5 cases (1, 3, 4 and 5) had an ovotestis and 2 of the patients, who were siblings, had bilateral ot - dsd . With regard to the frequency of gonadal distribution, it is reported that the most common form is ovotestis plus ovary followed by bilateral ovotestis and ovary on one side and testis on the other side, representing 34%, 29% and 25% of cases, respectively (8). Previous studies have reported that the risk of germ cell tumors, especially dysgerminomas, in patients with ot - dsd ranges from 4% among those with the 46,xx karyotype to up to 10% in those with 46,xy and 46,xx / xy chimerism (4, 5). Analysis of the chromosomal distribution of ot - dsd shows that about 5065% cases have a 46,xx chromosomal constitution followed by 46,xx/46,xy chimerism, while less than 10% have a 46,xy karyotype (1, 8, 9). The 46,xy karyotype is believed to be extremely rare and equally distributed throughout asia, europe and north america (3). In contrast to published reports (3, 4, 8, 9, 14), the predominant karyotype was 46,xy in 80% of the patients in this series . There is no apparent reason for 46,xy being the most common karyotype in this study other than the small number of patients studied, with possible over - representation of the 46,xy karyotype in this study sample, or it could be reflective of the pattern seen in the sri lankan population . Karyotypes such as 46,xx/46,xy, which was seen in only one patient, are thought to result from chimerism, possibly from double fertilization (involving two spermatocytes one x and one y) of either a binucleate ovum or of an ovum and its polar body (15). In some of the patients, proper investigation and accurate diagnosis had been delayed for many decades in spite of ambiguous genitalia being detected early in life, which led to clinical and psychological problems in their adult life . It is therefore recommended that in addition to cytogenetic evaluation, phenotypic males with ambiguous genitalia should be thoroughly investigated by means of either abdominal ultrasound scan and/or exploratory laparoscopy / laparotomy with histopathological examination to properly assess the exact nature of their internal genital system . Although considered a rare presentation, the cases reported in this series suggest that abnormal inguinoscrotal or abdominal masses may occur in ot - dsd and should be considered in the differential diagnosis of patients, especially in those with cryptorchidism and inguinal hernia having the 46,xy karyotype.
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Sialolithiasis is the most common disease of salivary glands caused by the obstruction of a salivary gland or its excretory duct by a calculus . It may occur at any age but there is a peak incidence in fourth, fifth, and sixth decades . Majority of salivary calculi (8095%) occur in the submandibular gland, whereas only 520% are found in the parotid gland . Sialoliths located in the duct are usually elongated, while those situated in the gland or hilus tend to be round or oval . The size of the salivary calculi may vary from <1 mm to a few cms in largest diameter . Most of the calculi (88%) are <10 mm in size, whereas only 7.6% are larger than 15 mm . They consist of mainly calcium phosphate with smaller amounts of carbonates in the form of hydroxyapatite, with smaller amounts of magnesium, potassium, and ammonia . A 45-year - old male reported to the outpatient department of vspm's dcrc, nagpur, with the chief complaint of pain and swelling in the floor of mouth on the left side since 1 month . Detailed history revealed that it has started as a small swelling which used to increase before meals 1 month back . Intraoral examination revealed a well - defined elongated swelling of approximately 2.5 cm 1 cm in size in the floor of the mouth in relation to lower left incisors to first molar region . Overlying mucosa was inflamed and the swelling was hard in consistency and tender on palpation [figure 1]. Radiographic evaluation included cross - sectional mandibular occlusal view which revealed a large well - defined elongated homogenous radio - opacity in the floor of the mouth on the left side in relation to lower left canine to first molar region . It was approximately 2.5 cm 1.5 cm in size [figure 2]. Swelling in the floor of mouth on the left side mandibular occlusal radiograph showing submandibular salivary duct calculus on the basis of clinical and radiological findings, a diagnosis of left submandibular duct sialolith was made . As it was a large sialolith, we elected to remove the sialolith surgically under local anesthesia [figure 3]. The sialolith removed measured 25 mm (i.e. 2.5 cm) in length [figure 4]. Removal of the sialolith from wharton's duct the removed sialolith measured 2.5 cm 1.5 cm in its largest dimension the sialolith was in the wharton's duct and the patient had pain before and during meals . For stone formation it is likely that intermittent stasis produces a change in the mucoid element of saliva, which forms a gel . This gel produces the framework for deposition of salts and organic substances creating a stone . Traditional theories suggest that the formation occurs in two phases: a central core and a layered periphery . The central core is formed by the precipitation of salts, which are bound by certain organic substances . Another theory has proposed that an unknown metabolic phenomenon can increase the saliva bicarbonate content, which alters calcium phosphate solubility and leads to precipitation of calcium and phosphate ions . A retrograde theory for sialolithiasis has also been proposed . Aliments, substances, or bacteria within the oral cavity might migrate into the salivary ducts and become the nidus for further calcification . Salivary stagnation, increased alkalinity of saliva, infection or inflammation of the salivary duct or gland, and physical trauma to salivary duct or gland may predispose to calculus formation . Submandibular sialolithiasis is more common as its saliva is (i) more alkaline, (ii) has an increased concentration of calcium and phosphate, and (iii) has a higher mucous content than saliva of the parotid and sublingual glands . In addition, the submandibular duct is longer and the gland has an antigravity flow . Sialolithiasis typically causes pain and swelling of the involved salivary gland by obstructing the food - related surge of salivary secretion . Calculi may cause stasis of saliva, leading to bacterial ascent into the parenchyma of the gland and therefore infection, pain, and swelling of the gland at meal time . Bimanual palpation of the floor of the mouth, in a posterior to anterior direction, reveals a palpable stone in a large number of cases of submandibular calculi formation and a uniformly firm and hard gland suggests a hypofunctional or nonfunctional gland . Other traditional diagnostic methods include sialography, ultrasound, computed tomography, and scintigraphy for sialoliths . As the lesions were observed clearly in occlusal radiographs, no further investigations were performed for diagnosis . The treatment objective for giant sialoliths, as for the standard - sized stones, is restoration of normal salivary secretion . There are three ways in which we can treat patients with salivary stones: removal through the oral cavity, interventional sialoendoscopy, and resection of the gland . Our choice depends on the site, size, shape, number, and quality of the stones . The giant sialolith should be removed in a minimally invasive manner, via a transoral sialolithotomy, to avoid the morbidity associated with sialadenectomy . Whenever the stone can be palpated intraorally, it is best to remove it through an intraoral approach . For giant sialoliths, transoralsialolithotomy with sialodochoplasty or sialadenectomy submandibular gland removal is indicated if (1) the gland has been damaged by recurrent infection and fibrosis, (2) there is a stone of substantial mass within the gland itself that is not surgically accessible intraorally, (3) there are small stones present in the vertical portion of wharton's duct from the comma area to the hilum, (4) the size of an intraglandular stone reaches 12 mm or more as the success of lithotripsy may be <20% in such cases . There are various methods available for the management of salivary stones, depending on the gland affected and stone location . Transoral sialolithotomy remains mainstay of the treatment for giant sialolith in the duct of submandibular gland . Also, patients should be followed up regularly as recurrence has been reported in the literature.
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Extracellular adenosine concentration increases under metabolically stressful conditions, notably in the tumor microenvironment, where hypoxia is frequently given [2, 3]. Such accumulation of adenosine mediates, through four distinct receptors (a1, a2a, a2b, and a3), complex and diverse effects that lead to tumor immunoescape . This includes cytoprotection and growth promotion of tumor cells [5, 6], angiogenesis increase [7, 8], and suppression of effector (antitumor) t cells . Although cells are provided with adenosine transporters, the main source of this nucleoside in the tumor interstitium is the hydrolysis of extracellular atp, which also accumulates in tumors, by membrane enzymes known as ecto - nucleotidases [6, 10]. Different families of these enzymes, acting extracellularly, are responsible for the generation of adenosine from adenine nucleotides (i.e., atp, adp, or amp): (1) the ectonucleoside triphosphate diphosphohydrolase (e - ntpdase) family, that includes four plasma membrane - bound members: ntpdase1 (cd39), ntpdase2, ntpdase3, and ntpdase8; these enzymes are differentially expressed and hydrolyze with different affinities nucleoside triphosphates and diphosphates to their monophosphate derivatives (e.g., atp and adp to amp); (2) the ectonucleotide pyrophosphatase / phosphodiesterase (e - npp) family, capable of hydrolyzing nucleoside triphosphates to monophosphates and pyrophosphate (ppi), such as atp to amp and ppi; (3) the alkaline phosphatase (ap) family, that includes ubiquitous enzymes degrading broad range of substrates, such as adenine nucleotides and ppi, releasing inorganic phosphate (pi); (4) the 5-nucleotidase family, with only one member attached to the outer plasma membrane, the ecto-5-nucleotidase (cd73), a glycosyl phosphatidylinositol - linked membrane - bound glycoprotein that efficiently hydrolyses amp to adenosine [1113]. Two members of ecto - nucleotidases families, the e - ntpdase cd39 and the 5-nucleotidase cd73, acting sequentially, seem to have a crucial role in tumor - immune cell interaction . They both are expressed not only by infiltrating immune cells but also by tumor cells, and their expression is regulated by hypoxia [10, 14]. Increased cd39 and cd73 expression has been described in various cancer types, mostly in correlation with a poor prognosis [1517]. Both molecules are considered promising therapeutic targets in oncology, and cd73 has already been proven to inhibit tumor growth and metastasis in a breast cancer model in mice [1820]. However, until now there were not available data concerning ecto - nucleotidases expression in endometrial cancer (ec). There are two clinicopathological variants: the estrogen - related, type i, endometrioid carcinoma, and the nonestrogen - related, type ii, nonendometrioid carcinoma . Although there are different molecular alterations that have been already identified in ec, with different prevalence between tumors [22, 23], there is need to decipher the complete molecular profile of ec pathogenesis to improve diagnosis and favor the design of new therapeutic strategies . The aim of the present work was to study the expression of cd39 and cd73 in endometrioid (type i) and serous (type ii) ec when compared with nontumoral endometrium . To achieve this objective, protein and gene expression experiments, as well as in situ enzyme activity assays, the ethical principles of this study adhere to the declaration of helsinki, and all the procedures were approved by the ethics committee for clinical investigation of bellvitge hospital . Endometrial samples from adenocarcinoma (endometrioid and serous types) and their corresponding nontumoral tissue (if present) were obtained from hysterectomy specimens at the service of gynecology of bellvitge hospital . Fresh samples were cut, embedded in o.c.t . Freezing media (tissue - tek; sakura finetek, zoeterwoude, the netherlands), snap - frozen in a shandon histobath 2 (neslab instruments inc ., usa) at the service of pathology, and stored at 80c until used . Alternatively, endometrial samples were obtained from the tumor bank of bellvitge biomedical research institute (idibell). Fifteen endometrioid adenocarcinomas (13 grade 1, 2 grade 2; all figo stage i) (5682 years old, median 61) and fourteen serous adenocarcinomas (grade 3; 9 figo stage i, 2 figo stage ii, and 3 figo stage iv) (6386 years old, median 77), and their adjacent nontumoral endometrium were used in this study . Touch preparations of endometrial cancer tissue samples were obtained by lightly pressing the freshly cut tumor surface on clean glass microscope slides, thus generating a tumor cell imprint . Briefly, tissue sections of 10 m thick and touch preparations were fixed in 10% phosphate - buffered formalin mixed with cold acetone (merck, darmstadt, germany) for 2.5 minutes . Fixed samples were rinsed with pbs and preincubated for 1 hour at room temperature (rt) with pbs containing 20% normal goat serum (gibco, paisley, uk) and 0.2% gelatin (merck). Samples were then incubated overnight at 4c with the following primary antibodies: anti - human cd39 clone bu61 (ancell corporation, minnesota, mn, usa) at 1/500, mouse monoclonal anti - human ecto-5-nucleotidase (cd73) clone 4g4 (abcam, cambridge, uk) at 1/50, and rabbit monoclonal anti - human cytokeratin 19 (ck19) clone epr1579y (abcam) at 1/200 . After three washes in pbs, samples were incubated for 1 hour at rt with the appropriate secondary antibodies: horseradish peroxidase - conjugated goat anti - mouse (envision + system; dako, carpinteria, usa), alexa fluor 488- or 555-goat anti - mouse or anti - rabbit (life technologies, paisley, uk). Nuclei were counterstained with haematoxylin or, alternatively, in fluorescence assays, to - pro-3 or dapi (life technologies) were used to visualize the nuclei . Samples were mounted with fluoromount aqueous mounting medium (sigma - aldrich, sant louis, missouri, mo, usa). The results were observed and photographed under a light leica dmd 108 microscope (leica microsystems, wetzlar, germany) or, in fluorescence assays, under a nikon eclipse e-800 microscope (nikon, tokyo, japan) or under a leica tcs - sl spectral confocal microscope (leica). Results from both tissue samples and touch preparations were independently evaluated by at least two observers . Label intensity was scored as negative (), intermediate (+), or strongly positive (+ +). For enzyme histochemistry, adpase and ecto-5-nucleotidase (ampase) activities were localized by using the wachstein / meisel lead phosphate method [24, 25] in tissue samples and in touch preparations . Briefly, fixed samples were preincubated for 1 hour at rt in 50 mm tris - maleate buffer, ph 7.4 containing 2 mm cacl2 and 0.25 m sucrose . Enzyme reaction was carried out for 1 hour at 37c in the same buffer supplemented with 5 mm mncl2, 2 mm pb(no3)2, 3% dextran t250, and 2.5 mm levamisole, as inhibitor of alkaline phosphatases, and in the presence of 200 m adp or 1 mm amp, as substrate . For cd39 and cd73 inhibition experiments, 1 mm nf279 (tocris bioscience, bristol, united kingdom) and 1 mm, -meadp (sigma - aldrich) were added, respectively, to both preincubation and enzyme reaction buffers . The reaction was revealed by incubating with 1% (nh4)2s v / v for exactly 1 minute . Samples were counterstained with haematoxylin, mounted with fluoromount aqueous mounting medium (sigma - aldrich), and observed and photographed as described above . 50100 g of human tumor (endometrioid and serous endometrial adenocarcinoma) and nontumoral tissue samples were homogenized in a buffer containing 20 mm hepes, 250 mm sucrose, 0.3 mm pmsf, 1 mm dtt, 1 mm egta, and 1 mm mgcl2 (ph 7.4) using a glass homogenizer (vidrafoc, barcelona, spain). After homogenization, samples were centrifuged at 600 g for 10 minutes at 4c in a beckman ja-20 centrifuge . The pellet was discarded and supernatants were centrifuged at 48,000 g for 20 minutes at 4c in a beckman ti-70 centrifuge . The resulting pellets were resuspended in a buffer containing 20 mm hepes, 0.3 mm pmsf, and 1 mm dtt (ph 7.4). Protein concentration was determined by the method of lowry et al . Using bovine serum albumin as a standard . Adpase and ampase activities were determined by measuring the amount of pi using the malachite green colorimetric assay, as previously described . Total rna from endometrial tumor tissue samples was isolated using the rneasy plus mini kit (qiagen, hilden, germany), following the manufacturer's protocol . Total isolated rna (2 g) was reversely transcribed into complementary dna (cdna) using the first strand cdna synthesis kit (fermentas, thermo scientific, chicago, il, usa). Quantitative real - time pcr (qrt - pcr) was performed to examine the expression of cd39, ntpdase2, and cd73 genes . Designed large - scale taqman low - density array (tlda) microfluidic cards (applied biosystems, foster city, ca, usa) were used . The 384 wells of each card were preloaded with predesigned fluorogenic taqman probes and primers for cd39, ntpdase2, and cd73 . Cdna (1 g) combined with taqman 2x universal pcr master mix (applied biosystems) were loaded into each sample - loading port . Qrt - pcr reactions were carried out using the abi prism 7900ht real - time pcr system (applied biosystems). Data were collected using the sds v2.1 software (applied biosystems) and analyzed by the comparative ct (ct) quantification method using the expression suite v1.0 software (applied biosystems). The relative expression levels of cd39, ntpdase2, and cd73 genes were determined using 18s mrna as an endogenous control for normalization . Results are expressed as the mean of the relative quantification (rq) of the tested transcripts (n = 7 serous adenocarcinoma samples; n = 7 endometrioid adenocarcinoma samples) the standard error of the mean (sem). Results were obtained from five independent experiments performed using 1 g of cdna, all with duplicate measurements . Student's t - test was used to compare the means of two independent groups of normally distributed data . Extracellular adenosine in tumors, mainly generated by the sequential action of ecto - nucleotidases, has immunosuppressive effects through a broad range of actions, including inhibition of antitumor t - cell function, modification of local interleukin levels, and inhibition of phagocytosis (reviewed in). In this section we show and discuss our results on the expression of the ecto - nucleotidases cd39 and cd73 in type i and type ii endometrial carcinomas . Cd39 was immunolocalized in the stroma of both nontumoral and tumoral endometria (figure 1(a)). For the nonpathological endometrium the expression of cd39 has already been previously described in association with stromal cells and blood vessels . Here we show that label score was significantly higher for both endometrioid and serous types of tumors when compared with the corresponding nontumoral coexisting endometrium (figure 1(b)). No cd39 labeling was found in endometrial adenocarcinoma epithelia, as demonstrated with the double staining performed with the anti - cd39 and anti - ck19 antibodies (see supplementary figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2014/509027). Strong in situ adpase activity was detected in the tumor stroma, coinciding with the cd39 immunolocalization (figure 2(a)). Equivalent results were obtained using atp as substrate for the in situ activity assay (not shown). Adpase activity measured in tumor tissue homogenates demonstrated that serous (grade 3) adenocarcinomas had significantly higher activity than endometrioid (grade 1) adenocarcinomas (figure 2(b)). Cd73 expression was strongly immunodetected in both types of tumors, in epithelial structures and in the stroma (figure 3(a)), thus partially colocalizing with ck19 (supplementary figure 2). Specific cd73 activity, demonstrated with the inhibitor, -meadp, matched the immunolabeled structures (figure 3(a)). We have already previously demonstrated that the expression and activity of cd73 are abundant in nonpathological endometrium [24, 25]. Consequently, due to the high expression of cd73 in tumoral and nontumoral endometria, label score comparisons were not possible . Moreover, no differences among tumors were observed with the enzyme assays either in tissue slices or in tissue homogenates (not shown). Immunolabeling and in situ enzyme activity results obtained with the touch prep technique were equivalent to those obtained with tissue slices (figures 2(c) and 3(b)). The usefulness of the touch prep technique for diagnosis has already been demonstrated in breast cancer with 100% sensitivity and specificity in the evaluation of tumor margins at the time of the surgery . This technique has also been previously used to demonstrate by immunolabeling a decreased expression of p2x7 atp receptor in endometrial cancer cells and also, recently, the relationship between p53 expression and the tumor grade . However, to our knowledge, this is the first report validating the use of touch preps for enzyme activity studies, therefore opening the possibility of performing such studies in cytological samples . In order to determine if the differences in cd39 protein and adpase activity between tumor types also involved gene expression changes, quantitative real - time pcr analyses were performed (figure 4). Cd39 gene expression was 2-fold higher in serous endometrial adenocarcinoma than in endometrioid, coinciding with the data obtained with the protein (figure 4(a)). These gene expression changes did not apply to ntpdase2 (figure 4(b)), indicating that cd39 upregulation is not a general feature of other members of ntpdase family . No changes were detected in cd73 gene expression between the two tumor types (figure 4(c)). These results on endometrial tumors add to the list of human cancers in which cd39 is overexpressed and support the growing body of evidence that cd39 is a potential therapeutic target for cancer immunotherapy . Antibody - based therapy and pharmacological approaches against cd73 have been reported to significantly inhibit tumor growth and improve antitumor immunity in mouse models [28, 32]. This also coincides with the higher grade of these tumors, but further studies are needed to establish statistical correlations with the tumor grade in the case of type i endometrioid tumors . The consequences of this high cd39 activity in endometrial tumors are increased levels of amp, the substrate for cd73, and also highly expressed in these tumors, which will, in turn, generate increased immunosuppressive levels of extracellular adenosine.
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The incidence of oral cancer especially squamous cell carcinoma accounts for nearly 2.4% of all cancers . Due to significant number of oral cancer cases raising rapidly in the developing regions this life style habits such as heavy smoking and alcoholism are the important risk factors for developing oral cancer that increases at least three- to fifteenfold especially in females and young people . In addition, marijuana, chewing beetle - leaf, human papilloma virus, ultraviolet radiations, iron deficiency anemia, candida infections, immunosuppression, and deletion or mutation of tumor suppressor genes are some of the other causes of oral cancer . Lack of public awareness regarding oral health and low intake of fruits and vegetables, older age, and poor oral hygiene are some of the implications for oral cancer . Majority of the oral cancer was detected at late stages (iii and iv) and early diagnosis is important to increase patient survivability and to delay its prognosis . In 2011, world health organization (who) reported the incidence of oral cancer deaths in malaysia to about 1.5% of the total deaths, with age adjusted death rate of 7.72 per 100,000 populations . Malaysia ranked 14 in the world with annual oral cancer deaths of 1,587 . Increasing the public awareness and early diagnosis, it is important to have sufficient knowledge and awareness among dentists for detection and early diagnosis . Initiatives were undertaken by university of malaya to increase the oral cancer awareness in malaysia such as malaysian oral cancer research initiative (mocri) and oral cancer research & coordinating centre (ocrcc). These publicity initiatives are crucial to improve the oral cancer awareness among general public and health professionals in malaysia . In addition, general dental practitioner's role is decisive in identifying the oral mucosal changes that may lead to oral cancer . Assessing the knowledge of dental students paves the way towards understanding their level of awareness in the early detection and prevention of oral cancer . To the best of our knowledge, previous researches on dental students' knowledge and awareness were conducted in the university of malaya (um) and universiti sains malaysia (usm) [9, 10]. Since there is a paucity of information regarding oral cancer awareness in undergraduate dental students in different other regions of malaysia, therefore it is pertinent to assess these characteristics in senior dental students (third, fourth, and fifth year) at international islamic university, kuantan, pahang, malaysia . The aim of the current research was to assess the knowledge and awareness of oral cancer towards early identification of risk factors among undergraduate dental students . This is a descriptive cross - sectional study to assess the oral cancer knowledge and awareness of senior undergraduate dental students using a survey questionnaire, adopted by carter and ogden and brzak et al . . Ethical permission to conduct the study was obtained from the respective deans, international islamic university, malaysia . The study was conducted via face - to - face interview at international islamic university malaysia during the period of february to march, 2015 . Sample size was determined using 95% confidence interval, with an accuracy of 60% for the total dental students being 300 studying in international islamic university given a confidence interval of 5.5; the recommended sample size is 155 or more . A systemic random sampling technique was used to select senior dental students which includes third, fourth, and fifth years . In general, as included in the curriculum, dental students receive information regarding oral cancer during their oral pathology and oral medicine sessions in their first and second year as well as oral examination during their clinical sessions . Students of both gender studying third-, fourth-, and fifth - year dentistry were included in the study . A 9-item pretested questionnaire was employed after explaining the purpose of the study and verbal consent was obtained from each study participant . The questionnaire constitutes 7 close - ended (yes / no) questions such as (1) oral mucosal examination (2 items), (2) advising current and future patients regarding risk factors for oral cancer, (3) opportunity to examine oral lesions, (4) knowledge regarding prevention and detection of oral cancer, (5) point of referral selection, and (6) desire for further information or teaching regarding oral cancer . Two open - ended questionnaires were asked to identify the risk factors for development of oral cancer and encouraged to select at least three to four options out of 10 options . In addition, interest of preferences for obtaining oral cancer information (1 out of 3 options). The wilcoxon rank - sum test and chi - square were used to identify the difference between groups . A total of 162 students were approached, and 114 questionnaires were returned with an overall response rate of 70.3% . Eighty - eight were females and twenty - six were males with a mean age (standard deviation) of 24.36 (7.12). Sex distribution with the number of respondents per year of course was shown in table 1 . When asked about the examination of oral mucosa of the patients, all the students answered yes during their clinical training . Of those who examine the oral mucosa routinely, a high majority of the students (97.3%) would not examine the oral mucosa of the patient with high risk of developing oral cancer . Significantly, 67.5% of the students did not get opportunity to examine the oral lesions (= 15.892, df = 2, and p = 0.000) (table 2). More than ninety percent of the participants preferred to refer patients with oral lesion as a point of care to dental specialties rather than doctors . However, significantly, two - thirds (65.7%) of students felt that they did not have sufficient knowledge about prevention and early detection of oral cancer . This was much higher observed in third - year student participants (42/45) than others (= 28.598, df = 2, and p = 0.000). Of note, most of the study participants (95.6%) requested further information regarding oral cancer prevention and early detection, with more than fifty percent preferred to obtain in the form of information package (52.6%), twenty - eight percent through seminars, and nearly twenty percent as lectures (figure 1). A majority of the dental students (93%) identified a number of different risk factors for oral cancer were shown in figure 2 . All the participants identified poor oral hygiene as a major risk factor, whereas 70.7% identified diet with low vitamin c levels as a risk factor for oral cancer . In addition, only 14.5% of the participants identified alcohol and chewing beetle leaves as a risk factors . However, other oral cancer risk factors such as immunosuppression, viral infections, and occupational hazards were poorly reported by the final year students, and none of the other students identified these as a risk factors . Squamous cell carcinoma accounts for 90% of oral cancer and it is a general practice of the dental students to examine the patients' oral mucosa . Providing opportunity to examine and early detection can reduce the morbidity of oral cancer especially in high risk patients . It is the prime responsibility of the dental schools to provide sufficient knowledge to students for early diagnose in asymptomatic patients and prevent prevalent oral diseases . Hence, this study was carried out to determine the level of knowledge and awareness of oral cancer among dental students at international islamic university, selangor, malaysia . The response rate of the current study was 70.3% which is much lower than the studies conducted on dental students in croatia (95%), india (90.6%), iran (88%), and brazil (75.1%) but fairly higher than the similar studies conducted in other specialties . Although the response rate was low, a comparable number of students from different academic years participated in the study . In the present study, further, it was identified that a large majority of the students had an opportunity to examine patients with oral lesions . But unfortunately, a high majority of these students claimed that they failed to screen the high risk patient groups, which implies the gaps in their knowledge regarding oral cancer risk factors . Poor knowledge is directly related to lack of awareness, and emphasis should be taken to provide more opportunities engaging undergraduates to take oral health histories and examine oral lesions in patients during clinical attachments that should be undertaken . It is arguable that majority of the oral cancer patients are asymptomatic and identifying the changes in their cancerous and precancerous lesions in the oral cavity would help them to apply their critical knowledge into practice, importantly needed in high prevalent countries like malaysia . For such reasons, ogden et al . Claimed to implement work - based assessments to know these gaps and specific test for oral cancer within the curriculum prior to dental students graduation . Regarding referral pattern for oral cancer, more than ninety percent felt that it is the dentist's responsibility to diagnose the oral malignancies . These results are encouraging as they demonstrate the recognition of dentistry, and it is their responsibility of dentists to diagnose and evaluate oral cancer . These results were consistent with other studies conducted by ogden and mahboobi, awan et al ., carter and ogden, and fotedar et al . But contradict with study by brzak et al . Where majority of the undergraduate dental students chose to refer oral cancer patients to a plastic surgeon specialist . A recent meta - analysis concluded that diagnosis delay is a potential risk factor for developing advanced stage oral cancer . In our study, the majority reported that they would advise their patients about oral cancer and associated risk factors after graduation . These findings were similar to the previous study performed in malaysia, uk, and croatia . It is crucial role of dentists to take a strong responsibility to offer advice to the patients on high - risk habits like cessation of smoking cigarettes and self - examination of oral mucosa to improve the oral hygiene . These counseling techniques also enhance early detection of oral mucosal changes in the oral cavity . Approximately, seventy percent felt that they are have insufficient knowledge (p <0.001) with regard to prevention and early detection of oral cancer . These numbers are higher in those who were in third year (36.8%). In mccready et al . 's study 77% of dental students from second year and fourth year reported that they were poorly informed regarding oral cancer, whereas in carter and ogden's study 93% of the final - year medical students also reported the same . A well - designed institutional - based clinical training by incorporating different dental specialties such as oral medicine, dental oncology, and oral and maxillofacial surgery to improve the knowledge about oral cancer however, almost all the students requesting further information regarding oral cancer which is similarly identified in studies by awan et al, brzak et al, and mccready et al where more than 90% of the students requested to receive more information regarding oral cancer . Further, majority of the students are interested in receiving further information in the form of information package which is also most preferred in other studies [1, 9, 10, 12]. Study assessing the oral cancer prevention and clinical attitude among spanish dentists highlighted that providing continuous education through scientific newsletters and journals can provide positive preventive attitude in oral cancer . In our study, 106 out of 114 participants identified the risk factors for oral cancer . Of these, all the students felt poor oral hygiene as the single most important risk factor . Only sixty percent of students identified tobacco smoking as a risk factor for development of oral cancer . Although previous studies revealed that smoking tobacco and alcohol consumption increase the incidence of oral cancer, these were unidentified by our third - year dental students . These findings were contradictory with other studies in the literatures, which show that around 90% of the dental and medical students identified tobacco smoking as an important risk factor 12,58 [1, 9, 10, 1214, 17, 19]. However, alarmingly, none of the third and fourth years identified alcohol, beetle - chewing, and immunosuppression as a risk factor . Thus the knowledge on risk factors was poor in both third and fourth years and also very minimal in final - year dental students . There was trend towards better identification of risk factors which was observed with progression of their academic years, which is similarly noticed in other studies [9, 11, 14]. All these findings identified different knowledge gaps in identification of risk factors among dental students, and there is a need of educational intervention by implementing training or workshop particularly focusing on oral cancer . We used a prevalidated questionnaire which is used in other surveys assessing oral cancer knowledge [9, 11] on dental students to reduce selection bias . Further, recent research identified that nearly 2030% of the oropharyngeal squamous cell cancer did not have traditional risk factors of smoking and cancer which may be falsely interpreted in the light of respondents' knowledge . The study was conducted on senior dental students in a single institution in malaysia and may not be generalized to other regions . In addition, the data presented here is self - reported, and some of the respondents may provide extreme responses than others, due to the motivations and beliefs of the participants, and might be subjected to recall bias . However, we believed that the participants were honest to provide appropriate responses conducted in a single institution, and national level multifaceted studies are further needed to assess dental students' knowledge about oral cancer . Lack of awareness about the risk factors initiates the need based educational interventions among future dental practitioners regarding early detection and prevention of oral cancer in malaysia.
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Globally, approximately 40% of households rely on solid fuels including wood, dung, grass, coal, and crop residues for cooking . The 2010 comparative risk assessment of the global burden of disease attributed 3.6 million deaths yearly to the harmful byproducts of solid fuel combustion for cooking and an additional 0.3 million deaths from contributions of household air pollution to ambient air quality . While the proportion of households using solid fuels appears to be declining, most efforts to mitigate this health burden have focused on providing biomass - burning stoves that vent pollution outdoors and/or improve combustion efficiency to reduce emission rates . Increasingly, some are focused on providing access to clean energy for cooking including electricity or liquefied petroleum gas . Several conditions must be met if household energy interventions are to improve health: continuous access to a low - emissions energy source for cooking, sustained usage of this energy source, and discarding of the more polluting traditional stoves . Stacking is well - documented through surveys, though little objective continuous monitoring of usage of multiple cooking appliances during intervention studies has occurred to date . In palwal district, haryana, we provided a fan - assisted, advanced cookstove, with modifications to improve combustion efficiency (not just improve fuel efficiency or vent pollutants outdoors), to pregnant women via local antenatal healthcare system workers . Preliminary research evaluating potential interventions and describing this community has been published . During this initial work the philips hd4012as suitable, despite requiring access to power for battery charging and the need to chop the biomass fuel into small pieces . Among other goals, this study evaluated the use of the intervention and primary traditional stoves over time and investigated predictors of usage . Monitoring usage and adoption of intervention stoves traditionally relied on simple metrics obtained through interviews or by a trained observer . Due either to recall bias or to the influence of an outsider in the home (the hawthorne effect). Recent work in rwanda, for example, highlighted that usage estimates obtained from surveys were biased upward relative to objective measures from electronic sensors . These biases have been well described in water and sanitation studies, including recent evidence showing significant effects of structured observation on behavior and attempts to address these issues using simple data - logging sensors . Previous studies of household energy identified maxim ic s ibutton technology as an objective, field - validated stove use monitor (sum). Ibuttons are small, coin - shaped thermometers that log time - resolved instantaneous temperatures at the surface upon which they are mounted . Properly placed, ibuttons offer both an objective measure of stove usage and a relatively unobtrusive way to monitor interventions over time . This paper describes time - trends in usage of the intervention and primary traditional stoves in rural indian homes . We examine how well short - term measures (1, 2, and 7 day mean measurements) of stove use predict study means, with the goal of optimizing sampling times and strategies for monitoring household energy interventions . We believe the data set described in this paper is the longest and deepest data set of measured stove usage generated to date, spanning over 15 months of monitoring at 10 min intervals on both intervention and primary traditional stoves in 200 homes (21 million data points). Measuring multiple stoves required creation of new metrics to characterize shifts in usage patterns over time . Our secondary focus on reducing total monitoring duration for assessing use, without compromising data quality informs strategies to optimize the conflicting goals of precise measurements and efficient fieldwork . This study took place approximately 80 km south of new delhi at the international clinical epidemiology network (inclen) somaarth demographic, development, and environmental surveillance site in palwal district, haryana, india beginning in november of 2011 and ending in march of 2013 . At the time of the study, inclen was carrying out demographic and environmental surveillance in 51 villages, covering a population of approximately 200 000 . During the study, ambient temperatures varied widely by season, reaching a maximum of 45 c in may and a minimum of 4 c in january (supporting information, si, figure s1). Temperature data were logged every minute by the project meteorological station (onset microstation, onset computer corporation) at the inclen field headquarters in palwal town, between 5 and 12 km from study villages . A metal sheet stamped with a unique identifier and machined with a hole was used to securely hold each stove use monitor . The current study focused on 7 rural villages, selected based on their use of biomass for cooking, total population, and their accessibility to the somaarth field headquarters . All households recruited into the study used dung, wood, and crop residues in a traditional hearth (figure 1a) as the primary means of cooking . Nearly all homes (n = 200) cooked outdoors . The philips hd4012 (figure 1b) is a top - loading, fan - assisted semigasifier stove fueled by small wood pieces 5 cm in length and up to 2.5 cm in diameter . It contains a rechargeable battery that powers a fan used to enhance combustion efficiency . Initial selection of the philips stove was based on its performance in laboratory testing by the u.s . Epa, which found it to be among the cleanest stoves evaluated using standard simulated cooking methods . Field emissions from this stove were evaluated by other research projects in india and our research team validated this stove s acceptability in the community prior to this project . At the time of the study, the stove was produced in ghaziabad, india, and sold for approximately 60 usd . Participants who received the philips stove were trained on proper stove use and maintenance by community health workers and inclen field staff . Contact information for inclen s field office, which was equipped with spare parts and had access to trained technicians and electricians, was provided to participants in case of any stove malfunction, error, or other user complaint . Complaints could be filed during regular household visits by inclen field staff, through calls to inclen, or by visiting the field headquarters . Upon receipt of a complaint, repair attempts were undertaken first by inclen support staff and then, if necessary, by electricians . A supply of replacement stoves was available to avoid prolonged interruption in homes with stove failures . Detailed logs of stove reliability, malfunction, and maintenance were maintained by inclen field staff (see the si). Upon enrollment into the study, field staff obtained informed consent, administered a baseline questionnaire, and installed a sum on the primary traditional stove in each participant s household . Sums were placed in a custom - made metal holder and plastered onto the traditional stove side wall with the same slurry of mud and water used to construct and repair stoves . The holder and a sum can be seen in the inset image in figure 1a . The selected sums placement location did not disturb standard cooking practices, was protected from overflow and spills, and captured variability in temperatures adequately . Stoves varied in shape and size between households; sums were placed in approximately the same location on each stove throughout the study . Within 4 weeks after preintervention monitoring began, the philips intervention stove, prefitted with a sum (visible in figure 1b), was delivered to the home . A custom - made metal bracket, stamped with a unique stove i d, was used to hold the sums in an identical location on all intervention stoves . Field workers visited homes every 2 weeks to inspect stoves and download data from the sums . Touch and hold probe connected to a usb to 1-wire rj11 adaptor (maxim integrated, san jose, ca, u.s.a . ). Data transfer took approximately 25 min per stove and involved holding the probe to the surface of the ibutton . Stove usage files were transferred to the field office, where they were inspected for errors and minimally processed . Filenames contained metadata, including stove type (philips or traditional), household i d, and download date . Raw files were archived at the field site and at inclen headquarters in new delhi . Cleaned files were transferred to a secure server in the school of public health at the university of california, berkeley, and analyzed using r 3.0 . Approximately 20.6 million sums data points were collected during the main study, representing 143 000 stove - days of data from 408 stoves . The number and duration of usage events, derived from raw sums temperature traces, were determined for each stove on each monitored day . Algorithms for processing sums data were created using an iterative process, beginning with recommendations from the literature that identify events by setting thresholds for the rate of increase and decrease in temperature . Due to the high variability in ambient temperatures in palwal, we took advantage of our continuous ambient temperature measurement to adjust for diurnal variation . To compensate for variability in temperatures between households and the field office, we calculated the mean and standard deviation of ambient temperature by each recorded hour during the study . These values were used to create thresholds for evaluating whether a stove was in use or not . For each stove, the daily recorded sums temperature range (drange) sums data were then merged with data for mean hourly ambient temperatures (hmean amb) and their standard deviations (hsd amb). A stove was considered in use when the sums temperature exceeded the mean ambient temperature plus 6 times its standard deviation . Any period detected for which the drange was less than 20 c was marked as a period of nonuse . To count the total number of daily uses, periods of use that occurred less than 40 min apart this clustering threshold was based on manual observation of temperature traces . For each stove, summarized data were analyzed to understand trends in usage of both the traditional and intervention stoves . All analyses were restricted to households for which we had at least 2 days of preintervention data (n = 177). Analyses were performed separately (1) for the entire data set for these households and (2) for days on which data were successfully collected from both traditional and intervention stoves (see the si). The proportion of stove use - time spent using the philips intervention stove was defined as follows:1where prop is proportion and dur is duration . All durations were calculated in minutes . While the proportion of time spent using an intervention is useful to track adoption, it does not take into account gains in efficiency of heat transferred to the pot by the intervention stove, leading to shorter cooking times, and thus, we linked durations of cooking derived from the sums with cooking power from laboratory studies to determine the utilized cooking energy (uce) in megajoules (mj):2where st is the stovetype . Calculation of uce allowed estimation of changes in total energy used before and after deployment of the intervention . Laboratory cooking power estimates were derived from controlled burning for water boiling using uniform wood fuel and may not be representative of conditions in the field, where multiple biomass fuels of varying moisture contents may be used . The metrics described above were used to create a log of daily household usage, including the number of uses, duration of use, and estimated energy used by each stove . Overall trends in use of the traditional stove before and after introduction of the intervention were compared using t tests . We evaluated the change in daily mean traditional stove use after introduction of the intervention using linear mixed models to partition the between- and within - household variance components and to calculate the intraclass correlation coefficient (icc, the proportion of variability explained by between subject differences). Models took the following form:3where yij is the i duration of use in household j, 0 is the overall intercept, bi is the random effect for household i, and eij is the leftover error . This baseline model was run first for the combined data set and then separately by period (preintervention and post intervention) for the traditional stoves . We additionally evaluated how well short measures of usage predicted the study average during stable periods of usage . This analysis was restricted to the traditional stove, which exhibited stable use patterns, and was performed independently for the pre- and postintervention periods . We calculated means from varying lengths (1 day, two consecutive days, two random days, 1 week, and 1 day per month) of usage data selected randomly from each household and study period and compared it to the mean duration of use for the entire study period . For these shorter measures, we calculated the probability of a random measurement falling within a precision interval (for instance, within 20% in either direction of the period mean). During the preintervention period, usage of the traditional stove was measured in 177 homes for, on average, 34 days (sd = 35, range = 3103). In this period, households used their primary traditional stove 1.4 times (sd = 0.8) for an average of 209 min (sd = 105) per day . After introduction of the intervention, the traditional stove was monitored for, on average, 251 days (sd = 97, range = 52426); the philips stove was monitored for, on average, 358 days (sd = 54, range = 139433). During the postintervention period, households exhibited a significant mean decrease in the use of their primary traditional stove to 144 min per day (p <2.2e-16, sd = 134) once daily . The intervention stove was used, on average, 0.6 times daily (sd = 0.8) for 60 min (sd = 87) after its introduction . Panels show temperature traces for the traditional stove (blue dashed line) and for the philips stove (solid red line). Figure 2 shows patterns of the transition between traditional stoves and the intervention stove, as illustrated by data from two study households . In both panels, the dotted blue line is the sums trace from the traditional stove; the solid red line is the trace from the philips . Pre- and postintervention patterns of use are shown . In the upper panel (mixed use), the philips is used upon introduction repeatedly over the course of a week concurrently with traditional stove use . Philips use declines and tapers off in the final week . In the lower panel (philips use), a third pattern, in which the philips was rarely or never used, was observed but is not displayed . These types of patterns were typical of the larger population during the first month after introduction of the stove . Use patterns during the first through third months postintervention in homes with sums data available on both stoves for at least 15 days per month are described in table 1 . During the first month with the philips, almost all homes used both stoves (n = 152). 6% of homes used the philips exclusively (n = 9); only one home did not use the philips . Among the homes using both stoves, the philips accounted for greater than 80% of cooking events in 17% of homes subsequent months exhibited wide variability between and within homes (see the si). Among the 9 homes that exclusively used the philips during the first month, average use of the philips decreased from 111 min daily during the first month postintervention to 78 min daily across the remaining months . Additionally, all households exhibited multiple days during later months in which neither stove was in use, suggesting that food was obtained by other means (from relatives or purchased), cooked in alternate locations, or cooked using stoves not fitted with sums . Similar trends were noted for homes exclusively using the philips in months two and three . No use of either stove recorded . The variability in usage of the intervention and the lack of displacement of cooking tasks from the traditional stove to the intervention is emphasized at the study population scale in figure 3 . Between introduction of the intervention and postintervention day 200, there is a significant and consistent decrease of 0.28 min / day in use of the philips (p <2e-16); between day 200 and the end of monitoring, usage stabilizes but continues to decrease by 0.04 min per day . Similar trends were noted for daily use event counts over time (see the si). Most of the total variability in usage across stove types was due to variability within homes: 66% across periods for traditional stoves and 78% for intervention stoves . The total variability was highest for traditional stoves in the postintervention period, perhaps indicative of either a shift first to and then from the philips or mixed use of both stoves . Si table s3 shows the means of use duration overall and by stove type and period and presents the calculated iccs, the proportion of variability explained by differences between subjects . Prior to the intervention, households utilized 15.5 mj of energy per day (sd = 1.5) from cooking with their traditional stoves (figure 4). After introduction of the intervention stove, utilized cooking energy from the monitored traditional stove decreased significantly to 10.6 mj per day (sd = 0.86, p <2.2 10). In the first month after introducing the intervention, however, total average utilized energy increased to 21 mj daily, due to use of both stoves . Counterintuitively, perhaps, decreasing usage of the more efficient philips in subsequent weeks led to decreasing total energy use . Assuming the rate of energy consumption of each stove remained constant throughout the study, the average daily utilized energy across the postintervention period increased to 16.3 mj (p = 0.003). The utilized cooking energy is presented separately for the traditional and intervention stoves (blue and red, respectively) and pre- and postintervention periods . Of cooking duration1 day, 1 day per study month, 2 random or 2 consecutive days, and one consecutive week to predict mean stove usage of the traditional stove during the pre- and postintervention periods . These periods for the traditional stove were selected because they exhibited relative stability over time, as compared to the philips . Short measurements had a low probability of predicting the study - wide mean of stove usage . Precision varied across the pre- and postintervention periods (si figure s5 and table s4). Short - term measures adequately predicted preintervention means with traditional stoves . During this period, a consecutive week of sampling had the highest probability (75%) of being within 20% of the long - term mean . Just 18% of random single days were within 20% of the long - term mean for the traditional stove . The mean of samples taken for 1 day per month postintervention had a 66% chance of being within 20% of the long - term average . We report on the usage of an intervention stove distributed to 177 pregnant woman and related changes in use of the traditional stove over approximately 60 weeks in rural india . The data set consists of one of the largest and longest objective measurement campaigns of stove usage to date . By deploying stove use sensors for over a year, we were able to track and report for the first time the changes in usage of an advanced cookstove intervention and the primary traditional stove over time . Few algorithms for converting temperature traces to event counts and durations of use have been published . We offer a novel analysis method: usage events defined as periods that deviate from ambient temperatures . This method does not rely on any additional assumptions about the distribution of the data and facilitates relatively fast analysis of large volumes of data . It does, however, require local measurement of ambient temperature, which can introduce additional cost . We focus on durations of use, as we believe this to be a more health - relevant metric and a better indicator of potential risk than number of events, which can be easily obtained from duration data if needed (see si). Further evaluation of this algorithm is ongoing on both previously collected and new sums data sets . We are additionally investigating the feasibility of household or village level ambient sums to aid with signal processing and to account for microclimatic variability not captured by a single, meteorological station . Finally, we are monitoring usage on many different stove phenotypes globally; these activities will help optimize sums placement practices, and evaluate and hone the described algorithm to determine its broader applicability . We see a need for standard methodologies for interpretation of ibutton signals that cater to specific research or programmatic goals . Daily time of use and number of uses are simple metrics obtainable from sums data through a number of methods . Interstudy comparisons of usage may be complicated, however, by the algorithm design decisions used to generate these metrics . For instance, time - of - use is impacted by the threshold at which the stove is no longer considered to be on; the number of uses is similarly affected by decisions about clustering of temperature peaks . Clear specification of algorithm parameters ideally in the form of open - source code and evaluation of algorithms in multiple studies can help clarify differences between methods . We found continuously decreasing population trends in usage of the intervention stove over time . This trend leveled off between 175 and 200 days postintervention . While usage of the intervention had not completely ceased at the end of data collection, the number of homes using the intervention stove regularly and the related durations of use were lower than immediately after stove distribution . Our findings are supported by other studies that have (1) indicated stacking of devices throughout the adoption process and (2) acknowledged a trial period during which the household evaluates the suitability of the intervention . Utilized cooking energy showed similar trends, with an increase in total uce following introduction of the philips followed by a leveling off and stabilization . Future studies should focus on similar calculations to understand if there is a setting, addition of the advanced stove seemed to increase overall energy use, perhaps because the users took advantage of an additional stove to provide more cooking services rather than substituting the philips for the traditional stove . Any future studies seeking to calculate uce should evaluate cooking power in the field, as laboratory and field stove performance parameters often vary widely . Because we relied on these laboratory estimates and applied them uniformly over the study period, we may be mis - estimating the actual utilized cooking energy . Our findings indicate that the philips may have temporarily offset a portion of measured traditional cookstove usage, albeit in a way that may have increased total energy use . Despite this continued use of the philips, however, it failed to become the dominant stove used in the home, as would be necessary to maximize health protection . Importantly, without measurement of usage of both the primary traditional and intervention stoves, we would have been unable to make any determinations about the role of the philips as an added cooking appliance in household cooking . Finally, we would not have observed the initial uptick and subsequent decrease in uce after introduction of the intervention . The high within - household variability of daily usage of both stoves especially in the postintervention period indicates that care must be taken when using short - term measures of usage to predict long - term means . This stands in stark contrast to previous work in guatemala, where the majority of variability was found to be between households . Most likely, this is due to the difference between the character of the intervention in guatemala, which was well - known to the community and locally created, and the intervention in india, which while vetted in the community was an engineered object brought in from elsewhere . Continuous measurements allowed us to evaluate the ability of short - term measurements to predict the long - term mean . Short - term measurements of one or two consecutive days did a poor job of predicting the long - term mean, with the majority of measurements deviating from the mean by over 20% . For instance, one 24 h measurement per month of the study were much closer to the long - term mean . These findings suggest that future intervention studies should measure stove usage regularly to capture inherent variability in household behavioral patterns and to best capture changes in usage over time . Given that short - term measures fail to accurately predict long - term means in relatively stable situations, their value in dynamic situations, such as the days and weeks following intervention introduction, is limited . Attempts to assess adoption and use must track behaviors consistently for longer periods of time . Although promoted by village health workers, the stoves were given to participants free - of - charge, which has been shown to impact perceptions of value . Participants were enrolled based on pregnancy during the initial phases of the study and may not represent the broader population . Cultural cooking practices related to pregnancy may impact adoption of an intervention stove; initial and long - term usage in households without a pregnant woman may be more consistent or significantly different from the patterns we observed . However, as our study population represents a particularly vulnerable group, indications on how they use this free intervention can inform future studies targeted toward similar communities . Second, we were unprepared to instrument the other traditional stove types found in many households . While we placed two sensors, one on the intervention stove and one on the participant - reported primary traditional cookstove, it is possible that other traditional stoves were also used during the study period . Further, there is possibility of the hawthorne effect: instrumentation of the primary traditional cookstove may have shifted usage to other, unmonitored traditional stoves . Among users who exclusively used the philips during month 1 or 2 of the study, we noted multiday periods of inactivity with both monitored stoves in subsequent months, indicative of cooking elsewhere or use of another stove . We believe either of these reasons may account for the higher levels, on average, of traditional stove usage in the preintervention period . As a result of these caveats, our study paints only a partial picture of the true usage patterns in the home . As these secondary and tertiary stoves were reported to be used only for simmering milk or cooking during inclement weather, we do not believe there were wide changes in their use as a result of introduction of the philips . We cannot, however, discount the possibility of use of unobserved and unmonitored stoves . The fieldworker burden for this study was high, with a small team of fieldworkers visiting each household every 2 weeks . Households were spread over a relatively wide area, leading to significant transit time and costs and fieldworker turnover . Similarly, the volume of data proved to be a logistical challenge to manage, clean, and transfer . Strict protocols and fieldworker assurances facilitated analyses but could not, inherently, decrease data transfer and processing times . Sums on traditional stoves were especially difficult to maintain over long periods of time due to challenges with placement related to overheating and exposure to water (see si). We are exploring alternate measurement techniques including infrared thermometers, thermocouple - based data - loggers, and wireless transmission of data to improve data completeness and fidelity for traditional stoves . Comparisons of data measured with sums to participant - reported stove use and perceptions of the philips as a replacement for the traditional stove are in preparation . Such comparisons have, in some cases, revealed that reported stove use is similar to measured use, while in other cases reported use exceeds measured use . Future intervention studies should focus on long - term objective measurement of stove use and seek a deeper understanding of the individual and community behaviors motivating use or nonuse of an intervention through qualitative methods from behavioral science . Stove usage is a critical link between the potential and delivered benefits of intervention programs . Monitoring of usage over time is necessary to fully understand the potential for delivery of those benefits; in this study, short - term measurements of benefits immediately after intervention distribution would have been misleading and potentially led to mistaken claims of benefits . The low long - term usage of the intervention stove, while disappointing, is informative . It indicates (1) that preliminary work, while valuable to assess initial feasibility of an intervention, will most likely not predict long - term viability; (2) that measurement of usage of both traditional and intervention stoves is required over time to fully understand and accurately characterize adoption of an intervention and changes in traditional habits; and (3) that a combination of more transformative, aspirational interventions that can fully displace the traditional stove and education and training, to sway participants away from the old stove, will be required to fully realize benefits.
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A 66-year - old caucasian woman with a medical history significant for chronic kidney disease (ckd) stage 3, hypertension, and morbid obesity was followed regularly at our nephrology and hypertension clinic . She was being treated aggressively for her hypertension, proteinuria, hyperuricemia, and secondary hyperparathyroidism (shpt). Her baseline creatinine fluctuated between 1.5 mg / dl and 2.0 mg / dl, and egfr (6 variable modification of diet in renal disease) between 24 and 34 ml / min/1.73 m. her home medications included paricalcitol, gemfibrozil, febuxostat, pantoprazole, furosemide, lisinopril, latanoprost ophthalmic, and ferrous sulfate . Our patient had never taken warfarin, vitamin d, or any oral phosphate binders . In 2009, her intact parathyroid hormone (pth) levels were noted to be rising despite her receiving oral paricalcitol 1 mcg daily . The dose was eventually adjusted up to 2 mcg daily without controlling her pth which climbed as high as 216 pg / ml . Her calcium levels climbed from 9.0 to 10.4 mg / dl but never higher . Her serum phosphorous levels ranged from 2.9 to 3.7 mg / dl before, during, and after subsequent treatment . Most importantly, the ca po4 solubility product was never greater than 33.7 . In august 2010, she presented with painful eruptions and ulcerations along the medial aspects of her thighs and intertriginous spaces of the lower extremities, slightly more on her right side . Initially, our patient was concerned that it represented a drug - related exanthem in response to a newly initiated uric acid - lowering agent . Accordingly, she had tried to self - medicate using over - the - counter preparations without success . The presence of violaceous ulcerations were identified along the aforementioned distributions, some of which had already begun to drain purulent material . She was prescribed oral doxycycline as well as cleocin - t gel and cultures and biopsy were procured while at the dermatology clinic . Complement levels were normal but serum protein electrophoresis found elevation of acute phase reactants suggesting acute inflammation . Cultures were unrevealing, however, the biopsy from the right medial thigh lesion revealed dermal necrosis with severe neutrophilic inflammation of the local blood vessels . The biopsy was nonspecific as calcium deposition was not identified, but the clinical picture was strongly suggestive of calciphylaxis . A repeat biopsy was not obtained due to our fear of inciting another ulcerating lesion . Furthermore, it has been shown that dermatopathology is most often nonspecific and often needs to be repeated several times to see the typical microvascular calcifications associated with calciphylaxis.1 for this reason, our nephrology clinic generally does not recommend biopsy of these ulcers . At this point she was sent for a three - phase technetium bone scan that was markedly abnormal on blood pool imaging (figure 1). It demonstrated widespread hyperintensities in the soft tissue of her thighs bilaterally corresponding to the suspect lesions, once again more so on her right side . The use of this imaging modality in calciphylaxis has been reported numerous times and has been shown to be abnormal with soft tissue uptake in up to 97% of patients with calciphylaxis, whether ulcerating or not.1,2 as such, she was referred for a technetium sestamibi parathyroid scan, which was suspicious for a parathyroid adenoma on the lower pole of the left lobe of the thyroid . This scan was repeated and confirmed by our surgeon who then performed a subtotal parathyroidectomy the following day . Intraoperatively, an enlarged parathyroid gland was identified, corresponding to the abnormality seen on imaging . It was later confirmed to be a hyperplastic parathyroid adenoma by pathology . In the weeks following surgery, the drainage from her wounds begun to wane, margins began to heal, and her pth level fell to 136 pg / ml, though did not fall lower on serial follow - up . It was at this point that her primary hyperparathyroidism was consider to have been definitively been treated, but the patient s background shpt had not . We were reluctant to give her oral vitamin d, or vitamin d receptor antagonists; instead, she was given cinacalcet . Within 3 months, her pth had fallen to 38 pg / ml and her wounds had entirely healed . Administration of the cinacalcet was not associated with hypocalcemia or significant change to our patient s ca po4 solubility product, which was low to begin with . Calciphylaxis (often referred to as calcific uremic arteriolopathy) is a disabling, and potentially life - threatening complication that has been reported to affect as many as 4% of patients with end - stage renal disease.3 it has been observed to occur primarily in patients receiving maintenance hemodialysis and peritoneal dialysis . However, syndromes resembling calciphylaxis have been reported in individuals with pre - end - stage renal disease - staged ckd and even more rarely in those without any evidence of renal dysfunction but with inflammatory bowel disease, malignancy, trauma, and primary hyperparathyroidism.47 this disease entity is characterized by calcification, intimal hypertrophy, and thrombosis of small vessels that slowly lead to tissue ischemia followed by infarction . Predominantly affecting subcutaneous tissue, this microvasculopathic process leads to the formation of painful nodules or plaques, which are often described as erythematous or violaceous foci that later erupt as necrotizing, nonhealing ulcers . These lesions are typically distributed in highly vascular regions with thick overlying adipose tissue such as the breast, abdomen, and thighs, although acral and even visceral organ involvement has been reported.4 the formation of these ulcerating necrotic lesions portends a grim prognosis as they are often accompanied by severe infectious complications resulting in mortality rates that can exceed 60%.3,8 several strategies aimed at treating and preventing this affliction in the nondialysis population have been reported in the literature . These include early parathyroidectomy,5,7 sodium thiosulfate,8,9 vitamin d analogs,9 bisphosphonates,10 cinacalcet,10,11 and hyperbaric oxygen.12 unfortunately, none of these maneuvers have been shown to be universally beneficial and the outcome for most patients afflicted with calciphylaxis remains quite poor . We describe a unique case of a patient with stage 3 ckd who presented with calciphylaxis when she developed primary hyperparathyroidism superimposed on background shpt that was actively being treated with paricalcitol . She developed her lesions in the face of escalating dosages of the vitamin d analog despite normal ca po4 solubility products . What is interesting in this case is that her wounds did not begin to heal until her parathyroid adenoma was surgically removed and did not completely heal until her pth level was normalized with cinacalcet . This is demonstrated in a straightforward way as pth is secreted in a constitutive fashion from the parathyroid cells without regard for ambient serum calcium levels, often driving the remaining parathyroid glands into dormancy in the process ., the kidney has a decreased ability to generate 1,25-dihydroxycholecalciferol (calcitriol) from its precursor ergocalciferol via the kidney 25(oh) 1--hydroxylase enzyme . Phosphate retention gradually occurs and this, in conjunction with reduced calcitriol levels, results in reduced intestinal calcium absorption and decreased serum calcium levels . As the degree of ckd progresses, the cellular expression of vitamin d receptors and calcium - sensing receptors (casrs) decrease within the parathyroid cells . Furthermore, the casrs are less sensitized to ambient calcium levels.13,14 these circumstances eventually lead to an increase in pth release and a resultant rise in calcium levels . In the long- term, this phenomenon is maladaptive as it leads to bone demineralization (renal osteodystrophy). In these scenarios, pth is believed by some investigators to enhance coronary artery calcifications15 and lead to the aforementioned microvascular calcification, intimal hypertrophy, and thrombosis, which in turn leads to ischemia and subsequent calciphylactic ulcers.16 more recently, fibroblast growth factor 23 (fgf-23) has been established as an important player in the regulation of phosphate - vitamin d homeostasis . Elevations in fgf-23 expression lead to the downregulation of residual renal 25(oh) 1--hydroxylase enzyme activity . This unfortunately potentiates the previously mentioned deficiency of calcitriol production, resulting in enhanced pth synthesis and subsequent parathyroid hyperplasia.17 in our patient, both pathways were operating concurrently . It also explains why she had an excellent initial response to a surgical intervention in the form of a subtotal parathyroidectomy . However, it was not until the calcimimetic agent cinacalcet was added that the pth level normalized followed by complete wound healing . Calcimimetic agents activate the casrs, thereby potentiating the effects of ambient extracellular calcium on parathyroid cell function.18 this, in effect, reengages a negative feedback loop and suppresses pth secretion . Calcimimetics also upregulate casr- and vitamin d - receptor expression and, in animal models, reduce parathyroid gland hypertrophy.19 in our patient, the use of cinacalcet allowed us to lower her secondary elevations in pth without having to provide her with calcitriol or vitamin d analogs, which theoretically may have worsened her calciphylaxis . In conclusion, calciphylaxis is a disabling and potentially life - threatening complication most often accompanying advanced degrees of renal insufficiency . A variety of interventions have been proposed to treat and/or prevent this morbid condition; unfortunately, the outcome for most patients afflicted with calciphylaxis remains poor . To the best of our knowledge, this case represents the first reported case of an individual with stage 3 ckd who developed calciphylaxis amidst mixed hyperparathyroidism (both primary and secondary) and was successfully treated with subtotal parathyroidectomy (for pathologically confirmed parathyroid adenoma) followed by cinacalcet (for background shpt). We feel this case is extraordinary because of the patient s development of calciphylaxis in spite of her relatively mild stage of ckd, as well as clinical response to specific therapy targeted at the pathophysiology of both primary and shpt as detailed . Based on our experience in conjunction with that reported by others, we feel that administration of calcimimetics should be considered as an important adjunct in the treatment of calciphylaxis.
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The success of this therapy is evident between 19401970, where twenty novel classes of antibiotics were discovered . These antibiotics vary, concerning their structure and mechanism of action . Today, many of these drugs are not so effective because bacteria develop resistance, revealing a major challenge for our society . These proteins cleave the antibiotic, so it cannot reach and interact with its target site . This is seen in streptococcus pneumonia and is possible because the bacteria obtains dna from other bacteria via recombinational events . The third way resistance to antibiotics occurs is by targeting new sites, e.g., methicillin - resistant staphylococcus aureus (mrsa). Instead of just relying on the original penicillin binding proteins to maintain bacterial membrane integrity, this strain of bacteria obtained dna from an unknown bacterial donor . It has a new gene called meca which codes for an alternative protein, called penicillin - binding protein 2a (pbp2a). Beta - lactam antibiotics are not capable of targeting these alternative proteins and thus, mrsa infections can be lethal . The last method involves a decreased uptake of the antibiotic, and if it does get into the cell, it is pumped out at a faster rate . These types of resistance are now prevailing in many species and strains of bacteria, in part, because of our propensity to use these agents too frequently . In time, bacteria with these resistant processes will emerge as the predominant form of the bacteria and will be difficult to kill . As we respond to these bacterial survival mechanisms we also inadvertently create drugs, which have the potential to influence other processes, e.g., human behavior . This occurs because our drug discovery process fine tunes itself as resistance develops and we simultaneously develop stereo specific overlaps with naturally occurring biochemicals, altering their actions downstream . In addition to the above concerns in antibiotic development, many of the agents exhibit toxic effects on the host . In part, we surmise, this is due to unique evolutionary relationships that link selective biochemical and molecular aspects of mitochondrial biology to primordial processes in bacterial progenitors . The mitochondrion is an enslaved bacterium, normally producing significant amounts of atp in comparison to glycolysis . It has a similar structure and function to that found in bacteria, and it has a higher level of mutations compared to the nuclear rrna . Antibiotics that are supposed to target pathogens will also bind to mitochondria with high affinity and cause side effects . The commonality of these antibiotic - induced side - effects lead physicians to create a term for this phenomenon called antimicrobial - induced mania, or antibiomania, since it can occur in neural tissues due to higher metabolic rates [1921]. We and others propose that mitochondria dysfunction may be part of the core problem for abnormal behaviors induced by antibiotic treatment, e.g., depression, autism, etc . [dysfunctional mitochondria have recently become a center of interest in explaining mental disorders [2833]. Ciprofloxacin induces a small percent of treated patients to develop psychosis [3436]. In this regard, gamma - aminobutyric acid (gaba) receptor binding is inhibited by ciprofloxacin . Importantly, the 18 kda translocator protein (tspo) localized to the outer mitochondrial membrane, previously designated as the peripheral - type benzodiazepine receptor, has been found to be temporally enhanced in the striatum and substantial nigra pars compacta in a neuro - inflammatory rat model of parkinson s disease or diffuse nerve injury . Interestingly, a reversal of repeated social stress - induced anxiety - like behavioral outcomes in rodents has been linked to the off - target peripheral effects of the widely used benzodiazepine lorazepam on tspo activation . More precisely, in vivo positron emission tomography (pet) scanning using the tspo - specific ligand [c]dpa713 has demonstrated enhanced signal in select brain areas due to in vivo microglial activation as a result of aging and neuronal degeneration . Interestingly, a subtype a of gaba receptor (gabaa) is regulated by the level of mitochondrial reactive oxygen species(mros) at inhibitory synapses of cerebellar stellate cells . Behavioral changes are not limited just to ciprofloxacin, but also occurs with exposure to metronidazole, ofloxacin, trimethoprim - sulfamethoxazole, cotrimoxazole, procaine penicillin and clarithromycin . Additional examples of mitochondrial dysfunction, which are antibiotic - induced, are extensive and not limited to psychiatric behavior . Aminoglycosides have been used for decades, and they are still considered to be effective for treating bacterial infections . However, there is a high risk of damage to sensory cells inside the inner ear when exposed to this antibiotic due to reactive oxygen species (ros) being released from the mitochondria [15,5155]. Another experiment demonstrated that binding of aminoglycosides to the human mitochondrial h69 hairpin is the most likely factor in causing the side effect . Moreover, tetracycline also works by manipulating gene expression via the tet - on / tet - off system . In addition to gene manipulation, it will also induce unnecessary stress upon the mitochondria by disrupting translation . Therefore, translation - targeted antibiotics must be used with extreme caution, especially in patients that have mitochondrial translation defects . Antibiotic - induced mitochondrial damage can be pronounced on neurons, as noted earlier for behavior, especially given their metabolism, which requires 20% of the oxygen entering the body . This phenomenon suggests that aspects of antibiotic activity and cancers may be connected via energy processing . Examples are erythromycin, tetracycline, and glycylcyclines, which have beneficial roles in eradicating some cancer stem cell lines while chloramphenicol, a broad spectrum antibiotic, exhibits conflicting results . This drug works through the jnk and pi3k pathways, which lead to a phosphorylated c - jun protein binding to the promoter region of the matrix metalloproteinase-13 region (mm-13). Vancomycin is a very potent antibiotic and is prescribed against resistant staphylococcus aureus (mrsa) infections . Hmox1, a gene that is associated with cellular oxidative damage is regulated upon vancomycin exposure . Exacerbating this event is the fact that antioxidant genes are down regulated, indicating that this potent drug could be increasing oxidative stress in nephrons . Despite the danger in administering this antibiotic to kill staphylococcus aureus, the benefit of this drug clearly outweighs the risk of damages that can occur . The long - term effect of this agent on mitochondria has yet to be determined . Pseudomonas aeruginosa destroys the cell by releasing pyocyanin, a permeable pigment that targets the mitochondrial respiratory chain . Activation of the sphingomyelinase acid and the release of cytochrome c from the mitochondria shortly follow . Staphylococcus, on the other hand, secretes a toxin (pvl) that creates holes in the mitochondrial outer membrane of neutrophils and stimulates apoptosis via bax genes . The prokaryotic bacterial organism has evolved, over millions of years, the ability to subvert the innate immune response via mitochondrial processes . This strategy, in all probability, is based on conserved common molecular knowledge . Clearly, a good part of the communication is within and external to the cell s organelles, whether it is the eukaryotic mitochondria or prokaryotic ribosomes, this occurs via conformational matching, providing the reason for the mechanism of action . Antibiotics can bind to the bacteria cell and cause changes in the bacterial physiological responses, and the efficacy of these antibiotics is limited or enhanced by environmental factors . The relationship that exists between antibiotics and induced ros have been studied through biochemical, biophysical and enzymatic assays . To further prove that ros, e.g., h202, is being produced, the promoters for oxidative stress regulator were analyzed and showed that there was significant activation of these genes due to the treatment of norfloxacin and ampicillin . These results prove extensive ros production is stimulated by antibiotics and strongly suggest that mitochondria, in general, can be involved in the response . The action of bactericidal drugs can be via interfering with the tricarboxylic acid cycle, destabilizing iron - sulfur clusters, so that iron will participate in the fenton reaction that occurs in the mitochondria, producing harmful hydroxyl radicals . Hydroxyl radicals from the fenton reaction also will damage nucleotides in bacteria and cause the mitochondria to undergo metabolic stress . A possible alternative to advance future antibiotic development involves targeting fatty acid biosynthesis because of differences found in eukaryotic and prokaryotic cells [8486]. Key proteins that can be inhibited in bacteria are, for example, acps, accbcd, fabd, and coaa . These proteins assist in enzymatic activities in simple prokaryotes and inhibit fatty acid synthesis gene expression . Platensimycin, platencin, and phomallenic acid appear to destroy gram - positive cocci, such as staphylococcus- aureus, -pneumonia, and enterococcus faecium . Relatively recent work demonstrates that some gram - positive bacteria are resistant to agents targeting fatty acid synthesis pathways . Problematically, studies show that certain bacterial strains grow better when they get an exogenous source of fatty acids . Interestingly, the large microbe population in the enteric system has not been examined for this phenomenon . These microbes may affect the activation state of white blood cells, which can enter the brain compartment and communicate with neurons . Since these agents are already in use, their approval status for fda evaluation can be either shortened or exempt . In this case this timely mini - review brings attention to the role that mitochondria play in establishing an environment for normal overall behavior to emerge . Pathological perturbations of this process via antibiotics, demonstrate the role this enslaved bacterium performs . A large amount of oxygen consumed, e.g., in the brain, testifies to its moment by moment critical activity . In the shared commonality of chemical communication with bacteria, antibiotic - induced mitochondrial interactions represent a critical factor in micro - environmental and organismic survival . Thus, an enhanced microbial presence or antibiotic level may alter the energy supply of a cell and thus enhance the occurrence of an induced behavior disorder . In this case, the potential to initiate mitochondrial dysfunction becomes clear, and this cascading type of action ends in stimulating abnormal behaviors . Clearly, antibiotics have an important place in medicine; despite the risk of damage to the host . In this scenario, one may expect alterations in behavior since they will emerge from high - level energy nerve cells . We speculate that in susceptible individuals and ones using these agents for extended periods of time and non - recommended doses, antibiotics may turn an acute stress response into one that is chronic.
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Pain in the upper trapezius (ut) region may be caused by the performance of repetitive tasks or continuous weight on the shoulder1, 2 . Brassieres may be a factor contributing to ut pain in women because the weight of the breast causes the brassiere strap to press on the ut muscle4, 5 . From their teenage years on, women continuously wear brassieres on a daily basis6, 7 . In most cases, the brassiere has parallel straps that go over the shoulders from front to back8 . The weight of the breasts causes the parallel straps to cut into the outer shoulder and lengthen the ut muscle, causing pain5 . Excessive ut muscle activation resulting from continuous weight on the ut region generates myofascial trigger points that cause pressure pain9 . Generally, clinicians concerned about upper trapezius region pain have overlooked whether or not wearing a brassiere leads to upper trapezius region pain . In addition, previous studies have suggested that wearing a brassiere may be a factor contributing to ut region pain due to the weight of the breasts4, 5, 9 . However, the influence of wearing a brassiere on the muscle activity of ut has not been scientifically proven . Therefore, the purpose of the present study was to examine the effect of wearing a brassiere on ut region pain and muscle activity during arm elevation by women . Fourteen female with ut region pain aged 25 to 47 years volunteered for this study . Inclusion criteria were history of ut region pain for at least 6 weeks and a visual analog scale (vas) score> 5 (severe pain) at rest . Exclusion criteria included past or present neurological pain, cervical spine fractures, radiating pain to an upper limb, and a history of unresolved cancer . The subject s mean age was 34.12 11.62 years, and their mean height and weight were 158.73 4.49 cm and 54.65 6.23 kg, respectively . All subjects read and signed an informed consent form approved by the inje university ethics committee for human investigations prior to their participation in this study . Surface electromyography (emg) data were recorded using a delsys trigno wireless emg system (delsys, inc ., emg data were collected from the right side ut muscle (approximately half the distance between the seventh cervical spinal process and the acromion). Sampling was performed at 1,000 hz, with a bandwidth of 20450 hz, and the root mean square was calculated using emg works 4.0 analysis software (delsys, boston, ma, usa). Each maximum isometric contraction maneuver was performed twice for 5 s, and the average muscle activity of the middle 3 s of the two trials was used to normalize the data . Pressure pain in the ut region was measured using a baseline dolorimeter (pain diagnosis and treatment, inc ., great neck, ny, usa). The instrument consists of a gauge attached to a hard rubber tip 1 cm in diameter . The dial gauge can be calibrated in kilograms (kg) or pounds (lb), with a range of 130 kg or 160 lb . At intervals of 0.25 kg or 0.5 lb . Inter - examiner reliability for the baseline dolorimeter is good to excellent (interclass correlation coefficient= 0.750.89)11 . Prior to testing, the subjects were instructed to indicate when the pressure point was painful . To measure the pressure pain in the ut, the subjects were instructed to sit upright on a chair with their feet on the floor looking straight ahead . The examiner stood and measured pressure pain in the middle of the muscle belly between c7 and the acromion . The right and left sides were each measured three times, and the average was calculated . The subjects were instructed to perform shoulder flexion with scaption . During upper limb elevation, the scaption plane was controlled using a vertical bar . Subjects were asked to hold the right side shoulder at an angle of 120 degrees for 5 s. the middle 3 s of muscle activity averaged over three trials was used in the analysis . The pressure pain and emg activity were measured with and without wearing of a brassiere in a randomized order . Ut muscle activity and pressure pain between with and without wearing brassiere were compared using the paired t - test . Statistical analyses were performed using spss (ver . 17.0; spss, chicago, il, usa). The emg signal amplitude (% mvic) of ut increased significantly (mean sd, 50.87 8.92 compared to 39.79 7.08) when wearing a brassiere compared to the no brassiere condition in the females with ut region pain (p <0.05). The ut pressure pain did not differ between two conditions (p> 0.05). Previous studies have suggested that wearing a brassiere may be a factor contributing to ut region pain in women9, 10 . However, no studies have reported the influence of wearing a brassiere on the emg activity of ut . To the best of our knowledge, this is the first study to demonstrate that a brassiere can increase ut muscle activity during shoulder scaption in women with upper trapezius region pain . Our results shows there was a significant 21.7% increase in ut muscle activity when wearing a brassiere . A brassiere transfers the weight of the breast from the pectoral fascia to the ut region, generating a downward force on the shoulder12 . To counterbalance the downward force, greater ut contraction is required to elevate the shoulder, resulting in increased ut muscle activity moreover, shoulder scaption resulted in greater contraction of the ut muscle due to the increased leverage of the arm in this study . Sustained and repeated ut muscle contraction would quickly generate ut muscle fatigue, resulting in ut tenderness10 . Therefore, when wearing a brassiere, the ut sustains the small weight of the breast with ischemia induced by the brassiere, which would lower the pressure pain threshold of ut . A previous study suggested that brassiere removal during a day (24 hours) was effective at reducing ut and pectoral girdle muscle pain12 . However, in this study, the pressure pain threshold showed no significant difference between the brassiere and no brassiere conditions, because subjects were asked to remove the brassiere for just one minute for no brassiere condition . Further study is needed to investigate the effects of brassiere removal during the day on the ut muscle activity and pressure pain threshold of the ut region in women with upper trapezius region pain . The investigation and comparison of various designs of brassiere on the ut muscle activity and pain threshold is also required . A limitation of this study was that we did not measure the breast weight or size of the subjects; different sizes of breast may affect the pain intensity of ut . Also, we did not control for the fit of the brassiere that each participant wore . In conclusion, wearing a brassiere can increase ut muscle activity, so clinicians should consider recommending brassiere removal for as long as possible, or suggest wearing a well - fitting and supportive brassiere when managing women with upper trapezius region pain.
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Orthotopic liver transplantation is an important treatment option for patients with end - stage liver disease . In the united states, 109,126 liver transplants were performed between january 1, 1988 and april 30, 2011 with 6291 transplants performed in 2010 . Indications for liver transplant include cirrhosis secondary to hepatitis c, hepatitis b, cryptogenic hepatitis, and autoimmune disease . Cholestatic liver diseases for which liver transplantation may be required include biliary cirrhosis, caroli's disease, and primary sclerosing cholangitis . Other reasons for transplant include biliary atresia, acute hepatic necrosis, metabolic disorders, malignant neoplasms in certain cases, and budd chiari syndrome . Advances in surgical techniques over the past several decades, along with improvements in organ preservation and immunosuppressive therapy have improved patient outcomes after liver transplantation . Post - operative complications, however, can limit this success, especially if not recognized early enough to allow for graft salvage . Among the most common and clinically significant complications to occur in the post - operative period are those of vascular origin . Ultrasound with doppler is the primary imaging modality for the evaluation of hepatic transplants, providing detailed assessment of vascular flow in the graft . However, a multimodality approach including ct angiography, mri, or conventional angiography may be necessary to diagnose the complications . This article describes the vascular anatomy of liver transplants and imaging appearances of the major vascular complications that may occur within the hepatic artery, portal vein, and venous outflow tract . All our images have been obtained from a single major urban medical center where all 52 transplants have been performed since our program began three years ago . All liver transplant patients undergo a liver sonogram with comprehensive doppler evaluation on post - operative day one . The sonographic examination algorithm includes gray - scale evaluation of the graft and biliary tree, color, and spectral doppler of the main hepatic artery, portal vein, hepatic veins and ivc, their anastomoses and intrahepatic branches . Further imaging is based on clinical follow - up including daily monitoring of laboratory values . If clinical parameters are abnormal, repeat sonography is performed and depending on the results, ct, mri, or angiography may be requested . Typically, the donor hepatic artery is anastomosed end - to - end to the recipient hepatic or celiac artery . In recipients with a diseased or small hepatic artery, a donor iliac artery or ptfe (polytetrafluoroethylene) interposition graft hepatic arterial complications may lead to graft ischemia and are associated with high patient morbidity and mortality . Common complications of graft ischemia include biliary stenosis or necrosis as the biliary tree relies solely on the hepatic arteries for its blood supply [figures 1 and 2]. (a) axial ct angiography image (cta) performed 1-month post - transplant shows a patent interposition graft (white arrow) between the abdominal aorta (black arrow) and donor hepatic artery . (c) coronal reformat cta demonstrates patent intrahepatic arterial collaterals (black arrows). (d) mrcp coronal thick slab demonstrates two biliary strictures in the common duct secondary to arterial ischemia (white arrows). 60-year - old woman status post - liver transplant with an interposition graft between the aorta and the donor hepatic artery with questionable findings for graft stenosis on sonography . (a) coronal mip reformatted image of contrast - enhanced mri reveals areas of interruption of the contrast column in the graft (white arrows) between the aorta (block arrow) and donor hepatic artery (black arrow) consistent with significant stenoses . Top image: long segment of turbulent flow in the graft with color aliasing (white arrow). Bottom right: spectral doppler reveals persistent graft stenosis post - angioplasty with elevated peak systolic velocity in the distal graft . (f) stenoses were treated with balloon angioplasty with excellent results (black arrows) and a stent was placed in the proximal graft (white arrow). The incidence of hepatic artery thrombosis is 4 - 12% in adults and 42% in children . Risk factors include donor and recipient arterial caliber discrepancy, use of an interposition conduit, acute rejection, and excessive cold ischemia time . Early thrombosis is a dreaded complication associated with liver failure, biliary leak, sepsis, and a high mortality rate of 20 - 60% . Prompt diagnosis is crucial, generally necessitating immediate graft revascularization . Even with early recognition of thrombosis, 60% of patients will still require retransplantation . Patients with hepatic artery thrombosis can occasionally present, many years post - transplant, with sepsis and chronic rejection . Hepatic artery stenosis occurs in 5 - 11% of liver transplant recipients usually in the first 100 days post - transplant . It is often at the anastomotic site, due to clamp injury and intimal trauma . The normal hepatic arterial spectral flow waveform demonstrates a low resistance pattern with a brisk systolic upstroke . The systolic upstroke acceleration time should be <0.08 s. the normal hepatic arterial resistivity index (ri) ranges from 0.50 to 0.80 . In the first 72 hours post - transplant, the ri may be greater than 0.80 due to vascular spasm and post - surgical swelling, although it generally returns to normal in a few days . In main hepatic artery thrombosis, the artery may not be seen on gray - scale . Abnormal doppler findings of hepatic artery thrombosis are absent color power doppler and spectral flow in the main or intra - hepatic arteries . Hepatic arterial collaterals may develop in chronic thrombosis and demonstrate low intrahepatic arterial ris, mimicking stenosis . Therefore, the sensitivity of ultrasound for the detection of hepatic artery thrombosis decreases as the interval following transplant increases . With hemodynamically significant hepatic artery stenosis, a segmental increase in flow velocity is found at the post - stenotic jet . A focal peak systolic velocity (psv) in the main hepatic artery of greater than 2.0 m / s indicates significant stenosis . Color doppler imaging can be used to rapidly identify the line of maximal velocity associated with the stenotic site, by carefully reducing the color doppler scale to produce the slightest degree of aliasing, which can be recognized as a change in doppler color at the brightest portion of the color spectrum . Intrahepatic arteries may have a tardus - parvus waveform with a prolonged systolic upstroke acceleration time and low intrahepatic ri(s) of less than 0.50[figure 2]. In a recent study by park et al, the use of post - stenotic hepatic arterial flow velocity in conjunction with the finding of a tardus - parvus wave pattern can significantly increase the positive predictive value of doppler for diagnosing hepatic artery stenosis . Pseudoaneurysms are a rare but potentially catastrophic complication of hepatic transplantation, often occurring at the anastomotic site . They may develop secondary to infection, graft biopsy, or transhepatic catheter / drain placement . They are often clinically occult or may present with nonspecific graft dysfunction or abnormally elevated serum liver enzyme levels . Contrast - enhanced ct is superior to sonography in identifying small pseudoaneurysms, as bowel gas may obscure a tortuous extrahepatic hepatic artery . Ct angiography or conventional angiography is performed if there is suspicion of graft - related bleeding or unexplained graft malfunction . The donor portal vein is usually anastomosed end - to - end to the recipient portal vein . However, if there is pre - existing thrombosis of the recipient portal vein, thrombectomy or placement of a jump graft between the donor portal vein and recipient superior mesenteric or splenic vein can be performed . Mild focal narrowing at the anastomosis is a common finding which does not necessarily imply stenosis . On doppler examination turbulent flow with elevated portal venous flow velocities may be present in the early post - operative period, decreasing over time in the absence of significant stenosis . Portal vein stenosis and thrombosis are rare complications with an incidence of 1 - 2% . Predisposing factors include previous portal vein surgery or thrombosis, high downstream resistance due to an inferior vena cava (ivc) stricture, hypercoagulable state, and technical problems during surgery such as mismatched vessel size or vascular kinking . Treatments include angioplasty, stent placement, thrombolysis, surgical repair with thrombectomy, venous jump graft or portosystemic shunt creation . Gray - scale findings of portal vein thrombosis may include a hyperechogenic clot; however, because acute thrombus can be isoechoic or hypoechoic, color doppler imaging should be performed . With acute thrombosis, the portal vein distends; in chronic thrombosis, the vessel caliber decreases . Very slow portal venous flow may simulate thrombosis, even with power doppler, and so further evaluation with other imaging modalities such as ct, mri or angiography may be appropriate . If arterial flow is detected within the clot, sonographic findings of portal vein stenosis include vascular narrowing with segmental color doppler aliasing and post - stenotic dilatation . Duplex imaging reveals at least a 3- to 4-fold velocity gradient across the stenosis[figure 3]. In a study by mullan et al, a peak portal vein flow velocity of 80 cm / s diagnosed portal vein stenosis with a sensitivity of 100% and specificity of 84% . In a study by chong et al, a peak portal vein velocity of greater than 125 cm / s had only 73% sensitivity but a specificity of 95% . With chronic portal thrombosis and cavernous transformation, an attempt should be made to differentiate large venous collaterals in the porta hepatis from the main portal vein and intrahepatic branches . (a) gray scale shows narrowing at the portal vein anastomosis (arrow). (d) spectral doppler with high velocity at the post - stenotic jet, which is 3.5 times higher than peak velocity in the pre - stenotic portal vein . Decreased portal venous flow velocity may be seen downstream from portal vein stenosis, but it can also be associated with increased hepatic resistance secondary to rejection or recurrent parenchymal disease . Vascular steal phenomenon directing portal flow away from the graft may occur when large pre - transplant portosystemic shunts, commonly splenorenal, are not ligated at the time of transplant . In vascular steal, the portal venous flow may have decreased velocity or variable direction . A pulsatile in orthotopic liver transplantation, there are two main surgical techniques for reestablishing the venous outflow tract . In one, the intrahepatic segment of the recipient inferior vena cava is explanted with the diseased liver and replaced en bloc with the donor intrahepatic cava and graft . Anastomosis usually is performed end - to - end, at both the suprahepatic and infrahepatic segments of the recipient's residual ivc [figure 4a]. (b) piggyback technique: the donor ivc or hepatic vein is anastomosed to the recipient common hepatic vein stump (arrow). An alternative transplant procedure focuses on maintaining an intact recipient inferior vena cava avoiding venovenous bypass intraoperatively . In the piggyback technique, a widely used form of caval - preserving surgery, a short segment of the donor ivc is anastomosed to the common trunk of the recipient's main hepatic veins [figure 4b]. In partial hepatic transplants, one of several techniques can be used to anastomose the donor hepatic vein(s) to the recipient ivc . Complications involving the venous outflow tract are uncommon, reportedly occurring in <1 - 10% depending on whether the procedure is orthotopic or partial . Patients can develop stenosis or thrombosis of the inferior vena cava or main hepatic veins, most commonly at an anastomotic site . Patients with inferior vena caval or hepatic venous complications may present with pain, ascites, hepatomegaly, pleural effusion, peripheral edema, or abnormal serum liver enzyme levels . Caval obstruction can result in renal failure . In the acute post - surgical period, caval stenosis is usually due to anastomotic stricture or caval compression from graft swelling, hematoma or other post - operative fluid collection . With pediatric or living - related donor grafts, vascular redundancy and graft rotation in the latter group may result in venous kinking or frank torsion . Delayed stenosis can result from patient growth or graft enlargement, fibrosis, chronic thrombus, or neointimal hyperplasia ., findings of venous outflow compromise may include caval or anastomotic narrowing with pre - stenotic dilatation of the ivc and/or hepatic veins . Color doppler imaging frequently reveals vascular narrowing and aliasing at the stenotic site where flow velocities are abnormally elevated . Isoechoic or hypoechoic thrombus, not visible on gray - scale imaging, may be identified as an intravascular filling defect with color doppler [figure 5]. 68-year - old man three months status post liver transplant with ivc clot demonstrated with color doppler (arrow). Duplex / spectral imaging typically demonstrates dampening with loss of pulsatility of the normally triphasic flow wave patterns in the pre - stenotic cava and/or hepatic veins . A monophasic wave pattern in the cava and hepatic veins is nonspecific, often seen in the acute post - surgical period [figure 6]. Even a persistent monophasic flow wave pattern in the hepatic veins is non - specific for venous complications, as it is also seen with graft rejection or recurrent parenchymal disease . Post - transplant caval or hepatic venous triphasic wave pattern has a high negative predictive value for downstream stenosis and graft rejection . With hemodynamically significant stenosis, there is an at least 3- to 4-fold flow velocity gradient across the stenosis [figure 7]. (b) same patient one month after surgery with spectral doppler showing normal triphasic hepatic venous flow . The spectral waveform is dampened with maximum flow velocity measured at 20 cm / s . (c) spectral doppler shows dampened waveform and increased flow velocity at the post - stenotic jet 4.8 times higher than the maximum flow velocity in the pre - stenotic ivc . No intravascular spectral flow is demonstrated in regions of venous thrombosis . In severe cases of venous outflow stenosis or in thrombosis, there can be reversed flow in one or more main hepatic veins or in the portal vein . Post contrast computed tomography / angiography (ct / cta) or magnetic resonance imaging / angiography / cholangiopancreatography (mri / mra / mrcp) are ancillary modalities used to evaluate vascular and nonvascular complications of hepatic transplants [figure 1]. These imaging techniques are especially valuable in patients with complex, atypical, or obscured post - surgical anatomy on sonography, or in patients with inconclusive or conflicting ultrasound findings . These include demonstrating nutmeg enhancement pattern of the congested liver in the setting of venous outflow obstruction, hepatic infarction [figure 8], and biliary abnormalities . These modalities also demonstrate the extent of post - operative fluid collections and other abnormalities, which may be partially obscured by gaseous interference on sonography [figure 9]. (b) axial pre - contrast fat saturated t1-weighted mri image demonstrates hemorrhagic infarction in the right hepatic dome evidenced by increased signal (arrows). (c) axial post - contrast t1-weighted mri image with subtraction showing no enhancement in the infarcted area (arrows). 52-year - old man s / p liver transplant with extrinsic portal vein compression by an inflammatory mass . Mri axial t2-weighted images shows (a) signal void of the pre - stenotic main portal vein (white arrow) at the confluence of splenic vein (black arrow) and superior mesenteric vein . (b) an inflammatory mass which is hyperintense on t2-weighted images (small black arrows) causes extrinsic compression of the main portal vein evidenced by decreased caliber of signal void (long white arrow). (c) signal void of the post - stenotic portal vein at the hilum (arrow). Conventional angiography is generally used for problem solving and in preparation for intended vascular interventions . During catheterization, subsequent balloon angioplasty and stent placement are frequently successful in salvaging the graft, especially if early diagnosis is made [figure 2]. All our images have been obtained from a single major urban medical center where all 52 transplants have been performed since our program began three years ago . All liver transplant patients undergo a liver sonogram with comprehensive doppler evaluation on post - operative day one . The sonographic examination algorithm includes gray - scale evaluation of the graft and biliary tree, color, and spectral doppler of the main hepatic artery, portal vein, hepatic veins and ivc, their anastomoses and intrahepatic branches . Further imaging is based on clinical follow - up including daily monitoring of laboratory values . If clinical parameters are abnormal, repeat sonography is performed and depending on the results, ct, mri, or angiography may be requested . Typically, the donor hepatic artery is anastomosed end - to - end to the recipient hepatic or celiac artery . In recipients with a diseased or small hepatic artery, a donor iliac artery or ptfe (polytetrafluoroethylene) interposition graft hepatic arterial complications may lead to graft ischemia and are associated with high patient morbidity and mortality . Common complications of graft ischemia include biliary stenosis or necrosis as the biliary tree relies solely on the hepatic arteries for its blood supply [figures 1 and 2]. (a) axial ct angiography image (cta) performed 1-month post - transplant shows a patent interposition graft (white arrow) between the abdominal aorta (black arrow) and donor hepatic artery . The superior mesenteric artery (sma) (c) coronal reformat cta demonstrates patent intrahepatic arterial collaterals (black arrows). (d) mrcp coronal thick slab demonstrates two biliary strictures in the common duct secondary to arterial ischemia (white arrows). 60-year - old woman status post - liver transplant with an interposition graft between the aorta and the donor hepatic artery with questionable findings for graft stenosis on sonography . (a) coronal mip reformatted image of contrast - enhanced mri reveals areas of interruption of the contrast column in the graft (white arrows) between the aorta (block arrow) and donor hepatic artery (black arrow) consistent with significant stenoses . Top image: long segment of turbulent flow in the graft with color aliasing (white arrow). Bottom right: spectral doppler reveals persistent graft stenosis post - angioplasty with elevated peak systolic velocity in the distal graft . (f) stenoses were treated with balloon angioplasty with excellent results (black arrows) and a stent was placed in the proximal graft (white arrow). The incidence of hepatic artery thrombosis is 4 - 12% in adults and 42% in children . Risk factors include donor and recipient arterial caliber discrepancy, use of an interposition conduit, acute rejection, and excessive cold ischemia time . Early thrombosis is a dreaded complication associated with liver failure, biliary leak, sepsis, and a high mortality rate of 20 - 60% . Prompt diagnosis is crucial, generally necessitating immediate graft revascularization . Even with early recognition of thrombosis, 60% of patients will still require retransplantation . Patients with hepatic artery thrombosis can occasionally present, many years post - transplant, with sepsis and chronic rejection . Hepatic artery stenosis occurs in 5 - 11% of liver transplant recipients usually in the first 100 days post - transplant . It is often at the anastomotic site, due to clamp injury and intimal trauma . The normal hepatic arterial spectral flow waveform demonstrates a low resistance pattern with a brisk systolic upstroke . The systolic upstroke acceleration time should be <0.08 s. the normal hepatic arterial resistivity index (ri) ranges from 0.50 to 0.80 . In the first 72 hours post - transplant, the ri may be greater than 0.80 due to vascular spasm and post - surgical swelling, although it generally returns to normal in a few days . In main hepatic artery thrombosis abnormal doppler findings of hepatic artery thrombosis are absent color power doppler and spectral flow in the main or intra - hepatic arteries . Hepatic arterial collaterals may develop in chronic thrombosis and demonstrate low intrahepatic arterial ris, mimicking stenosis . Therefore, the sensitivity of ultrasound for the detection of hepatic artery thrombosis decreases as the interval following transplant increases . With hemodynamically significant hepatic artery stenosis, a segmental increase in flow velocity is found at the post - stenotic jet . A focal peak systolic velocity (psv) in the main hepatic artery of greater than 2.0 m / s indicates significant stenosis . Color doppler imaging can be used to rapidly identify the line of maximal velocity associated with the stenotic site, by carefully reducing the color doppler scale to produce the slightest degree of aliasing, which can be recognized as a change in doppler color at the brightest portion of the color spectrum . Intrahepatic arteries may have a tardus - parvus waveform with a prolonged systolic upstroke acceleration time and low intrahepatic ri(s) of less than 0.50[figure 2]. In a recent study by park et al, the use of post - stenotic hepatic arterial flow velocity in conjunction with the finding of a tardus - parvus wave pattern can significantly increase the positive predictive value of doppler for diagnosing hepatic artery stenosis . Pseudoaneurysms are a rare but potentially catastrophic complication of hepatic transplantation, often occurring at the anastomotic site . They may develop secondary to infection, graft biopsy, or transhepatic catheter / drain placement . They are often clinically occult or may present with nonspecific graft dysfunction or abnormally elevated serum liver enzyme levels . Contrast - enhanced ct is superior to sonography in identifying small pseudoaneurysms, as bowel gas may obscure a tortuous extrahepatic hepatic artery . Ct angiography or conventional angiography is performed if there is suspicion of graft - related bleeding or unexplained graft malfunction . The donor portal vein is usually anastomosed end - to - end to the recipient portal vein . However, if there is pre - existing thrombosis of the recipient portal vein, thrombectomy or placement of a jump graft between the donor portal vein and recipient superior mesenteric or splenic vein can be performed . Mild focal narrowing at the anastomosis is a common finding which does not necessarily imply stenosis . On doppler examination turbulent flow with elevated portal venous flow velocities may be present in the early post - operative period, decreasing over time in the absence of significant stenosis . Portal vein stenosis and thrombosis are rare complications with an incidence of 1 - 2% . Predisposing factors include previous portal vein surgery or thrombosis, high downstream resistance due to an inferior vena cava (ivc) stricture, hypercoagulable state, and technical problems during surgery such as mismatched vessel size or vascular kinking . Treatments include angioplasty, stent placement, thrombolysis, surgical repair with thrombectomy, venous jump graft or portosystemic shunt creation . Gray - scale findings of portal vein thrombosis may include a hyperechogenic clot; however, because acute thrombus can be isoechoic or hypoechoic, color doppler imaging should be performed . With acute thrombosis, the portal vein distends; in chronic thrombosis, the vessel caliber decreases . Very slow portal venous flow may simulate thrombosis, even with power doppler, and so further evaluation with other imaging modalities such as ct, mri or angiography may be appropriate . If arterial flow is detected within the clot, tumor recurrence must be suspected . Sonographic findings of portal vein stenosis include vascular narrowing with segmental color doppler aliasing and post - stenotic dilatation . Duplex imaging reveals at least a 3- to 4-fold velocity gradient across the stenosis[figure 3]. In a study by mullan et al, a peak portal vein flow velocity of 80 cm / s diagnosed portal vein stenosis with a sensitivity of 100% and specificity of 84% . In a study by chong et al, a peak portal vein velocity of greater than 125 cm / s had only 73% sensitivity but a specificity of 95% . With chronic portal thrombosis and cavernous transformation, an attempt should be made to differentiate large venous collaterals in the porta hepatis from the main portal vein and intrahepatic branches . (a) gray scale shows narrowing at the portal vein anastomosis (arrow). (d) spectral doppler with high velocity at the post - stenotic jet, which is 3.5 times higher than peak velocity in the pre - stenotic portal vein . Decreased portal venous flow velocity may be seen downstream from portal vein stenosis, but it can also be associated with increased hepatic resistance secondary to rejection or recurrent parenchymal disease . Vascular steal phenomenon directing portal flow away from the graft may occur when large pre - transplant portosystemic shunts, commonly splenorenal, are not ligated at the time of transplant . In vascular steal, the portal venous flow may have decreased velocity or variable direction . A pulsatile portal venous waveform may be normal in the early post - operative period . If there is cardiac disease or arterioportal shunting, the pulsatile waveform will persist . In orthotopic liver transplantation, there are two main surgical techniques for reestablishing the venous outflow tract . In one, the intrahepatic segment of the recipient inferior vena cava is explanted with the diseased liver and replaced en bloc with the donor intrahepatic cava and graft . Anastomosis usually is performed end - to - end, at both the suprahepatic and infrahepatic segments of the recipient's residual ivc [figure 4a]. (b) piggyback technique: the donor ivc or hepatic vein is anastomosed to the recipient common hepatic vein stump (arrow). An alternative transplant procedure focuses on maintaining an intact recipient inferior vena cava avoiding venovenous bypass intraoperatively . In the piggyback technique, a widely used form of caval - preserving surgery, a short segment of the donor ivc is anastomosed to the common trunk of the recipient's main hepatic veins [figure 4b]. In partial hepatic transplants, one of several techniques can be used to anastomose the donor hepatic vein(s) to the recipient ivc . Complications involving the venous outflow tract are uncommon, reportedly occurring in <1 - 10% depending on whether the procedure is orthotopic or partial . Patients can develop stenosis or thrombosis of the inferior vena cava or main hepatic veins, most commonly at an anastomotic site . Patients with inferior vena caval or hepatic venous complications may present with pain, ascites, hepatomegaly, pleural effusion, peripheral edema, or abnormal serum liver enzyme levels . Caval obstruction can result in renal failure . In the acute post - surgical period, caval stenosis is usually due to anastomotic stricture or caval compression from graft swelling, hematoma or other post - operative fluid collection . With pediatric or living - related donor grafts, vascular redundancy and graft rotation in the latter group may result in venous kinking or frank torsion . Delayed stenosis can result from patient growth or graft enlargement, fibrosis, chronic thrombus, or neointimal hyperplasia ., findings of venous outflow compromise may include caval or anastomotic narrowing with pre - stenotic dilatation of the ivc and/or hepatic veins . Color doppler imaging frequently reveals vascular narrowing and aliasing at the stenotic site where flow velocities are abnormally elevated . Isoechoic or hypoechoic thrombus, not visible on gray - scale imaging, may be identified as an intravascular filling defect with color doppler [figure 5]. 68-year - old man three months status post liver transplant with ivc clot demonstrated with color doppler (arrow). Duplex / spectral imaging typically demonstrates dampening with loss of pulsatility of the normally triphasic flow wave patterns in the pre - stenotic cava and/or hepatic veins . A monophasic wave pattern in the cava and hepatic veins is nonspecific, often seen in the acute post - surgical period [figure 6]. Even a persistent monophasic flow wave pattern in the hepatic veins is non - specific for venous complications, as it is also seen with graft rejection or recurrent parenchymal disease . Post - transplant caval or hepatic venous triphasic wave pattern has a high negative predictive value for downstream stenosis and graft rejection . With hemodynamically significant stenosis, there is an at least 3- to 4-fold flow velocity gradient across the stenosis [figure 7]. (b) same patient one month after surgery with spectral doppler showing normal triphasic hepatic venous flow . The spectral waveform is dampened with maximum flow velocity measured at 20 cm / s . (c) spectral doppler shows dampened waveform and increased flow velocity at the post - stenotic jet 4.8 times higher than the maximum flow velocity in the pre - stenotic ivc . No intravascular spectral flow is demonstrated in regions of venous thrombosis . In severe cases of venous outflow stenosis or in thrombosis, there can be reversed flow in one or more main hepatic veins or in the portal vein . Post contrast computed tomography / angiography (ct / cta) or magnetic resonance imaging / angiography / cholangiopancreatography (mri / mra / mrcp) are ancillary modalities used to evaluate vascular and nonvascular complications of hepatic transplants [figure 1]. These imaging techniques are especially valuable in patients with complex, atypical, or obscured post - surgical anatomy on sonography, or in patients with inconclusive or conflicting ultrasound findings . These include demonstrating nutmeg enhancement pattern of the congested liver in the setting of venous outflow obstruction, hepatic infarction [figure 8], and biliary abnormalities . These modalities also demonstrate the extent of post - operative fluid collections and other abnormalities, which may be partially obscured by gaseous interference on sonography [figure 9]. (b) axial pre - contrast fat saturated t1-weighted mri image demonstrates hemorrhagic infarction in the right hepatic dome evidenced by increased signal (arrows). (c) axial post - contrast t1-weighted mri image with subtraction showing no enhancement in the infarcted area (arrows). 52-year - old man s / p liver transplant with extrinsic portal vein compression by an inflammatory mass . Mri axial t2-weighted images shows (a) signal void of the pre - stenotic main portal vein (white arrow) at the confluence of splenic vein (black arrow) and superior mesenteric vein . (b) an inflammatory mass which is hyperintense on t2-weighted images (small black arrows) causes extrinsic compression of the main portal vein evidenced by decreased caliber of signal void (long white arrow). (c) signal void of the post - stenotic portal vein at the hilum (arrow). Conventional angiography is generally used for problem solving and in preparation for intended vascular interventions . During catheterization, subsequent balloon angioplasty and stent placement are frequently successful in salvaging the graft, especially if early diagnosis is made [figure 2]. Sonography is the primary imaging tool for diagnosis of early and delayed vascular complications in liver transplant patients . After rejection, this review of the normal sonographic post - transplant vascular anatomy and the imaging appearances of the major hepatic artery, portal vein, and venous outflow tract complications that may occur will assist the radiologist in identifying post - transplant vascular pathology . Recognition of the doppler and cross - sectional imaging manifestations of normal and abnormal hepatic graft vasculature is essential for appropriate and timely management to ensure graft salvage . In cases of inconclusive ultrasound findings, a multimodality approach,
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Sodium valproate is a frequently used drug for the treatment of seizure disorders, bipolar disease and chronic pain . It is known to cause a serious complication, hyperammonemic encephalopathy which is characterized by acute onset of impaired consciousness, headache, vomiting, seizures, ataxia, generalized slowing on electroencephalography and rapid recovery after discontinuation . In this submission, we present a case of valproate - induced hyperammonemic encephalopathy where the major manifestation was restricted to bilateral cerebellar clinical features . A 19-year - old male presented with acute onset tremulousness, staggering gait and difficulty in walking and speech . He was a known case of idiopathic generalized epilepsy treated initially with carbamazepine (800 mg / day) and recently switched over to sodium valproate (1000 mg / day) before the onset of this acute complication . The patient showed bilateral cerebellar signs in the form of defective coordination in both upper and lower limbs with severe degree of gait ataxia and scanning speech . Investigations including hemogram, liver function tests, renal function parameters, serum electrolytes and blood sugar showed normal results . Thyroid function tests (t3, t4, thyroid stimulating hormone) and nutritional parameters including vitamin b12 levels were also found in normal range . However, serum ammonia level was raised to 106 mol / l (normal range 12 - 47 mol / l). The levels of serum valproate and carbamazepine were found to be in the normal range (serum valproate: 52 ug / ml [range: 50 to 100 gm / ml] and serum carbamazepine <1.25 gm / ml [range: 4 to 12 gm / ml]). Patient underwent magnetic resonance imaging of cranium, which turned out to be normal . With this background of clinical observations and laboratory assessment, a diagnosis of valproate - induced hyperammonemic encephalopathy the valproate was replaced by levetiracetam (500 mg) two times a day along with carnitine supplementation . The patient showed rapid recovery and was able to walk without support after a week . His serum ammonia level normalized on repeat serum ammonia estimation (20 mol / l). Valproic acid by virtue of its broad spectrum of action has application in various disorders like epilepsy, bipolar disorders, prophylaxis and treatment of migraine and neuropathic pain . Although valproate - induced hepatic dysfunction leading to encephalopathy is a well - recognized entity, less commonly the drug can also produce an encephalopathy of non - hepatic origin by producing hyperammonemia, and is called as valproate - induced non - hepatic hyperammonemic encephalopathy (vnhe). Other causes of non - hepatic hyperammonemia include inborn errors of metabolism (urea cycle enzyme defects, fatty acid oxidation defects), drugs like 5-fu, asparaginase, salicylate, halothane and enflurane, hematologic diseases like multiple myeloma and acute myeloblastic leukemia, hyperinsulinemia, hyperglycemia, distal renal tubular acidosis, parenteral nutrition and reye's syndrome . The clinical presentation of hyperammonemic encephalopathy constitutes irritability, drowsiness, coma and occasionally these patients have paradoxical seizures . The presence of significant hyperammonemia with cerebellar signs and reversal of hyperammonemia, encephalopathy and disappearance of cerebellar signs and symptoms after discontinuation of sodium valproate confirmed the diagnosis of valproate - induced hyperammonemic cerebellar ataxia with metabolic encephalopathy . This view is also supported by generalized slowing in eeg suggestive of metabolic encephalopathy which also normalized after discontinuation of valproate . We estimated serum valproate and carbamazepine levels which were within normal limits although there is little correlation between valproate level and clinical effects because of the variable absorption rate and short half - life of the drug . In our study, there was decline in ammonia levels after withdrawal of valproate and subsequent disappearance of cerebellar symptoms and features of encephalopathy . There is scarcity of literature on unusual symptoms of valproate - induced hyperammonemic encephalopathy presenting as mainly cerebellar ataxia . Cases have been reported where patients presented with acute onset of confusion, decline in cognitive abilities, and ataxia . We gave carnitine supplementation to our patient as valproate is known to enhance urinary excretion of l - carnitine, leading to depletion of its blood stores . Thus we conclude that valproate - induced hyperammonemic encephalopathy can present mainly with cerebellar ataxia and a high index of suspicion is required for an early recognition, investigation, and treatment of this potentially life - threatening condition.
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Aneurysmal bone cysts (abcs) are rare entities which cause expansile and destructive bone lesions characterized by reactive proliferation of connective tissue1,5). They are benign lesions and can occur in any part of the skeleton and the spine can be affected up to 30% of the cases . Abcs can cause symptoms such as back and/or dorsal pain, neurological deficitis, and pathological fractures . Although abcs mostly occur in the distal part of femur and/or proximal part of the tibia, some cases have been reported in pelvis and posterior elements2,4,5). Abcs can occur rarely in the sacrum and this location has some difficulties in treatment because of the relations with the sacral nerves . Togetherness of abc and fibrous dysplasia, giant cell tumors and/or osteoblastomas are defined as secondary abc by some authors and this association was seen aproximately in 20 - 30% of cases . Diagnosis can be verified by ct scans and mri . In this paper, authors reported two rare cases with thoracic and sacral aneurysmal bone cysts . A 14-year - old male admitted to neurosurgery department with suffering of being unable to walk for 15 days . Thoracic mri images and ct scans demonstrated a mass which causes mass effect on spinal cord (fig . 2 day he was mobilised and his neurological examination was enough for walking with arm holder on postoperative 7 day . On the 5 months control of the patient there were no suffering and no instable images on x - ray and mr images (fig . An 8-year - old male pateint admitted to us with right hip and back pain . In his history, he had fallen down from 2 meters . Spinal mri revealed abc on the right side of the 3 sacral vertebra with an enlargement of 2419mm(fig . The second case is under control and no surgical treatment is suggested because of the patient's age and observing no neurological deficits at the patient . A 14-year - old male admitted to neurosurgery department with suffering of being unable to walk for 15 days . Thoracic mri images and ct scans demonstrated a mass which causes mass effect on spinal cord (fig . 2 day he was mobilised and his neurological examination was enough for walking with arm holder on postoperative 7 day . On the 5 months control of the patient there were no suffering and no instable images on x - ray and mr images (fig . An 8-year - old male pateint admitted to us with right hip and back pain . In his history, he had fallen down from 2 meters . Spinal mri revealed abc on the right side of the 3 sacral vertebra with an enlargement of 2419mm(fig . The second case is under control and no surgical treatment is suggested because of the patient's age and observing no neurological deficits at the patient . Known as a non - neoplastic expansile bone lesion, abc has a consistance of blood - filled spaces separated by connective tissue septa containing bone and osteoclastic giant cells . Discussions about thoracic and sacral aneurysmal bone cysts are limited and only a few cases have been reported3,7). Papagelapoulos et al described 44 pelvis abcs and 12 of them were in the sacrum6). It is well known that total extirpation of sabc is so difficult and surgical results in treatment of sabc are excellent . These clinical findings include leg weakness and numbness, bowl or bladder dysfunctions . In our cases up to 60 - 70% of abc cases that occured in spine present with neurological deficits . The clinical aspects are pain due to the destruction and neurological deficits due to the fractures and compression . In our case the diagnostic problems are because of the expeditious growth of abc and its expansive destruction of bone . The diagnosis becomes more complicated if there is an extra - osseous and soft - tissue tumour mass . It also helps to evaluate the fluidfluid level, which is characteristic for abc on mr images1). Our first case was evaluated as a pathological fracture, and the patient underwent urgent surgery because of the compression on spinal cannal . But the second case was so typical for abc on the radiological examinations as if in the literature . The primary option for treatment is surgery . Enneking classified 3 surgical types: 1-intralesional (curettage and bone grafting), 2-marginal (en bloc) resection, 3-wide resection (segmental resection)3,4,5,6,7). The most important factors in preoperative planning are the location and the growing pattern of the abc . If abc is growing superficially and besides if it is not involving the one - third of the bone, in this case it will be more advantageous . This is really a good intervention for abc and local recurrences occur less com- monly . Besides this, if the abc is so large and located in pelvis or spine extraperitoneal excision and bone grafting could be more difficult and risky . In similar cases like this, curettage and bone grafting remain a choiceable surgical technique . Low - dose radiotheraphy can be performed after curettage and bone grafting as an other option for treatment5). Age, location, size and number of mitotic figures have been suggested for recurrence treatment of abc is difficult and histological examination should be done to prevent overlooked of an underlying more aggressive neoplasm.
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Hypertension is one of the most common if not the commonest medical complication in pregnancy.19 hyper - tensive disorders in pregnancy are found to be the greatest single cause of maternal mortality.911 it also causes a lot of prenatal mortality.12 most of the complications caused by this problem could be reduced by early detection and proper management.1314 studies on the incidence of hypertensive disease in pregnancy in most developing countries, including saudi arabia, are scarce.15 few of those studies, if any, have looked at the extent of knowledge, attitude or practice of doctors or nurses dealing with hypertensive pregnant mothers . Hence, the objective of this study was to determine the status of knowledge, attitude and practice (kap) of doctors and nurses in phc centers with regard to hypertension in pregnancy and to analyze factors affecting kap in the al - khobar area of the eastern province of the kingdom of saudi arabia . Terms used to describe hypertension and its complications in pregnancy differ according to its presentation, gestational age of discovery and the presence of previous history of hypertension before pregnancy . The one defined by the 1972 committee on terminology of the american obstetricians and gynecologists (acog)131519 was adopted for the purpose of this kap study . Doctors and female nurses working at all 8 phc centers in al - khobar area were enrolled for the study . Two self - administered questionnaires structured to examine the essential knowledge, attitude, and practice of doctors and nurses in dealing with hypertensive disorders during pregnancy . The first was intended to gather demographic characteristics and information on the respondent that might affect the kap . These variables included age, sex, nationality, year of graduation, certification, training and experience in obstetrics, duration of work in phc in the country and any in - service training received . The second part of the questionnaire consisted of 49 questions divided into three sections dealing with practice, attitude and knowledge . The practice sections included questions designed to test competence of doctors and nurses in the skills of measuring blood pressure, management of the hypertensive pregnant mother and practices of health education . The knowledge section was composed of 28 questions to test basic medical knowledge regarding blood pressure reading, management of the problem and its complications . Both questionnaires were given to three consultant obstetricians and two nurse educators for consent validity and elimination of non - essential questions . Both knowledge and practice areas were scored on a zero and one additive scoring system in which each correct answer or practice was given one score and no mark given for a wrong response . The attitude part included questions that measured attitudes of participants towards the seriousness and commonness of hypertension in pregnancy and their motivation to improve their knowledge on the subject . Attitude questions were scored using the five - point likert scaling system . In this scale, a high attitude was assigned to the answer if the respondent's answer was to agree or strongly agree to the question in the scale and a low attitude level for not sure or final scoring of the knowledge and practice section was satisfactory if the participant scored at least 60% of the total marks in these two sections . This was an arbitrary cut - off point based on the pilot study and judgment of the experts . On attitude, the questionnaire was handed over to five postgraduate doctors in general practice and their responses to attitude questions were used as a reference standard . The questionnaire for nurses both versions of the questionnaire were pilot tested on a pilot group of 10 doctors and 22 nurses, selected randomly from phc centers in a nearby city, al - dammam to assess level of difficulty, clarity, suitability and time required for their completion . Data were fed into a personal computer, cleaned and analyzed using epi info and spss statistical packages . Frequency distribution tables were constructed and appropriate tests including the multiple regression analysis to identify significant independent factors were applied . The total number of phc doctors in al - khobar area was 44 . Among those 36 (81.4%) who were present at time of data collection their ages ranged between 25 - 47 with mean of 37.1 years (sd 6.4) and 56% were in the 35 - 44 years group . Arab nationals other than saudis including egyptians, palestinian, and jordanian formed the majority of doctors (41.7%), while saudis constituted 22.2% of the group and the rest were from the indian subcontinent . The mean number of years in practice after graduation was 12.6 years (sd 7.38). Distribution of phc doctors according to years of practice after graduation the mean number of years working in phc was 4.7 years (sd 3.4) and 17 doctors (47.2%) had worked for over 5 years in phc centers . Only, 4 doctors (11.1%) had postgraduate qualifications; out of these only one held a diploma in obstetrics . Five doctors (13.9%) had had 3 - 6 months post - internship training in obstetrics but no certification . Of the entire group, only 2 doctors (5.6%) had had courses in hypertensive disorders in pregnancy in the course of their work . In describing their general attitude towards managing hypertension in pregnancy, a majority of doctors (80%) stated clearly that they did not feel confident in managing hypertensive pregnant mothers and would prefer to see children or adult male patients in their clinic . Nevertheless, the attitude of 34 doctors (94.4%) toward learning more about hypertension in pregnancy was positive . Out of the maximum attitude score of 45 marks, the mean score for the attitude questions for the group reached a reasonably high figure of 30.69 marks . Doctors attitudes towards hypertension in pregnancy doctors management of hypertension in pregnancy was quite deficient as 16 doctors (44.4%) reported that they had not actually seen any hypertensive pregnant mother during last year . Around 53% of the doctors depended on bp measurement taken by a competent staff and did not check it . The doctors scores for practice was generally low, as only 60% of them got the correct answers to the skill of blood pressure measurement, a basic skill necessary for any medical graduate . In addition, almost all doctors wrongly reported that they had advised their pregnant hypertensive patients to lose weight and reduce their salt intake . Another major mistake noted among 25% of the doctors was that they said they would start treatment of hypertensive cases immediately after diagnosis in their clinic without considering referral . The scores on practice of 75% of the doctors were below the satisfactory cut - of point . Again the doctors score of 58.3% on all areas of knowledge was unsatisfactory since this directly affected the diagnosis and quality of care for hypertensive mothers . Some of the responses dealt with normal and abnormal readings of blood pressure and the presenting symptoms of preeclampsia . Doctors mean score 12.1 marks out of a total score of 21 on knowledge was moderate . The total number of phc nurses in al - khobar area was 120 . Among these, the 91 (75.8%) who were present at the time of data collection answered the questionnaire . Their mean age was 29.8 years (sd 6.3) ranging between 20 - 44 years, 74% of them fell within the 20 - 34 age group . Indians formed the second largest group (37.4%) followed by filipinos (11%). According to their qualification, 14 nurses (15.4%) held bachelor degrees and 77 (84.6%) had diplomas . Mean years of practice after registration was 8.7 (sd 5.4) and their distribution is shown in table 3 . Mean duration of work in phc was 4.1 years (sd 2.8) and most nurses (69.2%) had spent <5 years working in phc in the country . Distribution of phc nurses according to years of practice after registration thirty - seven nurses (40.7%) had spent <6 months in obstetrics training and 10 nurses (11%) had spent> 1 year . Most of the group (48.3%), mainly the saudis, had minimal training of 3 weeks in obstetrics ward during their internship period as required by the saudi female health institution . A majority of the nurses 73 (80.2%) had not worked in a phc setting before their current positions . Out of those who had had previous phc experience, nine of them had had courses in hypertensive disorders in pregnancy during their work in phc . In describing their general attitude towards hypertension in pregnancy, 86% of them reported that it was a common health problem and they were all positive about learning more . They were all keen to talk to pregnant women about their problem and advice them to take bed rest . Out of the maximum attitude score of 35 marks, the highest mean score was 30.02 marks . Table 4 shows some of the attitude questions and their responses . Nurses attitude towards hypertension in pregnancy the nurses were quite good on practice in the area of hypertension in pregnancy, as 95.6% of them scored satisfactory . The mean score of this section on practice was quite high for nurses being 8.3 marks out of a total score of 12 marks . The nurses level of knowledge was very low since only five of them (5.5%) scored satisfactory on the questions in this section . The deficiencies were in areas dealing with diagnosis and quality of care for hypertensive mothers . Most of them (96.7%) wrongly considered salt restriction and weight reduction as important in the health education of hypertensive pregnant women . Mean scores on knowledge was 9.02 marks out of a total score of 20 marks which was quite low . In the regression analysis of independent factors associated with kap of doctors and nurses, all variables in the first part of the questionnaire such as age, nationality and certification were included in a series of multiple regression equations against each of the dependent variables, namely, scores on knowledge, attitude, and practice for doctors and nurses . There was a positive significant association of the doctors attitude with their sex i.e., male, nationality, being older and previous enrollment in a course on hypertension (r square = 0.26 p=0.02). Their knowledge was only associated with previous training in obstetrics (r square = 0.14, p=0.02), while practices of both doctors and nurses were not associated with any of the factors under study . The attitude of nurses, on the other hand, showed different associations as non - saudi nationals and training in obstetrics had a positive significant effect on their attitude (r square = 0.08, p=0.02). Their knowledge was only associated significantly with being non - saudi (r square = 0.19, p=0.001). Hypertension in pregnancy is one of the major causes of prenatal morality and morbidity.20 it is responsible for about 18% of maternal mortality,102122 maternal mortality in hypertensive disorders of pregnancy is primarily due to low standard of care and delay in referral.14 antenatal care (anc) is a major part of maternal and child services in phc . One of the most important functions of anc is to detect high - risk pregnancies and to give them the necessary care . Findings from this study will help to identify the status kap of workers in phc on hypertensive disorders in pregnancy . It was discovered in this study that the definition of raised blood pressure and its management was not standardized among doctors and nurses in phc . This is not surprising as previous study by bisson in bristol23 in which a large group of general practitioners, hospital doctors, hospital midwives, community midwives and student midwives who were questioned, gave variety of action plans according to their understanding of diagnostic criteria . They considered the reading of 90 mmhg the model value of diastolic bp at which further action would be taken, whether proteinuria was present or absent . Edema was considered a useful indicator by 93% of the respondents and 49% would use ankle edema in their assessment . Another questionnaire - based survey by hutton24 on the management of hypertension in pregnancy completed by 65 new zealand obstetricians found that 40 (61.5%) doctors considered the diastolic of 80 - 85 mmhg, the lowest abnormal reading, at 28 weeks, and by 18 (27.7%) doctors at 36-week gestation . However, 20 (30.8%) doctors considered the diastolic of 90 mmhg, at 28 weeks and 42 (64.6%) doctors at 36 weeks gestation, the lowest abnormal reading . Around 47% of phc doctors had spent more than 5 years in phc service in ksa and only 14% of them had had post - internship training in obstetrics but being males had not been involved in anc activity in phc . Only 2 had had courses in hypertensive disorders of pregnancy . About 44% of the doctors had not seen any cases of hypertensive pregnant mothers during the last year . Questions asked were concerned with four aspects namely, techniques of blood pressure measurements, history and physical examination, health education practice and action management to be taken by the phc doctors on discovered cases . It is obvious that having such little contact with hypertensive cases and lacking the basic training or refresher courses, their management skills will be inadequate . Since hypertension is one of the commonest medical complications in pregnancy, it is important to educate the pregnant mother about it . Almost all doctors report that they advise hypertensive mothers on the need for bed rest . This is an important non - pharmacological measure in the management of the problem.25 on the other hand, almost all doctors report that they advise their patients to restrict their salt intake and to go on a weight - reducing diet . Salt restriction in hypertensive non - pregnant women can be effective but in pregnancy may aggravate the condition . Moreover, diet restriction in pregnancy can lead to delivery of small - for - date fetus.25 similar findings of wrong advice were reported in other studies by trudinger26 and bisson.23 around 85% of phc doctors reported that they would not refer hypertensive cases after diagnosis and 25% of them would start medication immediately after diagnosis . In fact, the cornerstone of management of hypertensive cases starts in the phc center with accurate diagnosis and undelayed referral of these cases from the phc to the specialist or hospital.1621 phc doctors should be aware of their limitation in the management of these cases and not to jeopardize the health of the mother and her fetus . Around 31% did not recognize this is a common health problem in pregnancy, while 97% of them stressed the importance of taking blood pressure reading at each anc visit . That 53% of phc doctors depended on blood pressure measurement taken by clinical staff without confirming it, the reading themselves is an alarming negative attitude as well as wrong practice . The diagnosis of hypertension in general or in pregnancy in any patient is not an easy task and proper management and modifications depend on it . It is therefore, important that the person responsible for the management, namely, the doctor, should confirm the reading again . In looking for factors affecting the attitude of doctors, it was found that gender i.e., male, arab, older and previous training in a course on hypertension were factors associated with higher scores . Longer experience in phc work, being male and older and the absence of a language barrier for arabic speakers, including saudis, might explain to some extent their better attitude scores as compared to those of the females and younger doctors . The knowledge of about 58% of doctors on questions that bare on the diagnosis and quality of care of hypertensive mothers as well as nurses provide most of the vital anc services in the phc . Around 53% of the phc nurses were non - saudis and non - arabic speakers and the language barrier adversely affects the health education in hypertensive cases . It is obvious from the results that there is a shortage of specialized courses for nurses . None of the saudi nurses had more than a diploma in nursing and there did nt seem to be any arrangements for their further movement at universities . A period of 3 weeks in an obstetrics ward for new graduate nurses as part of an internship period is not enough to give clear, pragmatic information on anc services in a phc setting . About 31% of the nurses have spent> 5 years in the phc centers in the ksa, but only 10% had taken courses in hypertension . This clearly, demonstrates the necessity of a better arrangement for on - job training classes for them . Their responses to the question of history and physical examinations were 87% correct and about 90% of them responded correctly to the questions on health education . Their erroneous responses were on the items on restriction on salt intake and weight reduction . These good scores indicate that except for their wrong ideas on salt and diet restriction, which should be corrected at refresher courses, the correct procedure have been learnt . Although the phc nurses were very good with practice in general, around 95% of them had poor knowledge . Knowledge and facts, on the other hand, need to be updated by continuous education . Around 86% of them felt that it was a common health problem and reported that they needed to know more about it . The importance of a health education as a vital task for nurses cannot be ignored . The presence of these cases in the clinic, therefore, provides a good opportunity for the performance of this task . There was a strong positive association of attitude of nurses to non - saudi nationality and training in obstetrics . While all saudi nurses hold diplomas, about 30% of the non - saudi nurses have bachelors degrees . There is a masked difference in the type, duration and content of curriculum at the pre - graduate level for both groups and non - saudi nurses had higher knowledge scores than saudis . The implication of these findings clearly points out the need to improve the knowledge and attitude of phc nurses through refresher courses . Good in attitude questions but had low scores on the practice and knowledge component of the questionnaire . The nurses scored high on practice and attitude but had low scores on the knowledge component . It is recommended that appropriate regular refresher courses on common and serious problems like hypertension be organized for doctors and nurses in the phcs . There should be opportunities for effective training of reasonable duration with clearly defined objectives under proper supervision in good hospitals to improve their knowledge and practice . It would be also appropriate to offer saudi doctors extra incentives for postgraduate study in family medicine to deal with these common problems, and to institute a suitable program of continuing medical education within the health centers for both doctors and nurses . It is also vital to review the curriculum of the female nursing institutions to update both its theoretical and practical content, and extend the duration of training in such common problems as hypertension in obstetrics.
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The use of implantable cardioverter - defibrillators (icds) is important for preventing sudden cardiac arrest in patients at high risk of fatal ventricular arrhythmias.1) in the past decades, cardiologists paid attention primarily to medical problems and prolongation of the life expectancy of patients with heart failure . Recently, the quality of life as well as survival of such patients has been emphasized . Therefore, the demand for subpectoral implantation of cardiac implantable electronic devices (cieds) is increasing in young female patients concerned about their body image . We report a case of combined subpectoral implantation of icd and augmentation mammoplasty via the axillary approach in a young female patient with dilated cardiomyopathy and small breasts . A 20-year - old female patient presented to the emergency department because of dyspnea and chest discomfort . Transthoracic echocardiogram revealed an enlarged left ventricular dimension and severe global hypokinesia of the left ventricle (ejection fraction, 20%; fig . 1). Cardiac magnetic resonance images showed severely decreased left ventricular function and ill - defined delayed enhancement in the septum, both compatible with dilated cardiomyopathy . After 9 months of optimal medical treatment including perindopril, furosemide and spironolactone, cardiac function had not improved . The patient still complained of dyspnea on exertion of new york heart association functional class ii . Non - sustained monomorphic ventricular tachycardia was detected on 24 h electrocardiogram monitoring (fig . We proposed two options for icd implantation: subcutaneous or subpectoral implantation via the axillary incision . The patient preferred the latter option, and also requested augmentation mammoplasty for her small breasts . After consulting the plastic surgeon, we performed a combined subpectoral icd implantation and augmentation mammoplasty procedure via the axillary incision . Before augmentation mammoplasty, the volumes of right and left breasts were 46 and 56 ml, respectively, as measured anthropometrically . Under general anesthesia, skin incisions were performed on both axillary creases, and the plane between the pectoralis major muscle and the rib cage were dissected under endoscopic guidance . Two 185 g form - stable gel breast implants (natrelle; allergan, irvine, ca, usa) were implanted into the subpectoral plane of both breasts . Because the patient's blood pressure decreased in the sitting position, norepinephrine was infused intravenously to maintain mean arterial blood pressure above 80 mmhg . After bilateral breast augmentation by the plastic surgery team, the left axillary vein was punctured via the seldinger technique . A defibrillating ventricular lead (durata 7120q-58 cm; screw type, st . Jude medical, valley view court sylmar, ca, usa) was inserted into the right ventricle via the 9 fr guiding sheath and was stably anchored at the right ventricular apex . An atrial lead (tendril sts 2088tc-52 cm; screw type, st jude medical) the ventricular and atrial leads were connected to the icd generator (ellipse dr, st jude medical). The icd generator was implanted into the subpectoral plane immediately above the left breast implant ., volumes of the right and left breasts were 163 and 169 ml, respectively (fig . We present herein a successful case of combined subpectoral icd implantation and augmentation mammoplasty via axillary incisions in a young female patient who was dissatisfied with her body image . Cieds have shown to improve survival and symptoms of patients with heart disease, and their use has been increasing.1)2)3) the use of icd implantation in young patients due to congenital heart disease, cardiomyopathy, and genetic disorders, such as a long qt syndrome, has also increased.4) currently, quality of life as well as survival is important . As the number of young patients who require cied has increased, physicians should be concerned regarding not only patients' medical problems but also esthetic aspects and psychological fitness . In particular, young or female patients are more concerned about their body image and psychosocial distress associated with shock or sudden death rather than older or male patients.5)6) despite the reduction in the size of cieds in the last few decades, routine subcutaneous device implantation in the pectoral area still results in a visible scar and protrusion . Furthermore, protrusion at the anterior chest causes awareness of the device and discomfort with daily activities when using purse straps, bra straps, or seat belts . Recently, in the united states, the application of subpectoral cied implantation has increased in young female patients having cosmetic concerns.7)8) although there are several methods for cosmetic cied implantation, no nomenclature for these methods has been suggested . We suggest a nomenclature for cosmetic cied implantation based on a combination of three components: incision for lead insertion, incision for generator insertion, and layer of generator implantation . In previous studies9)10) and real - world practice, four types of cosmetic cied implantation have been reported: axillary - axillary - subpectoral, axillary - inframammary - submammary, infraclavicular - axillary - subpectoral, and infraclavicular - inframammary - submammary implantation (fig . 6). The submammary layer lies beneath the mammary glandular tissue and above the pectoralis major muscle . In the present case, we used axillary - axillary - subpectoral implantation . Implantation is superior at it results in minimal and invisible scarring . Complications of subpectoral or submammary cied implantation are not higher than that of those implanted subcutaneously . Obeyesekere et al.10) reported 20 cases of submammary icd implantation, and did not note complications related to the implantation site, such as infection and device migration . During a follow - up of 5 years, the incidence of appropriate and inappropriate shock due to the icd was similar to that reported in other studies . The overall risk of any pocket - related complications was not different between the two groups, and lead complications occurred more frequently in the subcutaneous group.11) patients' satisfaction and acceptance rates were higher in the submammary or subpectoral group than subcutaneous group.7)9) a generator is implanted between the pectoralis major muscle and the rib cage via an axillary incision . Although the device is implanted subpectorally or submammarily, esthetic concerns remain in female patients with low body weight and small breasts . A combined subpectoral icd implantation and breast augmentation surgery has been reported.12) breast augmentation helps conceal the remaining protuberant chest due to the device . A combined subpectoral cied implantation and augmentation mammoplasty procedure is feasible, because the layer of cied implantation is identical to that of breast implantation . This combined procedure is usually performed by cooperation between a cardiac electrophysiologist and a plastic surgeon under general anesthesia . Therefore, multidisciplinary care by a cardiac electrophysiologist, a plastic surgeon, and an anesthesiologist is important for combined surgery . In particular, vital signs and heart function monitoring during the operation are essential in patients with heart failure . Generator change, icd removal or repositioning from the subpectoral to subcutaneous area also requires general anesthesia . As mentioned above, the complication rates of cied implantation in the subpectoral area and other areas are similar . Indeed, in some case series, subpectoral cied implantations not combined with breast augmentation via an axillary approach were performed successfully.13) however, later procedures should be carefully managed, because the icd generator and breast implant were implanted in the same subpectoral layer . Cases of combined subpectoral icd implantation and augmentation mammoplasty in asians have not been reported previously . Combined subpectoral icd implantation and augmentation mammoplasty via the axillary incision is feasible in young female patients with icd indications and small breasts.
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Cerebral palsy (cp) is a non - progressive disease with symptoms that include neurological disorders or developmental disabilities . Children with cp have spasticity, musculoskeletal problems, mobility disturbances, and decreased pelvic movements that lead to awkward movement and sitting posture1, 2 . Symptoms also induce unstable posture control, imbalance, and aberrant control of movement . Postural control is the ability to control the body position in space, and it has a relationship with the sense of balance3 . Hippotherapy and horseback riding have been suggested as interventions for correcting the balance problems of children with cp4 . Two types of horseback riding are available: hippotherapy and therapeutic horseback riding (thr). Hippotherapy provides better pelvic, hip, and trunk movement and influences childrens posture, balance, and coordination5 . Hippotherapy has short - term beneficial effects on the muscle symmetry of the trunk and hip, whereas thr has no effect on muscle tone6 . The effects of these two types of horseback riding have been studied in children with cp, and both types have been demonstrated to improve gross motor function and promote better standing and bipedal balance7, 8 . A horseback riding simulator (joba, panasonic inc ., it was developed to overcome the primary limitations of hippotherapy, such as unavailability and high cost . This device offers an indoor experience of horseback riding and mimics the rhythmic movement of horseback riding, thereby promoting muscular strength and improving sense of balance . The objective of this study was to compare hippotherapy to the use of a horseback riding simulator (joba, panasonic inc ., japan) with respect to their effects on the static and dynamic balance of children with cp . Our results indicate that a horseback riding simulator has beneficial effects as an intervention for children with cp . This study included 26 children with cp who were receiving physical therapy at the h horseback riding center and the n horseback riding center in kyung - ki in korea . The selection criteria for the subjects were a modified ashworth scale (mas) grade less than + 1 . Children who could perform more than 10 m independent walking and were available for more than 30 min training per day were selected . The parents or guardians of all the participants provided their written informed consent in accordance with the ethical principles of the declaration of helsinki (table 1table 1 . General characteristics of subjectshippotherapyhrsgender (m / f)8/59/4age (year)10.81.610.02.2height (cm)125.812.6122.614.3weight (kg)25.26.425.55.7meansd, hrs: horseback riding simulator). Meansd, hrs: horseback riding simulator the children were randomly divided into two groups: a hippotherapy group that included 13 children, and a horseback riding simulator (joba, panasonic inc . The two groups participated in 1 hour of exercise per day, 3 times a week, for 12 weeks . The hippotherapy group carried out anterior - sitting, posterior - sitting, and side - sitting exercises for 10 min each while horseback riding at a walking pace (6 km / h). The course leader held the reins of the horse and two side - walkers held the child s legs to assist the child in sitting in the saddle in order to prevent the child from falling and to help the child to exercise during the horseback riding walk . The horseback riding simulator group exercised with the same protective equipment and were assisted by the same leader and side - walkers . Both groups received 20 min of conventional physical therapy before hippotherapy and performed stretching on the horse or horseback riding simulator for 5 min before and after the exercise . Three physical therapists, who had more than 3 years of experience in pediatric physical therapy and had received education in the experimental method, participated in this study . The subjects static balance ability was measured using bpm (software 5.3, sms healthcare inc ., uk) as the center of pressure sway length while standing for 30 seconds with the eyes open and looking to the front . Statistically significant differences between the measurements obtained before and after the intervention in each group were determined using the independent t - test . The children were divided into two groups and evaluated pre- and post - test using bpm (software 5.3, sms healthcare inc ., uk) and the pbs (pediatric balance scale) (table 2table 2 . Comparison of measurement values at pre - test and post - testvariablegroupprepostpbs (score)hippotherapy35.63.841.24.7*hrs35.84.738.55.3*sway length (mm)hippotherapy220.127.6135.014.3*hrs219.931.7142.818.8**p<0.05 meansd, pbs: pediatric balance scale, hrs: horseback riding simulator). * p<0.05 meansd, pbs: pediatric balance scale, hrs: horseback riding simulator the hippotherapy and horseback riding simulator groups showed significantly decreased sway lengths in the static balance test . Comparison of the pre- and post - test sway lengths showed that the hippotherapy group and the horseback riding simulator group showed significant decreases in sway length of 85 mm and 80 mm, respectively (p<0.05). Both groups showed improved static balance, but no significant difference was found between the two groups . Dynamic balance was also evaluated pre- and post - test using the pbs (pediatric balance scale). The hippotherapy group showed an increase in score of about 4 points, while the horseback riding simulator group showed an increase of 3 points . Both groups showed significant improvements in dynamic balance, but no significant difference was found between the two groups (p<0.05). Balance control is very important for children with cp, and maintaining balance requires three distinct sensory system inputs: visual, somatosensory, and vestibular9 . Generally, the dynamic sense of balance depends primarily on vestibular input on unstable surfaces; however, static balance primarily depends on somatosensory input . Many studies have investigated balance and clinical tools for children with cp . A systematic review identified 22 tools that can assess balance10 . Hippotherapy and thr are types of exercises that affect balance, coordination, and posture, contributing to the development of sensory and perceptual motor skills6, 11 . A horse s rhythmic movement, velocity, and variations can facilitate righting and equilibrium actions . During hippotherapy and thr, these facilitations improve muscular co - contraction and postural balance resulting in improvements in the gross motor function of children with cp12,13,14,15 . Many studies of hippotherapy and thr in children with cp have demonstrated they have positive effects on postural control and balance . The horseback riding simulator has also been reported to be beneficial for children with cp, improving their postural control and global function, and providing enjoyment16 . Haehl et al . Found no significant improvement of in hippotherapy and thr but indicated that hippotherapy had positive effects on the postural control and balance of children with cp17 . Other studies confirmed that hippotherapy and thr are suitable interventions for children with cp . The horseback riding simulator used in the present study offers several advantages over hippotherapy . The simulator is free from space limitations, has a low price, is easy to handle, and is non - affected by weather conditions16 . Use of the horseback riding simulator resulted in improved muscle strength and contraction in elderly people18 . A previous study of children with cp reported that use of a horseback riding simulator significantly improved their postural control and balance16 . Our data indicate that both hippotherapy and use of the horseback riding simulator improved the static and dynamic balance of children with cp . However, due to the fast learning characteristics of children, the long - term 12-week exercise program might have been responsible for the similar results seen in balance improvement . Many factors remain to be considered regarding exercise for children with cp . Therefore, if other tools or exercises were utilized, the results might not match the results presented here . Overall, our results indicate that children with cp may respond equally well to the use of a horseback riding simulator as they do to hippotherapy in terms of balance improvement . First, the number of subjects was small, and second, only static and dynamic balance were evaluated . In spite of these limitations, our results show that the use of a horseback riding simulator can be a good intervention for children with cp . Further studies incorporating larger sample sizes, various cp types, and full utilization of the programs of the horseback riding simulator might be needed . In conclusion, the horseback riding simulator could be a useful alternative to hippotherapy for improving of static and dynamic balance of children with cp.
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Synchronous multiple primary lung cancers are reported rarely and account for about 8% (0.2% to 20%) of all lung cancers [1, 2]. Patients with two or more lung tumours always require thorough diagnostics and an individually planned treatment procedure due to the nature of nodular lesions in the lung . Two primary lung cancers are rarely reported, but triple primary lung cancers are almost unheard of, and there has been just a single report from england describing a patient with this condition (2011). The authors successfully treated the patient surgically (right upper lobectomy), and post - operative histopathological examination revealed squamous cell carcinoma, adenocarcinoma, and adenomatous hyperplasia . The 74-year - old patient with multiple primary lung cancers within the left lung was admitted to the thoracic, general and oncology surgical clinic in 2013 . On interview, the patient reported fatigue and about 8 kg loss of body weight within 6 months . The patient did not report any other ailments, but had been suffering from hypertension and copd, and reported smoking for 60 years, 20 cigarettes per day . The patient underwent bronchoscopy, but it did not confirm any pathology suggesting a neoplastic process in the bronchial tree . The first lesion, 41 21 mm in size, was located in segments 1 and 2, and the second lesion, 31 19 mm in size, was found in segment 3 of the left lung (figs . 1 a, b). Tomographic imaging revealed lesions suspicious for cancer, group 5 lymph nodes up to 13 mm, and left hilum up to 12 mm . An image from the abdominal ultrasound scan revealed no significant abnormalities, apart from a left kidney cyst, 28 19 mm in size . The patient was referred for fdg - pet scanning (positron emission tomography with f fluorodeoxyglucose (fdg)) and for determination of the standard uptake value (suv), providing indirect diagnosis of pulmonary and other neoplastic tumours . In the upper lobe of the left lung investigations revealed a metabolically active tumour between segments 1 and 2, with a maximum diameter of 42 mm and suvmax fdg = 12.5 . The second metabolically active tumour was detected in segment 3 of the left lung and had a maximum diameter of 21 mm with suvmax fdg = 25.6 (figs . Similar to findings from computed tomography, metabolically active lymph nodes were detected with suvmax fdg = 6.6 . No abnormalities associated with elevated fdg metabolism were found in other scanned parts of the body . Fine - needle aspiration (fna) biopsy indicated non - small cell lung cancer . The patient underwent left upper lobectomy plus lymphadenectomy under general anaesthesia and single - lung ventilation . Intraoperative findings confirmed the presence of the previously described lesions within the upper lobe of the left lung, and small lymph node groups 5, 6, 10 and 11 l, which were resected . On the second and third day after surgery, drains were gradually removed from the pleural cavity and full lung expansion was achieved . On the fourth day after the surgery the patient was discharged and currently remains under the care of the thoracic surgery outpatient clinic affiliated with the hospital . Histopathological findings confirmed a tumour 30 20 20 mm in size in segment 3, with the histopathological structure of keratinizing squamous cell carcinoma g2, and with immunohistochemical profiles of p63 (+) and ttf1 (). The second nodular lesion, 45 35 20 mm in size, was described by histopathologists as two lung cancers . The first part of it (90%) consisted of neuroendocrine carcinoma g2 cells, with the immunohistochemical profile cd56 (+), ck7 (), chromogranin (), and synaptophysin (). The second part of the described nodular lesion (10%) consisted of acinar adenocarcinoma g2 cells, with the immunohistochemical profile p63 () and ttf1 (+). In total, 12 lymph nodes were resected; of these, in 5, 6 and 11 l groups no metastases were found, and one out of two resected lymph nodes from group 10 l was found to be metastatic . All the three identified lesions were synchronous primary lung cancers . Computed tomography scan nodular lesions in the left lung positron emission tomography scan the synchronous occurrence of two primary lung cancers in the same lung was described for the first time by beyreuther in 1924 . In the literature there is a single case report, dated 2011, describing the occurrence of triple synchronous primary lung cancers, i.e. Squamous cell carcinoma, adenocarcinoma, and adenomatous hyperplasia . The researchers estimated that two primary lung cancers in the same lobe were diagnosed in 15% of all analysed patients (n = 175). The precise diagnosis of nodular lesions in lungs is crucial for the choice of further treatment method . Procedures used in patients with multiple metastatic lung cancers differ from those used in patients with multiple primary lung cancers . Positron emission tomography (pet) and computed tomography (ct) are very helpful diagnostic techniques for the preoperative assessment of patients . These techniques allow for the determination of the number, type and location of tumours within the body . Multiple lung cancers are usually metastatic, but their status can be confirmed by pet / ct scans [6, 7]. Treatment options for patients with multiple and multifocal lung cancers remain controversial . In the described case examination results provided us with information on multiple metabolically active tumours within the same lung lobe . Nevertheless, the final confirmation of the lesion type is still provided by histopathological examination . Studies evaluating the survival of patients after procedural treatment for multiple lung cancers demonstrated much better outcomes in patients treated surgically for two synchronous primary lung cancers in the same lobe compared to patients with cancer at stage iiib or iv . The patient was classified as having stage t3n1m0 lung cancer (stage iiia) according to the latest, 7 edition of the tnm classification.
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Animals: all experiments were approved by the animal care and use committee of the faculty of agriculture at the university of tokyo, according to guidelines adapted from the consensus recommendations on effective institutional animal care and use committees by the scientists center for animal welfare . Experimentally nave male wistar rats (aged 8 weeks) were purchased from charles river laboratories japan (yokohama, japan). They were housed with three animals per cage in a room with an ambient temperature of 24 1 c and a humidity of 45 5% . The room had a 12-hr light /12-hr dark cycle (lights were switched on at 8:00). All rats were housed separately and were handled for 3 min twice daily, commencing 3 days before the conditioning day . Fear conditioning: fear conditioning was performed in an illuminated room between 9:00 and 18:00, as described in our previous studies [11, 23]. A subject from the conditioned group was placed in an acrylic conditioning box (28 20 27 cm) for 20 min, where it received 7 repetitions of a 3-sec tone (cs, 8 khz, 70 db) that terminated concurrently with a foot shock (0.5 sec, 0.75 ma). We also prepared the non - conditioned group by presenting the tone and foot shock separately during a 20-min period . Fear - expression test: we performed a fear - expression test as described in our previous studies [11, 14]. A test box (25 25 35 cm) was placed in a dark room illuminated with dim red light . The box had three acrylic walls, one wire mesh wall and a wire mesh ceiling . The wire mesh wall was constructed with 1-cm gauge mesh in the lower section (20 cm) and vertical bars spaced 1 cm apart in the upper section (15 cm), which prevented the rats from climbing up to the ceiling . The subject was placed in the box and kept undisturbed during a 3-min acclimation period . Then, a cs was presented five times for 3 sec each at 1-min intervals during the first half of the 10-min experimental period . The behavior of the subjects during the acclimation and experimental periods was recorded with a video camera (hdr - hc9; sony, tokyo, japan) and an hdd - bd recorder (dmr - bw770; panasonic, osaka, japan). Sixty minutes after the beginning of the acclimation period, each subject was deeply anesthetized with sodium pentobarbital and intracardially perfused with saline, followed by 4% paraformaldehyde in 0.1 m phosphate buffer . The brain was removed and immersed overnight in the same fixative, and then placed in 30% sucrose / phosphate buffer for cryoprotection . We used the avidin - biotin - peroxidase immunohistochemistry method to detect fos expression, as described previously [15, 16, 19]. Briefly, we collected successive 30-m sections from bregma 2.16 mm through bregma 3.24 mm . Half of the sections were stained with cresyl violet to confirm the location of the nucleus . The remaining sections were incubated with a primary antibody to c - fos protein (1:7,500; pc38, merck millipore, billerica, ma, u.s.a .) For 65 hr and a biotinylated anti - rabbit secondary antibody (ba-1000, vector laboratories, burlingame, ca, u.s.a .) For 2 hr . The sections were then processed using the abc kit (vector laboratories), and staining was developed by incubating the tissue in a diaminobenzidine solution with nickel intensification . Data analyses and statistical procedures: the data are expressed as means standard error of the means (sem). A researcher who was blind to the experimental conditions recorded the duration of freezing (immobile posture, with cessation of skeletal and vibrissae movement, except for that associated with respiration) and the frequency of walking (number of steps taken with the hind paws) using microsoft excel - based visual basic software to record the duration and number of key presses, as in our previous studies [17, 19, 24]. The behavioral data for the conditioned and non - conditioned subjects during the acclimation and experimental periods were compared using a manova followed by fisher s plsd post hoc test . For immunohistochemical analyses of the bla and cea, we counted the number of fos - immunoreactive cells in each bilateral nucleus in all sections . We then compared the total number of immunoreactive cells between the conditioned and non - conditioned groups using a student s t test . When we detected activation of the nucleus, we compared the total number of immunoreactive cells between the right and left sides in each group using a paired t test . In order to assess the relationship between fear and neural responses, the correlation between duration of freezing and number of fos - immunoreactive cells in the bla and cea was analyzed using pearson s correlation analysis . Rats were either fear - conditioned (conditioned group: n = 10) or non - conditioned (non - conditioned group: n = 11) to an auditory cs on the conditioning day . Then, 24 hr after the conditioning procedure, each subject was placed in the test box and kept undisturbed for 3 min as an acclimation period . Then, during a subsequent 10-min experimental period, five cs tones were presented . As summarized in table 1table 1.behavioral responses during the acclimation periodgroupfreezing (sec)walking (steps)non - conditioned10.9 5.545.9 5.4conditioned3.1 1.254.6 6.0data are expressed as means standard error of the mean ., we found no difference between the conditioned and non - conditioned groups during the initial acclimation period (f(2,18)=0.964, p=0.400). In contrast, behavioral responses during the experimental period were significantly affected by the conditioning procedure (f(2,18)=14.3, p<0.01). A post hoc test revealed that the conditioned group showed increased freezing (p<0.01) and decreased walking (p<0.01) compared with the non - conditioned group (fig . (a) duration of freezing and frequency of walking (mean + sem) and (b) the number of fos - immunoreactive cells (mean + sem) in the basolateral complex of the amygdala (bla) and central amygdala (cea) of fear - conditioned and non - conditioned subjects . * p<0.05 according to a manova followed by fisher s plsd post hoc test for behavioral results, and according to a student s t test for fos expression in the bla and cea . ). Along with these behavioral responses, the conditioned group showed increased fos expression in the bla (t19= 3.29, p<0.01) and cea (t19= 2.30, p<0.05) compared with the non - conditioned group (fig . (a) duration of freezing and frequency of walking (mean + sem) and (b) the number of fos - immunoreactive cells (mean + sem) in the basolateral complex of the amygdala (bla) and central amygdala (cea) of fear - conditioned and non - conditioned subjects . * p<0.05 according to a manova followed by fisher s plsd post hoc test for behavioral results, and according to a student s t test for fos expression in the bla and cea . In the bla (fig . 2.representative photomicrograph showing fos immunoreactive cells in the left (a) and right (b) basolateral complex of the amygdala and in the left (c) and right (d) central amygdala of fear - conditioned subjects . ), fos expression was similar between the right and left hemispheres, both in the conditioned (t9= 1.25, p=0.244) and non - conditioned groups (t10= 0.698, p=0.501) (fig . 3.number of fos - immunoreactive cells (mean + sem) in the right and left basolateral complex of the amygdala (bla) and central amygdala (cea) of fear - conditioned and non - conditioned subjects . * p<0.05 according to a paired t test . ). In the cea (fig . 2c and 2d), we found that fos expression in the conditioned group was greater in the right hemisphere compared with that in the left hemisphere (t9= 2.75, p<0.05) (fig . Fos expression in the non - conditioned group was similar between the right and left hemispheres (t10= 1.25, p=0.241) (fig . Representative photomicrograph showing fos immunoreactive cells in the left (a) and right (b) basolateral complex of the amygdala and in the left (c) and right (d) central amygdala of fear - conditioned subjects . Number of fos - immunoreactive cells (mean + sem) in the right and left basolateral complex of the amygdala (bla) and central amygdala (cea) of fear - conditioned and non - conditioned subjects . * p<0.05 according to a paired t test . In the fear - conditioned group, we further assessed the correlation between the duration of freezing during the experimental period and fos expression in the bilateral bla (p=0.265), bilateral cea (p=0.683), right bla (p=0.445), left bla (p=0.160), right cea (p=0.557) and left cea (p=0.979). When fear - conditioned subjects were re - exposed to the auditory cs, they showed fear responses, such as increased freezing and decreased walking . Along with these behavioral responses, fear - conditioned subjects exhibited activation of the bla and cea, as assessed by fos expression . These results confirmed that the fear - conditioning was successful in our subject group . When we compared fos expression between the hemispheres, fos expression in the cea, but not in the bla, was greater in the right hemisphere compared with the left hemisphere . These results suggest that the right cea is more strongly activated in response to the auditory cs . In the present study, fear - conditioned subjects showed fear responses and an increment of fos expression in the amygdala simultaneously . Given that the amygdala plays a critical role in fear responses [21, 22], we assume a causal relationship between these 2 phenomena . However, we cannot deny an alternative possibility that the observed difference in fos expression was ascribed to the activation of the neurons that are not related to fear responses, because we did not confirm the type of neurons that showed fos expression . To clarify this however, the correlation was not significant possibly due to the small number of the subjects . Therefore, further analyses are crucial to clarify this point . Based on the findings in the present and previous studies, we hypothesize that the cea is the region of the amygdala that plays a lateralized role in auditory fear conditioning . In the present study, we found stronger activation in the right hemisphere in the cea, but not the bla . In addition, in our previous study in which we obtained results suggesting a lateralized role of the amygdala, the lesioned brain area included the cea . This hypothesis may also explain why previous studies have not reported a lateralized role of the amygdala . In rats with pre - training or post - training [2, 20] electrical lesion of the unilateral amygdala, differences in fear responses did not occur according to the lesioned side . However, the lesions in these studies appear to have been placed mainly on the bla, such that the damage to the cea was likely minimal . Given that the cea mediates fear responses to the auditory cs independently from the bla, it is possible that the residual cea mediated fear responses, which prevented the observation of a lateralized role of the amygdala in these studies . In contrast to auditory fear conditioning, contextual fear conditioning has been reported to be right amygdala dominant . This may be attributable to the facts that an intact basal amygdala is indispensable for contextual fear conditioning . In an above - mentioned study, freezing in response to a contextual cs was reduced when a post - training, but not pre - training, electrical lesion was placed mainly on the right as compared with the left bla . Specifically, rats received an electrical lesion of the entire amygdala in one hemisphere and a small chemical lesion of each sub - nucleus of the amygdala in the contralateral hemisphere . Then, these rats underwent fear conditioning and were re - exposed to the contextual cs . The authors found that rats showed reduced freezing when the left amygdala and right bla were lesioned as compared with when the right amygdala and left bla were lesioned . Given that the cea requires an intact bla, specifically an intact basal amygdala, to mediate fear responses to a contextual cs, it is possible that lesioning the bla was sufficient to impair the function of the ipsilateral cea, which enabled us to observe the lateralized role of amygdala in the present study . Consistent with this notion, fos expression in the basal amygdala and cea in response to the contextual cs was greater in the right hemisphere than in the left hemisphere . One possible explanation for the lateralized role of the amygdala is that the conditioning procedure induces more synaptic plasticity in the right cea than in the left cea . Some of the neurons that induce fear responses receive both synapses transmitting foot shock information and synapses transmitting the cs information . The foot shock information and cs information strongly and weakly activate the neurons, respectively . When these activations occur simultaneously during fear conditioning, the synapses transmitting the cs information are strengthened, which enable the cs alone to strongly activate the neuron . As a result previous findings suggest that this synaptic plasticity can be more easily established in the right cea than in the left cea . For example, neurons in the right cea show greater activation to pain than those in the left cea . In addition, pain activates the extracellular signal - regulated kinases required for synaptic plasticity associated with auditory fear conditioning in the right, but not the left, cea . Therefore, during fear conditioning, it is possible that the pain caused by foot shocks activates a greater number of neurons that induce fear responses in the right compared with the left cea, which increases the number of the neurons that receive cs information simultaneously . As a result, the number of strengthened synapses in the right cea becomes higher than that in the left cea, leading to right hemisphere dominance in fear responses to the cs . In summary, we found that fos expression in the cea, but not the bla, was greater in the right hemisphere compared with the left hemisphere . These results suggest that the right cea shows a greater degree of activation in response to an auditory cs . Although lateralized brain function has been reported for both fear conditioning and other phenomena, the biological significance of lateralization has yet to be clarified.
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Many lines of evidence support the proposal that visceral obesity is strongly associated with features of the metabolic / insulin resistance syndrome, and that obesity predicts the development of both type 2 diabetes and cardiovascular disease (cvd). Obesity is associated with a chronic low - grade inflammation, as evidenced by an increase in circulating inflammatory markers, such as c - reactive protein, serum amyloid a (saa), and interleukin 6 . The presence of systemic inflammation in visceral obesity has been linked to an increased risk of developing cvd and type 2 diabetes . Obesity results when there is an imbalance between energy ingested and energy expended . A relative excess of energy (either genetic or diet - induced) results in two major cellular features; adipocyte expansion and infiltration of inflammatory cells into adipose tissue in both mice and humans . Adipocytes and macrophages both generate inflammatory molecules, which lead to insulin resistance and systemic inflammation . Certain saturated free fatty acids (sfas; laurate, myristate, and palmitate) increase inflammatory genes in adipocytes . These events are associated with the generation of reactive oxygen species (ros) and nuclear factor b (nf-b) transactivation . Treatment with the antioxidants, n - acetyl cysteine, catalase and superoxide dismutase (sod) repressed ros generation and nf-b translocation stimulated by excess glucose 4tate, and decreased inflammatory gene expression . Thus, glucose- and palmitate - stimulated ros generation appears to play an important role in adipocyte inflammation . Although several mechanisms such as endoplasmic reticulum (er) stress, hypoxia, and adipocyte death have been reported to be related to adipocyte inflammation during obesity, ros generation is upstream of er stress and apoptosis of adipocyte occurs at later stages of obesity . Ros is a key - modulator that activates the initial the sequence of events that leads to adipose tissue inflammation . The relative contribution of ros derived from different sources in adipocytes during the progression of obesity remains unknown . Therefore, as a first step, our review has been chosen to focus on the role of various sources of ros generated in adipose tissue on inflammation and insulin resistance . Chronic ros production has recently been suggested to be an important contributor to the pathogenesis of obesity - associated insulin resistance . Our review focuses on the pathophysiological roles of ros in adipocytes inflammation; i.e., the effects of prolonged ros generation by excess nutrients and the effect of ros unregulated by insulin signaling . Ros in visceral adipose tissue are significantly increased in genetically obese mice and mice made obese by consumption of a high - fat diet . Ros also can be generated by macrophages, which accumulate in adipose tissue in obesity . Therefore, it is important to understand the source of ros during the progression of obesity . In the early insulin sensitive state, energy flux from nutrient excess flows into lipogenesis, in which excess glucose and free fatty acid (ffa) are used for triglyceride synthesis by adipocytes . In the insulin sensitive state, insulin activates the insulin receptor tyrosine kinase which leads to stimulation of insulin receptor substrate proteins and phosphatidylinositol 3-kinase (pi3-kinase). Consequently, these events increase intracellular glucose uptake into adipocytes via insulin - stimulated translocation of glucose transporter 4 molecules to the cell membrane . In this state, ffa are stored as triglyceride by activating the production of proteins involved in lipid metabolism / uptake, including lipoprotein lipase, fatty acid transport protein, and acetyl coa - synthase . However, it is totally unknown whether these ros casually trigger the insulin resistance, or are a casual bystander of insulin resistance in adipocytes . Thus, although much is known about energy flux in adipocytes during the progression of obesity, little is known as to whether this energy flux leads to differences in the generation of ros in the early and late stages of obesity, nor is there a good understanding of the potential pathophysiological roles of ros derived in these stages . We previously have shown that excess glucose and palmitate are not metabolized to a major extent via mitochondrial oxidation . Instead excess nutrients activate nicotinamide adenine dinucleotide phosphate (nadph) oxidases (nox). Members of the nox family are membrane - bound enzyme complexes that transfer electrons from nadph to oxygen, generating superoxide . This short lived, non - membrane permeable ros is converted, to a longer lived membrane permeable ros, hydrogen peroxide by sod or spontaneously . The nox family has seven isoforms, nox1, nox2, nox3, nox4, nox5, dual oxidase 1 (duox1), and duox2, which are strongly conserved in mammals and are widely expressed in various tissues . Nox1 to 3 require additional cytosolic activators (p47, p67, nadph oxidase organizer 1) whereas nox4 is constitutively active and independent of an activator protein (ap). It is therefore assumed that nox1 to 3 mediates short - term effects, while nox4 is responsible for long term effects . We have found that nox4 is the major nox isoform in cultured murine and human adipocytes . Moreover, silencing nox4 decreased ros generation stimulated by excess glucose as well as palmitate, leading to inhibition of monocyte chemoattractant protein-1 and saa3 expression in vitro . Thus, nox4-derived ros may be a common mediator induced by both excess glucose and palmitate in adipocytes (fig . Other studies showed that nox4 activity increases in the adipose tissue with diet - induced obesity (dio), and nox inhibitor, apocynin treatment reduces ros generation . However, whether nox4-derived ros itself can promote the onset of insulin resistance in adipocytes during the progression of obesity is unknown and needs to be investigated . The pentose phosphate pathway (ppp) generates nadph and pentose from the 6 carbon glucose and is a major source of cellular nadph . Glucose-6 phosphate dehydrogenase (g6pd) is the rate - limiting enzyme in the ppp . Recent studies indicate that g6pd expression is upregulated in adipose tissue in genetic and dio, and that its overexpression is associated with increased adipocyte inflammation and ros generation . Other studies have shown that treatment with dehydroepiandrosterone (dhea), a g6pd inhibitor, reduces obesity in zucker diabetic fatty rats . We found that nadph content and ppp activity were increased by excess glucose, but not by palmitate in 3t3-l1 adipocytes . Moreover, g6pd inhibitors, dhea and 6-aminonicotinamide, or silencing g6pd all inhibited ros generation and monocyte chemotactic factor gene expression by both high glucose and palmitate in 3t3-l1 adipocytes . These studies support the concept that ppp and g6pd could be modulators or mediators of adipose tissue inflammation (fig ., we hypothesize that adipocytes will continue to actively store triglycerides derived from excess nutrients, and will demonstrate increased ppp activity and nadph content, which lead to nox4-derived ros generation (fig . However, whole body nox4 deficiency has been reported to worsen adipose tissue inflammation in a model of dio in mice . Since nox4 activity is essential for pre - adipocyte differentiation to adipocytes, blunted adipogenesis in the absence of nox4 would reduce the number of adipocytes, allowing the remaining adipocytes to become more hypertrophic, thereby leading to adipose tissue inflammation . Indeed, expression of adipogenesis genes (peroxisome proliferator - activated receptor [ppar] and ccaat - enhancer - binding protein [c / ebp]) was inhibited in whole body nox4 knockout mice . Therefore, it is imperative to investigate the alteration of nox4 activity in mice during the pathophysiological progression of obesity where adipogenesis is intact, and to study the effect of adipocyte - specific deficiency of nox4 . Another potentially important source of ros in obesity is from macrophages that are recruited and accumulate in obese adipose tissue . Obesity provokes changes in t - cell subsets and increases the infiltration and accumulation of activated macrophages in adipose tissue . These recruited and activated immune cells can promote the generation of ros by nox2, which is predominately expressed activated in t - cells and macrophages . Whole body deficiency of nox2 shows attenuation of adipose tissue inflammation and insulin resistance in mice fed a high fat diet . This implies that nox2 from immune cells may play a role in adipose tissue inflammation in the intermediate stages of obesity . However, whether these nox2-dervied ros itself has effects in adipose tissue inflammation and can promote the onset of insulin resistance in adipocytes during the progression of obesity is unknown and needs to be investigated . Ros generation is accelerated when the flow of electrons through the electron transport chain is overload, resulting in leakage of electrons . Overloaded mitochondria mainly produce superoxide, which can signal intracellularly, leading to a pro - inflammatory signaling cascade that includes nf-b and ap-1 activation . Although glucose excess presumably drives the overflow of electrons in mitochondria, other energy sources in adipocytes are preferred in the late stages of obesity since uptake of glucose is limited by the insulin resistance of adipocytes . When glucose consumption is reduced by insulin resistance, adipocytes start to use ffa from triglyceride stores for energy (fig . This alteration of energy flux into -oxidation could overwhelm the capacity of mitochondria, leading to leakage of electrons (fig . When pro - inflammatory macrophages accumulate in adipose tissue as a result of the nox4-mediated generation of macrophage chemoattractants by adipocytes, the inflammatory environment induced by these macrophages will be expected to result in adipocyte insulin resistance . With the advent of sufficient insulin resistance, adipocytes will stop storing additional triglycerides, will utilize stored fatty acids from triglycerides, and will show evidence of decreased ppp activity and nadph content . At such times, adipocyte - derived nox4 might have a limited role in ros generation and adipose tissue inflammation . Instead, mitochondria will now take the place of nox4 and play a pivotal role in ros generation and adipose tissue inflammation (fig . 2). In support of this hypothesis are studies that show the production of pro - inflammatory cytokines and macrophage chemoattractants by adipocytes at an early time point (1 week), while adipose tissue insulin resistance occurs much later (after 14 weeks) during the development of dio in mice . These studies also show that even though some infiltration of macrophages occur at early time points, massive infiltration of macrophage happens much later (after 12 weeks). Thus, these studies strongly suggest that adipocytes become insulin resistant during the later stages of obesity, while adipocytes are insulin sensitive during the early stages of obesity . Recent studies also have reported that fasting and caloric restriction in mice in which insulin signaling is disturbed and -oxidation of ffa is increased in adipocytes, results in the generation of chemoattractants and an increase of macrophage accumulation in adipose tissue . These studies support that mitochondria and -oxidation of fatty acids might lead to ros generation . Therefore, we hypothesize that mitochondria - derived ros accounts for massive macrophage accumulation, worsening insulin resistance and systemic inflammation during the late stages of obesity (fig . 2). Also, we hypothesize that a temporal transition of the source of ros between nox4 and mitochondria as obesity progresses is responsible for conversion to a more insulin resistant phase of obesity . Chronic ros production has recently been suggested to be an important contributor to the pathogenesis of obesity - associated insulin resistance . Our review focuses on the pathophysiological roles of ros in adipocytes inflammation; i.e., the effects of prolonged ros generation by excess nutrients and the effect of ros unregulated by insulin signaling . Ros in visceral adipose tissue are significantly increased in genetically obese mice and mice made obese by consumption of a high - fat diet . Ros also can be generated by macrophages, which accumulate in adipose tissue in obesity . Therefore, it is important to understand the source of ros during the progression of obesity . In the early insulin sensitive state, energy flux from nutrient excess flows into lipogenesis, in which excess glucose and free fatty acid (ffa) are used for triglyceride synthesis by adipocytes . In the insulin sensitive state, insulin activates the insulin receptor tyrosine kinase which leads to stimulation of insulin receptor substrate proteins and phosphatidylinositol 3-kinase (pi3-kinase). Consequently, these events increase intracellular glucose uptake into adipocytes via insulin - stimulated translocation of glucose transporter 4 molecules to the cell membrane . In this state, ffa are stored as triglyceride by activating the production of proteins involved in lipid metabolism / uptake, including lipoprotein lipase, fatty acid transport protein, and acetyl coa - synthase . However, it is totally unknown whether these ros casually trigger the insulin resistance, or are a casual bystander of insulin resistance in adipocytes . Thus, although much is known about energy flux in adipocytes during the progression of obesity, little is known as to whether this energy flux leads to differences in the generation of ros in the early and late stages of obesity, nor is there a good understanding of the potential pathophysiological roles of ros derived in these stages . We previously have shown that excess glucose and palmitate are not metabolized to a major extent via mitochondrial oxidation . Instead excess nutrients activate nicotinamide adenine dinucleotide phosphate (nadph) oxidases (nox). Members of the nox family are membrane - bound enzyme complexes that transfer electrons from nadph to oxygen, generating superoxide . This short lived, non - membrane permeable ros is converted, to a longer lived membrane permeable ros, hydrogen peroxide by sod or spontaneously . The nox family has seven isoforms, nox1, nox2, nox3, nox4, nox5, dual oxidase 1 (duox1), and duox2, which are strongly conserved in mammals and are widely expressed in various tissues . Nox1 to 3 require additional cytosolic activators (p47, p67, nadph oxidase organizer 1) whereas nox4 is constitutively active and independent of an activator protein (ap). Nox5 and duox1/2 need intracellular calcium to activate ros generation . It is therefore assumed that nox1 to 3 mediates short - term effects, while nox4 is responsible for long term effects . We have found that nox4 is the major nox isoform in cultured murine and human adipocytes . Moreover, silencing nox4 decreased ros generation stimulated by excess glucose as well as palmitate, leading to inhibition of monocyte chemoattractant protein-1 and saa3 expression in vitro . Thus, nox4-derived ros may be a common mediator induced by both excess glucose and palmitate in adipocytes (fig . Other studies showed that nox4 activity increases in the adipose tissue with diet - induced obesity (dio), and nox inhibitor, apocynin treatment reduces ros generation . However, whether nox4-derived ros itself can promote the onset of insulin resistance in adipocytes during the progression of obesity is unknown and needs to be investigated . The pentose phosphate pathway (ppp) generates nadph and pentose from the 6 carbon glucose and is a major source of cellular nadph . Glucose-6 phosphate dehydrogenase (g6pd) is the rate - limiting enzyme in the ppp . Recent studies indicate that g6pd expression is upregulated in adipose tissue in genetic and dio, and that its overexpression is associated with increased adipocyte inflammation and ros generation . Other studies have shown that treatment with dehydroepiandrosterone (dhea), a g6pd inhibitor, reduces obesity in zucker diabetic fatty rats . We found that nadph content and ppp activity were increased by excess glucose, but not by palmitate in 3t3-l1 adipocytes . Moreover, g6pd inhibitors, dhea and 6-aminonicotinamide, or silencing g6pd all inhibited ros generation and monocyte chemotactic factor gene expression by both high glucose and palmitate in 3t3-l1 adipocytes . These studies support the concept that ppp and g6pd could be modulators or mediators of adipose tissue inflammation (fig ., we hypothesize that adipocytes will continue to actively store triglycerides derived from excess nutrients, and will demonstrate increased ppp activity and nadph content, which lead to nox4-derived ros generation (fig . However, whole body nox4 deficiency has been reported to worsen adipose tissue inflammation in a model of dio in mice . Since nox4 activity is essential for pre - adipocyte differentiation to adipocytes, blunted adipogenesis in the absence of nox4 would reduce the number of adipocytes, allowing the remaining adipocytes to become more hypertrophic, thereby leading to adipose tissue inflammation . Indeed, expression of adipogenesis genes (peroxisome proliferator - activated receptor [ppar] and ccaat - enhancer - binding protein [c / ebp]) was inhibited in whole body nox4 knockout mice . Therefore, it is imperative to investigate the alteration of nox4 activity in mice during the pathophysiological progression of obesity where adipogenesis is intact, and to study the effect of adipocyte - specific deficiency of nox4 . Another potentially important source of ros in obesity is from macrophages that are recruited and accumulate in obese adipose tissue . Obesity provokes changes in t - cell subsets and increases the infiltration and accumulation of activated macrophages in adipose tissue . These recruited and activated immune cells can promote the generation of ros by nox2, which is predominately expressed activated in t - cells and macrophages . Whole body deficiency of nox2 shows attenuation of adipose tissue inflammation and insulin resistance in mice fed a high fat diet . This implies that nox2 from immune cells may play a role in adipose tissue inflammation in the intermediate stages of obesity . However, whether these nox2-dervied ros itself has effects in adipose tissue inflammation and can promote the onset of insulin resistance in adipocytes during the progression of obesity is unknown and needs to be investigated . Ros generation is accelerated when the flow of electrons through the electron transport chain is overload, resulting in leakage of electrons . Overloaded mitochondria mainly produce superoxide, which can signal intracellularly, leading to a pro - inflammatory signaling cascade that includes nf-b and ap-1 activation . Although glucose excess presumably drives the overflow of electrons in mitochondria, other energy sources in adipocytes are preferred in the late stages of obesity since uptake of glucose is limited by the insulin resistance of adipocytes . When glucose consumption is reduced by insulin resistance, adipocytes start to use ffa from triglyceride stores for energy (fig . This alteration of energy flux into -oxidation could overwhelm the capacity of mitochondria, leading to leakage of electrons (fig . 2). When pro - inflammatory macrophages accumulate in adipose tissue as a result of the nox4-mediated generation of macrophage chemoattractants by adipocytes, the inflammatory environment induced by these macrophages will be expected to result in adipocyte insulin resistance . With the advent of sufficient insulin resistance, adipocytes will stop storing additional triglycerides, will utilize stored fatty acids from triglycerides, and will show evidence of decreased ppp activity and nadph content . At such times, adipocyte - derived nox4 might have a limited role in ros generation and adipose tissue inflammation . Instead, mitochondria will now take the place of nox4 and play a pivotal role in ros generation and adipose tissue inflammation (fig . 2). In support of this hypothesis are studies that show the production of pro - inflammatory cytokines and macrophage chemoattractants by adipocytes at an early time point (1 week), while adipose tissue insulin resistance occurs much later (after 14 weeks) during the development of dio in mice . These studies also show that even though some infiltration of macrophages occur at early time points, massive infiltration of macrophage happens much later (after 12 weeks). Thus, these studies strongly suggest that adipocytes become insulin resistant during the later stages of obesity, while adipocytes are insulin sensitive during the early stages of obesity . Recent studies also have reported that fasting and caloric restriction in mice in which insulin signaling is disturbed and -oxidation of ffa is increased in adipocytes, results in the generation of chemoattractants and an increase of macrophage accumulation in adipose tissue . These studies support that mitochondria and -oxidation of fatty acids might lead to ros generation . Therefore, we hypothesize that mitochondria - derived ros accounts for massive macrophage accumulation, worsening insulin resistance and systemic inflammation during the late stages of obesity (fig . 2). Also, we hypothesize that a temporal transition of the source of ros between nox4 and mitochondria as obesity progresses is responsible for conversion to a more insulin resistant phase of obesity . An overall role of distinct sources of ros for adipose tissue inflammation is that (1) in the early stages of obesity, nox4-derived ros from adipocytes provoke the onset of insulin resistance and initiates the recruitment of immune cells in adipose tissue, (2) in the intermediate stages of obesity, nox2-derived ros from infiltrated immune cells worsens adipocyte insulin resistance and adipose tissue inflammation, and (3) in the late stages of obesity, mitochondria - derived ros from adipocytes maintain the adipose tissue inflammation and insulin resistance . We summarize how these three distinct sources of ros might affect adipocyte insulin resistance and adipose tissue inflammation in the early, intermediate, and late stages of obesity (fig . Several large clinical trials have failed to show any beneficial effects of consumption of antioxidant supplements in the prevention of insulin resistance . Nevertheless, from studies in obese mice there is increasing evidence that antioxidant might be beneficial in attenuating insulin resistance and restoring insulin signaling . For example, the antioxidant manganese tetrakis porphyrin and the cell permeable small - peptide antioxidant ss31 (d - arg-2',6'-dimethyltyrosine - lys - phe - nh2) improve insulin sensitivity without altering body weight in a genetic and dio mice . Moreover, transgenic mice overexpressing sod2 exhibit improvements in glucose tolerance and insulin sensitivity resulting from consumption of a high fat diet . A discrepancy between basic research and clinical studies may be due to the fact that we have yet to address the importance of the timing of ros generation, the source of ros, and tissue specific effects of ros . Findings out these discrepancies would be likely to have important translational implications related to the development of antioxidants targeting nox4- or mitochondria - derived ros in different stages of obesity.
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We reviewed outbreak reports to identify outbreaks associated with an imported food from the inception of the surveillance system in 1973 through 2014, the most recent year for which data were available . We obtained additional data for some outbreaks (e.g., country of origin) from the us food and drug administration (fda) and the us department of agriculture food safety and inspection service . We categorized implicated foods by using the schema developed by the interagency food safety analytics collaboration (3). We conducted a descriptive analysis of the number of outbreaks over time, by food category, and by region of origin . During 19962014, a total of 195 outbreak investigations implicated an imported food, resulting in 10,685 illnesses, 1,017 hospitalizations, and 19 deaths . Outbreaks associated with imported foods represented an increasing proportion of all foodborne disease outbreaks where a food was implicated and reported (1% during 19962000 vs. 5% during 20092014). The number of outbreaks associated with an imported food increased from an average of 3 per year during 19962000 to an average of 18 per year during 20092014 (figure). Number of outbreaks caused by imported foods and total number of outbreaks with a food reported, united states, 19962014 . Reporting practices changed over time; 19731997, imported foods anecdotally noted in report comments; 19982008, contaminated food imported into u.s . Included as a location where food was prepared; 20092014, reporting jurisdictions could indicate whether each food is imported (yes / no) and the country of origin . The most common agents reported in outbreaks associated with imported foods were scombroid toxin and salmonella; most illnesses were associated with salmonella and cyclospora (table). Aquatic animals were responsible for 55% of outbreaks and 11% of outbreak - associated illnesses . Outbreaks attributed to produce had a median of 40 illnesses compared with a median of 3 in outbreaks attributed to aquatic animals . Most of the salmonella outbreaks (77%) were associated with produce, including fruits (n = 14), seeded vegetables (n = 10), sprouts (n = 6), nuts and seeds (n = 5), spices (n = 4), and herbs (n = 1). Other agents implicated were tetrodotoxin (3 outbreaks) and campylobacter, chaconine, paragonimus, other virus, sulfite, and trichinella (1 outbreak each). Foods implicated were a chicken dish, crab cake, creampuff, beer, and a wheat snack (1 outbreak each). Latin america and the caribbean was the most common region implicated, followed by asia (technical appendix). Thirty - one countries were implicated; mexico was most frequently implicated (42 outbreaks). Other countries associated with> 10 outbreaks were indonesia (n = 17) and canada (n = 11). Fish and shellfish originated from all regions except europe but were most commonly imported from asia (65% of outbreaks associated with fish or shellfish). Produce originated from all regions but was most commonly imported from latin america and the caribbean (64% of outbreaks associated with produce). All but 1 outbreak associated with dairy products involved products imported from latin america and the caribbean . Outbreaks in this analysis were reported from 31 states, most commonly california (n = 30), florida (n = 25), and new york (n = 16). The number of reported outbreaks associated with imported foods, although small, has increased as an absolute number and in proportion to the total number of outbreaks in which the implicated food was identified and reported . Although many types of imported foods were associated with outbreaks, fish and produce were most common . Many outbreaks, particularly outbreaks involving produce, were associated with foods imported from countries in latin america and the caribbean . Because of their proximity, these countries are major sources of perishable items such as fresh fruits and vegetables; mexico is the source of about one quarter of the total value of fruit and nut imports and 45%50% of vegetable imports, followed by chile and costa rica . Similarly, our finding that many outbreaks were associated with fish from asia is consistent with data on the sources of fish imports (6). One quarter of the outbreaks were multistate, reflecting the wide distribution of many imported foods . Systems like pulsenet have helped to improve detection and investigation of multistate outbreaks, resulting in an increased number of multistate outbreaks (7,8). The increasing number of outbreaks involving globally distributed foods underscores the need to strengthen regional and global networks for outbreak detection and information sharing . The importance of having standard protocols for molecular characterization of isolates and systems for rapid traceability of implicated foods to their source was illustrated during the investigation of a listeriosis outbreak linked to italian cheese imported into the united states in 2012 (9). Newer tools like whole genome sequencing can also help to generate hypothetical transmission networks and in some instances facilitate traceback of foods to their origin (10). Moreover, new tools that aid visualization of supplier networks facilitate the investigation of outbreaks involving the increasingly complex global economy (11). Only a small proportion of fda - regulated foods are inspected upon entry into the united states . New rules under the food safety modernization act of 2011, including the preventive controls rule for human food, produce safety rule, foreign supplier verification program, and accreditation of third party auditors, will help to strengthen the safety of imported foods by granting fda enhanced authorities to require that imported foods meet the same safety standards as foods produced domestically (12). Although data collection has improved in recent years, these findings might underestimate the number of outbreaks associated with imported foods because the origin of only a small proportion of foods causing outbreaks is reported . Similarly, because of how data are collected and reported, the relative safety of imported and domestically produced foods cannot be compared . Because of changes in surveillance and changing import patterns, changes over time should be interpreted cautiously . Our findings reflect current patterns in food imports and provide information to help guide future outbreak investigations . Prevention focused on the most common imported foods causing outbreaks, produce and seafood, could help prevent outbreaks . Efforts to improve the safety of the food supply can include strengthening reporting by gathering better data on the origin of implicated food items, including whether imported and from what country . Region and country of origin of imported foods implicated in outbreaks, by food category, united states, 19962014.
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This whole genome shotgun project has been deposited at ddbj / embl / genbank under the accession jpul02000000 (http://www.ncbi.nlm.nih.gov/nuccore/jpul00000000.2). The stramenopile blastocystis is a common anaerobic protist living in the digestive tract of several animal groups . Its prevalence in human often exceeds 5% in industrialized countries and can reach 100% in developing countries . Although the role of blastocystis as a human pathogen remains unclear, it has been associated with acute or chronic digestive disorders and some epidemiological surveys have suggested an association with irritable bowel syndrome (ibs), . In patients with ibs, blastocystis seems to be associated with a decrease of the fecal microbiota protective bacteria, bifidobacterium sp . And faecalibacterium prausnitzii . The life cycle of the parasite is poorly documented . Among the parasitic forms described in the literature, the vacuolar stage which is maintained in vitro in axenic culture, blastocystis exhibits an extensive genetic diversity and seventeen subtypes (st1st17) have been identified based on the gene coding for the small - subunit ribosomal rna among which the first nine are found in humans . Briefly, it consists of an 18.8 mbp nuclear genome with 6020 predicted genes and a circular genome of 29 kbp located within mitochondria - like organelles (mlo). Other mlo genomes with conserved gene synteny we report the sequencing of the blastocystis st4-wr1 genome from an isolate of a laboratory rodent and cultured axenically . Genomic dna was isolated using a qiagen dneasy blood and tissue kit and sequencing was performed with the illumina hiseq 2000 system (genoscreen, lille, france). A total of 43.855.085 of 100-bp high quality paired - end reads were generated and were de novo assembled using the idba - ud algorithm . The output was then scaffolded using sspace and gaps were filled by gapfiller software . In total, 1301 scaffolds from 494 bp to 133,271 bp were obtained, with a scaffold n50 of 29,931 bp . The draft genome sequence of blastocystis st4 has a deduced total length of 12.91 mbp and a g + c content of 39.7% . Assembly also provided a circular dna molecule of 27,717 bp in size with a g + c content of 21.9% corresponding to the whole mlo genome sequence . The maker pipeline was set with the results of ab initio gene prediction algorithms augustus and snap, the 6020 protein - coding genes of blastocystis st7, ests of both blastocystis st7 and st1 and 414 manually - designed genes of the st4-wr1 isolate . Basic information about the assembled genome and predicted genes are shown in table 1 . Gene functions were annotated by blast2go and blast analyses with ncbi (http://www.ncbi.nlm.nih.gov/). The preliminary annotation data revealed that blastocystis st4-wr1 nuclear genome harbors 5713 protein - coding genes . The presence of proteases was determined using blast against merops database, and secreted proteases were identified using signalp 3.0 and wolf psort . This comparative analysis revealed that the st4 genome contains less duplicated genes than st7 and that more than 30% of st4 genes have no ortholog in the st7 genome at an e value cutoff of 10 . This also led to the identification of new candidate genes, in particular some potential virulence factors, including 20 secreted proteases that may be involved in the physiopathology of this parasite . Among these proteases, 7 seem to be specific to st4 as no ortholog has been found in the st7 genome . Sequencing and annotation of additional st (st1, st2, st3 and st8) genomes are under progress and should be helpful for a better understanding of the genetic diversity, pathogenesis, metabolic potential and genome evolution of this highly prevalent human parasite.
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Factor v is a coagulation protein that is present in blood plasma as a single - chain polypeptide (approximately 80%) and in platelet alpha - granules (approximately 20%).1 factor v is cleaved after binding to activated platelets and serves as a cofactor for factor xa in the prothrombinase complex . This complex forms on the platelet surface and has limited proteolytic activity, converting prothrombin to thrombin to aid in blood clotting.2,3 acquired inhibitors to factor v were first reported in 195524 and develop in extremely rare cases (0.090.29 cases per million persons) via development of alloantibodies or autoantibodies against factor v. patients with acquired factor v inhibitors generally present with hemorrhagic manifestations . Here, we report a unique case of acquired factor v inhibitor in a patient with mantle cell lymphoma presenting with hematuria followed by thrombosis . A 64-year - old man initially presented to us with complaints of fatigue and joint discomfort for several months prior to december 2008 . He denied experiencing any fever, night sweats, and weight loss, and did not notice any swelling, recurrent infection, easy bleeding, or bruising . His past medical history included hernia repair, renal colic, gastroesophageal reflux, and osteoarthritis . Upon examination, one left axillary lymph node was swollen to approximately 12 cm, and was firm and slightly tender . His spleen was palpable on inspiration, and castell s sign was positive; however, his liver was not enlarged . A complete blood count showed normal platelet and hemoglobin levels of 206 10/l and 155 g / l, respectively, but a high white blood cell count of 18 10/l . A leukocyte differential indicated the following: lymphocytes 10.3; neutrophils 6.75; monocytes 0.58; eosinophils 0.23; and basophils 0.05 . Peripheral blood flow cytometry revealed a cd19, cd20, cd5, cd23, cd10 clonal b - cell population . The proportion of this fmc7-positive population reduced to 17% during a second flow analysis one month later, suggesting the possibility of mantle cell lymphoma or cd23-negative chronic lymphoid leukemia . Given that the patient was asymptomatic and showed no signs of bone marrow failure, a strategy of watchful waiting was implemented . In august 2009, 8 months after his initial visit, the patient was admitted to the emergency room with a 2-day history of hematuria . Evaluation of his blood plasma revealed a prolonged international normalized ratio (inr) of 6 and an activated partial thromboplastin time of 160 seconds . Except for some minor skin bruising on his face, the patient had no other bleeding or bruising, nor was any area abnormal on examination . Further, he had no past history of bleeding, with uneventful surgical procedures and tooth extractions in the past . On careful investigation of his coagulopathy, a prothrombin time mixing study was abnormal at 43 partially corrected to 30 seconds only (normal 12.515.7 seconds), suggesting the presence of an inhibitor . The thrombin time was 15.8 seconds (normal 15.518.3 seconds), serum fibrinogen level was 5.8 g / l), factor v level was <0.01 u / ml (normal 0.51.5 u / ml), factor viii level was 3.23 u / ml (normal 0.51.5 u / ml), factor x level was 1.08 u / ml (normal 0.51.5 u / ml), and factor ix level was 1.17 u / ml (normal 0.51.5 u / ml). Based on these results, a factor v inhibitor test was performed and indicated a factor v inhibitor titer of 80 bethesda units . The patient was started on prednisone (1 mg / kg, 80 mg daily), and after 2 weeks of treatment, the bleeding had stopped and serial measurement of factor v levels showed a dramatic increase; moreover, within 7 weeks, factor v levels were normal and factor v inhibitor was undetectable . Surprisingly, 7 weeks after initiation of steroid treatment, the patient developed an unprovoked severe pain with swelling in his right leg . A week prior to this event, his prothrombin time was 17 seconds, activated partial thromboplastin time was 28 seconds, inr was 1.4, and the factor v assay value was 0.62 bethesda units / ml, but factor v inhibitor was undetectable . Doppler ultrasound revealed deep venous thrombosis, and low - molecular - weight heparin (dalteparin, 12,500 u) and coumadin (7.5 mg) were administered orally once daily . The patient s condition continued to improve with no further bleeding or thrombotic events, and anticoagulation was successfully discontinued after 8 months . The use of prednisone was gradually decreased over several weeks after confirmation of the disappearance of the inhibitor, and was discontinued completely in december 2009 (figure 1). Screening assays to rule out inherited thrombophilia were negative for antithrombin, protein c, protein s, factor v leiden, and prothrombin 20210 gene mutations . Fluorescence in situ hybridization analysis of a peripheral blood sample showed igh / ccnd1 t(11;14), which confirmed the presence of a clonal b - cell population characteristic of mantle cell lymphoma . The clinical presentation of factor v inhibitors can range from asymptomatic hematological laboratory abnormalities to life - threatening hemorrhage . Factor v inhibitor has been associated with a number of conditions, including antibiotic use, sepsis, malignancies,10 autoimmune illnesses, vasculitis, valproic acid use, systemic amyloidosis, cold agglutinin disease, human immunodeficiency virus infection, hematopoietic stem cell transplantation, and liver transplantation, as well as use of amiodarone and warfarin.2,1113 if bleeding occurs, it predominantly involves the mucous membranes of the gastrointestinal, airway, and urinary tracts.2 in contrast with other coagulation factor inhibitors, the level of factor v inhibitors does not correlate with the degree of clinical bleeding, and streiff et al noted that patients who developed spontaneous factor v inhibitors had more significant bleeding than those with bovine thrombin - related factor v inhibitors.14 in addition, the risk of bleeding does not seem to correlate with prolongation of prothrombin time or activated partial thromboplastin time, factor v activity, factor v inhibitor levels, or the duration of presence of factor v inhibitor.14 because factor v is an anticoagulant, reports of thrombosis in patients with acquired factor v inhibitor are extremely rare.59 activated factor v (fva) is subsequently inactivated by activated protein c (apc), which cleaves fva at arg506, arg306, and arg679 . After fva is cleaved at arg506 (fvac), it further interacts with apc and protein s to enhance the inactivation of activated factor viii (fviiia) by the aps / protein s complex, thereby indirectly acting as an anticoagulant.15,16 factor v inhibitor can hinder this cascade by decreasing the level of fvac, resulting in sustained activation of factor viii and leading to procoagulant diathesis . Interestingly, an anti - factor v antibody isolated from a patient inhibited the cofactor activity of factor v during the apc / protein s complex s fviiia inactivation cascade by impairing the proper cleavage of fva by apc and inhibiting the inactivation of fva.17 this confirms that factor v inhibitors can lead to procoagulant diathesis by both direct and indirect mechanisms . Given that the factor v inhibitor was undetectable in our patient, it is unlikely that it elicited the development of thrombosis during the course of treatment, but we hypothesize that the significantly elevated factor viii might have played a role . Additionally, underlying hematological malignancy is another possible provoking factor . The patient presented in this case report had a unique clinical manifestation of acquired factor v inhibitor in that thrombosis occurred after factor v was no longer detectable by an assay measuring bethesda units; however, the patient still exhibited coagulopathy with high prothrombin time and inr, suggesting the continued presence of a small amount of factor v inhibitor . At first presentation, his factor viii level was significantly higher than normal at 3.23 u / ml (normal 0.51.5 u / ml) which might have caused this thrombotic event . To our knowledge, this is the first report of acquired factor v inhibitor in a patient with mantle cell lymphoma simultaneously presenting with this unusual coagulopathy.
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Von willebrand disease (vwd) is the most common inherited bleeding disorder, with a prevalence of approximately 1% according to population studies,1 but clinically relevant cases have a tenfold lower prevalence.2 the 2011registro nazionale delle coagulopatie congenite [national registry of congenital coagulopathy],3 which reports data relative to 51 of 54 hemophilia centers in italy, indicated that a total of 8,411 subjects are affected by coagulation disorders 25% by vwd; 43% by hemophilia a; 9% by hemophilia b; 14% by disorders of other coagulation factors; and 9% by platelet disorders, carrier hemophilia a / b, or other disorders . Vwd is caused by a deficiency or abnormality of the von willebrand factor (vwf), a multimeric adhesive glycoprotein with a key role in platelet adhesion; it is also the carrier and stabilizer of the factor viii coagulant moiety (fviii: c), thus indirectly contributes to the coagulation process.4 type 1 and 2 vwd usually display a mild hemorrhagic phenotype (partial deficiency and qualitative defect, respectively), whereas patients with type 3 vwd (complete deficiency) are affected by a severe bleeding tendency . The goal of the therapy for vwd is to correct the dual defects of hemostasis, abnormal platelet adhesion (due to low vwf adhesive activity) and abnormal intrinsic coagulation pathway (due to low fviii: c).4 two main options are available to manage vwd patients: desmopressin acetate (ddavp), which induces the release of endogenous vwf from endothelial compartments type 1 patients and a fraction of type 2 patients usually respondreplacement therapy, which involves the transfusion of exogenous vwf contained in plasma - derived fviii concentrates enriched with vwf (vwf / fviii concentrates). Vwf / fviii concentrates are the first choice for the treatment of patients with type 3 vwd, for patients with type 2 b (because ddavp can induce transient thrombocytopenia), and for those patients with type 1 and 2 who are unresponsive to ddavp or have contraindications to its use . Desmopressin acetate (ddavp), which induces the release of endogenous vwf from endothelial compartments type 1 patients and a fraction of type 2 patients usually respond replacement therapy, which involves the transfusion of exogenous vwf contained in plasma - derived fviii concentrates enriched with vwf (vwf / fviii concentrates). Vwf / fviii concentrates are the first choice for the treatment of patients with type 3 vwd, for patients with type 2 b (because ddavp can induce transient thrombocytopenia), and for those patients with type 1 and 2 who are unresponsive to ddavp or have contraindications to its use . Therapy with vwf / fviii concentrates can be administered either on demand, through infusion of the amount of factors determined by the severity of the disease and by the patient s body weight, in order to stop occasional bleedings, or as prophylaxis in the more severe forms of the disease, through multiple weekly infusions, in order to control recurrent bleeding and to prevent life - threatening hemorrhages.5 patients with severe forms of vwd may have frequent bleeding episodes, especially in those cases with fviii levels below 20 iu / dl, occurring in type 3 vwd and in some cases with severe forms of type 1 and 2 . In these cases large doses of vwf / fviii concentrates are required to control the bleeding . Prophylaxis with vwf / fviii concentrates is considered a potential approach for those patients with severe bleeding tendency.58 the wide heterogeneity of the bleeding tendency that impacts heavily on quality of life (qol) contributes to the uncertainties about the potential candidates, the optimal dose of concentrates, and regimes for prophylaxis . Furthermore, patients with vwd have an intact endogenous production of factor viii (fviii: c) and long - term exposure to vwf - enriched fviii concentrates may confer thromboembolic potential to prophylaxis.912 this observation suggests a particular care in the use of vwf / fviii concentrates in patients with severe vwd and the need for regular fviii: c level monitoring in order to maintain an appropriate hemostatic balance . In this regard, there is a growing interest in replacement therapy with vwf concentrates almost devoid of fviii, especially in severe vwd patients, in whom therapies with ddavp and dual concentrates are not effective, not tolerated, or contraindicated (when a rise in fviii concentration could predispose the patient to thrombotic events) in order to allow the control of bleeding episodes . The objective of the analysis presented here is to assess the cost benefit ratio of vwf with a low fviii content (wilfactin, laboratoire francais du fractionnement et des biotechnologies, les ulis, france) when compared with the vwf / fviii concentrates treatments (ie, haemate p, zlb behring, marburg, germany, and fanhdi, instituto grifols, barcelona, spain) currently applied in italy for long - term prophylaxis in patients with severe vwd, based on data from four case reports . The decision to use single case reports for the pharmacoeconomic analysis depended on two aspects: there are very few patients requiring long - term prophylaxis due to the difficulty in controlling bleeding episodes, and there are even fewer cases in which treatment with vwf with a low fviii content has been utilized in italy . A cost consequence analysis was adopted in order to assess the economic impact of the treatment of vwd from the perspective of the national health service (nhs) and society . The use of a cost consequence approach depends on the fact that this analysis measures the health consequences without combining them into one summary effectiveness measure . This kind of analysis describes the value of a health care intervention and has the advantage of being more readily understandable and more likely to be applied by health care decision - makers, leaving the decision regarding the relative importance of different outcomes to the decision - maker.13,14 the analysis described in this paper was conducted on case reports in disaggregated form, as a pooled analysis would not give a robust statistical approach . The analysis has been based on four case reports of type 3 (n=1), type 2 m (n=1) and type 1 (n=2) vwd from hemophilia centers at the molinette hospital of turin and university hospital of catania on the long - term treatment of vwd with vwf / fviii concentrates and vwf with a low fviii content . These centers have clinical experience with patients with severe bleeding tendency receiving long - term prophylaxis (started at least 6 months before) who were switched from vwf / fviii concentrates to vwf with a low fviii content because of frequent recurrence of bleeding at the same sites, in order to obtain a better control of bleedings and a better efficacy of the therapy . Patients were included in the analysis if they had severe forms of vwd with periodic bleeding episodes, generally presented a low basal level of fviii: c and fviii: ristocetin - cofactor (ri.cof) before starting prophylaxis with vwf with a low fviii content, and had been treated with vwf / fviii concentrates . The analysis includes direct costs associated with drug acquisition, hospital admissions, monitoring visits, and the cost of transfused units of red blood cell concentrates (packed red blood cells), and indirect costs evaluated as the number of working days lost per bleeding episode (loss of productivity). In order to collect real - world evidence, a questionnaire addressed to clinicians experienced in the treatment of bleeding disorders was developed . In the analysis, outcomes were evaluated in terms of treatment of minor or major bleeding episodes and related costs . The comparators used in the pharmacoeconomic evaluation were vwf / fviii concentrates (haemate p and fanhdi), as these are the most used vwf / fviii concentrates in the italian clinical practice setting for long - term prophylaxis in the treatment of vwd . Both medicinal products, vwf / fviii concentrates and vwf with a low fviii content, are authorized in italy for vwd . The concentrates were administered for prophylaxis at the following dosage: vwf / fviii concentrates (fandhi) 40 iu / kg three times per week; vwf / fviii concentrates (haemate p) from 35 to 50 iu / kg three times per week; vwf with a low fviii content from 30 to 50 iu / kg twice per week, and, in one case, 30 iu / kg three times per week . A single bleeding episode was treated with replacement therapy that is, 3050 iu / kg / day per 3 days of concentrates, according to national guidelines and summary of product characteristics.1 the unitary cost of concentrates was obtained from telematic pharmaceutical compendium 2013 http://www.farmadati.it/ and is equivalent to the ex - factory price that represents the maximum cost for the nhs structures . The cost of outpatients visits, transfusion of blood components, and laboratory and diagnostic tests are set in the national outpatient tariffs ministerial decree [dm] 1997 . The absorption of health care resources was calculated considering the diagnosis related group (drg) version 24 in order to assess drg refund value for day hospital and for ordinary hospitalization . The cost of hospitalization and day hospital was derived from national hospital tariffs dm 18th october, 2012 . The human capital method was adopted for the determination of the cost due to loss of productivity . Based on this approach, each day of absence from work, results in a simultaneous reduction in production . On the basis of this methodology, a value equivalent to the value generated from work for the same time period should be given to the working days lost due to illness.15 here, all working days lost (in 1 year) due to disease - related medical and diagnostic investigations, are considered . The number of working days lost was estimated on the basis of the average annual income.16 pharmaceutical cost includes both the yearly cost of treatment for long - term prophylaxis and the cost of treatment of bleeding episodes with concentrates (replacement therapy). Health care cost includes monitoring visits, diagnostic and laboratory tests, transfusion requirements, and hospitalizations (table 1). Although the cost of long - term prophylaxis was found to be higher with vwf with a low fviii content, the cost per bleeding episodes per year was considerably reduced (in case 1) or eliminated (in cases 24) with vwf with a low fviii content with respect to vwf / fviii concentrates (table 1, column 6: cost of bleeding episodes per year). Indeed, in these four cases, vwf with a low fviii content was able to reduce the number of bleeding episodes and consequently to minimize the cost of replacement therapy . In all cases, health care costs were found to be substantially lower with vwf with a low fviii content with respect to vwf / fviii concentrates (table 1, column 17: health care cost per year without cost of treatment). Indirect costs were evaluated in terms of number of working days lost (table 2). These data show that replacement therapy with vwf with a low fviii content was able to reduce the number of working days lost per bleeding episode together with the discomfort related to bleedings and, consequently, to improve the qol in all these patients . In one case report (case 4), the cost of loss of productivity was not calculated, as the patient was retired . Despite this, the days of well - being lost due to illness may be considered and have an impact on the qol of this patient . The results show data of pharmaceutical costs, health care costs, and indirect costs (loss of productivity) relative to each case (tables 36 and figures 14). These results show that the replacement therapy, both with vwf / fviii concentrates and vwf with a low fviii content, accounts for most of the total cost . Although the dose to achieve full control of bleeding episodes depends on many factors, including in particular patient - related factors, it is possible to observe a minimization of doses of concentrates after the switch to vwf with a low fviii content (table 1, column 3: concentrates iu / year). In the four cases, long - term prophylaxis with vwf with a low fviii content allowed the achievement of good control of bleedings, minimizing the cost of hospital admittances and transfusions of blood components . The reduction of these events with prophylaxis with vwf with a low fviii content was found to afford a reduction in the health care costs per patient per year in all the case reports analyzed (table 7, column 2: total health care costs without pharmaceutical costs avoided with vwf with a low fviii content per patient / year). Long - term prophylaxis with vwf with a low fviii content, allowed in all cases the use of a lower quantity of this concentrate with respect to the vwf / fviii concentrates . Nevertheless only in one case report (case 2) was it possible to observe a reduction in pharmaceutical costs per patient per year (table 7, column 3: pharmaceutical cost avoided with vwf with a low fviii content per patient / year). Furthermore these results show a reduction in indirect costs, evaluated as a reduction of the number of working days lost per bleeding episode, with a related positive impact on qol (table 7, column 4: indirect cost avoided with vwf with a low fviii content per patient / year). The analysis of these four case reports shows that vwf with a low fviii content seems be a cost effective treatment option for patients with severe vwd phenotype, in whom therapies with ddavp and dual concentrates are partially or not effective, non - tolerated or contra - indicated (when a rise of fviii concentration could predispose the patient to thrombotic events), aimed to obtain a better control of bleeding episodes and consequently to improve the qol . The treatment of a bleeding episode in vwd patients requires correction of the vwf deficiency (the defect in primary hemostasis) and the impaired secondary hemostasis (to increase the potentially low fviii: c level). Apart from the administration of ddavp in mild / moderate cases, replacement therapy with concentrates is the therapy of choice for bleeding situations or for short - term prophylaxis during surgery or clinical interventions,1719 while for long - term prophylaxis few retrospective or prospective data are available to date.2026 the greatest experience with secondary long - term prophylaxis in italy has been with prophylaxis implemented in a cohort of italian patients with vwd . In the study of federici et al,24 among 89 patients who needed treatment with vwf / fviii concentrates during the previous 2 years because of one or more bleeding episodes, eleven were included in a prophylaxis program because of frequent recurrence of bleeding at the same sites . When prophylaxis was compared with previous on - demand regimens, in all the eleven cases, the annual total consumption of concentrates, the number of transfused blood units, and the number of days spent in hospital were found to be significantly reduced . Similar results were obtained in a swedish study by berntorp and petrini.22 in that study, 35 patients with mostly type 3 vwd, prophylaxis was associated with a substantial decrease in the annual number of bleeding events . As regards vwf with a low fviii content, as far as we are aware, only one prospective study is available that includes, as additional experience, data on long - term prophylaxis in four patients with type 3 vwd . The study of borel - derlon et al26 investigated the efficacy and safety of vwf with a low fviii content to treat patients with clinically severe vwd by merging the results of two comparable protocols conducted prospectively in five european (italy is included) and twelve french centers . In that study, four patients received secondary long - term prophylaxis (of 17 months duration) with vwf with a low fviii content in order to prevent skin and musculoskeletal bleedings . After the long - term prophylaxis, the number of bleedings was substantially reduced . In our analysis, we report on four adult patients (two with type 1 vwd, one with type 2 m vwd, and one with type 3 vwd) who switched the long - term prophylaxis from vwf / fviii concentrates to vwf with a low fviii content in order to obtain better control of bleeding episodes and better efficacy of the treatment . In all these cases, vwf with a low fviii content treatment was found to be an effective tolerated modality that was highly beneficial for the patients both in terms of a reduction in bleeding episodes and in terms of cost benefit ratio . To the best of our knowledge, this is the first analysis to have investigated the impact of secondary long - term therapies with vwf with a low fviii content and the most used vwf / fviii concentrates (ie, haemate p and fanhdi), in the italian clinical practice setting, on clinical outcome (ie, bleeding frequency, hospitalizations) and indirect / social outcomes (reduction in number of working days lost per bleeding). As no previous studies have addressed this aspect, it is difficult to make any comparison . However long - term prophylaxis with vwf with a low fviii content was found to be associated with a significant reduction of the number of days spent on patient care (hospitalizations, outpatient visits, laboratory and other diagnostic examinations) together with a decrease in total concentrate consumption for bleedings, in all case reports considered . The main limitation of the analysis is that it was based on single case reports, because few patients are in long - term prophylaxis with vwf / fviii concentrates or vwf with a low fviii content in the italian clinical practice setting . Further case reports of long - term prophylaxis treatment with concentrates in vwd are necessary to support the results obtained in this primary analysis . Despite the limitations of the use of single case reports for the analysis, it seems that long - term prophylaxis with vwf with a low fviii content is likely to be a cost - effective approach with a favorable impact on the reduction of health care resource consumption and that also allows the patient to resume their normal life and work activities (the number of working days lost was found to be considerably reduced after switching to vwf with a low fviii content [table 2, column 2]). These data are consistent with the improvement in the qol of the patients, which is a primary objective of any health care intervention . This analysis shows that vwf with a low fviii content was more able to control bleeding episodes and, consequently, to reduce the health care interventions necessary, with respect to vwf / fviii concentrates, in four patients with severe vwd . Although these are preliminary data, they are likely to contribute to establishing the role of specific concentrates for the long - term prophylaxis of severe cases of vwd . Nevertheless, a large collection of clinical data is needed so as to assess regimens, comparative effectiveness, and the cost - effectiveness of long - term prophylaxis treatment in patients with vwd, in order to evaluate which of the available concentrates has a better cost benefit ratio in patients with severe vwd.
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A 64-year - old man visited our hospital with the chief complaint of microscopic hematuria for the past 2 months . Abdominal computed tomography (ct) revealed a homogeneous solid tumor with slightly low attenuation in the left renal sinus (fig . Altogether, these abdominal ct findings were suggestive of invasive urothelial carcinoma arising in the left pelvis with involvement of renal sinus soft tissue . Nephroureterectomy was performed, and the cut surface of the renal sinus revealed a grayish - white solid tumor of rubbery consistency . The tumor was primarily located in the renal pelvis, but extensions to the renal calyces and proximal ureter were suspicious . The pelvic tumor mass measured 42.5 cm and exhibited peripelvic infiltrative features (fig . 2). However, the mucosal surfaces of the renal pelvis, ureter and calyces were smooth, and there was no evidence of urothelial malignancy . Additionally, the mucosal layers of the renal pelvis, calyces and ureter were intact without abnormal urothelial changes . Microscopically, the tumor occupying the renal sinus contained widely scattered, hyperplastic lymphoid follicles, characteristic of a lymphoproliferative lesion . The follicles varied in size and showed polarized germinal centers; moreover, interfollicular spaces were markedly infiltrated by mature plasma cells, highlighted by cd138 immunostaining (fig . 3). Immunostaining for and immunoglobulin light chains indicated that the plasma cells were polyclonal in origin . Immunostaining for human herpes virus 8 (hhv-8) was performed, the results of which were negative . These histological and immunohistochemical findings were compatible with a diagnosis of castleman's disease of the plasma cell type . Three months after the operation, there was no evidence of disease recurrence on follow - up abdominal ct . The occurrence of castleman's disease in the renal sinus is exceedingly rare.1 to the best of our knowledge, only a few cases have been reported.1,3 - 6 the clinicopathological characteristics of the reported cases, involving six males and one female, are summarized in table 1 . Although castleman's disease generally shows no gender preference,2 cases involving the renal sinus appeared to show a male predominance . The median age for castleman's disease is in the fourth decade.2 however, the mean age of the cases involving the renal sinus was 65 years . The main presenting symptoms included abdominal pain, weight loss, anorexia, and microscopic hematuria . Five cases were of the plasma cell type, one of the hyaline vascular type and one of mixed type . The patient in the present case, a 64-year - old male, presented with symptoms of microscopic hematuria, and his disease was histologically classified as plasma cell type and clinically as unicentric type . Although the exact pathophysiology of castleman's disease is unknown, recent reports suggest that hhv-8 infection may stimulate b lymphocytes to induce interleukin 6 (il-6) production in the mantle zone; il-6 overproduction has been shown to be associated with the systemic manifestations of castleman's disease, especially in cases of multicentric disease.7 in our case, immunostaining for hhv-8 was performed, but the result was negative . Preoperative diagnostic imaging methods are not useful in differentiating castleman's disease arising in the renal sinus from other diseases because of a lack of tumor - specific imaging features . Previously, nishie et al.1 described the ct and magnetic resonance imaging features of three cases of castleman's disease involving the renal sinus . They reported that the renal sinus lesions formed in castleman's disease can appear as homogeneous masses with mild enhancement, such as is often observed in malignant lymphomas . Thus, it is difficult, on the basis of radiological findings, to differentiate castleman's disease from malignant lymphomas infiltrating the renal sinus . Other differential diagnoses may include invasive urothelial carcinoma, granulomatous diseases, sarcomas, and metastases . Consequently, pathological evaluation is currently the only method of identifying castleman's disease of the renal sinus . Here we have described an extremely rare case of unicentric castleman's disease of the plasma cell type arising in the renal sinus . Although preoperative diagnosis of castleman's disease is difficult and accurate diagnosis is only possible by histopathologic evaluation, it should be considered in the differential diagnosis of renal sinus tumors.
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Ethical approval was acquired and informed consent was obtained from the patient or the family of the patient . In addition to patient samples, control pancreatic blocks and isolated islet sections were prepared from five deceased donors without diabetes (three women; age 2461 years; bmi 2534 kg / m). Sections were stained with hematoxylin and eosin in addition to sirius red collagen staining using standard procedures . Indirect immunofluorescence staining was performed on 4-m sections after deparaffinization, rehydration, and heat - mediated antigen retrieval using citrate buffer . After blocking with 10% fcs, sections were incubated with guinea pig anti - insulin (1:500; abcam, cambridge, u.k . ), rabbit antivimentin (1:250; abcam), or mouse antiglucagon (1:1,000; sigma - aldrich, gillingham, u.k .) Overnight . Sections were incubated with anti - guinea pig fluorescein isothiocyanate, anti - mouse af543, or anti - rabbit af488/af543 secondary antibodies (invitrogen, paisley, u.k . ). For negative control subjects, patient 1 was a 65-year - old woman whose pancreas was procured during deceased organ donation after brain death after intracranial hemorrhage . Bmi was 32 kg / m, with random plasma glucose of 8.1 mmol / l . Patient 2 was an 81-year - old woman who underwent distal pancreatectomy for an intraductal papillary mucinous neoplasm . She had experienced two episodes of pancreatitis 12 months and 7 years before pancreatic resection but had no chronic symptoms or evidence of pancreatic exocrine deficiency . Patient 3 was a 52-year - old woman whose pancreas was procured for clinical islet isolation during deceased organ donation after brain death after intracranial hemorrhage . There was no history of known diabetes, but a diagnostic hba1c test performed on admission indicated hba1c of 63 mmol / mol (hba1c 7.9%) with random glucose of 8.7 morphological analysis after hematoxylin and eosin staining of pancreatic sections showed islet size, distribution, and integrity comparable with those of nondiabetic control subjects . There was no evidence of fibrosis in islets or exocrine pancreatic tissue with patterns of collagen deposition comparable with those of control samples on sirius red staining . Immunofluorescence staining clearly demonstrated cells within intact islets expressing both insulin and vimentin in the cytoplasm . D . As shown, cells expressing insulin and vimentin were present in 40% of islets, constituting 5% of insulin - positive cells in affected islets . In cells expressing both phenotypic markers, confocal imaging confirmed coexpression within individual cells and maintained characteristic cytoplasmic insulin and filamentous vimentin staining patterns . The boxed area in panel a indicates the region that is magnified in panels b d . Arrows indicate cells expressing both insulin (b, c) and vimentin (b, d). E: cytofluorogram derived from this image confirming colocalization of insulin and vimentin in islet -cells . Comparative cytofluorogram from stained, normal, nondiabetic pancreas (f) confirming no colocalization of insulin (green) and vimentin (red). I: representative images show islet -cells from patient 1 coexpressing the -cell marker glucagon . The boxed area in panel g indicates the magnified region in panels h and i, with arrows indicating cells expressing both insulin (h) and glucagon (i). The boxed area in panel j indicates the region that is magnified in panels k and l. arrows indicate cells expressing both glucagon (k) and vimentin (l). In contrast, no coexpression of vimentin in insulin - positive cells within or outside islets was detected in nondiabetic control sections . Islet cells coexpressing insulin and glucagon within the cytoplasm were identified in pancreatic sections from patient 1 (fig . L). Both of these phenotypes were rare, constituting 1% of all islet cells . In contrast, neither of these mixed phenotypes could be detected on pancreatic sections from nondiabetic control subjects . Macroscopic examination of resected pancreatic tail demonstrated a cystic area with a maximum diameter of 8 mm . Staining of pancreatic sections confirmed intraductal papillary mucinous neoplasm without high - grade dysplasia or evidence of malignancy . There was evidence of lobular atrophy and granulomatous inflammation in the surrounding pancreas, but islet endocrine morphology was reported as being within normal limits . Islets stained positive for insulin, with categorical evidence of cells coexpressing insulin and vimentin, insulin and glucagon, and vimentin and glucagon (supplementary fig . 5% of insulin - positive cells in affected islets coexpressed insulin and vimentin . In patient 2, although cells expressing only insulin, glucagon, or vimentin could be clearly identified, many cells coexpressed insulin and glucagon and vimentin and glucagon in virtually all islets . Immunofluorescence staining of isolated islets enabled clear differentiation of individual cells, particularly at the periphery . Cells coexpressing insulin and vimentin, insulin and glucagon, and vimentin and glucagon within the cytoplasm were identified (supplementary fig . 2). Absence of any of these phenotypes in control islets isolated from nondiabetic donors was confirmed . Patient 1 was a 65-year - old woman whose pancreas was procured during deceased organ donation after brain death after intracranial hemorrhage . Bmi was 32 kg / m, with random plasma glucose of 8.1 mmol / l . Patient 2 was an 81-year - old woman who underwent distal pancreatectomy for an intraductal papillary mucinous neoplasm . She had experienced two episodes of pancreatitis 12 months and 7 years before pancreatic resection but had no chronic symptoms or evidence of pancreatic exocrine deficiency . Patient 3 was a 52-year - old woman whose pancreas was procured for clinical islet isolation during deceased organ donation after brain death after intracranial hemorrhage . There was no history of known diabetes, but a diagnostic hba1c test performed on admission indicated hba1c of 63 mmol / mol (hba1c 7.9%) with random glucose of 8.7 morphological analysis after hematoxylin and eosin staining of pancreatic sections showed islet size, distribution, and integrity comparable with those of nondiabetic control subjects . There was no evidence of fibrosis in islets or exocrine pancreatic tissue with patterns of collagen deposition comparable with those of control samples on sirius red staining . Immunofluorescence staining clearly demonstrated cells within intact islets expressing both insulin and vimentin in the cytoplasm . D . As shown, cells expressing insulin and vimentin were present in 40% of islets, constituting 5% of insulin - positive cells in affected islets . In cells expressing both phenotypic markers, confocal imaging confirmed coexpression within individual cells and maintained characteristic cytoplasmic insulin and filamentous vimentin staining patterns . The boxed area in panel a indicates the region that is magnified in panels b d . Arrows indicate cells expressing both insulin (b, c) and vimentin (b, d). E: cytofluorogram derived from this image confirming colocalization of insulin and vimentin in islet -cells . Comparative cytofluorogram from stained, normal, nondiabetic pancreas (f) confirming no colocalization of insulin (green) and vimentin (red). I: representative images show islet -cells from patient 1 coexpressing the -cell marker glucagon . The boxed area in panel g indicates the magnified region in panels h and i, with arrows indicating cells expressing both insulin (h) and glucagon (i). The boxed area in panel j indicates the region that is magnified in panels k and l. arrows indicate cells expressing both glucagon (k) and vimentin (l). In contrast, no coexpression of vimentin in insulin - positive cells within or outside islets was detected in nondiabetic control sections . Islet cells coexpressing insulin and glucagon within the cytoplasm were identified in pancreatic sections from patient 1 (fig . L). Both of these phenotypes were rare, constituting 1% of all islet cells . In contrast, neither of these mixed phenotypes could be detected on pancreatic sections from nondiabetic control subjects . Macroscopic examination of resected pancreatic tail demonstrated a cystic area with a maximum diameter of 8 mm . Staining of pancreatic sections confirmed intraductal papillary mucinous neoplasm without high - grade dysplasia or evidence of malignancy . There was evidence of lobular atrophy and granulomatous inflammation in the surrounding pancreas, but islet endocrine morphology was reported as being within normal limits . Islets stained positive for insulin, with categorical evidence of cells coexpressing insulin and vimentin, insulin and glucagon, and vimentin and glucagon (supplementary fig . 5% of insulin - positive cells in affected islets coexpressed insulin and vimentin . In patient 2, although cells expressing only insulin, glucagon, or vimentin could be clearly identified, many cells coexpressed insulin and glucagon and vimentin and glucagon in virtually all islets . Immunofluorescence staining of isolated islets enabled clear differentiation of individual cells, particularly at the periphery . Cells coexpressing insulin and vimentin, insulin and glucagon, and vimentin and glucagon within the cytoplasm were identified (supplementary fig . 2). Absence of any of these phenotypes in control islets isolated from nondiabetic donors was confirmed . Morphological analysis after hematoxylin and eosin staining of pancreatic sections showed islet size, distribution, and integrity comparable with those of nondiabetic control subjects . There was no evidence of fibrosis in islets or exocrine pancreatic tissue with patterns of collagen deposition comparable with those of control samples on sirius red staining . Immunofluorescence staining clearly demonstrated cells within intact islets expressing both insulin and vimentin in the cytoplasm . D . As shown, cells expressing insulin and vimentin were present in 40% of islets, constituting 5% of insulin - positive cells in affected islets . In cells expressing both phenotypic markers, confocal imaging confirmed coexpression within individual cells and maintained characteristic cytoplasmic insulin and filamentous vimentin staining patterns . The boxed area in panel a indicates the region that is magnified in panels b d . Arrows indicate cells expressing both insulin (b, c) and vimentin (b, d). E: cytofluorogram derived from this image confirming colocalization of insulin and vimentin in islet -cells . Comparative cytofluorogram from stained, normal, nondiabetic pancreas (f) confirming no colocalization of insulin (green) and vimentin (red). I: representative images show islet -cells from patient 1 coexpressing the -cell marker glucagon . The boxed area in panel g indicates the magnified region in panels h and i, with arrows indicating cells expressing both insulin (h) and glucagon (i). The boxed area in panel j indicates the region that is magnified in panels k and l. arrows indicate cells expressing both glucagon (k) and vimentin (l). In contrast, no coexpression of vimentin in insulin - positive cells within or outside islets was detected in nondiabetic control sections . Islet cells coexpressing insulin and glucagon within the cytoplasm were identified in pancreatic sections from patient 1 (fig . L). Both of these phenotypes were rare, constituting 1% of all islet cells . In contrast, neither of these mixed phenotypes could be detected on pancreatic sections from nondiabetic control subjects . Macroscopic examination of resected pancreatic tail demonstrated a cystic area with a maximum diameter of 8 mm . Staining of pancreatic sections confirmed intraductal papillary mucinous neoplasm without high - grade dysplasia or evidence of malignancy . There was evidence of lobular atrophy and granulomatous inflammation in the surrounding pancreas, but islet endocrine morphology was reported as being within normal limits . Islets stained positive for insulin, with categorical evidence of cells coexpressing insulin and vimentin, insulin and glucagon, and vimentin and glucagon (supplementary fig . 5% of insulin - positive cells in affected islets coexpressed insulin and vimentin . In patient 2, although cells expressing only insulin, glucagon, or vimentin could be clearly identified, many cells coexpressed insulin and glucagon and vimentin and glucagon in virtually all islets . Immunofluorescence staining of isolated islets enabled clear differentiation of individual cells, particularly at the periphery . Cells coexpressing insulin and vimentin, insulin and glucagon, and vimentin and glucagon within the cytoplasm were identified (supplementary fig . 2). Absence of any of these phenotypes in control islets isolated from nondiabetic donors was confirmed . Consistent with a role for dedifferentiation in the pathogenesis of -cell dysfunction in diabetes, we describe previously unreported coexpression of mesenchymal and -cell phenotypic markers in insulin - positive cells in two patients with recently diagnosed noninsulin - requiring diabetes and in one patient with previously undiagnosed diabetes . It recently has been postulated from a series of -cell fate - marking studies of transgenic mice that dedifferentiation is the primary mechanism underlying -cell failure in nonautoimmune diabetes (5). Specifically, the investigators proposed that metabolic stress leads to activation of mesenchymal markers in endocrine cells, a phenomenon well described in human -cells after establishment in adherent proliferative culture but not previously described in vivo in preclinical studies or in situ in humans (7,8). (5) demonstrated that a number of dedifferentiated -cells undergo conversion to other endocrine phenotypes, leading them to suggest that -cell reprogramming to -cells may explain the apparent reciprocal association of insulinopenia with hyperglucagonemia in early type 2 diabetes (9). The presence of cells coexpressing glucagon and insulin in the patients reported here is in keeping with this hypothesis . We also detected cells expressing both glucagon and vimentin, a phenotype reported in the preclinical studies . Dedifferentiation of nonendocrine pancreas with coexpression of epithelial and mesenchymal markers has been recognized in human sections, with occasional cells coexpressing vimentin and glucagon within the ducts of patients with type 2 diabetes (10). Our data provide circumstantial evidence for the recently reported phenomenon of -cell dedifferentiation and possible reprogramming to -cells in humans . Whether this process is reversible in vivo, contributing to the rapid recovery of -cell function after calorie restriction or bariatric surgery, requires further study . If so, then this may provide a new target for the development of disease - modifying drugs that restore -cell mass and function in type 2 and secondary diabetes through redifferentiation.
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Posterior urethral valve (puv) is the commonest cause of renal impairment in boys during early childhood . Despite a systematic approach in deciding choice of therapy in each case, renal failure antenatal diagnosis of hydronephrosis is possible since 1980s but sensitivity and specificity of ultrasound for antenatal diagnosis of puv remains low . Specific diagnosis is required as puv is associated with a poorer prognosis as compared to other causes of hydronephrosis which are diagnosed antenatally, such as pelvi - ureteric junction obstruction (pujo) and vesico - ureteric reflux (vur). Antenatal diagnosis helps in parental counseling and considering options for antenatal intervention if the diagnosis could be made with precision & foetuses with puv and poorer prognosis could be identified antenatally . Renal damage in puv occurs as a result of activation of renin - angiotensin system pathway, in which renin is an early marker . Therefore, the levels of renin must be raised early in those cases with puv who progress to renal damage . This study was designed to study the role of cord blood plasma renin activity (pra) and ultrasonography in antenatal diagnosis and prognostication in puv . This was a prospective observational study conducted over a period of 1.5 years between january 2013 to june 2014 . Patients with parental refusal to consent and disappearance of hydronephrosis on 3 trimester ultrasound were excluded . Ethical clearance was obtained and patients were enrolled from obstetrics and gynecology outpatient department (opd). All the patients were followed up with serial ultrasounds till delivery and severity of hydronephrosis was assessed using the society of fetal urology (sfu) grading [table 1]. In patients with bilateral hydronephrosis, two milliliter of cord blood was collected for pra estimation at the time of cord clamping in ethylenediaminetetraacetic acid (edta). Ultrasonography kidney ureter, bladder (usg kub) to confirm hydronephrosis and baseline renal function tests (rft) were done at 48 hours of birth . Diagnosis of puv was confirmed by micturating cystourethrogram (mcu) done soon after birth . Accordingly, patients were divided into two groups: puv and those with hydronephrosis due to vesico - ureteric reflux and pelvi - ureteric junction obstruction (non - puv). Patients diagnosed as puv were managed according to our current clinical practice and published protocol . All puv patients were followed up with mcu, glomerular filtration rate (gfr) and tc-99 m dimercaptosuccinic acid (dmsa) scan at 1 and 6 months post - operatively . Puv patients were divided into two groups based on gfr values at 6 months post - operatively those with a gfr <60 and those with a gfr 60 ml / min/1.73 m bsa . Society of fetal urology (sfu) grading of hydronephrosis blood sample collected from peripheral vein was immediately transported to laboratory in an ice pack . Upon arrival in the laboratory, pra was measured by radioimmunoassay of generated angiotensin - i using diagnostics biochem canada inc (dbc) enzyme - linked immunosorbent assay (elisa) kit (can - ra-4600) and values expressed as ng the records of all patients were studied regarding gestational age at detection of hydronephrosis, surgical intervention, antenatal ultrasound findings, cord blood and follow - up pra values, vur, and renal cortical scarring on dmsa . Data analysis was done using ibm statistical package for the social sciences (spss) statistics 20 (ibm, armonk, new york, united states). Values were reported as number (%) or mean standard deviation (range) as appropriate, unless otherwise reported . Blood sample collected from peripheral vein was immediately transported to laboratory in an ice pack . Upon arrival in the laboratory, pra was measured by radioimmunoassay of generated angiotensin - i using diagnostics biochem canada inc (dbc) enzyme - linked immunosorbent assay (elisa) kit (can - ra-4600) and values expressed as ng / ml / hr . The records of all patients were studied regarding gestational age at detection of hydronephrosis, surgical intervention, antenatal ultrasound findings, cord blood and follow - up pra values, vur, and renal cortical scarring on dmsa . Data analysis was done using ibm statistical package for the social sciences (spss) statistics 20 (ibm, armonk, new york, united states). Values were reported as number (%) or mean standard deviation (range) as appropriate, unless otherwise reported subsequently, 21 of them were excluded as hydronephrosis disappeared in the third trimester scan . The data of remaining 25 patients (10 puv and 15 non - puv) were analyzed . In the non - puv group, 9 (60%) had pujo including two with bilateral pathology while 6 patients had non - obstructive hydronephrosis . The mean gestational age at detection of hydronephrosis was 154.6 15.9 days (range: 132 - 191 days). The mean age was 150.9 12.8 days (range: 134 - 167 days) and 157.1 17.6 days (range: 132 - 191 day) in puv and non - puv patients, respectively (p = 0.349). Overall, 19 (76%) patients had bilateral and 6 (24%) had unilateral hydronephrosis on initial ultrasound . All 10 puv patients had bilateral hydronephrosis . In the non - puv group, 9 (60%) had bilateral while 6 (40%) had unilateral hydronephrosis . The distribution of grades of hydronephrosis was not significantly different among these two groups (p = 0.669) [table 2]. Grades of hydronephrosis on initial ultrasound (n = 25, p = 0.669) the keyhole sign was seen in 6/25 patients on initial antenatal ultrasound . It was observed in 1/15 (6.7%) non - puv patients and 5/10 (50%) puv patients (p = 0.023). The sensitivity and specificity of keyhole sign for diagnosing puv were 50% and 93.33%, respectively . The mean amniotic fluid index (afi) on initial ultrasound was 12.3 3.4 cm (range: 4.6 - 19.8 cm). It was 10.1 2.8 cm (range: 4.6 - 16.2 cm) and 13.8 2.9 cm (range: 9.0 - 19.8 cm) in puv and non - puv group, respectively (p = 0.005) [table 3]. Oligohydramnios was observed in only one (10%) patient in the puv group and none in the non - puv group on initial ultrasound . Amniotic fluid index on initial ultrasound (n = 25) the mean cord blood pra value for controls and patients were 3.95 1.57 ng / ml / hr (range: 2.26 - 8.00 ng / ml / hr) and 10.71 5.57 ng / ml / hr (range: 1.51 - 20.71 mean pra was 7.08 3.28 ng / ml / hr (range: 1.51 - 12.58 ng / ml / hr) and 16.15 3.36 ng / ml / hr (range: 11.05 - 20.71 ng / ml / hr) for non - puv and puv group, respectively [figure 1]. The difference in mean cord blood pra values between the controls and non - puv group (p = 0.004), puv and control groups (p <0.0001), and puv and non - puv groups (p <mean pra was10.47 3.26 ng / ml / hr (range: 6.32 - 16.08 there was a significant fall in pra in all patients of puv post valve ablation (p <0.0001). Mean cord blood plasma renin activity (pra) values in various groups, noh: non - obstrcutive hydronephrosis, all pra values are expressed as ng / ml / hr mean plasma renin activity (pra) values in posterior urethral valve (puv) patients: pre- (cord blood) and post - valve ablation, all pra values are expressed as ng / ml / hr baseline serum creatinine (at 48 hours of birth) was within normal limits in 2/10 (20%) puv patients while it was raised in 8/10 (80%) patients . The mean baseline creatinine was 1.33 0.96 (range: 0.30 - 3.20 mg / dl). Vesicoureteric reflux was seen in 9 (90%) of 10 puv patients at the time of diagnosis . Renal cortical scan (dmsa) done 1 month post ablation showed presence of scars in 6 (60%) out of 10 puv patients . It was unilateral in 4 (66.6%) and bilateral in 2 (33.3%) patients . At 6 months post ablation, scars were unilateral in 6 (60%), bilateral in 2 (20%), and absent in 2 (20%) patients . The mean gfr was 37.3 18.21 ml / min/1.73 m (range: 12 - 70 ml / min/1.73 m) and 61.0 24.44 ml / min/1.73 m (range: 29 - 89 ml / min/1.73 m) at 1 and 6 month post valve ablation . Four patients had gfr <60 while 6 had gfr 60 ml / min/1.73 m bsa at last follow - up . Mean cord blood pra among puv patients with gfr <60 and those with gfr> 60ml / min/1.73 m bsa was 19.73 0.81 ng / ml / hr (range: 18.72 - 20.71 ng / ml / hr) and 13.77 1.73 ng / ml / hr (range: 11.05 - 15.57 ng / ml / hr), respectively (p <0.0001) [table 4]. Correlation between glomerular filtration rate (gfr) and plasma renin activity (pra) in posterior urethral valve (puv) patients (n = 10) gestational age at detection of hydronephrosis, bladder wall thickness, amnitotic fluid index, and presence of cortical cysts did not significantly correlate with renal impairment among puv patients . Posterior urethral valve is one of the most serious congenital urinary tract anomalies that can lead to deleterious effect on future bladder and renal function . Despite improvement in survival as many as 25 - 60% of these patients may have significant impairment in renal function in long - term follow - up . The gold standard for post - natal diagnosis is micturating cystourethrography, while pre - natal diagnosis is dependent on routine screening ultrasonography which has a low specificity and is operator dependent . Despite the ability to identify features of bladder outlet obstruction early in fetal development, there is no consensus on how to incorporate early detection into current screening protocols . In centers where antenatal salvage therapies in the form of amnio - infusion and vesico - amniotic shunts are being used, the correct diagnosis of puv would improve the efficacy of these procedures with respect to case selection . With our current screening strategy, mortality and long term morbidity from puv will likely remain unchanged until it is possible to intervene prior to the onset of irreversible renal damage . New biologic markers will allow for more effective diagnosis and intervention at earlier stages of fetal development . Bajpai et al ., first documented the activation of renin angiotensin system (ras) using pra, in patients with posterior urethral valves . They found that mean pra in patients with renal damage was significantly higher as compared to patients with normal renal function . In the present study, cord blood pra was raised in all patients of puv, whereas no other parameter showed such consistency . Cord blood pra was raised in 2/3 of the patients with pujo and none of the patients with non - obstructive hydronephrosis . Also, the levels were significantly higher in cases of puv than those without puv (p <0.0001). Keyhole sign was seen on initial antenatal ultrasound in higher proportion of puv patients (50% versus 6.7%) (p = 0.023). The sensitivity of this sign for diagnosis of puv was 50% and specificity was 93.3% . These values were higher compared to those reported in a series published by bernardes et al . Oligohydramnios did not significantly correlate with diagnosis of puv (p = 0.40). On the other hand, mean afi values were significantly lower in patients with puv when compared to non - puv patients . Renal function deterioration has been linked to age at presentation, gfr, prenatal diagnosis, renal dysplasia, vur, renal scarring, nadir creatinine during 1 year of life, upper tract obstruction, bladder dysfunction, and urinary tract infection (uti). In the recent decades, the choice of therapy in puv could be easily discerned by a step - wise approach using the step - ladder protocol . Endoscopic valve ablation has become the mainstay of treatment for puv . In the present study, age at presentation has been suggested as a predictor of renal function in children with puv . Prenatal diagnosis was initially thought to improve the outcome, but earlier studies failed to show that the long - term outcome in prenatally detected puv patients is better than symptomatic patients detected postnatally . Hutton et al ., reported that antenatal detection before 24 weeks of gestation predicted a poorer prognosis than later detection, with more than 50% of the earlier diagnosed group dead or in renal failure within 4 years of follow - up . In the present study, there was no significant difference in gestational age at detection among patients who ultimately developed renal impairment and those who did not . The decline in renal function after valve ablation is accompanied by activation of ras reflected in a gradual rise in pra . In an earlier study, fall in gfr, high grade vur, scars, and raised serum creatinine were not consistent in detecting renal damage in puv patients but plasma renin activity was found to be high in all such patients . It has been shown that increase in plasma renin activity (pra) precedes all the other presently accepted criteria of renal damage . In a recent study of 58 patients with puv, mean pra was high in all but four patients and the fall in gfr was preceded by a rise in pra by a significant interval, implying that ras is activated much earlier before the fall in gfr becomes evident . Therapy with angiotensin converting enzyme - inhibitors stabilizes and then improves renal function, thereby, retarding the pace of renal damage . In the present study mean cord blood pra values were higher in patients who had gfr <60 compared to those with a gfr 60ml / min/1.73 m bsa (p <0.0001). This study highlights the diagnostic significance of cord blood pra in infants with puv . In this pilot study, we have noted that utrasound parameters are not very useful in the antenatal diagnosis of puv and measurement of pra in the fetuses may have the potential to differentiate poor from better prognosis puv patients . On antenatal ultrasound keyhole sign, bladder cycling, oligoamnios, and cortical cysts are not consistent findings in posterior urethral valves . In congenital hydronephrosis cord blood pra is significantly higher in cases with puv than those without puv and falls significantly after valve ablation.
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The classical golgi method for staining neurons in the brain was first developed by camillio golgi (golgi, 1873 in mazzarello, 1999). Subsequently, the great neuroanatomist ramon y cajal (1909) applied the technique to demonstrate previously unimagined neuronal morphology virtually in all parts of the nervous system . However, the main drawback of the method is inconsistency of impregnation of the stain into the neurons resulting in reduced specificity, reproducibility, and success rate (globus and scheibel, 1966; pasternak and woolsey, 1975; zhang et al ., 2003). Several modifications of the method have been tried which include variations in ph (bertram and ihrig, 1957; gonzalez - burgos et al ., 1992; angulo et al ., 2003), use of microwaves (armstrong and parker, 1986; marani et al ., 1987; zhang et al ., 2003) and variations in temperature of the tissue incubating medium (armstrong and parker, 1986; berbel, 1986; marani et al ., 1987; angulo et al ., 1994 these modifications were aimed at faster staining process by decreasing the time for staining, reducing precipitation, promoting uniform crystallization, increasing reliability, and reproducibility of staining neurons by the golgi method . Cox method has been frequently used to stain neurons in both less myelinated, younger as well as in more myelinated, older rat brains . As compared to golgi method the advantage of golgi cox method include increased probability of staining more number of neurons (scheibel and tomiyasu, 1978); it is also reported to be better than the rapid golgi method for studying neuronal dendritic morphology (buell, 1982). However, the duration of impregnation of stain using golgi cox method has been reported to be between 14 and 80 days (rutledge et al ., 1969; glaser and van der loos, 1981; zhang et al ., 2003), depending on whether whole brain or smaller blocks of brain were used for staining, i.e., primarily depending on the thickness of the tissue block (sample). Thus, apart from inconsistency and lack of uniform, reproducible results, requirement of exceptionally long time to achieve neuronal staining is another major disadvantage of both the golgi and golgi cox methods, which practically limits the use of these methods . Cox method mentioned above could be by increasing random motion of the metallic ions in the staining solution facilitating their influx and deposition within the neurons; thus reducing the time required as well as increasing the success rate of more stained neurons and reproducibility of results . Here we describe a simple and inexpensive modification where we raised the temperature of the golgi cox solution in which moderately thick slices of brain were incubated only for 24 h and that gave us significantly more impregnation of stain and consistently excellent results in terms of stained neurons . Experiments were conducted on inbred male wistar rats (250300 g) maintained in their standard home cages under 12:12 h light / dark cycle with food and water ad libitum . The experiments were approved by the institutional animal ethics committee (iaec) and every effort was made to minimize the use of number of animals and their sufferings . Four rats were deeply anesthetized with over dose of ketamine xylazine (8032 mg / kg, i.p .) (chandra bhagat pharma pvt ltd, india) and then intracardially perfused with 0.1 m phosphate buffered saline (pbs, ph 7.4). For comparison and to differentiate if blood and perfusion play any role in staining, another four rats were sacrificed by cervical dislocation followed by decapitation . All the brains were removed, washed with distilled water followed by with freshly prepared golgi the brains were placed in a 1-mm brain slicer (wpi, usa) and three 5 mm thick coronal blocks a, b, and c were prepared (figure 1). Each of those coronal blocks was further cut into two equal mirror halves by giving a longitudinal cut along the midline . Thus, a total of six blocks labeled as a1, a2, b1, b2, c1, and c2 were obtained (figure 1). A rat brain showing how four coronal and one sagittal cuts were given to obtain six blocks from each rat brain . Each tissue block was placed in separate cotton lined dark colored glass bottle containing 2530 ml freshly prepared golgi cox solution . Bottles containing blocks a1, b1, and c1 were maintained in an incubator at 37 1c for 6, 12, and 24 h, respectively, while bottles containing blocks a2, b2, and c2 were incubated at 26 1c for 6, 12, and 24 h, respectively . Thus, all other conditions, except temperature, were identical in the two groups; hence the groups served as control for each other . At each time point one block (say a1) from 37c and the corresponding contralateral block (in this case a2) from 26c were taken and processed simultaneously; blocks b1, c1 and b2, c2 were processed similarly . This strategy ensured comparisons between sections from comparable brain areas from the same animal treated under identical conditions except that of change in the incubation temperature . As it will be seen later, since the results of intracardial perfused brain samples and that of brains taken out without intracardial perfusion were comparable after 24 h incubation, only the latter samples were treated for 6 and 12 h. we treated the three separate blocks of tissues for 6, 12, and 24 h in the golgi cox solution and four such repetitions were conducted . Further, to confirm if the temperature was affecting primarily the permeability of the stain resulting in increased staining efficiency, we kept separate brain blocks in golgi cox solution containing sodium dodecyl sulfate (sds) or triton x-100 . In separate sets, brain blocks as mentioned above were incubated at 37 1 and 26 1c in golgi cox solution containing either 0.1 or 0.2% sds or 0.5% triton x-100 (table 1). Since the results of perfused brain after 24 h incubation in golgi cox solution were similar (figure 2) to those of non - perfused brain, rest all the studies were conducted on the latter only . Completely filled neurons in 200 m thick sections prepared from 5 mm brain blocks treated under various conditions . + + + indicates completely filled neurons in cortical and sub - cortical regions, denotes some nucleation centers and very few partially filled cell bodies scattered in some cortical areas . Stained neurons in sections prepared from brain blocks incubated at 37 1c for 24 h. sections (a) and (b) were prepared without perfusion of the brain, while sections (c) and (d) were prepared after intracardial perfusion of the brain with 0.1 m phosphate buffered saline (pbs) at ph 7.4 . (a) and (c) at 100 magnification, while (b) and (d) at 400 magnification of the area marked on (a) and (c), respectively, showing completely filled neurons . At the end of incubation of the brain tissue in the staining solution for varying period, 200 m thick sections were prepared from each of the treated and control blocks using a vibratome (3000 series, evergreen blvd - st . It may also be noted that in pilot studies uneven hand cut (90350 m) sections also gave us comparable staining . Rinsed twice (5 min each) in distilled water to remove traces of impregnating solution . 3 . Kept in ammonia solution (3:1, ammonia: distilled water) for 510 min . Dehydrated twice (510 min each) in 70, 80, 95% ethanol and 99% 1-butanol, cleared in toluene and mounted in dpx on gelatinized slides . The slides were allowed to dry at room temperature and were observed under a microscope (nikon eclipse e400, japan and olympus bx51, japan) at low and high magnifications . Images were captured using a charged coupled device (ccd) digital camera (jvc, tokyo, japan and mbf cx9000) attached to the microscope using dedicated software image - pro plus 5.1.1 (media cybernetics, silver spring, usa) and/or neurolucida 9 (mbf biosciences, usa). The intensity of black staining of neurons in the sections was visually compared under microscope . In our pilot studies no nucleation of black stain was observed until 4 h incubation; however, significant number of completely filled neurons was seen after 24 h of incubation at 37c . Hence, in this study we chose to incubate the brain slices at 37c and 26c for 6, 12 and 24 h in the staining solution . I) staining was considered to have been initiated if visible black spot (nucleation) could be distinctly identified inside the neurons without spilling over to the surrounding; the latter (not many though) was considered due to neuronal damage or non - specific artifact . Ii) neurons were considered completely stained if the soma, axons, and dendrites could be seen well demarcated by the impregnated black stain without spilling outside the neurons . For quantitative estimation, five to six sections from each category (6, 12, 24 h in 26 and 37c) were observed under microscope (100 magnification). Every section was divided into two equal zones from the center; outer one was considered cortical while adjacent inner part was considered as sub - cortical (figure 3). In each section four to six frames (figure 3) were taken at random from cortical and sub - cortical regions (640 480 pixels or 220 170 m) using ccd camera (jvc, japan) attached to the microscope (nikon eclipse e400). On an average we counted the stained neurons under various conditions as defined above using dedicated software image - pro plus 5.1.1 (media cybernetics, silver spring, usa). Stereoscopic view of sample neurons was also observed by tracing soma, dendrites, and axons in 3-dimension (3-d) using neurolucida software (mbf biosciences, usa). The neurons were viewed in different planes and were captured using a digital camera (mbf cx9000) attached to the microscope (olympus bx51, japan). Dedicated software neurolucida 9 and neurolucida explorer (mbf biosciences, usa) were used to reconstruct 3-d view (x1000) of the neurons from the captured pictures, which may be conveniently used for qualitative and quantitative analysis . Photomicrograph (20) of golgi cox stained section prepared from a block incubated for 24 h at 37 1c . Dashed line defines the boundary between cortical (c) and sub - cortical (sc) zones . Stained neurons were counted within randomly selected rectangular areas as shown in the figure and explained in the text . At the end of incubation of the brain tissue in the staining solution for varying period, 200 m thick sections were prepared from each of the treated and control blocks using a vibratome (3000 series, evergreen blvd - st . It may also be noted that in pilot studies uneven hand cut (90350 m) sections also gave us comparable staining . Rinsed twice (5 min each) in distilled water to remove traces of impregnating solution . 3 . Kept in ammonia solution (3:1, ammonia: distilled water) for 510 min . Dehydrated twice (510 min each) in 70, 80, 95% ethanol and 99% 1-butanol, cleared in toluene and mounted in dpx on gelatinized slides . The slides were allowed to dry at room temperature and were observed under a microscope (nikon eclipse e400, japan and olympus bx51, japan) at low and high magnifications . Images were captured using a charged coupled device (ccd) digital camera (jvc, tokyo, japan and mbf cx9000) attached to the microscope using dedicated software image - pro plus 5.1.1 (media cybernetics, silver spring, usa) and/or neurolucida 9 (mbf biosciences, usa). The intensity of black staining of neurons in the sections was visually compared under microscope . In our pilot studies no nucleation of black stain was observed until 4 h incubation; however, significant number of completely filled neurons was seen after 24 h of incubation at 37c . Hence, in this study we chose to incubate the brain slices at 37c and 26c for 6, 12 and 24 h in the staining solution . I) staining was considered to have been initiated if visible black spot (nucleation) could be distinctly identified inside the neurons without spilling over to the surrounding; the latter (not many though) was considered due to neuronal damage or non - specific artifact . Ii) neurons were considered completely stained if the soma, axons, and dendrites could be seen well demarcated by the impregnated black stain without spilling outside the neurons . For quantitative estimation, five to six sections from each category (6, 12, 24 h in 26 and 37c) were observed under microscope (100 magnification). Every section was divided into two equal zones from the center; outer one was considered cortical while adjacent inner part was considered as sub - cortical (figure 3). In each section four to six frames (figure 3) were taken at random from cortical and sub - cortical regions (640 480 pixels or 220 170 m) using ccd camera (jvc, japan) attached to the microscope (nikon eclipse e400). On an average we counted the stained neurons under various conditions as defined above using dedicated software image - pro plus 5.1.1 (media cybernetics, silver spring, usa). Stereoscopic view of sample neurons was also observed by tracing soma, dendrites, and axons in 3-dimension (3-d) using neurolucida software (mbf biosciences, usa). The neurons were viewed in different planes and were captured using a digital camera (mbf cx9000) attached to the microscope (olympus bx51, japan). Dedicated software neurolucida 9 and neurolucida explorer (mbf biosciences, usa) were used to reconstruct 3-d view (x1000) of the neurons from the captured pictures, which may be conveniently used for qualitative and quantitative analysis . Photomicrograph (20) of golgi cox stained section prepared from a block incubated for 24 h at 37 1c . Dashed line defines the boundary between cortical (c) and sub - cortical (sc) zones . Stained neurons were counted within randomly selected rectangular areas as shown in the figure and explained in the text . Early sign of nucleation (filling of neurons with black products) started appearing after 6 h of incubation of the blocks at 37 1c, while no such sign was visible in the sections prepared from blocks incubated at 26 1c (figure 4). Stained neurons in cortical sections at 20 (left panel) and 100 (right panel, magnified view of the marked area on 20) prepared from brain blocks incubated in golgi cox solution for 6 h (a d), 12 h (e h), or 24 h (i l) at 37 1c (a, b, e, f, i, j) or at 26 1c (c, d, g, h, k, l) showing temperature and duration of incubation are critical for staining . By 12 h of incubation at 37 1c although many significantly filled (black) neurons could be seen in the cortical region of the sections; in very few sections partially filled neurons could be occasionally seen in the sub - cortical regions only . After 24 h of incubation almost all the stained neurons were completely impregnated with black stain in both the cortical and the sub - cortical regions of the sections prepared from tissue blocks incubated at 37 1c . However, the number of completely filled neurons in the sub - cortical region was significantly lesser than those in the cortical region (figures 46). Under higher magnification (1000), nice and clear dendritic spines could be seen against a clear background in sections obtained from the blocks incubated at 37 1c for 24 h (figure 6). Such results from blocks incubated at 37 1c were uniformly distributed throughout the sections and were consistent in all the sets of experiments irrespective of intracardial perfusion or not . Stained neurons (100) in sections through cortical (a and c) and sub - cortical (b and d) areas after incubation of the brain blocks for 24 h at 37 1c (a, b) and at 26 1c (c, d) are shown . All the sections showed completely filled neurons at the end of 24 h incubation at 37 1c; however, very few neurons can be seen after incubation at 26 1c . Stained neurons or parts in sections from cortical areas of brain blocks incubated at 37 1c for 24 h are shown under various magnification in (a c); while after incubation for 24 h at 26 1c are shown in (d f). (a) and (d) are at 100; the areas marked on (a) and (d) are shown at 400 in (b) and (e), respectively . The areas marked on (b) and (e) have been shown at 1000 in (c) and (f), respectively; spines can be clearly seen in (c) but not in (f). Photomicrographs in one focal plane at 100 of stained neurons incubated at 26 1c (a) and 37 1c (b) for 24 h are shown . Several such traces taken in different planes have been reconstructed automatically by neurolucida 9 and neurolucida explorer 9 and are shown in (c) and (d) as 3-d solid images . Magnified view of neuron in (a) and (b) are shown in (c) and (d), respectively . In contrast, the brain tissue blocks kept at 26 1c showed none or negligible staining after 12 h of incubation . However, after 24 h incubation at 26 1c, inconsistent between experiments and incomplete staining of a few neurons could be seen occasionally, discretely spread mostly in the cortical regions of the sections; insignificant number of partially stained neurons could be seen in the sub - cortical region (figures 46). Quantitative analysis of number of neurons stained in different brain regions under various conditions has been shown in figure 8 . Stereoscopic 3-d view of a neuron with its morphology and projections are shown in figure 7, which may be conveniently used for further analysis . Effects of varying duration and temperature of incubation of the brain tissues on impregnation of golgi cox stain into the neurons . Total number of completely filled neurons in cortical and sub - cortical brain areas in 26 2 frames (as described in text) after incubation of the brain blocks at 37 1c and 26 1c for 6, 12, and 24 h are shown . The staining quality was comparable when the tissues were incubated at higher (37 1c) or lower (26 1c) temperature as described above, irrespective of whether they were incubated in medium containing sds or triton x-100 (table 1). Similarly, perfusion of the brain with pbs was also of little consequences and gave comparable results when incubated at the higher (37 1c) or lower (26 1c) temperature . These observations suggested that the improvement of staining was primarily due to the increased temperature of the tissue incubation medium . Cox stain is one of the powerful techniques to study the neuronal morphology in brain sections (ramon y cajal, 1909). However, its major drawbacks are it is much time consuming to get the results, taking several weeks to more than a month; the results are not uniform, inconsistent and prone to high failure rates (globus and scheibel, 1966; zhang et al ., 2003; cox solution at different temperatures and observed that incubation of the tissues at normal body temperature 37 1c overcame those drawbacks . The effects due to the presence of blood and its constituents or due to perfusion with pbs were ruled out by using the brain taken out with or without perfusion with pbs . Similarly, to rule out the effect of membrane damage the brain tissues were incubated at various temperatures in medium with or without sds and triton - x 100 and the results were comparable . Thus, the findings suggest that the only factor to reduce the time for the stain to impregnate into the neurons was the incubation temperature at 37 1c . Incubating brain blocks in the golgi cox solution at 37c dramatically reduced the time required to achieve complete and uniform staining of neurons . While excellent staining was achieved within 24 h in blocks incubated at 37c, very few completely stained neurons could be seen in homotopic areas in blocks kept at room temperature (26 1c). Since perfusion of the brain with pbs, and incubation of the tissues in medium containing sds or triton - x100 produced comparable results when incubated at the same temperature, neuronal membrane damage causing influx of the stain may be ruled out . A possible rationale for such efficient staining is likely to be due to faster movement of the metallic molecules present in the golgi cox stain solution at the higher incubation temperature . At higher temperature the rate of movement and kinetic energy of the metallic ions in the golgi variations in incubating temperature have been tried earlier but those did not yield better results . Earlier studies used various temperatures in the form of heat (berbel, 1986; angulo et al ., 1994), microwave (armstrong and parker, 1986; marani et al ., 1987; zhang et al ., 2003), etc; however, temperature around 37c for shorter time as that of in this study was not tested . Although a critical temperature (37c) improved staining, we cannot comment from this study what specific role the temperature might be playing in this process . It may be noted that the body fluids are composed of a large number of metallic and non - metallic ions, which exchange through the cellular membranes . Also, the body temperature in the homeotherms is set at a unique (magic) number close to 37c . Whether optimum ion transport through biological membrane has any bearing on evolutionary and physiological significance with the set temperature for thermoregulation in warm blooded animals is an open question . Several methodological modifications of golgi cox staining process have been attempted in the past, e.g., varying duration of incubation of the tissues in the golgi solution and multiple impregnations (millhouse, 1969), changes in ph of chromatin solutions (bertram and ihrig, 1957; gonzalez - burgos et al ., 1992; 1994), vacuum immersion of the tissues (friedland et al ., 2006), use of single sections for staining (landas and phillips, 1982; gabbott and somogyi, 1984), use of microwave energy (armstrong and parker, 1986; marani et al ., 1987; zhang et al ., 2003), and changes in temperature (berbel, 1986; angulo et al ., 1994). Instead of blocks, use of individual sections (landas and phillips, 1982; gabbott and somogyi, 1984) are tedious and suffer from additional drawback that neurons stained in one section cannot be followed in the next section as they may remain unimpregnated . In other methods (table 3), the earliest complete staining of blocks could be obtained after at least 5 days unlike within 24 h in this study . Although in one study using solution (not golgi cox) at 60c, staining within 24 h has been reported (marani et al ., 1987) with some other shortcomings though, since, it was not physiological temperature, it has more chance to alter architecture of neurons after staining . In all those methods up to 3 mm thick brain blocks were incubated in the staining solution, where as in our study we have used 5 mm thick blocks . Further, in most of those modified methods 80150 m sections were studied under microscope . However, in our study we could use 200 m or thicker sections, which allow successfully following the extent of projections of stained neurons to increased depth . Thus, our method is significantly more advantageous for tracing neuronal projections and to study their synaptic connections for longer depth in 3-d . These properties and advantages could be used to study the connections between neurons located in areas which have been physiologically identified, e.g., in sleep, wake, and rem sleep modulation . Comparison of impregnation of golgi stain into the neurons under various conditions used by different authors . Ahb, adult human being; rt, room temperature as mentioned by the authors; sn, stained neurons seen . The results obtained in this study were reproducible and were not seen in the controls . Further, the findings were independent of tissue, animal, and area of the brain tissue being stained; hence, they cannot be due to non - specific artifacts . Another possibility of faster staining of neurons could be due to longitudinal sectioning of the brain tissue . However, such argument cannot be supported by the observation that the cortical neurons were stained first than the sub - cortical neurons; this study does not allow us to comment on this aspect . Incubating the brain tissue around body temperature at 37 1c improved golgi cox staining efficiency and reduced the time of staining, which were the primary drawbacks of using golgi cox method . This modified protocol should allow the researchers to significantly increase their turnover in studying the anatomical connections between neurons located in different anatomical regions and whose functions might have been known . It will facilitate studying arborizations as well as spine counting on neurons, including whose anatomical connections and physiological identifications, e.g., wake, sleep, rem sleep, memory, etc has been established . Additional significant advantage of this modified method lies in the fact that it does not require any specialized equipment or chemicals . 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.
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The experimental works of macklin and macklin provided insights into its pathophysiology [4, 5]; alveolar rupture occurs because of a pressure gradient between the alveolus and the surrounding tissues . This gradient develops either through overinflation of the alveolus or a reduction of interstitial pressure . The air that subsequently leaks into the interstitial tissue diffuses toward the peribronchial and perivascular tissue, and then towards the mediastinum, the neck and into the subcutaneous tissue . However, due to pressure equalisation between the affected and adjacent alveoli in the lungs, the interalveolar walls remain intact and the lungs inflated . The diagnosis of pm is confirmed by frontal chest roentgenogram, including the cervical region . Typical radiological signs of pm include the continuous diaphragm sign (interposition of air between the pericardium and the diaphragm, which becomes visible in the central mediastinal part) and linear bands of mediastinal air parallelling the left side of the heart and the descending aorta (pleura is shown as a fine opaque line) with extension superiorly along the great vessels into the neck . In infants, the spinnaker sign (an upwards and outwards deviation of thymic lobes) can be seen when the thymus is raised above the heart by pneumomediastinal air that elevates the thymus and separates it from the cardiac silhouette beneath . Various causes of pm are found in the literature, such as airway obstruction (e.g. Foreign body aspiration), iatrogenic (e.g. Mechanical ventilation), infections (e.g. Pneumonia), obstructive lung disease (e.g. Asthma), toxic effects (e.g. Smoking), trauma (e.g. Chest trauma), valsalva manoeuvres (e.g. Vomiting) and the weakness of tissue (e.g. Anorexia nervosa). In spontaneous pm, the underlying lung is healthy and the air leak is thought to be atraumatic . In neonates, known predisposing factors are mixed lung diseases, such as pneumonia or meconium aspiration syndrome, with coexisting atelectasis and airway obstruction ., we retrospectively analysed the incidence, severity and causalities of pm in neonates and children> 4 weeks of life admitted to our intensive care unit, and we investigated the possible differences between the groups . We retrospectively reviewed all records of children diagnosed with pm who were hospitalised in the interdisciplinary neonatal and paediatric intensive care unit of the university children s hospital in zrich, switzerland, between january 2000 and september 2006 . The patients were divided into two groups according to their age: neonates (under 4 weeks of age) and children (over 4 weeks of age). We were interested in the causes of pm as documented by the treating physicians, the types and results of radiologic investigations performed, any invasive interventions used to treat pm, the severity of the pm and the length of stay in the intensive care unit . The incidence of pm in our intensive care unit was 0.08% for children> 4 weeks of age and 0.1% for neonates . In all patients, pm was diagnosed by chest x - ray and all had a positive outcome related to the pm . All five patients with pneumopericardium (pp) did not suffer from any complications (e.g. Pericardial tamponade). In addition to pm, two children of this group had subcutaneous emphysema (se), two a pneumothorax (pt) and two a pp . Different causes were found for the air trapping . There were two traumatic aetiologies (rib fracture after a severe car accident, lesion in the hypopharynx after a fall). Two children were diagnosed with obstructive bronchitis and in one child, barotrauma occurred intraoperatively due to a clamped expiratory tube during mechanical ventilation (fig . 1). One child had exercised vigorously three days before hospitalisation, which may have caused the pm . In one adolescent, pm occurred spontaneously . All children were hospitalised in the intensive care unit for one to seven days, depending on the severity of their underlying disease . Diagnostics for the pm other than chest x - rays were performed in four patients . All of these had received a thoracic ct scan . In one child, who also had a huge subcutaneous emphysema and dysphagia, the reason for the air trapping could only be found by means of a laryngotracheoscopy, which showed a traumatic lesion in the hypopharynx . The patient s history revealed that she had fallen onto a piece of wood by her neck . Table 1results for the group of children> 4 weeks of lifeair leakaetiologyage (years)intubation (after diagnosis)pleural drainagediagnostic testsdays in icupm, pp, sespontaneous/3 days earlier intensive sport15.8nonochest x - ray 32pm, pttraumatic7.53 daysyesct / chest x - ray 35pm, se traumatic (lesion in hypopharynx)1.3nonoct / chest x - ray 3, oesophagogram with contrast medium, laryngotracheoscopy4pm, ppiatrogenic: equipment failure with barotrauma during mechanical ventilation2.3nonochest x - ray 22pmspontaneous15.4nonochest x - ray / ct1pm obstructive bronchitis5.6nonochest x - ray 2/ct2pm, ptobstructive bronchitis1.97 daysyeschest x - ray 77fig . 1pneumomediastinum (pm), subcutaneous emphysema (se) and pneumopericardium (pp) in a 2-year - old intubated patient results for the group of children> 4 weeks of life pneumomediastinum (pm), subcutaneous emphysema (se) and pneumopericardium (pp) in a 2-year - old intubated patient the group of children older than 4 weeks stayed in the intensive care unit for a mean of 3.2 days (range 17 days), depending on the severity of the pm and the underlying disease . Compared to the neonatal group, the length of stay in the intensive care unit was shorter . However, most neonates stayed in the intensive care unit longer, primarily because of comorbid conditions and not because of the pm . We found nine neonates who were diagnosed with pm (table 2); two premature and seven term infants, all of whom presented with signs of respiratory distress . Three neonates were also diagnosed with a pp, one with se and five with a pt . Birth weight ranged from 2,150 g to 4,140 g (mean 3,340 g). All children were vigorous at birth and none required resuscitation with bag mask ventilation or surfactant . Before arriving in the intensive care unit, where the diagnosis of pm was confirmed by chest x - ray, two infants had received ventilatory support by cpap and one of the premature infants had to be intubated for respiratory failure . During hospitalisation in the intensive care unit, two children deteriorated and required mechanical ventilation for three and four days, respectively, and two other children needed cpap for a few hours . The age at admission to the intensive care unit ranged from a few hours to four days . One neonate was admitted to the intensive care unit due to convulsions and developed a pm on day six of life . The treating physicians felt that the pm may have been associated with a valsalva manoeuvre, which occurred during the seizure . Other causes of pm were a pulmonary infection due to maternal infection and a possible barotrauma due to peak inspiratory pressure of 25 cm h2o in a mechanically ventilated premature neonate . Two newborns had pm related to cpap and four neonates were diagnosed with spontaneous pm . Neonates stayed in the intensive care unit for 313 days (mean 5.6 days), depending on the severity of the underlying diseases . Table 2results for the group of neonatesair leakaetiologybirth weightgestational age (weeks)mode of deliverymechanical ventilation before diagnosisduration of ventilatory support after diagnosispleural drainagedays in icupmspontaneous4,140 g40 0/7vaginalnonono4pm, pp, sepremature lungs, barotrauma2,150 g34 4/7caesarian section pip max . 25 cm h203 days (intubation)no4pm, pt, pppremature lungs, spontaneous or cpap2,480 g35 6/7vaginalcpap4 days (intubation)yes6pm, ptspontaneous3,485 g38 1/7caesarian sectionnonono3pm, ptspontaneous3,440 g37 5/7vaginalno6 hours (cpap)no3pm, ppspontaneous2,830 g39 1/7caesarian sectionnonono13pm, ptspontaneous3,970 g38 5/7caesarian sectionnonono9pm, ptpulmonary infection due to maternal infection3,440 g38 5/7vaginalcpap1 day (cpap)no5pmconvulsions or spontaneous4,130 g40 5/7vaginalnonono4 results for the group of neonates all children with pm had a good outcome without any complications due to air trapping . In the group of children older than 4 weeks, only two children developed a respiratory insufficiency, leading to mechanical ventilation . In both of them, respiratory failure was related to their underlying condition (polytrauma with haematothorax and severe obstructive bronchitis, respectively). All other children were treated with oxygen only and stayed in the intensive care unit until they improved clinically and radiographically . Regarding radiologic diagnostics, four patients of the group of children> 4 weeks of life had ct scans (three of them had been done in outside clinics from where the patients had been admitted to our intensive care unit). Retrospectively, the utility of the ct scans was put into question, as these scans did not change the patient management . The only patient in whom the ct scan changed management was the child with polytrauma . In this patient, other intrathoracic injuries needed to be ruled out . In the group of neonates, it was much more difficult to find the aetiology of the pm, since all neonates had presented with respiratory disease, and radiologic investigations were partially performed only after the use of cpap or tracheal intubation . Five of the nine neonates had a spontaneous pm without risk factors, such as mechanical respiratory support (bag mask ventilation after birth, cpap, mechanical ventilation) or restrictive lung disease . Three of these five babies were delivered by caesarean section . In the remaining four newborns, possible mechanical incidents leading to the air leak could be revealed: mechanical ventilation with high inspiratory pressure, cpap, pulmonary infection and convulsion . Further investigations are needed to find the aetiology of spontaneous pm in healthy, term neonates . In conclusion, it is diagnosed by chest x - ray alone . Whereas in older children mechanical events leading to the airway rupture can be revealed in most cases, about half of the neonates in our series suffered from pm without obvious reason.
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Leri - weill's dyschondrosteosis (lwd) is anautosomal dominant condition with variable penetrance characterized by mesomelic short stature and with madelung deformity . It is due to deletion or mutation in short stature homeobox (shox) gene . Here, there is deficient growth of a volar and ulnar aspect of distal radialphysis, triangulation of corpus with proximal and volar shift of the lunate . A 13-year - old girl referred to us with a history of deformity of bilateral forearm from the age of 5 to 6 years . There was no history of pain in the joint of hands, trauma to the wrist and no history of similar illness in the family . Patient was moderately built and significantly short for her age [figure 1]. With height of 130 cm (less than 3 percentile). Upper segment was 69 cm, lower segment was 61 cm, weight of 35 kg and body mass index of 20.71 kg / m . Secondary sexual characteristic tanner - whitehouse staging - b4 (breast), p4 (pubic hair), a3 (axillary hair) her elder brother had normal height for age and no any hand deformities and so were her parents . Investigation showed hemoglobin of 12.5 g / dl, liver function test and renal function test were normal ca= 9.0 mg / dl alkaline phosphates 260 iu and phosphorus = 3.5 mg / dl . X - ray bilateral wrist showed malformed medial radial epiphysis with dorsal and ulnar shift and with increased length of phalanges suggestive of madelung deformity [figure 2]. Her ultrasound abdomen showed no hepatomegaly or splenomegaly and had normal sized uterus and ovary x - ray spine and x - ray elbow were normal [figure 3]. Her luteinizing hormone = 1.1 miu / ml follicle stimulating hormone = 2.4 miu / ml and karyotype was normal as was urine mucopolysaccharide screening . X - ray bilateral wrist based on the history, clinical examination, and investigations findings, the final diagnosis of lwd as a cause of madelung deformity was made . It is the most common cause of mesomelic deformity and is inherited as an autosomal dominant trait with variable penetrance . It is often seen in girls and becomes apparent in late childhood or early adolescence . Madelung deformity, beside is also found in turner's syndrome, pseudohypoparathyroidism, mucopolysaccharidosis and achondroplasia of distal radial epiphysis . Madelung deformity was first described by malgaigne in 1885 and later by madelung in 1878 as a spontaneous forward subluxation of the hand . Madelung deformity is an ulnar and dorsal curvature of the distal radius due to deficient growth of the volar and ulnar aspect of distal radialphysis, increased inclination of the distal radial joint surface, triangulation of the corpus with proximal and volar migration of and a prominent dorsal subluxation of ulnar head . Recently, two subtypes of madelung deformity have been described, one with short stature and mesomelia consistent with lwd and the other with severe involvement of the entire radius with limited range of motion of extremity, markedly bowed appearance of the forearm and conspicuous radiographic deformity of the forearm and distal radius . The pathogenesis of lwd is linked to deletion or mutation in shox gene, present in the pseudoautosomal region of the sex chromosomes - xp23 and yp11 . In early human embryos it is expressed in the developing limbs (particular elbow, knee, distal radius / ulnar and wrist) as well as first and second pharyngeal arches and plays an important role in bone growth as well as development . Shox gene is also associated with short stature in turner's syndrome and also some causes of growth retardation like lws and langers mesomelic dysplasia . Management is usually conservative . Persistent pain and or severe deformity call for orthopedic surgery involving radial osteotomy . In addition, ulnar shortening in skeletally immature patient or excision of distal ulnar head in skeletally mature patients are done . Surgical prophylaxis by distal resection of the abnormal part of distal radial epiphysis and its replacement by autologous fat (also known as physeolysis) has recently been shown to restore growth and minimized deformity . Growth hormone supplementation is found to increase final height in turner's syndrome, lws but not in langers mesomelic dysplasia . Hand deformity is corrected by orthopedic surgery and for shox gene related short stature food and drug administration has recently approved growth hormone therapy.
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Age - related maculopathy (arm), also known as age - related macular degeneration, affects approximately 30 million people worldwide . In the united states, there are an estimated 13 million people with some vision loss from arm, including two million with the most severe late - stage of the disease . Fortunately, there are an increasing number of effective treatments for late - stage disease, such as pegaptabnib (macugen; pfizer) and ranibizumab (lucentis; genetech). However, treatment of advanced arm often results in less than optimal patient outcomes because moderate to severe irreversible vision loss has already occurred before stabilization . The treatment of the disease in its earliest stages is the best strategy to preserve the patient s vision, which may be only mildly disturbed as measured by high contrast visual acuity measured under high luminance conditions . The disease s slow progression hampers the feasibility of clinical trials because currently accepted endpoints of acuity and fundus appearance are relatively insensitive to the disease s progression through its earliest stages . These endpoints require clinical trials to have large sample sizes or long follow - up durations to evaluate efficacy of a potential treatment . Clinical trials evaluating early treatments are impractical because of the constraints current endpoints impose on trial design . While patients with early arm typically have good best - corrected visual acuity, impaired night vision is a prominent self - reported problem [13]. Anatomical studies of donor eyes have documented significant rod dysfunction and photoreceptor drop out in comparison to cone photoreceptors . Histopathological studies have found arm - related lesions in the rpe and bruch s membrane, which are generally invisible to clinical inspection [5, 6]. Not surprisingly, these insults to the photoreceptors and the rpe, which supports the photoreceptors, disrupt vision . Early arm patients exhibit moderate to severe impairment of rod - mediated dark adaptation and scotopic visual sensitivity even in the absence of visual acuity loss [7, 8]. Rod - mediated dark adaptation appears to be more impaired in early arm patients than photopic visual sensitivity, scotopic visual sensitivity, acuity, and contrast sensitivity . Advanced arm patients exhibit impaired cone - mediated dark adaptation [912] and early arm patients exhibit photostress impairment [13, 14]. However, rod - mediated dark adaptation appears to be more affected than cone - mediated dark adaptation at least outside the fovea in early arm patients . Rod - mediated dark adaptation impairment is modestly reversible with vitamin a supplementation, whereas cone - mediated dark adaptation impairment is not . A pilot retrospective study suggests that the rod - mediated dark adaptation impairment is detectable at least 4 years before the lesions associated with the disease are clinically apparent (unpublished data). These findings suggest that dark adaptation impairment is a sensitive marker of early arm and raises the possibility that it may used to predict progression . The utility of dark adaptometry as a clinical outcome measure or practical diagnostic tool is hampered by a long test duration, high participant burden, and lack of standardized dark adaptometers . Dark adaptation protocols used in prior research require up to 90 minutes, and typically more than 100 threshold estimates are made . The long duration and number of thresholds measurements the goal of this study was to develop a short - duration dark adaptation protocol that minimized patient fatigue, increased operator ease of use, and maintained the high sensitivity and specificity of research protocols . The protocol evaluated in this study has three distinct differences compared with prior research protocols: (1) the bleaching light intensity was significantly reduced to shorten the duration necessary to measure sensitivity recovery; (2) the stimulus was located within the area of greatest rod dysfunction to enhance the sensitivity of the test for detection of early disease; and (3) the speed of dark adaptation was estimated using a parameter that is robust to the shape of the dark adaptation function, which can be variable with disease severity . This protocol was implemented using a new dark adaptometer, the adaptdx, developed in collaboration with apeliotus technologies, inc . The institutional review board of the university of alabama at birmingham approved this study . Written informed consent was obtained from all participants after the nature and possible consequences of the study were explained . Normal adults and early to intermediate arm patients were recruited from the comprehensive ophthalmology and retina services of the department of ophthalmology, university of alabama at birmingham . Inclusion criteria were as follows: (1) participants were 20 to 45 years old for the young normal group and participants were at least 55 years of age for the old normal and arm groups; and (2) best - corrected distance visual acuity of 20/100 or better in at least one eye . If both eyes qualified, the eye with the better acuity was enrolled as the test eye . If the acuities were the same, the right eye was enrolled as the test eye . Exclusion criteria were as follows: (1) medical record or a general health interview indication of glaucoma, optic neuropathy or any ocular conditions other than arm, or refractive error (spherical equivalent) having an absolute value> 6 diopters; (2) neurological diseases such as alzheimer s disease, parkinson s disease, history of stroke or multiple sclerosis; (3) diabetes; and/or (4) inability to perform the psychophysical task used to measure dark adaptation . Best - corrected distance visual acuity was measured for each eye using the edtrs chart and expressed as logmar . The patient s test eye is dilated to 6 mm diameter, and corrective lenses are introduced as appropriate for the 30-cm viewing distance to correct for blur . An infrared camera located behind the fixation light continuously monitors the patient s test eye and displays an image of the eye on a personal computer - based operator control screen . The operator centers the patient s test eye to the red fixation light with the help of a reticule displayed on the image of the eye . The patient s test eye is bleached by exposure to a photoflash (0.25 ms duration, 6.38 log scot td second intensity) while the patient is focused on the fixation light . The flash of light passes through a diffuser and 4-diameter aperture centered at 5 on the inferior visual meridian to provide a uniform focal bleach surrounding the area to be tested during sensitivity recovery measurements . The patient focuses on the fixation light, and responds that a stimulus light is present by pushing a button . The stimulus light is a 1.7 circular test spot located at 5 on the inferior visual meridian . To focus on rod - mediated function, a stimulus wavelength of 500 nm was used, which is near the peak of rod sensitivity . Sensitivity is estimated using a three - down / one - up modified staircase threshold estimate procedure . Starting at a relatively high intensity (5.00 cd / m), stimulus lights are presented every two or three seconds for a 200-ms duration . If the patient does not respond within 2 seconds of stimulus onset, the stimulus light intensity remains unchanged on successive stimulus presentations until the patient responds . If the patient indicates that the stimulus is visible, the intensity is decreased for each successive presentation in steps of 0.3 log units until the patient stops responding that the stimulus is visible . After the patient indicates that the stimulus light is invisible by not pressing the button while the stimulus light is present, the intensity of the target is increased for each successive presentation in 0.1 log unit steps until the patient responds that the stimulus light is once again visible . Successive threshold measurements start with the stimulus intensity 0.2 log units brighter than the previous threshold measurement . Threshold measurements are made about once a minute for the duration of the measurement protocol; thus about 20 threshold measurements are made during the 20-minute test . The bleaching light (not shown) is presented through an aperture co - localized with the stimulus light the patient s view of the adaptdx . The bleaching light (not shown) is presented through an aperture co - localized with the stimulus light because arm patients can exhibit several differently shaped dark adaptation functions, the rod intercept, the rod intercept is the amount of time after bleach offset required for the patient s sensitivity to recover to a stimulus intensity of 5 10 cd / m (i.e. ; 4.0 log units). This light level was chosen because it typically is achieved late in the second component of rod - mediated dark adaptation and is therefore completely mediated by rod function . Each subject s dark adaptation function is plotted and the rod intercept is estimated by linear interpolation . Patients with normal rod recovery will have shorter rod intercepts than those with impaired dark adaptation . Stereoscopic 30 photographs were taken with a ff450 plus zeiss fundus camera for all participants more than 54 years old . Photographs were evaluated using the age - related eye disease study (areds) severity scale for age - related macular degeneration by two independent graders who were masked to the clinical and functional characteristics of the participants . Group assignment to normal or arm was based on the areds severity score of the test eye . These steps are indicative of normal (step 1), borderline arm (our terminology) (step 2), early arm (steps 36), intermediate arm (steps 79), and advanced arm (steps 10 and 11). The study population consisted of eight normal young adults (mean age = 32.6 years old), nine normal old adults (mean age = 73.1 years old) and 17 arm patients (mean age = 75.1 years old). The old normal and arm groups had similar ages (p = 0.42) and test eye acuities (p = 0.14) (table 1). Fellow eye acuity was higher on average for old normal adults than arm patients (p = 0.01). Arm patients exhibited substantially slower dark adaptation compared with normal old adults; whereas the dark adaptation speed was essentially the same for the young and old normal participants . Representative dark adaptation functions for a young normal adult, an early arm patient and an intermediate arm patient are shown in fig . 2 . The rod intercept for arm patients (mean = 17.20 min, sd = 3.8) was on average twice as long as the rod intercept for normal old adults (mean = 8.73 min, sd = 1.9) (p <0.0001). Nine of the 17 arm patients failed to obtain the criterion sensitivity within the 20-minute protocol, and were assigned a rod intercept of 20 min; thus the substantial difference between these averages actually understates the relative impairment between the two groups . The rod intercept for normal young adults (mean = 8.2 min, sd = 1.4) was statistically indistinguishable from normal old adults (p = 0.76). 2representative dark adaptation curves of a normal adult (closed circles), early arm patient (open triangles), and intermediate arm patient (open circles)table 1participant demographics young normal (n = 8)old normal (n = 9)arm (n = 17)age (years), mean (sd)32.6 (5.3)73.1 (4.8)75.1 (6.4)gender,% (n)female100 (8)66.7 (6)52.9 (9)male0 (0)33.3 (3)47.1 (8)race,% (n)white62.5 (5)88.9 (8)100 (17)african american25.0 (2)11.1 (1)0 (0)asian12.5 (1)0 (0)0 (0)visual acuity, logmar mean (sd)tested eye.02 (0.4).04 (.12).14 (.19)fellow eye.05 (0.1).14 (.21).52 (.40) representative dark adaptation curves of a normal adult (closed circles), early arm patient (open triangles), and intermediate arm patient (open circles) participant demographics each subject s dark adaptation was classified as normal or impaired based whether the participant s speed of dark adaptation as measured by the rod intercept fell within the normal reference range (mean 2 sd) of the normal old adults . The upper limit of the normal reference range was 12.5 minutes . Individuals with rod intercepts participants with a rod intercept 12.5 were classified as having normal dark adaptation . Using this criterion, 15 of the 17 arm patients all nine normal adults fell within the normal reference range for a diagnostic specificity of 100% . The sensitivity and specificity of our 20-minute study protocol compares favorably to that of a previously used 90-minute research protocol (85% sensitivity; 100% specificity), despite the considerable shortening of the test . Furthermore, it is useful to examine the two false negative results (those arm patients classified with normal dark adaptation). Both were for participants with an areds fundus grade of 2 . One of these patients exhibited three drusen of 63 m scattered throughout the central 3,000 m of the macula . The sensitivity of the test is 100% for those arm patients with an areds grade 3 and higher . The rod intercept for borderline arm patients (areds fundus grade 2) was, on average, 5.1 minutes slower than the normal old adult s rod intercept . The rod intercept for early arm patients (areds fundus grades 3 to 5) was, on average, yet another 3.4 minutes slower than borderline arm patients . Finally, all of the intermediate and late arm patients (areds fundus grades 6) failed to reach the rod intercept within the 20-minute test time . Table 2dark adaptation impairment increased with disease severity young normalold normalborderline armearly armintermediate armadvanced armsample size895651areds fundus graden / a123,4,56,7,8,910rod intercept, min mean (sd)8.2 (1.4)8.7 (1.9)13.8 (4.4)17.2 (2.7)20 () 20 () fundus photography and grading was not performed on young normal group.all subjects failed to reach the rod intercept within the 20-minute test duration . Dark adaptation impairment increased with disease severity fundus photography and grading was not performed on young normal group . Using a 20-minute, rod - mediated dark adaptation protocol, arm patients exhibited a dramatic slowing of dark adaptation in comparison with normal old adults . The magnitude of the impairment was similar to that previously measured using a 90-minute research protocol . As expected, reducing the bleaching light shortened the duration of the test, and moving the stimulus to an area of greater rod dysfunction preserved the sensitivity and specificity of the protocol . The magnitude of the dark adaptation impairment exhibited by early arm patients suggests that a rapid diagnostic screening test having a duration of less than 10 minutes is feasible . Retest reliability was found to be quite high (intraclass r = 0.95) in a separate, preliminary study of 40 normal adults and arm patients (data not shown). Perhaps more intriguing, the increase in the amount of rod - mediated dark adaptation impairment with increasing disease severity as assessed by the areds severity system for arm allows the possibility of using dark adaptation as a clinical outcome measure . Previously, we evaluated another short - duration protocol developed for the sst-1 dark adaptometer (lkc technologies, inc) for the detection of early arm . The sst-1 is a manual dark adaptometer that uses an abbreviated protocol, typically 20 to 30 minutes . The adaptdx and the sst-1 exhibit markedly different response characteristics . Whereas the adaptdx shows no difference with aging but is highly sensitive to arm, the sst-1 discriminates aging effects but is insensitive to arm . An important difference between the protocols is that the sst-1 measures the sensitivity recovery of the entire retina, instead of just the macula or a portion of the macula . We have developed a short duration protocol that can sensitively and specifically detect early arm . The impaired night vision encountered in early arm is a clinically significant problem similar to acuity impairment encountered in late arm . As such, we believe dark adaptation is a suitable primary endpoint to evaluate efficacy of treatments aimed at early arm.
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The greatest burden of malaria morbidity and mortality occurs in children under 5 years of age in africa . Travel and migration increasingly transport malaria to the usa and other industrialized countries, up to 30,000 cases per year by some estimates . In 2005, surveillance from the centers for disease control identified 1528 cases of malaria in the usa, a 15% increase from the previous year and the greatest number since 1980 . Plasmodium falciparum, the species responsible for severe malaria, accounted for 48% of the cases and caused all 7 fatalities . Severe malaria refers to malaria with signs of end organ dysfunction, as manifested by coma, pulmonary edema, renal failure, circulatory collapse, or severe anemia . In general, 5% of imported cases progress to severe malaria, which in turn carries 20% mortality . In non - endemic countries, malaria poses a significant challenge to medical education . Despite its worldwide importance it is a complex and lethal condition with the potential for rapid deterioration from nonspecific fever to multiorgan failure . Managing malaria thus requires time - critical actions, often on the basis of a presumptive diagnosis . One can also suspect that discontinuation of quinidine as an emergency antiarrhythmic drug further reduces prescribing experience with the only widely available treatment for severe malaria in the usa . Lack of physician familiarity with the diagnosis and treatment of malaria contributes heavily to preventable malaria deaths in non - endemic countries [3, 57]. This paper proposes that the challenge of teaching severe malaria in non - endemic areas is particularly amenable to the use of high - fidelity simulation . The objective was to develop a novel teaching tool for severe malaria, using high - fidelity simulation that emphasizes predefined learning objectives for the disease . High - fidelity simulation refers to simulation scenarios with mannequins that can reproduce a wide range of patient characteristics . According to a recent review of simulation in emergency medicine, the high fidelity depends on (1) the realism of the mannequin and its software (i.e., the simulator), (2) the realism of the environment, and (3) a psychological realism as perceived by the participant . The simulation center is equipped with the meti emergency care simulator (meti ecs), a high - fidelity patient simulator that blinks, breathes, has palpable pulses, and speaks through a speaker linked to an instructor in the control room . (additional information is available from medical education technologies, inc, sarasota, fl, http://www.meti.com/.) The meti ecs is located in a simulated emergency department resuscitation room complete with monitors, a code cart, and standard intravenous equipment and medications . The software of meti ecs permits the creation of multiple physiologic states, which can be programmed to worsen with time and to respond to therapeutic maneuvers . Certain physical findings that are not easily simulated on a mannequin, such as dry mucous membranes, can be communicated as observations from the patient's nurse, who is one of the simulation instructors . The simulation is directed by a lead instructor in the control room and facilitated by an in - scenario, role - playing nurse . The authors observed several simulation scenarios in progress to learn the capabilities of the simulation center and to adapt a malaria case scenario to the simulation environment . Learning objectives were derived from a selection of recent publications searched for in medline under malaria and severe malaria, with limits review, and from their respective reference lists [2, 4, 915], as well as from the authors' prior experience in teaching malaria . Special emphasis was placed on developing learning objectives that would address typical pitfalls in the diagnosis and management of malaria in non - endemic countries . A simulation scenario was designed and pilot - tested to address the learning objectives below . The summarized learning objectives were as follows (see the online emergency medicine simulation scenario library for full learning objectives at http://www.emedu.org/simlibrary or at http://www.aamc.org/mededportal). Objective 1: recognize high - risk groups for malariaany traveler to an endemic area in the year prior to presentation should be considered at risk for the disease . Immigrants originally from endemic countries are at particularly high risk, because they are much less likely to take malaria prophylaxis when they travel to visit family abroad . The travel history is crucial to identify the susceptible patient, as any travel to the tropics constitutes a risk . Any traveler to an endemic area in the year prior to presentation should be considered at risk for the disease . Immigrants originally from endemic countries are at particularly high risk, because they are much less likely to take malaria prophylaxis when they travel to visit family abroad . The travel history is crucial to identify the susceptible patient, as any travel to the tropics constitutes a risk . Objective 2: establish a definitive or presumptive diagnosis of malariafever is the most common initial manifestation of malaria, accompanied variably by headache, myalgias, jaundice, vomiting, abdominal pain, cough, and diarrhea . The non - specific nature of symptoms in early, uncomplicated malaria necessitates a focused differential diagnosis that includes other life - threatening infectious diseases that benefit from prompt antibiotic treatment . Blood smears, however, are not always immediately read by trained personnel, and false - negatives occur . A presumptive diagnosis of malaria is warranted in any seriously ill patient who is at risk for malaria and treatment should never be delayed in the wait for a positive blood smear . This frequent reliance on a presumptive scenario further highlights the importance of obtaining recent travel history on all patients presenting with fevers: unless health care workers habitually ask febrile patients about travel to the tropics, they will miss an opportunity to diagnose and treat malaria and other life - threatening tropical diseases . Additionally, some institutions may have rapid diagnostic tests (rdts) for diagnosing malaria . A rapid diagnostic test (rdt) is an alternate way of quickly establishing the diagnosis of malaria infection by detecting specific p. falciparum antigens in a person's blood . These rdts, whether negative or positive, must always be followed up by microscopy, as they will miss nonfalciparum malaria, and cannot measure levels of parasetemia . Fever is the most common initial manifestation of malaria, accompanied variably by headache, myalgias, jaundice, vomiting, abdominal pain, cough, and diarrhea . The non - specific nature of symptoms in early, uncomplicated malaria necessitates a focused differential diagnosis that includes other life - threatening infectious diseases that benefit from prompt antibiotic treatment . Blood smears, however, are not always immediately read by trained personnel, and false - negatives occur . A presumptive diagnosis of malaria is warranted in any seriously ill patient who is at risk for malaria and treatment should never be delayed in the wait for a positive blood smear . This frequent reliance on a presumptive scenario further highlights the importance of obtaining recent travel history on all patients presenting with fevers: unless health care workers habitually ask febrile patients about travel to the tropics, they will miss an opportunity to diagnose and treat malaria and other life - threatening tropical diseases . Additionally, some institutions may have rapid diagnostic tests (rdts) for diagnosing malaria . A rapid diagnostic test (rdt) is an alternate way of quickly establishing the diagnosis of malaria infection by detecting specific p. falciparum antigens in a person's blood . These rdts, whether negative or positive, must always be followed up by microscopy, as they will miss nonfalciparum malaria, and cannot measure levels of parasetemia . Objective 3: identify and treat the complications of severe malaria antimalarial therapy should be initiated on the basis of a presumptive diagnosis in any seriously ill patient who is at risk for malaria . Coma and acidosis are the most common manifestations of severe malaria in adults . Renal failure, seizures, hypoglycemia, severe malaria, disseminated intravascular coagulation (dic), and noncardiogenic pulmonary edema (ards) are other important complications of severe malaria . The most important step in managing the complications of severe malaria is the prompt initiation of antimalarial drugs . All complications of malaria should be treated as they would for any critically ill patient, with the caveat that malaria patients are more prone to fluid overload than septic patients and thus require cautious fluid administration [12, 13]. Antimalarial therapy should be initiated on the basis of a presumptive diagnosis in any seriously ill patient who is at risk for malaria . Renal failure, seizures, hypoglycemia, severe malaria, disseminated intravascular coagulation (dic), and noncardiogenic pulmonary edema (ards) are other important complications of severe malaria . The most important step in managing the complications of severe malaria is the prompt initiation of antimalarial drugs . All complications of malaria should be treated as they would for any critically ill patient, with the caveat that malaria patients are more prone to fluid overload than septic patients and thus require cautious fluid administration [12, 13]. Objective 4: know the treatment for severe malaria and its complicationsintravenous artesunate is now the world health organization- (who-) recommended first - line treatment for severe malaria . Artesunate has proved itself superior to intravenous quinine in both adults and children, dropping treatment with quinine to second line [1820]. Artesunate and other related compounds have a very good safety profile with few adverse effects: mild gastrointestinal symptoms may occur, while hypersensitivity reactions are on the order of 1 in 3000 . Artesunate was approved as a new investigational drug in the united states on june 21st, 2007 (ind protocol no . 76,725, entitled intravenous artesunate for treatment of severe malaria in the united states) and makes a new class of antimalarial medication, artemisinins, available in the united states . High - quality intravenous artesunate is available only to malaria patients hospitalized in the united states who need intravenous treatment because of severe malaria disease, high levels of malaria parasites in the blood, inability to take oral medications, lack of timely access to intravenous quinidine, quinidine intolerance or contraindications, or quinidine failure . The drug can be provided to hospitals upon request and on an emergency basis by the cdc (see contact details below) through the dcd drug service or by one of the cdc quarantine stations located around the united states . In the usa clinicians located in these major cities might therefore receive artesunate within minutes of calling the cdc, though delivery may take several hours in other cities or in other locations . The cdc reports an average call to infusion time of 7 hours (personal communication february 2011). Because parenteral forms of artesunate and quinine are not always immediately available in the united states and other non - endemic countries and because timely initiation of an effective anti - malarial is crucial, quinidine gluconate may still occasionally be the drug of choice . Quinidine is a more effective anti - malarial than quinine but is more cardiotoxic and thus requires continuous electrocardiographic monitoring . Since quinidine too has been in short supply in the usa, one might consider initiating a drug with anti - malarial activity (such as intravenous clindamycin or doxycycline) while awaiting delivery of artesunate . In cases of severe malaria with elevated parasitemia (> 10% of red blood cells on a thin smear) that do not respond promptly to antimalarials, one may consider an exchange transfusion, although there is no strong clinical evidence to support its use [6, 15]. Intravenous artesunate is now the world health organization- (who-) recommended first - line treatment for severe malaria . Artesunate has proved itself superior to intravenous quinine in both adults and children, dropping treatment with quinine to second line [1820]. Artesunate and other related compounds have a very good safety profile with few adverse effects: mild gastrointestinal symptoms may occur, while hypersensitivity reactions are on the order of 1 in 3000 . Artesunate was approved as a new investigational drug in the united states on june 21st, 2007 (ind protocol no . 76,725, entitled intravenous artesunate for treatment of severe malaria in the united states) and makes a new class of antimalarial medication, artemisinins, available in the united states . High - quality intravenous artesunate is available only to malaria patients hospitalized in the united states who need intravenous treatment because of severe malaria disease, high levels of malaria parasites in the blood, inability to take oral medications, lack of timely access to intravenous quinidine, quinidine intolerance or contraindications, or quinidine failure . The drug can be provided to hospitals upon request and on an emergency basis by the cdc (see contact details below) through the dcd drug service or by one of the cdc quarantine stations located around the united states . In the usa clinicians located in these major cities might therefore receive artesunate within minutes of calling the cdc, though delivery may take several hours in other cities or in other locations . The cdc reports an average call to infusion time of 7 hours (personal communication february 2011). Because parenteral forms of artesunate and quinine are not always immediately available in the united states and other non - endemic countries and because timely initiation of an effective anti - malarial is crucial, quinidine gluconate may still occasionally be the drug of choice . Quinidine is a more effective anti - malarial than quinine but is more cardiotoxic and thus requires continuous electrocardiographic monitoring . Since quinidine too has been in short supply in the usa, one might consider initiating a drug with anti - malarial activity (such as intravenous clindamycin or doxycycline) while awaiting delivery of artesunate . In cases of severe malaria with elevated parasitemia (> 10% of red blood cells on a thin smear) that do not respond promptly to antimalarials, one may consider an exchange transfusion, although there is no strong clinical evidence to support its use [6, 15]. Objective 5: identify resources for assistance in the management of malariaemergency medicine physicians and other critical care providers should know what resources are rapidly available to assist in the management of malaria . The website of the centers for disease control (cdc) at http://www.cdc.gov/malaria is a resource, and a cdc malaria specialist is also available 24 hours a day for telephone consultations (770 - 488 - 7788 daytime, and 770 - 488 - 7100 after working hours and weekends). The same cdc malaria specialist can authorize and organize an emergency delivery of artesunate (http://www.cdc.gov/malaria/diagnosis_treatment/artesunate.html). Although a consult to an infectious disease expert as well as an inpatient pharmacist for dosing assistance can be of use, the cdc has the expertise and the means to provide up - to - date and rapid advice in treating these patients and is highly recommended by these authors . Emergency medicine physicians and other critical care providers should know what resources are rapidly available to assist in the management of malaria . The website of the centers for disease control (cdc) at http://www.cdc.gov/malaria is a resource, and a cdc malaria specialist is also available 24 hours a day for telephone consultations (770 - 488 - 7788 daytime, and 770 - 488 - 7100 after working hours and weekends). The same cdc malaria specialist can authorize and organize an emergency delivery of artesunate (http://www.cdc.gov/malaria/diagnosis_treatment/artesunate.html). Although a consult to an infectious disease expert as well as an inpatient pharmacist for dosing assistance can be of use, the cdc has the expertise and the means to provide up - to - date and rapid advice in treating these patients and is highly recommended by these authors . After the initial pilot test, the scenario was conducted 5 times between march 13 and september 4, 2008 in the simulation center described above . The simulations were run during weekly simulation days for medical students, nurses, and emergency medicine residents working in the emergency department . Some participants were directly involved in the simulated patient care, while others watched from a video - linked conference room . The scenario involves a 34-year - old female who complains of fever, vomiting, headache, and malaise within a week after visiting family in nigeria . (for the full scenario, along with simulation case manager instructions, see the online simulation scenario library at http://www.emedu.org/simlibrary or http://www.aamc.org/mededportal). She did not take malaria prophylaxis but took all of her son's left - over erythromycin after becoming ill . She initially presents with a normal mental status but is febrile, tachycardic, tachypneic, and ill appearing . Laboratory tests reveal a significant anion - gap acidosis, but the blood smear is not immediately available . An ekg will show sinus tachycardia with qt prolongation . If participants recognize severe malaria and treat with quinidine, the patient will develop torsades de pointes . In order to prevent a premature end to the scenario, the role - playing nurse who is part of the simulation center staff will cue participants as necessary to take actions that will keep the scenario moving and the patient alive . The objective - related critical actions are as follows: to recognize that the patient is at high risk (objective 1) and to order the appropriate tests for malaria and other diseases in the differential diagnosis (objective 2); to identify seizure, coma, and acidosis as manifestations of severe malaria, and also to remember to look for or treat hypoglycemia (objective 3); to provide supportive care, including fluid resuscitation, anticonvulsants, and endotracheal intubation as necessary (objective 3); to start empiric therapy for severe malaria with intravenous artesunate if available (by calling the cdc if in the usa), quinidine, quinine, or a temporizing alternative; to recognize the complications of treatment (objective 4); to seek outside help for malaria care, including specialist consultation . After the scenario, participants received a standardized debriefing on the case, including discussion of the 5 learning objectives for severe malaria and their associated critical actions . Participants then took a 20-question survey, which included 8 multiple choice test questions relevant to the 5 learning objectives . 29 learners, 16 participants, and 13 observers were included in 5 simulation sessions . Trainees included 19 3rd year medical students, 8 3rd year em residents, and 2 nurses (table 1). There was no relationship between learner group (observer or participant) and trainee position (p = .33). Among all learners (observers and participants), simulation was rated a very effective instructional method by 66% (95% ci 48, 83), equivalent to actual patient care by 67% (95% ci 36, 97), and higher than problem - based learning by 6% (95% ci 0, 18) and lecture by 3% (95% ci 0, 10). There were also no differences in effectiveness ratings or test scores among the three trainee groups (tables 2 and 3). Our findings indicate a high learner satisfaction rate with high - fidelity simulation as a teaching tool for severe malaria . The majority of the participants in our study rated the simulation to be as effective or superior to other teaching methods . Additionally, our results suggest that not only is simulation an effective method of teaching in this case but that it was an equally effective learning tool for both observers and participants . These findings are important as they indicate that we can effectively utilize this expensive technology to educate all learners involved in the process, even if they are just watching . This allows us to increase the number of participants or observers in one simulation setting knowing they are being effectively educated, thus maximizing our educational investment . Clinical simulations, including high - fidelity simulations, are increasingly used as an assessment tool in medical education to test knowledge and competence . High - fidelity simulation has often been used in teaching the management of acute situations, particularly in emergency medicine, critical care, and anesthesia . One literature review of the use of high - fidelity simulation in emergency medicine found 11 papers directly relevant to the teaching of emergency medicine . Simulation applications included airway management, trauma management (atls), team performance training, presentation of ethical dilemmas, management of disaster / crisis scenarios, and management of medical errors . A survey of centers using simulation in anesthesia training investigated the specific reasons for the use of simulation: rare events (75%) and crisis management (80%) constituted the top two reasons for use at the postgraduate level . Another study, also out of the anesthesia literature, found that simulation - based training is superior to problem - based learning in acute care scenarios . It is likely that physicians in training in industrialized countries will encounter malaria during their training or later in their career, whether at home or abroad . The february 2008 issue of academic medicine is entirely devoted to the development of global health programs at academic medical centers . According to the results of an aamc survey, 26% of medical students graduating in 2007 had had a global health experience during medical school, and 43% felt their global health training to be inadequate . International rotations are now an important component of many post - graduate training programs in multiple specialties [2729] and have an impact on recruitment [30, 31]. It is therefore absolutely relevant to teach malaria in academic centers in non - endemic countries . Each year, patients in industrialized countries with advanced medical care die preventable deaths from malaria, attributable to physicians' lack of familiarity with malaria and its treatment [3, 57]. . How can medical educators better prepare trainees to recognize and manage malaria? High - fidelity simulation is a teaching tool well suited to the acuity and time - dependent management of severe malaria . The rarity of malaria in non - endemic countries also supports the use of simulation, since bedside teaching is not usually feasible . In this manner, high - fidelity simulation for malaria is similar to its application in bioterrorism preparedness, another rare and life - threatening situation [32, 33]. One can easily imagine the use of simulation in teaching the management of other infectious diseases and intoxications that may be rare but provide little margin for error in diagnosis and treatment . This paper does not address the long - term effectiveness of the teaching intervention, that is, its impact on the retention of knowledge and skills that have real - world applicability in the treatment of severe malaria: the evaluation was given immediately after the debriefing that reemphasized the learning objectives and critical actions . The subgroup of practicing clinicians (8 emergency medicine residents) most likely to use and benefit from new knowledge in malaria was small and perhaps too small a sample size to reach conclusions applicable to this group . One general limitation to simulation in medical education is the lack of evidence of its impact on patient outcomes . In general, there is good evidence that simulation - based training improves performance in simulated scenarios themselves . In the particular case of manual skills and procedures high - fidelity simulation constitutes a novel tool for teaching severe malaria that can meet specific learning objectives by demonstrating the need for time - dependent critical actions . In non - endemic countries it may be rare to witness a case of this disease, but, given its life - threatening nature, it is imperative that our learners are aware of this disease, know how to treat it, and have experience with its presentation, whether real or simulated, so they can manage it quickly and effectively as health care providers.
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To increase awareness of the limitations of high - risk human papillomavirus (hrhpv) laboratory - developed testing (ldt) widely used in us cervical cancer screening . A young woman in her 30s was diagnosed and treated for stage 1b1 cervical squamous cell carcinoma in which hpv 16 dna was detected using polymerase chain reaction testing . Both 1 month before and 42 months before cervical cancer diagnosis, the patient had highly abnormal cytology findings; however, residual surepath (becton, dickson and company, franklin lakes, nj) vial fluid yielded negative hybrid capture 2 (hc2; qiagen nv, hilden, germany) hrhpv ldt results from each of the two specimens . A review of the available data indicates that (1) purification of dna from surepath specimens requires complex sample preparation due to formaldehyde crosslinking of proteins and nucleic acids, (2) hc2surepath hrhpv testing had not been food and drug administration - approved after multiple premarket approval submissions, (3) detectible hrhpv dna in the surepath vial decreases over time, and (4) us laboratories performing hc2surepath hrhpv ldt testing are not using a standardized manufacturer - endorsed procedure . Recently updated cervical screening guidelines in the us recommend against the use of hrhpv ldt in cervical screening, including widely used hc2 testing from the surepath vial . The manufacturer recently issued a technical bulletin specifically warning that use of surepath samples with the hc2 hrhpv test may provide false negative results and potentially compromise patient safety . Co - collection using a food and drug administration - approved hrhpv test medium is recommended for hpv testing of patients undergoing cervical screening using surepath samples . Since 2001, adjunctive high - risk human papillomavirus (hrhpv) testing has become increasingly integrated along with cytologic testing as a part of routine us cervical cancer screening, initially as a preferred reflex test after atypical cells of undetermined significance liquid - based cytology results and on a more widespread basis after 2003 food and drug administration (fda) approval for routine cytology and hpv cotesting of women 30 years and older.1,2 recently updated cervical screening guidelines from the american cancer society, the american society for colposcopy and cervical pathology, and the american society for clinical pathology have proposed significantly lengthened screening intervals, particularly for patients with negative hrhpv test results and either negative or equivocally abnormal (atypical cells of undetermined significance) cytology findings.3 for women 30 years and older with either hrhpv - negative atypical cells of undetermined significance or double negative results, a screening interval of 5 years has for the first time been recommended.3 the guidelines, however, emphasize that the new extended screening intervals following negative hrhpv test results are based on hpv tests with performance characteristics similar to hpv tests used in the supporting evidence.3 since at least one - third of all us hrhpv tests use laboratory - developed test (ldt) methodology, largely exempt from regulatory oversight by the fda and unlikely to have undergone rigorous evaluation using grade 3 + or grade 2 + cervical intraepithelial neoplasia clinical endpoints in properly designed trials,3,4 the guidelines publications specifically recommend against the use of hpv ldts for cervical cancer screening.3 the most common form of hrhpv ldt to date has been hybrid capture 2 (hc2; qiagen nv, hilden, germany) performed on residual surepath vial fluid (becton, dickinson and company, franklin lakes, nj).57 although hc2 hrhpv testing is fda - approved from both the digene (qiagen) specimen transport medium tube (qiagen) and the methanol - based preservcyt vial (hologic, inc, bedford, ma), hc2 hrhpv testing from the surepath vial to date has not been able to obtain fda approval, despite multiple premarket approval submissions, beginning in 2002.8 in a 2002 press release, the manufacturer stated: we remain hopeful that resolution of the fda s issues will not significantly alter our prior expectations for introduction in 2003.8 qiagen investigators have acknowledged that purification of dna from surepath specimens requires complex sample preparation due to the formaldehyde crosslinking of proteins and nucleic acids.9 three additional hrhpv tests have now also gained fda approval from either the preservcyt vial or also from proprietary manufacturer s collection media, but none of these newer fda - approved hrhpv tests have been approved using the surepath vial.1012 recently, the authors encountered a patient diagnosed with invasive cervical cancer with two prior significantly abnormal pap tests and two negative hrhpv ldt cotest results . Since virtually all cervical cancers are now thought to be due to persistent carcinogenic hrhpv infections,13,14 this case was investigated to better understand the possible causes of negative hrhpv ldt in screened patients developing cervical cancer . The patient was a young woman in her 30s, a gravida 3, para 3 cigarette smoker with a long history of abnormal pap test results and inconsistent follow - up due to medical appointment cancellations which the patient attributed to intermittent lack of insurance coverage . Forty - two months before her diagnosis of cervical cancer, the patient had a surepath pap test interpreted as atypical squamous cells, cannot rule out a high grade squamous intraepithelial lesion (figure 1). Because the patient s gynecologist had ordered routine cytology and hpv cotesting in a woman 30 years and older, the residual surepath vial fluid the hrhpv test result was reported as not detected . In that report, an additional comment stated that patients without hrhpv rarely have cervical cancer . A cervical biopsy obtained 4 months later reported koilocytosis and an endocervical curetting as benign . High - grade squamous intraepithelial lesion . The patient failed to return for scheduled colposcopic evaluation . Eighteen months later, the patient presented with irregular painful periods that were getting worse . A third surepath pap test was obtained and reported as high - grade squamous intraepithelial lesion (figure 2). The patient s gynecologist had again ordered routine cytology and hrhpv cotesting in a woman 30 years or older, and therefore the residual surepath vial fluid was again sent to the regional laboratory that had previously performed hrhpv testing . The hrhpv test, which utilized hybrid capture with signal amplification, was as before reported as not detected . In this report, however, an additional comment stated that the analytical performance characteristics of this assay, when used to test surepath or vaginal specimens, have been determined by (the laboratory). A cold knife conization was performed 1 month later and the presence of a poorly differentiated squamous cell carcinoma (scc) measuring 1 1 0.5 cm and extending to multiple biopsy margins was documented . One month later at an outside cancer referral center, the patient underwent a radical hysterectomy, bilateral pelvic lymph node dissection, bilateral salpingectomy, and left oophorectomy . Final pathologic diagnosis was of a cervical scc (1.5 cm maximum tumor dimension) invading the upper third of the cervix with no lymphovascular invasion identified, and negative lymph nodes twenty - six months later, at last follow - up, the patient was reported as alive and well with no evidence of disease . Paraffin sections of scc samples from the patient s cold knife conization specimen were used for hrhpv testing by polymerase chain reaction methods.15 hpv in tumor sections was initially tested for using m09/m11 pcr primers, which amplify an approximately 450 base pair conserved region of the l1 gene of hpv . Hpv type - specific pcr was also performed by pcr amplification of portions of e6 and e7 followed by automated dna sequencing of the amplified products . Using these methods, presence of hpv 16 type - specific e6 fortunately, in the case of this patient, the false negative results did not affect her course or management . Confirmation of the presence of hpv 16 dna by pcr in this patient s invasive cervical scc is consistent with the current understanding that persistent infections with a group of approximately a dozen carcinogenic hpv genotypes cause virtually all cases of cervical cancer worldwide . However, negative hc2 hrhpv test results from residual surepath vial fluid 42 months and 1 month before tumor diagnosis were unexpected . Since both pap tests contained highly abnormal cells (atypical squamous cells, cannot rule out a high - grade squamous intraepithelial lesion and high - grade squamous intraepithelial lesion), the discrepancy cannot be attributed to a failure to sample lesional cells . In fact, it has been argued that one advantage of hc2 cotesting is that hc2 may detect hrhpv dna in patients with occult invasive cervical cancer, even when lesional cells are not sampled.16 in the largest study of hc2 tests collected in fda - approved specimen transport medium, positive hrhpv hc2 results were reported in 185 of 198 (93.4%) samples collected from patients with simultaneous histopathologic diagnoses of invasive cervical cancer.17 in the same study, similar positive hrhpv hc2 test results were reported in 246 of 264 (93.2%) specimen transport medium tubes collected from patients with simultaneous histopathologic diagnoses of grade 3 cervical intraepithelial neoplasia.17 in contrast, the very limited data from the most widely cited us laboratory self - validation study of hpv testing from the surepath vial showed positive hrhpv hc2 results for patients with cancer in only 33% (one of three tests) or 50% (one of two patients). Also in contrast to the above cited data, the authors puzzlingly asserted that false negative hrhpv screening test results in patients with invasive cervical cancer are not surprising.7 hc2 uses a positive cut point of 1.0 relative light units per positive control, a cut point which corresponds to greater than or equal to 5000 hpv dna copies per test well, based on a receiver operating characteristic curve analysis versus cervical intraepithelial neoplasia grade 2 +, to minimize the detection of lower viral load hpv infections that are mostly benign.1820 nevertheless, a subset of invasive cervical cancers associated with low viral load have been described,21 and low viral loads in patients with developing invasive cervical cancer may fall below the detection cut point of fda - approved hrhpv tests such as hc2.17,22 in one of the authors own laboratories (rma), three of 31 (10%) patients diagnosed with invasive cervical scc and tested within the prior 12 months for hrhpv by hc2 from fda - approved preservcyt vial fluid had negative hc2 results.23 all three patients had hpv 18 detected by pcr in scc sampled in paraffin sections, and two also had detectible hpv 16.23 hpv testing from the non - fda - approved surepath vial is thought to be more challenging, primarily due to the formaldehyde crosslinking of proteins and nucleic acids.9,24 although recovery of dna and ribonucleic acid is largely unaffected by long - term storage in preservcyt,25,26 storage in surepath preservative fluid (becton dickinson) has been shown to affect the recovery of both dna and ribonucleic acid.27,28 upon exposure to surepath media, recovery of both dna and ribonucleic acid rapidly diminished . This reduction was most apparent in the 0150 hours range (ie, up to around 6 days).27 the websites of four large national laboratories that offer hc2 testing of referred surepath samples all indicated that surepath samples are stable at room temperature for hc2 testing for 1 month compared to 90 days/3 months for the fda - approved thinprep vial (hologic) (table 1). Interestingly, none of the laboratories, when queried by telephone, could produce independent surepath laboratories referred to the fda - approved becton dickinson package insert which states that surepath preservative fluid preserves cells (for cytologic testing) for up to 4 weeks at room temperature (15c30c).29 even in the most widely cited validation study mentioned previously, the authors referred to the digene package insert for specimen stability parameters.7 accurun 372 series 400; (seracare life sciences, inc, milford, ma),31 the hpv proficiency testing vendor for the college of american pathologists has reported that hpv 16-positive control samples shipped in surepath fluid degraded so rapidly that detectible hpv dna was lost after 1 day.30 the vendor concluded that only use of a two tube methodology, separating the hpv 16 sample from the surepath sample until the time of testing, could be used for laboratory hpv proficiency testing.31,32 there is at present no standardized surepath hpv protocol that all laboratories use and no literature with sufficient detail to represent an agreed upon standard.6,7,3336 furthermore, the manufacturer cannot under current regulations recommend standardized procedures for non - fda - approved hrhpv testing . As a result of continued ongoing widespread off - label hpv ldt use and related patient safety concerns, on june 8, 2012 the manufacturer of surepath released a technical bulletin which stated: the becton dickinson surepath sample medium has not been approved by the fda for use with the hc2 test and use [...] may under certain conditions provide false negative results . The becton dickinson surepath sample medium has not been approved by the fda for use with the hc2 test and use [...] may under certain conditions provide false negative results . False negative results could lead to inappropriate patient management and potentially compromise patient safety . The authors echo the cautions of the new us screening guidelines that emphasize that extended screening intervals following negative hrhpv test results be based on hpv tests with performance characteristics similar to hpv tests used in the supporting evidence . Given those new cervical screening guidelines and manufacturer communications that caution against non - fda - approved ldt hrhpv testing from the surepath vial, it is reasonable to view continued widespread use of this nonstandardized off - label testing as a patient safety issue . With new, extended 5-year screening intervals proposed for many women, an increasing number of women an avoidable increase in false negative hrhpv results in women with both precancer and early invasive cervical cancer will place patients at unnecessary risk . The use of screening methods that have not been validated should be strongly discouraged.37,38 with four fda - approved alternatives, it is difficult to justify the use of anything but rigorously clinically validated specimens . The college of american pathologists should discontinue offering its current form of laboratory proficiency testing for hpv testing out of the surepath vial, as it could mislead participants to believe that their methodology is currently safe and acceptable . For laboratories that use surepath for cytology, co - collection of a second sample for hrhpv testing in an fda - approved collection medium
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Here, we describe a case of a hispanic male with thrombotic thrombocytopenic purpura induced by nonsteroidal anti - inflammatory drugs (nsaids). The patient was a 21-year - old latino male who presented to the emergency department reporting a five day history of nausea, vomiting, and decreased urine output . The patient reported that symptoms began shortly after a purposeful consumption of a large quantity of ibuprofen (600-mg pills) in a suicide attempt . The exact amount of ibuprofen is unclear, but it was predicted that he consumed approximately 18 grams of ibuprofen . The past medical history was only notable for hypertension diagnosed three years prior, for which he was not receiving any medical therapy . The patient noted a prior recreational use of alcohol, marijuana and cocaine use, but he denied any use in the past few months . Family history was unremarkable for blood disorders and heart or renal disease . On initial exam, the patient had a temperature of 35.8c, a heart rate of 56 beats per minute, a blood pressure of 122/70 mmhg, a respiratory rate of 18 breaths per minute and his oxygen saturation was 100% on room air . The patient was noted to be alert and oriented despite appearing drowsy . On physical exam, pertinent findings included jaundice, icteric sclera, dry mucous membranes, and generalized petechiae . Heart and lung examination were unremarkable . The abdomen was found to be soft but slightly tender to palpation at the right upper quadrant . No rebound or guarding was appreciated . Mmol / l, potassium of 5.1 mmol / l, chloride of 83 mmol / l, bicarbonate of 21 mmol / l, blood urea nitrogen of 184 md / dl and creatinine of 19.1 mg / dl, and glucose of 95 mg / dl . Serum osmolality was 323 mosm / kg, with an anion gap calculated at 26 . White blood cell count 13.4 4 (10(3)/mcl), hemoglobin was 11.9 (10(6)/mcl) with an mcv of 73 fl, hematocrit was 34%, and platelets were 31,000/mcl . Total bilirubin was 24.6 mg / dl (direct bilirubin, 20 mg / dl), liver function tests were elevated with aspartate aminotransferase was 105 units / l, alanine aminotransferase was 167 units / l, and alkaline phosphatase was 464 units / l . International normalized ratio and prothrombin time were within normal limits . Lactate dehydrogenase (ldh) was 8747 units / l, and haptoglobin was less than 10 mg / dl . On the peripheral smear, schistocytes, helmet cells, and large platelets were noted . Ultrasound of the abdomen was unremarkable and hepatomegaly and atrophy of kidneys was not appreciated . The patient immediately underwent plasma exchange for a total duration of 5 days and simultaneously started on oral glucocorticoids (prednisone 1 mg / kg). Following treatment, platelet count recovered and remained above 150,000 after 2 days . The initiation of intermittent hemodialysis, creatinine and blood urea nitrogen improved significantly . Additionally, the ldh returned to baseline . Psychiatry also evaluated the patient and concluded that the suicidal incident was rooted to an adjustment disorder . A few weeks later, the patient was seen in the outpatient setting where he continued to report resolution of all symptoms and normal urination . Thrombotic thrombocytic purpura is a rare blood disorder characterized by clotting of small blood vessels and low blood platelet counts . A pentad often characterizes ttp that includes neurological deficits (often in the form of altered mental status), thrombocytopenia, fever, renal dysfunction, and evidence of microangiopathic hemolytic anemia . Not all features must be present for a diagnosis, but important primary criteria include thrombocytopenia and microangiopathy.3 the diagnosis of ttp is made after an exclusion of more prevalent multisystem diseases such as disseminated intravascular coagulation, sepsis, malignant hypertension, systemic lupus erythematosus, and hematologic malignancies.2 ttp is a rare disease with approximately 3.7 cases per million per year in the united states . In approximately 90% of patients, the cause of ttp is unknown.4,5 known conditions that are associated with ttp include a postpartum state, postinfection (escherichia coli 0157:h7, e. coli 0111, e. coli 0104:h4), pancreatitis, malignancy, post - surgical, hiv, post - pneumococcal infection, and drug - induced . For the association with drugs, quinine is the most commonly identified.6 other documented drugs include oral contraceptives, extended - release opioids, valacyclovir and chemotherapeutic agents such as mitomycin c, gemcitabine, cisplatin, oxaliplatin, pentostatin, bevacizumab, and sunitinib . Additionally, the chemotherapeutic agents are typically dose - dependent in their involvement with ttp, and therapies such plasma exchange have been found to be ineffective.4,6 other widely used medications such as the anti - platelet agents ticlopidine and clopidogrel have also been associated with development of ttp . It is not clear if there is a direct effect of the drug or its metabolites on the vasculature or if a secondary immune response is responsible.3 however, the underlying mechanism for how platelet consumption occurs and the thrombotic microangiopathy that ensues is well - understood . Ttp is associated with a deficiency of a protein known as adamts13 (a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13). The gene for the adamts13 protein is located on chromosome 9q34 . For the congenital form, this protein functions to cleave a large von willebrand factor (ul - vwf) multimer (derived from endothelial cells) into a smaller form.7 if the ul vwf - multimer cannot be cleaved, it remains in circulation and activated platelets adhere to it . Platelets are then consumed in the process and the microvascular is further injured from the high shear stress.7 the platelet - uf vwf complexes, if large enough, can also thrombose in the microvasculature of multiple organs . Clinically, the adamts13 protein level is a useful tool in reaching the diagnosis of ttp . It is important to recognize that levels of this protein can be variable and even normal in several of the drugs associated with ttp.8 in one study, the variability in ttp ranged from 1370%.9 therefore, a decrease in adamts13 activity and the presence of its antibody is more complementary to the diagnosis and overall clinical picture . A decrease in activity can be associated with ttp if less than 510% (detection range 0.5100%). The overall consensus is that the presence of an adamts13 deficiency is a critical underlying risk factor, but requires a secondary trigger to develop ttp.4 in clinical practice, if a diagnosis is even suspected, it is imperative to consider initiating plasma exchange (plasmapheresis with infusions of fresh frozen plasma) immediately.3 plasma exchange therapy for acquired ttp is effective because it removes adamts13 autoantibodies and ul - vwf multimers, as well as prevents further supply of adamts13.7 disease activity can then be tracked by monitoring ldh and platelets . Notably, prior to the availability of plasma exchange, patient survival was about 10% . With the advent of plasma exchange technology, the overall response rate is now estimated at 8090%.3,4 the therapy options include plasma exchange, and recent studies have also shown a benefit in using rituximab as a first line therapy of acute acquired ttp.7,10 the rituximab was used in conjunction with plasma exchange and corticosteroids and administered at 375 mg / m intravenously within the first three days of admission and diagnosis with shown clinical benefit.10 the chemical compound 2-(4-isobutylphenyl) propionic acid, also known as ibuprofen, is a nonsteroidal anti - inflammatory agent used in the management of pain, inflammation, fever, etc . In the the us alone about 30 billion doses are utilized each year.11 the drug inhibits both cyclooxygenase 1 and 2 that prevents the production of thromboxanes and prostaglandins upon inhibition induce an anti - inflammatory, analgesic, and antipyretic effect . It is well characterized that the inhibition of the cox-1 pathway is responsible for most of the unwanted side effects.12 these commonly include nausea, dyspepsia, gastrointestinal bleeding, transaminitis, and, less likely, jaundice, fatal fulminant hepatitis, liver necrosis, and renal papillary necrosis and occasionally hepatic failure . Ibuprofen is plasma protein bound with a large volume of distribution (0.110.19 l / kg). A single 400-mg dose in adults produces a peak plasma level at 12 hours after ingestion and is eliminated via the kidneys within the next 8 h and then is practically undetectable by 12 h. its maximum recommended daily dose is 12003200 mg.1214 anemia is the only known hematological effect.15 more serious effects associated with long - term use and can manifest as esophageal ulceration and chronic renal failure and can worsen heart failure.16 nsaid overdose is generally a benign process . In 2010, the annual report of the american association of poison control centers national poison data system (npds): 28th annual report reported 65,699 cases of overdose with 9,169 of those characterized as intentional . Forty - seven cases were classified as a serious toxicity that manifested as status epilepticus, respiratory failure, ventricular arrhythmias, or cardiac arrest . No deaths due to ibuprofen were reported in 2010 . The high index of safety centers around ibuprofen s inability to accumulate in the body regardless of subsequent doses.17 there have been no recent cases attributing the development of ttp to patients exposed to nsaids . One case report from 1974 described a 55-year - old italian female who developed ttp after ingesting 900 mg of ibuprofen . No other alternative etiologies could be accounted for and the patient eventually expired as no plasma exchange therapy was available at that time.2 however, there have been some reports of nsaids causing hemolytic uremic syndrome (hus), a disease process very similar to ttp.18 paradoxically, antiplatelet therapy was at one point considered as potential treatment for ttp . The theory stipulated that by preventing platelet aggregation with antiplatelet agents, further endothelial injury and thrombosis could be prevented . Data from this study demonstrated that at least in certain cases, antiplatelet drugs probably play only a limited role in the treatment of patients with ttp.19 in another trial, prostacyclin (pgi2) infusion, again with the same principle, failed to reverse platelet aggregation caused by ttp.20 our patient presented with clinical symptoms that included altered mental status, acute renal dysfunction, thrombocytopenia, and evidence of red blood cell fragmentation . All symptoms evolved a few hours after ingesting a large amount of ibuprofen . In our assessment, other possible disease entities were considered but excluded such as hemolytic uremic syndrome (no history of diarrhea and negative e. coli 0157h7 study), autoimmune hemolytic anemia (coombs test was negative), disseminated intravascular coagulation (coagulation factors were normal), hiv, and absence of other known drugs . Adamts13 activity was less than 10% and solidified our assessment that ttp was the clear diagnosis . Ibuprofen, its metabolites or even components of the drug formula may have caused this condition . Nonetheless, because of the widespread use of nsaids and the high mortality associated with undiagnosed ttp, we present this case - report to shed light into a possible, unknown association between ibuprofen and ttp.
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Mscs can be extracted from adult fat and bone marrow, as well as peripheral blood,8 9 and can be induced to differentiate into various mesenchymal tissues including bone, cartilage, and muscle.10 11 in vitro induction can be accomplished by growth factor supplementation and creating culturing conditions that are favorable for the preferred differentiation . Specifically, adipogenic differentiation from mscs has been successfully accomplished in dulbecco's modified eagle medium supplemented with isobutylmethylxanthine, indomethacin, and either dexamethasone and insulin or with hydrocortisone.11 12 successful induction is then verified by the identification of lipid vacuoles within the cell and various other adipose cell markers . In comparison, in vitro chondrogenic differentiation has been induced by centrifuging the mscs into micromass pellets and culturing in a medium containing dexamethasone and transforming growth factor -3.13 chrondrocytes are then detected by the presence of secreted extracellular matrix components such as type ii collagen and aggrecan, among others.14 osteogenic differentiation may be induced in a culture of dexamethasone, ascorbic acid phosphate, -glycerol phosphate;11 12 14 15 successful differentiation is identified by the expression of alkaline phosphate and using cell - specific antibodies . Successful models used to expand the msc population and induce osteogenic differentiation have also included substances such as transforming growth factor- and fibroblast growth factor-2.16 17 bone morphogenetic protein has also been shown to successfully expand the osteogenic cell population;7 18 however, limitations exist regarding cost and safety . The authors refer the interested reader to some of the early studies inducing msc differentiation for additional explanations as to the nature and function of the specific growth factor supplements.11 12 13 14 19 20 21 in vivo induction occurs through site - specific differentiation and is often implanted within scaffolds.7 10 in addition to requiring sufficient numbers of mscs implanted for bone regeneration,22 an osteoconductive matrix is also necessary for osseous growth.18 the scaffold provides a structural support for cell cell interactions, extracellular matrix formation, and new tissue formation . The osteoconductive scaffold occupies the site of the fusion, but also provides an environment conducive for the osteoinductive factors and tissue growth . Various biomaterials have been investigated as scaffolds including hydroxyapatite, tricalcium phosphate, calcium sulfate, metals, and biodegradable polymers.23 serving as the initial structure for tissue growth and blood vessel formation, the biodegradable types of scaffolds eventually resorb as new bone is formed.24 isolating human mscs has traditionally been accomplished by using their selective adherence to plastic surfaces, which the hematopoietic cells less commonly do.25 this method, however, typically leads to heterogeneous cell isolation, in which only ~30% of the cells are multipotent mscs.26 although the mscs do not express the cell surface markers normally found on hematopoietic cells, such as cd34 and cd45, there are cell markers that are unique to the mscs.27 stro-1 was one of the earlier monoclonal antibodies developed to isolate mscs,28 and this was followed by additional cell - surface markers such as cd146, cd200, and cd271.29 30 31 use of these markers alone may not be sufficient to isolate pure samples of mscs . Gronthos and colleagues28 described a method whereby they used stro-1 monoclonal antibodies and then isolated the cells using immunomagnetic cell sorting (macs). This resulted in a milieu of cells with varying intensities of stro-1 fluorescence intensity, which was then further purified for the high - intensity fluorescing cells using fluorescence - activated cell sorting (facs). Further purification was accomplished using dual - color facs to isolate those cells with the surface markers vam-1, which are also expressed on mscs.32 another recent study utilized macs to separate cells that were cd45 (as this marker is not expressed on mscs) and then subsequently used facs to isolate cells that were cd146.30 the most effect cell identification technique seems to be a combination of facs and macs, though the cell surface markers of choice vary between laboratories . In an effort to identify the most efficient marker of mscs, delorme and colleagues31 used microarrays and flow cytometry to culture a pure sample of mscs that expressed 113 transcripts and 17 proteins not found on other hematopoietic cells . They found that cd146 and cd200 were among the most efficient markers to purify mscs . Following isolation, mscs can be cultured in either fetal calf serum or human serum, which show no difference in their effects on the cells to proliferate and differentiate.33 for bone formation, mscs are then directed to differentiate into osteoblast lineage cells via the aforementioned factors (e.g., transforming growth factor-) or via selective genetic expression (e.g., osx, zip1).34 35 allogeneic transplantation of mscs can be done in the site of fusion, due to their hypoimmunogenic and even immunosuppressive nature . Flow cytometry experiments have shown that mscs express intermediate levels of hla class i and little to no hla class ii or costimulatory molecules (e.g., cd40).11 36 neither undifferentiated nor differentiated major histocompatibility complexes elicit lymphocyte proliferation when transplanted,36 37 and in fact, they tend to alter the cytokine profile to an anti - inflammatory state by decreasing tumor necrosis factor- and interferon - gamma and increasing interleukin-10, interleukin-4, and regulatory t cells.38 mscs have even been used for their immunosuppressive actions in treating acute graft - versus - host disease.39 for these reasons, host - versus - graft disease, or graft rejection, does not appear to be a problem with msc use in spine fusion . The use of msc therapy in bone regeneration has been and is currently investigated both in animal models and in the clinical setting . This is being done through both local implantation of the stem cells and via gene therapy, as well as through autologous transfer of engineered extracted mscs . Following the original discoveries of mscs by friedenstein et al40 41 and then of owen in 1988 that mscs could differentiate into bone,42 43 several groups demonstrated successful autologous transfer of mscs in healing long bone lesions in various animal models.44 45 46 for example, arinzeh and colleagues used allogeneic mscs loaded in a ceramic hydroxyapatite - tricalcium phosphate scaffold to treat large femur defects in adult dogs . They showed that not only did a callus of lamellar bone fill the lesion within 8 weeks, with complete new bone form by 16 weeks, histologically, there was no immune response detected.46 in a rat posterior spinal fusion model, cui and colleagues showed that cloned osteoprogenitor cells implanted in the fusion bed led to successful spine fusion in all animals, compared with only 50% fusion success in animals that were implanted with mixed marrow stromal cells.47 similarly, muschler et al, in a canine model of dorsal spinal fusion, demonstrated the superiority of the marrow - derived osteoblastic progenitors in promoting spine fusion versus the growth factor and cellular milieu found in a bone marrow clot.48 the effectiveness of mscs in promoting spinal fusion has been shown in progressively larger animals, including rabbit,49 50 51 52 53 ovine,54 55 56 57 and primate models.58 59 specifically, wang and colleagues58 performed anterior lumbar interbody fusions in nine rhesus monkeys, with two fusion sites each, that either utilized autologous bone - marrow derived mscs on a calcium phosphate scaffold, icbg, or a control ceramic graft treatment . They found that the msc group had equivalent biomechanical strength as compared with the icbg group, and that they were both biomechanically and histologically superior to the control ceramic graft group . Orii et al59 performed posterolateral lumbar spine fusions in nine macaque monkeys, which received either marrow - derived mscs with a -tricalcium phosphate graft, autogenous bone, or a control tricalcium phosphate graft treatment . Using both x - ray and manual palpation to identify fusion status, they found that the group receiving the mscs had the highest fusion rate (83.3%) compared with the autogenous bone group (66.7%) and the control group (0% fused). The aforementioned studies used bone marrow - derived mscs; however, another approach with potentially fewer complications and less morbidity may be the use of adipose - derived mscs (ascs). Ascs were first identified in 2001 by zuk and colleagues60 61 who showed that ascs can differentiate into multiple cell types including osteogenic and chondrogenic cells, thereby providing an potential therapeutic avenue for bone regeneration . These cell types were subsequently shown to be capable of adhering to a bioengineered scaffold, as well as remaining viable, proliferating, and differentiating under various conditions.62 rodbell and jones standardized the first protocol for asc isolation.63 64 65 cowan et al66 and later levi et al67 used ascs to regenerate bone in large mouse calvarial defects . Lopez and colleagues68 performed dorsolateral spinal fusions in 56 fischer rats, with either no graft, only a scaffold, a scaffold with allogeneic ascs, or a scaffold with syngeneic ascs . Similar to the studies investigating marrow - derived mscs, they found that when ascs were used there were fewer inflammatory infiltrates compared with the control groups, as well as superior bone formation and fusion when using the ascs . Clinical trials are currently investigating ascs in treatments ranging from type i diabetes mellitus to liver disease69 70 and may soon be used for spine fusion treatments as well . Although there has been much success demonstrated in the animal models, there remain barriers prior to this therapy's translation into the clinical setting . This includes identification of the optimal number and concentration of mscs, as well as the ideal preparation and implantation techniques needed.71 minamide and colleagues50 compared the use of differing number of marrow - derived mscs in a rabbit posterolateral spine fusion model . The low dose contained one million cells per milliliter and resulted in the fusion of zero of seven spines . In contrast, the high dose was one hundred million cells per milliliter and led to the fusion of five of seven rabbit spines . Perhaps an even greater concentration would have led to 100% fusion . Wang et al58 compared bone marrow - derived mscs to autogenous icbgs in rhesus monkey spine fusion models and found that although the fusions were equivalent in stiffness, the autograft produced greater bone volume . This can be explained by the fact that only three million cells per milliliter were used for the msc - treated group, in contrast to the 100 million that was used successfully in the study by minamide et al . Finally, gan and colleagues,72 used bone - marrow - derived mscs for posterior spine fusion in 41 patients . Using a slightly different quantification method, they recovered and implanted 16.1 million nucleated bone marrow cells per milliliter, of which they measured an average of 213 mscs per milliliter (mscs were defined as colony - forming units expressing alkaline phosphatase). Despite the relatively small number of implanted cells, 95% of their patients achieved complete fusion by 34 months' follow - up . It is clear that we have yet to determine the optimal number of mscs needed for complete fusion prior to the translation of this technology to the clinical setting . Although some experimental data suggest that increased cell concentrations are required to repair bony defects,58 73 others have suggested that the cell concentration is not sufficient but rather the delivery type and biological environment of the graft will significantly affect success of the fusion.48 for example, the study by minamide et al50 used a three - dimensional culture, as compared with the more traditional two - dimensional monolayer culture, as they believed that it enhanced proliferation and differentiation . However, there is literature on the osteogenic potential of mscs in various other skeletal defects as well as ongoing clinical investigations of mscs in spine surgery with various commercial products such as neofuse (mesoblast ltd ., melbourne, austrialia) and osteocel (nuvasive, san diego, ca the earliest clinical studies involving mscs involved small case studies of autologous mscs used for bone regeneration . Quarto et al74 showed successful and abundant callus formation in three patients with tibial, ulnar, or humeral fractures using autologous mscs; lendeckel and colleagues75 reported a case study where autologous ascs were successfully used to treat a large calvarial injury . Subsequent trials, including some larger ones, involved autologous bone marrow successfully used to promote bone fusion in tibial nonunions,76 77 autologous mscs for femoral head osteonecrosis,78 79 80 81 and allogeneic mscs to treat osteogenesis imperfect,82 all of which showed the clinical feasibility of therapeutic application of mscs to promote bone growth . The 2011 study by goldschlager and colleagues,55 which demonstrated superior bone formation in ewes with the use of allogeneic ovine mscs (mesoblast limited, melbourne, australia) compared with autograft or stand - alone scaffold, was the precursor study to the ongoing clinical trials (nct01290367, nct00996073, nct01097486, nct00810212, nct00549913, nct01106417) using this msc allograft product in both cervical and lumbar spine fusions . Results on safety and preliminary efficacy in patients will likely be revealed in the coming years . Much of the preliminary research done in animal models has shown potential for the use of mscs, both bone marrow and adipose derived, for bone regeneration . Although there have been limited systematic clinical trials, small case studies have shown that msc use in humans may have successful bone growth and long - term durability.74 some current limitations include decreased bone growth compared with autograft,58 weaker mechanical stability of the implanted graft and poor resorption of the bioceramic constructs,74 and ambiguity surrounding the optimal cell concentration and delivery method . More studies examining optimal msc concentrations are needed in larger animals, which are more comparable to humans, considering the fact that there is decreased potential for bone growth as compared with smaller animals (e.g., rabbits, rats, etc. ).58 83 84 this also demonstrates the need for methods to maximize the number of mscs collected, as well as techniques that can be feasible in the operating room setting.85 other options can include obtaining somatic cells and converting them into pluripotent cells,86 using minimally invasive approaches to collect and culture bone marrow- or adipose - derived mscs prior to surgery, and potentially even using recombinant forms of mscs . The present hurdles to clinical use include optimization of osteoinductive and osteoconductive properties of mscs in bone grafts . Vascularization of the implant and integration of the vasculature with the host will prove to be important; additionally the long - term mechanical strength and durability, particularly at the load - bearing sites such as the lower lumbar spine regions will need to be comparable to native bone.87 allogeneic msc therapy for spine fusion and other skeletal treatments is still in its infancy . There has been a surge of interest in the various msc formulations commercially available for clinical use in spinal surgery . This enthusiasm, and clinical use, must be tempered with the understanding that there are no clinical data that had defined the efficacy or safety profiles in spine surgery patients . Therefore, it is imperative that the spine surgery community carefully evaluate the use of msc in spine fusion through well - designed and executed studies . Although more than a decade of preclinical animal research that has shown promising results, the safety and efficacy of these products in randomized controlled trials must be ascertained . With the rapidly growing number of spine fusion surgeries performed annually, further study into fusion - enhancing compounds becomes increasingly necessary.
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The influenza viruses include three genera, a, b, and c within the family orthomyxoviridae . Avian influenza viruses (aivs), all of which are contained in the genus influenza virus a, are an economically important cause of disease in fowl and occasionally affect humans, pigs, and horses [1, 2]. Each genus of the virus is further subdivided into serotypes based on the surface proteins, consisting of 16 different hemagglutinin (h) and 9 neuraminidase (n) subtypes . While only a limited number of h and n subtypes are circulating in humans and other mammalian species, all the h and n subtypes are found in avian species [3, 4]. The genome of influenza a viruses consists of 8 unique segments of single - stranded negative sense rna . The viral rna segments encode 10 recognized gene products, pb1, pb2, and pa polymerases, h, np, n, m1, m2, ns1, and ns2 proteins . The h6 subtype is one of the most commonly recognized subtypes in domestic ducks in southern china [6, 7] and in migratory birds in north america and in europe [810]. H6 viruses have caused several outbreaks in commercial poultry worldwide that resulted in decreased egg production and increased mortality [1113]. During the hong kong h5n1 incident in 1997, an h6n1 avian influenza virus, teal / hong kong / w312/97 (w312), was isolated from birds in a live poultry market . Genetic characterization of this virus revealed that except for the h gene, the remaining 7-gene segments were closely related to those of highly pathogenic avian influenza h5n1 viruses (hpaivs) found in both poultry and humans . The present study reports on the outbreak of avian influenza (ai) caused by an h6n1 subtype of aiv isolated during january 2009 in kibbutz gvulot, in the southern part of israel . The present isolate, a / turkey / israel/09 (h6n1), is the third h6 aiv obtained in israel since the year 2000 . Therefore, circulation of these viruses was concurrent with that of the highly prevalent h9n2 aiv in israel [15, 16]. While the former h6 aiv isolates, a / duck / israel/289/01 (h6n2) and a / mallard / israel/320/01 (h6n2), were isolated from water birds, the present isolate was obtained from domestic turkey poults . 2) located in one farm, kibbutz gvulot, exhibited an increased daily mortality of approximately 1% over a two - day period . The clinical signs were swollen infraorbital sinuses, nasal discharge, respiratory rales, rattles, and lethargy . Birds in the affected poultry house had developed aiv antibodies, as detected by elisa and aiv h6 haemagglutination inhibition (hi) tests . The elisa test was performed with the flockchek avian influenza antibody test kit (idexx, usa) according to the manufacturer's instructions, and the hi test employed a panel of reference antisera to h5, h6, h7, and h9 . Blood samples were tested for antibodies by the hi test using 4 (ha) units of the h6 subtype antigen . Turkey poults from the six houses were distributed to 4 secondary farms, mishmeret, ramon, ein zurim, and evron . Tracheal and cloacal swabs of clinically - affected turkeys were assayed for the presence of aiv by pcr and virus isolation in 11-day - old embryonated eggs . The presence of aiv was initially detected in the allantoic fluids (af) of dead eggs after 2 - 3 days of incubation, by the hemagglutination (ha) assay . Testing of ha - positive af was performed by reverse transcriptase polymerase chain reaction (rt - pcr). In parallel, rt - pcr was performed on rna extracted directly from tracheal and cloacal swabs . Viral rna was extracted directly from the tracheal and cloacal swabs and from af using the qiaamp viral rna mini kit (qiagen, valencia, ca) according to the manufacturer's instructions . Firstly, aiv was detected using a primer pair specific for the m gene of influenza a virus, mf (5-ttctaaccgaggtyraaacgt-3) and mr (5-ctgggcacggtgagcgt-3) to amplify a 200 bp product (panshin a., kimron veterinary institute, bet dagan, israel, unpublished). Rt - pcr was performed using a one step rt - pcr kit, (qiagen ltd . ). A 25 l mixture containing 5 l of buffer, 1 l of dntp mix, 1 l of each primer, 1 l of one step enzyme mix, 13 l nuclease - free water, and 3 l rna template was used in the assay as follows: 30 min at 50c, 10 min at 95c, 30 cycles at 94c for 30 sec, then 30 sec at 54c, 30 sec at 72c, 5 min at 72c, and 15 min at 4c using the dna engine thermal cycler (bio rad, waltham, ma, usa). Secondly, molecular subtyping was performed with a set of specific primers for genes of h subtypes h1h4, h6, h9, and h10 and h5, h7, and n subtypes n1 n2, n3, n7, n9 (panshin a., kimron veterinary institute, bet dagan, israel, unpublished). The primers used to amplify a 623 bp segment of the n2 gene were n2f (5-ytgyacagtrgtaatgacbgatggr-3) and n2r (5-cratrctryttgadgtccaccabac-3); the primers to used to to amplify a 470 bp product of the n3 gene were n3f (5-aayagrccwtggrtgagrat-3) and n3r (5-cccgatccaggttcattgt-3). The primers used to amplify a 509 bp segment of the n7 gene were n7f (5-ttggrtggtcragyacaagctgcc-3) and n7r (5-accaggrctyccagtdatwggattg-3), the primers used to amplify a 520 bp segment of n9 gene were n9f (5-caaccaatgcaagccaaacaata-3) and n9r (5-tttcgggcccatgtgttgatttc-3). The amplification conditions for h and n genes were similar to those of the m gene . The pcr products were purified with the megaquick - spin pcr & agarose gel extraction system (intron biotechnology, inc, gyeonggi - do, korea) according to the manufacturer's instructions . Sequencing reactions were performed by the weizmann institute of science, rehovot, israel, using a 3700 dna analyzer (perkin - elmer, applied biosystems, foster city, ca ., u.s.a .) And analysed by capillary electrophoresis . The bioedit package, version 7.3, and dnastar system software were used for sequence analysis and alignment . Nucleotide sequences of all gene segments and available sequence data from genbank were used to generate phylogenetic trees . The intravenous pathogenicity index (ivpi) of aiv isolates was determined as described in the oie manual of standards for diagnostic tests and vaccines . The test employs a standard volume of virus which is inoculated intravenously into spf chickens . The ivpi test yields an index of virulence valued from 0 to 3, which is calculated according to illness severity and the viability period following inoculation . The mishmeret farm received poults from the original poultry houses nos . 1 and 4 and developed a low level of hi antibodies (geometric mean titer (gmt) = 1.8); the evron farm received poults from the original poultry houses nos . 5, and 6 and did not develop hi antibodies (gmt = 0); the ramon farm received poults from the original poultry houses nos . 1, 4, 5 and 6, and did not develop hi antibodies (gmt = 0); the ein zurim farm received poults from the original poultry house no . 2, that was infected with the a / turkey / israel/09 (h6n1) isolate, and from house no ., birds on the ein zurim farm developed hi antibodies to h6 and showed clinical signs (gmt = 6.3). To identify the aiv h and n gene, specificity amplifications and hi assays the virus was isolated and characterized by classical methods (oie) and genome was detected by pcr . A / turkey / israel/09 (h6n1) was characterized as low pathogenic avian influenza virus (lpai) with an ivpi of 0.00 . Infection in the chickens used in the ivpi assay was confirmed by the hi test . The amino acid sequence of the h gene cleavage site was examined to support the virulence determination of the a / turkey / israel/09/(h6n1) isolate . The amino acid sequence at the cleavage site was pqietr*glf, indicating that the isolate was of low pathogenicity, since it did not contain multiple basic amino acids, characteristic of hpaivs . Most lpaivs have a single arginine at the cleavage site, whereas hpaivs usually exhibit a multibasic amino acid motif (r and k) flanking the cleavage site [22, 23]. The genome of influenza a viruses consists of eight unique segments of single - stranded rna, which are of negative polarity (i.e., complementary to the mrna sense). The 8 gene segments of the a / turkey / israel/09 (h6n1) isolate were sequenced . The nucleotide sequences obtained in the present study were compared to those of aivs from the genebank (figures 18). Figure 1 shows the phylogenetic tree of the complete h gene, subtype h6, of representative recent aiv h6 isolates reported worldwide, including the previous two israeli h6 isolates, which were not reported to the genebank . It seems that the h gene of the present isolate, a / turkey / israel/09 (h6n1), differed from the h genes of the two previous israeli isolates, as they belonged to different clades, although the differences in the nucleotide content were about 3.7 - 3.8% . It is notable that the h6 aiv, that shaped the clade to which the present isolate belonged, originated from waterbirds . Figure 2 shows the phylogenetic tree of the n gene of n1 subtypes, based on the analysis of nucleotides 204 to 1149 . The analysis includes representative aiv n1 genes compared on the respective gene fragment . While the compared sequences differed up to 31%, the present isolate, a / turkey / israel/09 (h6n1), was closest to the n1 from a / mute swan / aktau/06 (h5n1) and a / swan / mangystau/06 (h5n1). It is notable to mention that the hong kong avian a / teal / hong kong / w312/97 (h6n1) and the human influenza virus a / hong kong/156/97 (h5n1) showed a very high nucleotide homology in the 6 aiv internal genes, and especially in the n1 gene sequence [14, 24]. That similarity might indicate a common precursor and that the present virus could become a potential source of novel pathogenic aiv strains . Figures 3, 4, 5, 6, 7, and 8 show the phylogenetic comparison of the a / turkey / israel/09 (h6n1) isolate complete internal genes, ns, m, np, pa, pb1 and pb2, respectively, to genes of representative aivs . The main feature revealed from these phylogenetic trees was the close similarity between the former two h6n2 aiv isolates, a / duck / israel/289/01 and a / mallard / israel/320/01, as compared to the phylogenetic divergence of the present h6n1 isolate, a / turkey / israel/09 . The phylogenetic distances between the 2009 h6n1 isolate and the two previous h6n2 isolates were calculated . While the two previously described h6 isolates, a / duck / israel/289/01 (h6n2) and a / mallard / israel/320/01 (h6n2), were identical in their ns, m, np, pa, pb1, and pb2 genes, the a / turkey / israel/09 (h6n1) isolate differed from the former two isolates by 3%, 2.3%, 6%, 2.5%, 3.6%, and 5%, respectively . In conclusion, the present paper describes a third h6 aiv isolate (h6n1) in israel, which unlike the two previous h6n2 isolates that were obtained from ducks and mallard, was now detected in turkeys . This h6n1 lpaiv was shown to possess a low - pathogenic amino acid sequence at the cleavage site and had a low ivpi . A / turkey / israel/09 (h6n1) was not highly transmissible among commercial flocks . That phenomenon was supported by two observations; (a) the absence of clinically - affected cases in adjacent poultry houses and (b) the detection of only three cases of aiv h6 in israel.
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The squat is one of the most frequently used exercises in the field of strength and conditioning . The squat is an exercise that increases hip and knee extensor muscle strength which then indirectly improves the quality of life in athletic and nonathletic populations . Muscle forces also vary depending on joint positions (moment arm, length - tension relationship), whether the muscle acts as a prime mover or stabilizer, and whether the task is dynamic or static . Though evidence suggests that architecture, position, and function drive muscle performance during the squat, little is known about the neuromuscular changes that occur from a muscle activation standpoint . Elucidating how muscle activation patterns change in the monoarticular and biarticular knee and hip extensors during squatting at different knee angles would thus enhance our understanding of how one could capitalize on maximizing muscle activation and the best position to specific evaluations and semg normalization . This would be a first step to then apply the knowledge during exercise prescription that includes the squat . Considering that the squat exercise is a multijoint task, a large number of muscle groups can be activated simultaneously in a more complex way . Several studies have shown that manipulating features of the squat exercise resulted in altered muscle activity . These manipulations include changes in foot position [2, 3], barbell position, stability of the surface on which the exercise is performed [59], different levels of intensity of load, range of motion [1012], and different equipment . As a multijoint exercise, the knee extensors (e.g., rectus femoris, rf; vastus lateralis, vl; and vastus medialis, vm) and hip extensors (e.g., gluteus maximus, gm; biceps femoris, bf; and semitendinosus, st) are considered to be the prime movers during the squat exercise, with other muscles acting in a secondary capacity [1, 11, 14]. Measured the relative contributions of gm, bf, vm, and vl muscles of ten experienced lifters while performing dynamics squats at 3 depths (partial squat (the angle between the femur and the tibia was ~2.36 rad at the knee joint), parallel squat (the angle between the femur and the tibia was ~1.57 rad at the knee joint), and full - depth squat (the angle between the femur and the tibia was ~0.79 rad at the knee joint)), using 100125% of body weight as resistance . The results suggested that the gm was most active, rather than the bf, the vm, or the vl, during a concentric contraction as squat depth increases . On the contrary, robertson et al . Robertson et al . Also showed that the biarticular muscles functioned mainly as stabilizers of the ankle, knee, and hip joints by working eccentrically to control descent or transferring energy among the segments during ascent . Whether monoarticular and biarticular hip and knee extensors have different muscle activation during an isometric squat and whether activation changes during different knee angles are unclear . Consequently, the rationale of the present study was to evaluate, indirectly, the muscle activation in different mechanical positions related to differences in the joint - angle - torque diagram and the sticking region effect in all three joint angles (20, 90, and 140). Finally, differences in muscle activity during dynamic and isometric squat exercise have received less attention in the physical education and rehabilitation area . Others have shown the isometric squat (90 and 120 of knee join position) as a reliable test to provide an indicator of changes in dynamic strength (1-repetition maximum barbell back squat, 1rm) and power performance [15, 16]; however, whether muscle activity changes as an isometric squat is manipulated is unknown . Although motor units are recruited differently during dynamic movements, they generate the same relative force / torque during a static contraction . Despite inherent neural and mechanical differences between isometric and dynamic contractions, the isometric squat exercise performed in different knee joint angles may be used to understand changes in muscle activation patterns without confounding any other external effects such as the stretch - shortening cycle from dynamic movements . Therefore, the purpose of this study was to evaluate the maximal isometric muscle activation of the lower limbs during three different knee joint - angle positions in the back squat exercise . We collected the peak amplitude of the root mean square (rms) from vl semg data during a pilot study to drive this power analysis . Based on a statistical power analysis derived from these data (rms vl emg), it was determined that twelve subjects would be necessary to achieve an alpha level of 0.05, effect size of 1.41, and a power (1) of 0.80 . Therefore, we recruited fifteen young, healthy, resistance - trained men (age: 30 7 years, height: 174 6 cm, and total body mass: 76 9 kg, with 5 1 years of experience on the back squat exercise) to participate in this study . Subjects had no previous lower back injury, no surgery on the lower extremities, and no history of injury with residual symptoms (pain, giving - away sensations) in the lower limbs within the last year . This study was approved by the university research ethics committee and all subjects read and signed an informed consent document . Prior to data collection, subjects were asked to identify their preferred leg for kicking a ball, which was then considered their dominant leg . Volunteers attended one session in the laboratory, and they reported to have refrained from performing any lower body exercise other than activities of daily living for at least 48 hours prior to testing . Subjects performed a 5-minute cycle warm - up and a familiarization session with all isometric conditions . The familiarization session was performed in all joint angles used during the experimental procedure (20, 90, and 140) for 1 set of 3 seconds each . After the warm - up and familiarization, all subjects performed three trials of 10-second maximal isometric contractions against a locked smith machine under three different knee joint - angle positions in a randomized, counterbalanced order: back squat at 20 degrees (20); back squat at 90 degrees (90); and back squat at 140 degrees (140). The knee joint - angle positions were evaluated by a goniometer (plastic 12 goniometer 360 degree isom), and, for all angles, full knee extension was considered the zero position . The subjects' feet were always positioned at hip width and vertically aligned with the barbell position . The barbell was positioned on the shoulders (high - bar position) for all subjects and experimental conditions . A rest period of 15 minutes was provided between conditions with 3 minutes afforded between sets . All measures were performed at the same hour of the day, between 9 and 12 am, and by the same researcher . The subjects' hair was shaved at the site of electrode placement and the skin was cleaned with alcohol before the semg electrode was affixed . Bipolar active disposable dual ag / agcl snap electrodes were used which were 1 cm in diameter for each circular conductive area with 2 cm center - to - center spacing . Electrodes were placed on the dominant limb along the axes of the muscle fibers, according to the seniam / iseki protocol: gluteus maximus (gm) at 50% of the distance between the sacral vertebrae and the greater trochanter; vastus lateralis (vl) at 2/3 of the distance between the anterior spine iliac and the superior aspect of the lateral side of the patella; rectus femoris (rf) at 50% on the line from the anterior spine iliac to the superior part of patella; vastus medialis (vm) at 80% on the line between the anterior spine iliac superior and the joint space in front of the anterior border of the medial ligament; biceps femoris (bf) at 50% on the line between the ischial tuberosity and the lateral epicondyle of the tibia; and semitendinosus (st) at 50% on the line between the ischial tuberosity and the medial epicondyle of the tibia . The semg signals were recorded by an electromyographic acquisition system (emg832c, emg system do brasil, so jos dos campos, brazil) with a sampling rate of 2000 hz using a commercially designed software program (emg system do brasil, so jos dos campos, brazil). Emg activity was amplified (bipolar differential amplifier, input impedance = 2 m, common mode rejection ratio> 100 db min (60 hz), gain 20, noise> 5 v) and converted from an analog to digital signal (12 bits). Emg signals collected during all conditions were normalized to a maximum voluntary isometric contraction (mvic) against fixed strap resistance . Then, two trials of five - second mvics were performed for each muscle with one - minute rest between actions for the dominant leg . The first mvic was performed to familiarize the participant with the procedure . For gm mvic, subjects were in the prone position with their knee flexed at 90 and resistance placed on the distal region of the thigh with the pelvis stabilized . For vl, vm, and rf mvics, subjects were seated with their knee flexed at 90 and resistance placed on the distal tibia . For bf and st mvics, subjects were seated with their knee flexed at 90 and resistance placed on the distal tibia . The digitized semg data were band - pass filtered at 20400 hz using a fourth - order butterworth filter with zero lag . For muscle activation time domain analysis, rms (150 ms moving window) isometric back squat data was then normalized to the rms peak of the two peak mvics, the first second was removed from rms normalized, and the following 3 seconds of each trial were integrated (iemg). The normality and homogeneity of variances within the data were confirmed with the shapiro - wilk and levene's tests, respectively . To test differences for each muscle activity (iemg), cohen's formula for effect size (d) was calculated, and the results were based on the following criteria: <0.35 trivial effect; 0.350.80 small effect; 0.801.50 moderate effect; and> 1.5 large effect, for recreationally trained subjects . Interrater reliability was assessed for the researcher who positioned and evaluated iemg tracings for all muscles and conditions . Reliability was operationalized using the following criteria: <0.4 poor; 0.4<0.75 satisfactory; 0.75 excellent . There was a significant main effect of vl (p <0.001), vm (p = 0.030), rf (p = 0.018), and gm (p <0.001) for muscle activity during three different knee joint - angle positions (20, 90, and 140) in the isometric back squat . The vl activity was significantly less in 140 than 20 (p = 0.027,% = 24.4) and 90 (p <0.001,% = 37.5). The vm activity was significantly less in 140 than 90 (p = 0.036,% = 30). The rf activity was significantly less in 20 than 90 (p = 0.015,% = 36). The gm activity was significantly less in 140 than 90 (p <0.001,% = 80.4) and 20 (p <0.001,% = 80) (table 1 and figure 1). The purpose of this study was to evaluate the maximal isometric muscle activation of the lower limbs during three different knee joint - angle positions in the back squat exercise . The architecture, position, and function influence muscle forces during the squat; however, little is known about the neuromuscular changes that occur from a muscle activation standpoint . The primary finding of this investigation was that, during isometric squatting, a position of 90 of knee joint angle demonstrated the overall highest muscle activation of the quadriceps and gluteus maximus, whereas the 140 knee joint - angle position presented the lowest muscle activation values for almost all muscles that act as prime movers . Interestingly, the activation of the hamstring did not differ among knee joint - angle positions and the three quadriceps muscles responded differently as the knee went from a relatively extended position to a more flexed position . Given the close chain nature of the squat, as the knee joint changes position, the hip joint angles also change positions . Consequently, the squat exercise simultaneously utilizes several muscles with different morphologies (monoarticular and biarticular) in a manner that produces muscle coordination . A multijoint task to strengthen the knee and hip extensors is more complex for the neuromuscular system as two joints work in concert to achieve the task . Also, since some muscles cross more than one joint, the complexity increases compared to open chain terminal knee extension or isolated hip extension exercise . During the squat exercise, biarticular muscles such as rf, bf, and st have intermediate activation when the muscles have agonistic action at one joint and antagonistic action at the other joint; this is in contrast with the high activation seen when a biarticular muscle works as an agonist for both joints simultaneously . Lombard suggested that biarticular muscles of the lower extremity act in a paradoxical fashion when the movement is constrained or controlled (named lombard's paradox); it is observed when rf, bf, and st contract concurrently when rising from a chair . The extension seen from both the hip and the knee is the result of the differential moment arms of the two muscles at each joint . The present results showed low muscle activation for bf and st in all knee positions, probably because these muscles act more like a joint stabilizer at the knee and a prime mover at the hip . Both bf and st have the longer moment arm at the hip thereby creating a hip extensor moment . Thus, the bf and st muscles allow for the extension of both the knee and the hip . Since the rf has a greater moment arm across the knee, due to the patella, it creates an extensor moment at the knee joint . Considering the present results, the rf showed higher muscle activation at 90 when compared to 20; however, it was similar to 140 of knee angle . This may represent a higher effect on muscle activation during the initial phase of the squat movement (between 20 and 90) than after 90 since the muscle activation did not change . On the other hand, monoarticular muscles act on one specific joint . During the squat exercise, several monoarticular muscles contribute to movement including the soleus, vasti (lateralis, medialis, and intermedius), and gm . The present results showed that muscle activation for all monoarticular muscles (e.g., vm, vl, and gm) did not differ between 20 and 90. additionally, the highest muscle activation was observed at 90 when compared to 20 and 140; on the other hand, 140 presented the lowest activation for vl and gm muscles . Interestingly, the vm behaved differently from the other monoarticular muscles, even the vl, as the muscle activation of the vm did not differ between the 20- and 140-degree knee joint angles . Usually, when monoarticular muscles perform as agonists, the activation increases as the joint moment increases . Additionally, monoarticular muscles are affected by the sticking region which is considered a poor mechanical force position in which the mechanical advantage of the muscles involved is such that their capacity to exert force is reduced and where the lifter experiences difficulty in exerting force against the external load [2630]. Displayed that the higher muscle activation during the squat exercise occurs at 90 of knee joint - angle position, which is considered the sticking region . The present results support this finding for all monoarticular muscles analyzed (vl, vm, and gm). Our findings support this theory as all monoarticular muscles presented lower values of activation at 140 of knee joint - angle position when compared to 90. in this specific position (at 140), it is feasible to speculate that changes in muscle length modify muscle contractile abilities and, in turn, modify semg - force and semg - moment relationships [18, 24]. Alternatively, afferent signals from muscles could decrease motoneuronal firing frequency (i.e., golgi tendon reflex) during isometric contractions when the muscle fibers are in an elongated position . Others have also investigated muscle activation during the squat by comparing different knee joint angles, yet previous studies compared knee positions during a dynamic squat, not an isometric squat . Measured the relative contributions of gm, bf, vm, and vl muscles of ten experienced lifters while performing dynamics squats at 3 depths (partial squat (the angle between the femur and the tibia was ~2.36 rad at the knee joint), parallel squat (the angle between the femur and the tibia was ~1.57 rad at the knee joint), and full - depth squat (the angle between the femur and the tibia was ~0.79 rad at the knee joint)), using 100125% of body weight as resistance . . Found that, during the concentric phase of the dynamic squat, the gm activation was higher during full - depth (35.4%) squat compared to the partial (16.9%) and parallel (28.0%) squat exercise and that the bf, the vm, and the vl did not change . The results suggested that the gm, rather than the bf, the vm, or the vl, becomes more active in concentric contraction as squat depth increases . Others have also shown superior muscular hypertrophy when squatting throughout a full versus a partial range of motion [32, 33]. Our findings of less muscle activation at 140 do not support bloomquist and colleagues' findings . The greater cross - sectional area of the muscles found by bloomquist et al . May be more related to time under tension than the muscle activation . When our subjects performed an isometric squat in different positions, the gm activity was the highest in the 90-degree position, not the deeper knee flexion position . Perhaps the change in 100125% body weight load during the dynamic trials and our maximum isometric load in all three conditions influence the lack of agreement between the studies . Reported that the gm muscle activity level was reduced at maximum full (deep - knee) squat depth . Robertson et al . Also concluded that the biarticular muscles (bf, st, and rf) functioned mainly as stabilizers of the knee and hip joints during the eccentric and concentric phases of a dynamic squat . The authors presumed that the reduced gm activity level at maximum squat depth was because the gm was not needed to maintain stability or perhaps it permitted an extra degree of hip flexion that created a deeper countermovement immediately before the ascent phase . From an activation standpoint, our findings suggest a diminished benefit from squatting beyond 90. the reason for these seemingly contradictory findings among studies remains to be elucidated . A limitation of this study includes the use of healthy, well - trained men only, and, therefore, our findings are not generalizable to other conditions, populations, or women . We also have a small sample size; thus, this study may be underpowered to identify differences between knee joint positions . An isometric back squat at 90 generates the highest overall muscle activation, yet an isometric back squat at 140 generates the lowest overall muscle activation of the vl and gm only . Thus, we recommend performing an isometric squat at 90 to maximize neuromuscular recruitment of the knee and hip extensors.
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Since the beginning of the 1990s cases of illicit trafficking and other unauthorised acts involving nuclear material from various stages of the nuclear fuel cycle have taken place . Due to these incidents nuclear materials were started to be analysed in the context of criminal investigations, and a new branch of forensic science nuclear forensics was born . The iaea defines the nuclear forensics as the analysis of intercepted illicit nuclear or radioactive material and any associated material to provide evidence for nuclear attribution . The aim for nuclear forensic scientists is therefore to identify indicators which represent relationships between measurable parameters of the material and its production history . These nuclear forensic indicators, so - called signatures, can be various properties of the material in question, such as structure, morphology, major and minor elements, isotopes and impurities . Among these properties there are only few exclusive parameters, which would give straightforward information about an unknown sample without the need to use in interpretation comparison samples or data . For example, the enrichment and concentration of uranium (u) of an investigated sample could lead us easily to the intended use of the sample or to the stage of the nuclear fuel - cycle from which the sample originates . However, the majority of the nuclear signatures belong to the so - called non - exclusive, comparative parameters . This means that the analytical results have to be compared with those of measured for known samples or reference data in databases in order to draw conclusions about the possible origin of the sample . In order to support the investigations of unknown seized nuclear materials, besides the new method developments, the improvement of existing methodologies is important as well . Up to now the isotopic patterns of o, s, pb, sr, and u have been investigated and found to be valuable signatures [26]. Besides these signatures the nd / nd isotope ratio was investigated recently, which is commonly used in geology for chronometry and provenance measurements . It was found a promising candidate for a new nuclear forensic signature, since the ratio is indicative of the age and types of the minerals present [7, 8]. Although the nd / nd isotope ratio in most of the uranium ore concentrates (uoc) samples was possible to be measured with a sufficiently low uncertainty (~0.05% rsd) there were still a few samples which contained so small amounts of nd that the measurement was not possible with the standard method (i.e. Tru extraction chromatography) or it could be performed only with too large uncertainty . This work presents an improved procedure developed for trace - level analysis of nd / nd isotope ratio in uoc samples by inductively coupled plasma mass spectrometry (icp - ms). The aim of the study was to develop an effective pre - concentration method prior to the chromatographic separations, which enables the measurement of nd isotope ratio in uranium samples . Co - precipitation as the most effective pre - concentration method was selected to achieve the required limits of detection in the low pg g range . For high - purity uranium materials the removal of uranium is of necessity as relatively large amounts of samples (100500 mg of u) are required to yield measurable quantities of the analytes, i.e., lanthanides (ln). For such sample quantities the standard methods (e.g. Direct extraction chromatography separation) cannot be applied, since the high amount of u precludes their use . Applying co - precipitation for the preconcentration of traces of ln from larger amounts of environmental samples (e.g. Sea water [1012], geological samples [13, 14]) has been studied and it has been proved to be an effective method . Our procedure involves a co - precipitation of rare - earth elements as fe(oh)3 in the presence of fe(iii) carrier, followed by an extraction chromatographic group separation of ln and a sequential separation of nd, sm, and other heavy lanthanides . Though the primary purpose of the study is the separation of ln, the methodology can be extended for the pre - concentration of other important elements for nuclear forensics present at trace - levels, such as th, am or pu . Thorough cleaning of all labware is necessary before use for trace - level measurement of nd isotope ratio . This was performed with dilute ethanol, followed by dilute nitric acid, and finally with high purity water rinsing . For all the dilutions high - purity water was used (uhq system, usf elga, germany). Hydrochloric and nitric acids were of suprapur grade (merck, darmstadt, germany), although the nitric acid was further purified by sub - boiling distillation . Analytical grade fe(no3)3 salt was used as carrier for the co - precipitation (alfa aesar, karlsruhe, germany). Analytical grade sodium - hydroxide and ammonium - carbonate used for the precipitation were purchased from sigma aldrich (st louis, mo, usa). Ammonium carbonate was further purified prior the use by adding about 10 mg of fe and precipitating fe(oh)3 to remove the trace - level lanthanide impurities still present in the analytical grade ammonium carbonate solution . For the lanthanide group separation, the tru extraction chromatographic resin supplied by triskem (triskem international, bruz, france) was used . For the preparation of columns, 1.6 ml of the resin was placed in plastic bio - rad holders (diameter 8 mm) and plugged with porous teflon frit (reichelt chemietechnik heidelberg, germany) on the top of the resin to avoid mixing . For nd separation, the ln resin for the extraction chromatographic separation was purchased from triskem (triskem international, bruz, france). For the preparation of columns, 400 l of the resin was placed in plastic bio - rad holders and plugged with porous teflon frit . For the optimization of the chemical separation procedure and the measurements by icp - ms, lanthanide standard solution and monoelemental nd and sm standard solutions (alfa aesar, karlsruhe, germany) were prepared by the dilution from 1,000 to 100 g ml standard solutions, respectively . The u3o8 certified reference material, morille (cetama, france) was used for the validation of the co - precipitation method as it is certified for four lanthanides content (dy, gd, eu and sm). The mass spectrometric analysis of aliquots from the co - precipitation step was carried out using an element2 (thermo electron corp ., bremen, germany) double - focusing magnetic sector inductively coupled plasma mass spectrometer (icp - sfms). Measurements were carried out in low resolution mode (r = 300) using a low - flow microconcentric nebulizer (flow rate was about 100 l min). Instrument was tuned using a 1 ng g multielement solution (merck, darmstadt, germany). The optimization was carried out with respect to maximum uranium sensitivity and low uo / u ratio . For the nd isotope ratio measurements nuplasma (nu instruments, oxford, united kingdom) double - focusing multi - collector inductively coupled plasma mass spectrometer (mc - icp - ms) was used . The sample introduction was done by a low - flow teflon micro - concentric nebulizer in combination with a dsn-100 desolvation unit (nu instruments, oxford, united kingdom). Instrument optimisation with respect to maximum sensitivity was carried out using a 100 ng g nd monoelemental solution (alfa aesar, karlsruhe, germany). The sensitivity was ~500 mv for nd in 100 ng g nd standard solution . The distribution of u and th during the co - precipitation was followed by gamma spectrometric measurements using a well - type hpge detector (gcw 2022 model) with ~20% relative efficiency and a resolution of <1.7 kev at 185.6 kev (canberra industries inc . The measurement time varied between 600 and 5,400 s. all gamma spectrometric measurements were performed as relative measurements to the original starting material before and after the separation at fixed geometry . All uncertainties quoted are given as expanded uncertainty using a coverage factor of k = 2 with last significant digits in parenthesis . Approximately 0.5 g of samples were weighed into a teflon erlenmeyer and dissolved in 6 ml of 8 mol l ultra - pure nitric acid while heating to 90 c on a hot - plate for 12 h covered with a pe lid . After cooling to room temperature about 3 ml of the stock solution, corresponding to about 200 mg of uranium, was transferred into a 50 ml polyethylene centrifuge vial . Ln, th and u were precipitated as hydroxides (ph 1214) with 40% sodium hydroxide in the presence of 2 mg fe(iii) carrier . The supernatant, containing most of the alkali - soluble matrix elements (e.g. Alkali metals) were carefully discarded after thorough centrifugation . Selective re - dissolution of uranium from the precipitate was done with 10 ml 1% (nh4)2co3 as uranium forms soluble di- and tri - carbonato complexes between ph 58 . This step was repeated three to five times until clear solution was obtained assuring that u was removed from the sample to the highest extent possible . Representative aliquots of the supernatant were collected after each separation step in order to (i) control uranium decontamination and th recovery factors by gamma spectrometric measurements parallel to the separation and (ii) use the achieved relatively pure uranium solution for other purposes (e.g. Uranium isotope ratio measurement). The decontamination factor of the order of 1010 achieved for u was sufficiently high to use extraction chromatography afterwards . The precipitate containing the ln and th was dissolved in 2 ml of 3 mol l nitric acid to be in suitable form for further concentration . From this final solution 100 the nd separation was performed in two steps: first a lanthanide group separation followed by the nd separation . The nd purification step is necessary for the removal of sm, which interferes otherwise with the icp - ms analysis . In the first step the lanthanide content of the sample aliquots was separated using extraction chromatography by the selective retention of trivalent lanthanides on the tru resin in 3 mol l nitric acid medium . In the second step, ln resin was used in 0.05 mol l hcl medium for the nd separation . This solution was evaporated to almost complete dryness and the residue was dissolved in nitric acid for mass spectrometric analysis . A method blank was processed through the entire dissolution and separation procedure parallel to the samples . The measured signal of the method blank was <2% for all samples . The simplified scheme of the entire separation procedure can be seen in fig . 1 . The detailed extraction chromatographic separation procedure can be found elsewhere [9, 16]. The method was validated by the measurement of reference material (morille, cetama), the recovery for the certified rare - earth elements (sm, eu, gd, dy) being better than 90% .fig . Thorough cleaning of all labware is necessary before use for trace - level measurement of nd isotope ratio . This was performed with dilute ethanol, followed by dilute nitric acid, and finally with high purity water rinsing . For all the dilutions high - purity water was used (uhq system, usf elga, germany). Hydrochloric and nitric acids were of suprapur grade (merck, darmstadt, germany), although the nitric acid was further purified by sub - boiling distillation . Analytical grade fe(no3)3 salt was used as carrier for the co - precipitation (alfa aesar, karlsruhe, germany). Analytical grade sodium - hydroxide and ammonium - carbonate used for the precipitation were purchased from sigma aldrich (st louis, mo, usa). Ammonium carbonate was further purified prior the use by adding about 10 mg of fe and precipitating fe(oh)3 to remove the trace - level lanthanide impurities still present in the analytical grade ammonium carbonate solution . For the lanthanide group separation, the tru extraction chromatographic resin supplied by triskem (triskem international, bruz, france) was used . For the preparation of columns, 1.6 ml of the resin was placed in plastic bio - rad holders (diameter 8 mm) and plugged with porous teflon frit (reichelt chemietechnik heidelberg, germany) on the top of the resin to avoid mixing . For nd separation, the ln resin for the extraction chromatographic separation was purchased from triskem (triskem international, bruz, france). For the preparation of columns, 400 l of the resin was placed in plastic bio - rad holders and plugged with porous teflon frit . For the optimization of the chemical separation procedure and the measurements by icp - ms, lanthanide standard solution and monoelemental nd and sm standard solutions (alfa aesar, karlsruhe, germany) were prepared by the dilution from 1,000 to 100 g ml standard solutions, respectively . The u3o8 certified reference material, morille (cetama, france) was used for the validation of the co - precipitation method as it is certified for four lanthanides content (dy, gd, eu and sm). The mass spectrometric analysis of aliquots from the co - precipitation step was carried out using an element2 (thermo electron corp ., bremen, germany) double - focusing magnetic sector inductively coupled plasma mass spectrometer (icp - sfms). Measurements were carried out in low resolution mode (r = 300) using a low - flow microconcentric nebulizer (flow rate was about 100 l min). Instrument was tuned using a 1 ng g multielement solution (merck, darmstadt, germany). The optimization was carried out with respect to maximum uranium sensitivity and low uo / u ratio . For the nd isotope ratio measurements nuplasma (nu instruments, oxford, united kingdom) double - focusing multi - collector inductively coupled plasma mass spectrometer (mc - icp - ms) was used . The sample introduction was done by a low - flow teflon micro - concentric nebulizer in combination with a dsn-100 desolvation unit (nu instruments, oxford, united kingdom). Instrument optimisation with respect to maximum sensitivity was carried out using a 100 ng g nd monoelemental solution (alfa aesar, karlsruhe, germany). The sensitivity was ~500 mv for nd in 100 ng g nd standard solution . The distribution of u and th during the co - precipitation was followed by gamma spectrometric measurements using a well - type hpge detector (gcw 2022 model) with ~20% relative efficiency and a resolution of <1.7 kev at 185.6 kev (canberra industries inc . The measurement time varied between 600 and 5,400 s. all gamma spectrometric measurements were performed as relative measurements to the original starting material before and after the separation at fixed geometry . All uncertainties quoted are given as expanded uncertainty using a coverage factor of k = 2 with last significant digits in parenthesis . Approximately 0.5 g of samples were weighed into a teflon erlenmeyer and dissolved in 6 ml of 8 mol l ultra - pure nitric acid while heating to 90 c on a hot - plate for 12 h covered with a pe lid . After cooling to room temperature about 3 ml of the stock solution, corresponding to about 200 mg of uranium, was transferred into a 50 ml polyethylene centrifuge vial . Ln, th and u were precipitated as hydroxides (ph 1214) with 40% sodium hydroxide in the presence of 2 mg fe(iii) carrier . The supernatant, containing most of the alkali - soluble matrix elements (e.g. Alkali metals) were carefully discarded after thorough centrifugation . Selective re - dissolution of uranium from the precipitate was done with 10 ml 1% (nh4)2co3 as uranium forms soluble di- and tri - carbonato complexes between ph 58 . This step was repeated three to five times until clear solution was obtained assuring that u was removed from the sample to the highest extent possible . Representative aliquots of the supernatant were collected after each separation step in order to (i) control uranium decontamination and th recovery factors by gamma spectrometric measurements parallel to the separation and (ii) use the achieved relatively pure uranium solution for other purposes (e.g. Uranium isotope ratio measurement). The decontamination factor of the order of 1010 achieved for u was sufficiently high to use extraction chromatography afterwards . The precipitate containing the ln and th was dissolved in 2 ml of 3 mol l nitric acid to be in suitable form for further concentration . From this final solution 100 the nd separation was performed in two steps: first a lanthanide group separation followed by the nd separation . The nd purification step is necessary for the removal of sm, which interferes otherwise with the icp - ms analysis . In the first step the lanthanide content of the sample aliquots was separated using extraction chromatography by the selective retention of trivalent lanthanides on the tru resin in 3 mol l nitric acid medium . In the second step, ln resin was used in 0.05 mol l hcl medium for the nd separation . After nd was stripped from the column with 0.2 mol l hcl . This solution was evaporated to almost complete dryness and the residue was dissolved in nitric acid for mass spectrometric analysis . A method blank was processed through the entire dissolution and separation procedure parallel to the samples . The measured signal of the method blank was <2% for all samples . The simplified scheme of the entire separation procedure can be seen in fig . 1 . The detailed extraction chromatographic separation procedure can be found elsewhere [9, 16]. The method was validated by the measurement of reference material (morille, cetama), the recovery for the certified rare - earth elements (sm, eu, gd, dy) being better than 90% .fig . Six uranium ore concentrate samples were chosen to evaluate the capabilities of the method developed . Three of them (rabbit lake, mary kathleen and nabarlek) were analysed to compare the ree pattern with and without pre - concentration step in order to verify that no interferences were introduced to samples by the used reagents . As one can see from fig . 2 the high yb level of mary kathleen sample is possibly related to isobaric interference if only tru separation is applied . Our result of mary kathleen uranium ore concentrate sample is also in good agreement with the recently published work of keegan et al . . Note that ree patterns of the investigated uranium ore concentrate samples are presented after chondrite normalisation and in logarithmic scale .fig . 2ree patterns of investigated uranium ore concentrate samples obtained with two different separation procedures: extraction chromatography with tru resin (tru) and in this work developed pre - concentration (preconc) ree patterns of investigated uranium ore concentrate samples obtained with two different separation procedures: extraction chromatography with tru resin (tru) and in this work developed pre - concentration (preconc) the nd / nd isotope ratio for three uoc samples (rssing, shirley basin and can esi) and bcr-2 geological standard was measured (table 1). The measured nd / nd value of the bcr-2 standard was 0.512598(78), which agrees with the certified value 0.512629(8) within uncertainty . The nd isotope ratio of rssing sample had been measured previously using only extraction chromatography without pre - concentration and it resulted in a nd / nd ratio of 0.51363(230) with high uncertainty . Comparing to our new result 0.51346(34), obtained with the improved sample pre - concentration procedure, one can see that the values are in good agreement.table 1measured nd / nd isotope ratios in the investigated uranium ore concentrate samplessamplecountrydeposit typecnd (ng g) nd / ndtru nd / ndpreconc esicanadaphosphate90<dl0.51225(9)rssingnamibiaintrusive150.51363(230)0.51346(34)shirley basinusasandstone8<dl0.51356(61)all uncertainties quoted are given as expanded uncertainty using a coverage factor of k = 2 with last significant digits . Nd / ndtru corresponds to the results obtained by using only tru separation, while nd / ndpreconc are the results of the present method with preconcentration measured nd / nd isotope ratios in the investigated uranium ore concentrate samples all uncertainties quoted are given as expanded uncertainty using a coverage factor of k = 2 with last significant digits . Nd / ndtru corresponds to the results obtained by using only tru separation, while nd / ndpreconc are the results of the present method with preconcentration moreover, the uncertainty of the new result is almost an order of magnitude better . For shirley basin and can esi the nd isotopic composition could nt be measured previously when using only the tru separation because of the nd concentration being under detection limit . However, with the new sample preparation procedure precise results were obtained and they fit well in the previously found 0.5100.515 range . With the improved nd isotopic information and the lower uncertainties the uranium deposit type, and therefore the origin of an unknown nuclear material can be assessed with higher reliability . The sample amount in nuclear forensic investigations is of crucial importance, not just because the available sample amount is often limited as an evidence specimen, but also due to the need of relatively high amount of sample for the high precision elemental or isotopic analysis . Therefore, careful planning and sequencing of the measurements are required to perform comprehensive analyses . The proposed fe(oh)3 co - precipitation in 1% ammonium carbonate combines the effective pre - concentration of the trace - level constituents with the removal of the uranium matrix . The presented method is not just useful for the trace - level nd isotope ratio analysis as demonstrated, but it is also a versatile and straightforward sample preparation procedure, which can be applied to pre - concentrate and separate other elements of interest, such as th, pu or am from a single sample aliquot . These are just few examples of the promising potential of the newly developed pre - concentration procedure.
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The association of pancreatic disease with fat necrosis at distant foci was first described by chiari in 1883 (1). The most common pancreatic disorders associated with pancreatic panniculitis are acute or chronic pancreatitis, especially the alcohol - related types, and pancreatic carcinoma . Other pancreatic disorders have been infrequently reported to be associated with pancreatic panniculitis, and these include post - traumatic pancreatitis, pancreatic pseudocyst, pancreas divisum, and hemophagocytic syndrome (2). To the best of our knowledge, one case was associated with pancreatic adenocarcinoma, and the other two were associated with acute and chronic pancreatitis, respectively (1, 3, 4). We report here on a case of pancreatic panniculitis associated with acute pancreatitis that had a fatal outcome . Physicians should be aware of this disease entity because this rare cutaneous manifestation may be associated with major morbidity and significant mortality . A 50-yr - old man with a history of alcohol abuse presented with increasing fatigue, generalized weakness, decreased appetite, and abdominal distension and discomfort for the past 2 weeks . He was admitted to the department of internal medicine under the impression of acute pancreatitis, and he was referred to the department of dermatology for the multiple painful subcutaneous nodules on his legs, which had suddenly developed 3 weeks before (fig . His hemoglobin value was 11.4 g / dl (reference range: 13 - 18 g / dl), the total count of white blood cells was 31,300/l (reference range: 4,000 - 10,000/l) with 90.7% segment neutrophils, but the coagulation profiles and platelet counts were normal . The serum amylase was 1,909 u / l (reference range: 20 - 120 u / l), and the lipase was 2,306 u / l (reference range: 5 - 51 u / l), an alanine aminotransferase level of 24 u / l (reference range: 7 - 40 u / l), and a lactate dehydrogenase level of 665 u / l (reference range: 200 - 400 the fasting glucose level was 133.9 mg / dl (reference range of 70 - 110 mg / dl) and the electrolytes were unbalanced . The calcium level was 7 mg / dl (reference range: 8.6 - 10 mg / dl), and the sodium level was 125 the blood urea nitrogen and creatinine were 40.1 mg / dl and 2.1 mg / dl, respectively . After 48 hr, the blood urea nitrogen was increased to 62.7 mg / dl after intravenous fluid administration . As his leukocytosis, elevated serum ldh at admission, and hypocalcemia, hypoalbuminemia, increase in blood urea nitrogen during initial 48 hr were poor prognostic factors in ranson criteria, increased risk of complications was predicted . The chest radiograpy showed mild pleural effusion . On the abdominal computed tomography scan and magnetic resonance (mr) imaging taken on the second day of admission, a swollen pancreas with an dilated pancreatic duct, a loculated fluid collection in the left anterior perirenal space, multiple hepatic cysts, and massive ascites u / l, the serum - ascites albumin gradient was calculated to be 1.07, and there was no evidence of malignancy . A skin biopsy performed on the 4th day of admission from the nodule on the left lower leg showed a diffuse subcutaneous fat necrosis and ghost - like cells with thick shadowy walls and no nuclei . There was a fine granular basophilic material deposited within and around the necrotic fat cells (fig . After 10 days of intensive medical care for the pancreatic disease, the patient's condition began to worsen; he and his family began to refuse any further treatment . Despite a strong warning by physicians the pathogenesis of pancreatic panniculitis is still unknown, but the released pancreatic enzymes such as trypsin may increase the permeability of the microcirculation . Lipase or amylase is then involved in the process of fat degradation, which results in the liberation of free fatty acids that combine with calcium to form soap (2). Although elevation of the pancreatic enzymes is common in pancreatitis patients, pancreatic panniculitis is a very rare malady . Mullin et al . (5) identified only one case in a retrospective review of 893 patients who had suffered with pancreatic disease from various causes . Furthermore, well documented cases of fat necrosis with normal serum lipase levels have also been described (6). These reports, suggest that there would be some other factors that allow the pancreatic enzymes to escape from the circulation and act on the subcutaneous fat . Zellman (7) suggested that some damage to the blood vessels via inflammation, edema, or altered immunity may act as the initiating factor . As for the two cases in the korean literature, one case was associated with chronic pancreatisis, in which the cutaneous lesions had occurred 3 months later than the other systemic symptoms, and they involuted as the underlying pancreatitis was ameliorated (4). The other case was associated with acute pancreatitis with pancreas divisum, in which his skin lesions had appeared 2 - 3 days after his admission due to acute pancreatitis; the skin lesions also resolved as the underlying pancratitis subsided with conservative treatment (1). On the contrary, in our patient, the skin lesions preceded the other systemic symptoms . Although the underlying pancreatic pathologic conditions can vary, the clinical features of pancreatic panniculitis are similar . The legs were the most commonly affected area, but the lesions can also occur on the arms, thighs, and trunk . The lesions usually began as erythematous or red - brown subcutaneous nodules with a tendency to show central softening . In the mild form if the fat necrosis is severe, individual nodules may break down and extrude necrotic material (8). Although patients with other panniculitides such as erythema nodosum, erythema induratum, lupus panniculitis, weber - christian pannicultitis, or alpha-1 antitrypsin deficiency - associated panniculitis can have similar clinical lesions, the diagnosis of pancreatic panniculitis is suggested by the presence of pancreatic disease and this the pathognomonic findings are collections of' ghost cells' and anucleated adipocytes containing intracytoplasmic fine basophilic granular material from the saponification of fat . The resistance of the fat cell membrane to lipase produces the shadowy walls and the fatty acids combine with calcium to form calcium soap (10). Sometimes conservative care can ameliorate the pancreatitis, and this then results in the resolution of the skin lesions . In isolated cases, surgical correction of anatomic ductal anomaly or pancreatic pseudocyst, or the removal of gallstones has resulted in complete resolution (8). However, disseminated fat necrosis is associated with major morbidity and mortality . In a review of 27 patients with pancreatic panniculitis, all 8 patients with pancreatic carcinoma and 42% of the 19 patients with pancreatitis died of their disease, as was seen in our case (9). Pancreatic panniculitis is a pathognomic finding of pancreatic disease, and as in our case, the cutaneous lesions may precede the usual manifestations of underlying pancreatic disease by several weeks to months (11).
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, several trauma scoring systems have been validated for prediction of patient survival, new injury severity score (niss) is defined as the sum of the squares of the abbreviated injury scale (ais) of each of the patient's most severe ais injuries irrespective of the body region in which they occur . The pediatric trauma score (pts) was devised specifically for the triage of pediatric trauma patients . The pediatric big score can be performed rapidly on admission to evaluate the severity of illness and to predict mortality in children with traumatic injuries . International normalization ratio (inr) is a measure of tissue factor - activated arm of the coagulation cascade, coagulopathy, as characterized by increased fibrin degradation products, has been shown to predict mortality in children with head trauma while base deficit is a measure of shock and acidosis . A big score of <12 points suggests a mortality of <5%, whereas a cutoff of> 26 points corresponds to a mortality of> 50% . The limitation of the big score is that it requires laboratory values to calculate it . This study aimed at evaluation of pediatric trauma big score in comparison with niss and pts in tanta university emergency hospital . The study was conducted as a prospective comparative study in a university emergency hospital in egypt for 1 year from february 2014 to february 2015, on fifty multiple trauma pediatric patients admitted to the pediatric emergency department . All pediatric patients between 1 and 18 years old and within 24 h after trauma were included in the study . Patients <1 year old, patients who came to the hospital after 24 h of occurrence of trauma, burn, and electric shock without polytrauma, and patients with chronic diseases such as chronic renal failure, hepatic, hematologic, or neurologic diseases were excluded from the study . Data collected for each multiple trauma patient attended to emergency department, include history taking stressing on mechanism of injury, thorough clinical examination, especially systolic blood pressure (sbp), pulse rate, respiratory rate, glasgow coma scale (gcs), and laboratory investigations including arterial blood gas, prothrombin time, partial thromboplastin time, and inr . Pts was calculated by pts was calculated by summation of these variables [table a]. Variables for calculation of pediatric trauma score(pts) (citated from emergency medical journal 2011) niss was calculated by summation of square of ais score of the most three affected regions of body regardless site of injury as follows: niss = (ais1) + (ais2) + (ais3) [table b]. Abbreviated injury scale of new injury severity score (niss) (citated from emergency medical journal 2011) pediatric trauma big score then was calculated as follows: pediatric big score equation = (admission base deficit) + (2.5 inr) + (15 gcs). This equation can then be implemented into a mortality - predicting formula: predicted mortality = 1/(1 + e), where x = 0.2 (big score) 5.208 . Testing the mortality prediction of the big score on our trauma patients and compared observed and expected mortality by big score . Comparing between big score, pts, and niss as regard validity and observed outcome . Trauma management at patient arrival includes primary then secondary survey in the first 1015 min . Initial observations of the patient as he or she was being wheeled in by emergency medical service (ems), airway was evaluated with cervical spine precautions, assisted bag valve mask ventilation may be indicated, assess the breathing . Evaluation of chest wall motion, lung sounds, respiratory rate, and oxygen saturation was done . Early intubation was done for poor respiratory effort, inadequate oxygenation, or gcs score of eight or less . For assessment of the circulation, examination of focused assessment sonography in trauma(fast) and two large bore aintravenous acess were established . Routine imaging studies include chest x - ray, anteroposterior, pelvis, and lateral cervical spine . Computed tomography scan of the cervical spine, fast or extended - fast to evaluate for intraperitoneal blood, pneumothorax, and hemothorax as well . A foley catheter was placed for evaluation for hematuria as well as monitoring urine output . Rectal examination was done before placement to evaluate for signs of urethral injury such as high - riding prostate as well as blood at the urethral meatus . Secondary assessment is an organized, head - to - toe assessment, especially difficult to see areas such as the axillae and perineum . Signs / symptoms, allergies, medications, past medical history, last meal, and events history was obtained from the patient, family, or ems . By this time, the initial resuscitation and stabilization of most patients were completed, and the patient was ready to leave the trauma bay to (a) the operating room if immediate surgery was indicated, (b) the imaging suite if he or she was stable, (c) the intensive care unit if observation was the planned course of action, or (d) a regular gurney / stretcher in the emergency department for observation before discharge home . The collected data were analyzed using spss version 20 software (spss inc ., chicago, il, usa) and microstat - w software (india, cnet download.com). Categorical data were presented as number and percentages whereas quantitative data were expressed as a mean standard deviation . Chi - square test, fisher's exact test, z - test, student's t - test, mann whitney u - test, and mcnemar's test were used as tests of significance . Receiver operating characteristic (roc) curve was used to determine cutoff values of the studied mortality scores with optimum sensitivity and specificity . Binary logistic regression model was used to detect the significant predictors of mortality and to design an equation for predicted mortality from big score . The accepted level of significance in this work was stated at 0.05 (p <0.05 was considered significant). P> 0.05 is nonsignificant, p <0.05 is significant, and p 0.001 is highly significant . All pediatric patients between 1 and 18 years old and within 24 h after trauma were included in the study . Patients <1 year old, patients who came to the hospital after 24 h of occurrence of trauma, burn, and electric shock without polytrauma, and patients with chronic diseases such as chronic renal failure, hepatic, hematologic, or neurologic diseases were excluded from the study . Data collected for each multiple trauma patient attended to emergency department, include history taking stressing on mechanism of injury, thorough clinical examination, especially systolic blood pressure (sbp), pulse rate, respiratory rate, glasgow coma scale (gcs), and laboratory investigations including arterial blood gas, prothrombin time, partial thromboplastin time, and inr . Pts was calculated by pts was calculated by summation of these variables [table a]. Variables for calculation of pediatric trauma score(pts) (citated from emergency medical journal 2011) niss was calculated by summation of square of ais score of the most three affected regions of body regardless site of injury as follows: niss = (ais1) + (ais2) + (ais3) [table b]. Abbreviated injury scale of new injury severity score (niss) (citated from emergency medical journal 2011) pediatric trauma big score then was calculated as follows: pediatric big score equation = (admission base deficit) + (2.5 inr) + (15 gcs). This equation can then be implemented into a mortality - predicting formula: predicted mortality = 1/(1 + e), where x = 0.2 (big score) 5.208 . Testing the mortality prediction of the big score on our trauma patients and compared observed and expected mortality by big score . Comparing between big score, pts, and niss as regard validity and observed outcome . Trauma management at patient arrival includes primary then secondary survey in the first 1015 min . Initial observations of the patient as he or she was being wheeled in by emergency medical service (ems), airway was evaluated with cervical spine precautions, assisted bag valve mask ventilation may be indicated, assess the breathing . Evaluation of chest wall motion, lung sounds, respiratory rate, and oxygen saturation was done . Early intubation was done for poor respiratory effort, inadequate oxygenation, or gcs score of eight or less . For assessment of the circulation, examination of focused assessment sonography in trauma(fast) and two large bore aintravenous acess were established . Routine imaging studies include chest x - ray, anteroposterior, pelvis, and lateral cervical spine . Computed tomography scan of the cervical spine, fast or extended - fast to evaluate for intraperitoneal blood, pneumothorax, and hemothorax as well . A foley catheter was placed for evaluation for hematuria as well as monitoring urine output . Rectal examination was done before placement to evaluate for signs of urethral injury such as high - riding prostate as well as blood at the urethral meatus . Secondary assessment is an organized, head - to - toe assessment, especially difficult to see areas such as the axillae and perineum . Signs / symptoms, allergies, medications, past medical history, last meal, and events history was obtained from the patient, family, or ems . By this time, the initial resuscitation and stabilization of most patients were completed, and the patient was ready to leave the trauma bay to (a) the operating room if immediate surgery was indicated, (b) the imaging suite if he or she was stable, (c) the intensive care unit if observation was the planned course of action, or (d) a regular gurney / stretcher in the emergency department for observation before discharge home . Chicago, il, usa) and microstat - w software (india, cnet download.com). Categorical data were presented as number and percentages whereas quantitative data were expressed as a mean standard deviation . Chi - square test, fisher's exact test, z - test, student's t - test, mann whitney u - test, and mcnemar's test were used as tests of significance . Receiver operating characteristic (roc) curve was used to determine cutoff values of the studied mortality scores with optimum sensitivity and specificity . Binary logistic regression model was used to detect the significant predictors of mortality and to design an equation for predicted mortality from big score . The accepted level of significance in this work p> 0.05 is nonsignificant, p <0.05 is significant, and p 0.001 is highly significant . All pediatric patients between 1 and 18 years old and within 24 h after trauma were included in the study . Patients <1 year old, patients who came to the hospital after 24 h of occurrence of trauma, burn, and electric shock without polytrauma, and patients with chronic diseases such as chronic renal failure, hepatic, hematologic, or neurologic diseases were excluded from the study . Data collected for each multiple trauma patient attended to emergency department, include history taking stressing on mechanism of injury, thorough clinical examination, especially systolic blood pressure (sbp), pulse rate, respiratory rate, glasgow coma scale (gcs), and laboratory investigations including arterial blood gas, prothrombin time, partial thromboplastin time, and inr . Pts was calculated by pts was calculated by summation of these variables [table a]. Variables for calculation of pediatric trauma score(pts) (citated from emergency medical journal 2011) niss was calculated by summation of square of ais score of the most three affected regions of body regardless site of injury as follows: niss = (ais1) + (ais2) + (ais3) [table b]. Abbreviated injury scale of new injury severity score (niss) (citated from emergency medical journal 2011) pediatric trauma big score then was calculated as follows: pediatric big score equation = (admission base deficit) + (2.5 inr) + (15 gcs). This equation can then be implemented into a mortality - predicting formula: predicted mortality = 1/(1 + e), where x = 0.2 (big score) 5.208 . Testing the mortality prediction of the big score on our trauma patients and compared observed and expected mortality by big score . Comparing between big score, pts, and niss as regard validity and observed outcome . Trauma management at patient arrival includes primary then secondary survey in the first 1015 min . Initial observations of the patient as he or she was being wheeled in by emergency medical service (ems), airway was evaluated with cervical spine precautions, assisted bag valve mask ventilation may be indicated, assess the breathing . Evaluation of chest wall motion, lung sounds, respiratory rate, and oxygen saturation was done . Early intubation was done for poor respiratory effort, inadequate oxygenation, or gcs score of eight or less . For assessment of the circulation, examination of focused assessment sonography in trauma(fast) and two large bore aintravenous acess were established . Routine imaging studies include chest x - ray, anteroposterior, pelvis, and lateral cervical spine . Computed tomography scan of the cervical spine, fast or extended - fast to evaluate for intraperitoneal blood, pneumothorax, and hemothorax as well . A foley catheter was placed for evaluation for hematuria as well as monitoring urine output . Rectal examination was done before placement to evaluate for signs of urethral injury such as high - riding prostate as well as blood at the urethral meatus . Secondary assessment is an organized, head - to - toe assessment, especially difficult to see areas such as the axillae and perineum . Signs / symptoms, allergies, medications, past medical history, last meal, and events history was obtained from the patient, family, or ems . By this time, the initial resuscitation and stabilization of most patients were completed, and the patient was ready to leave the trauma bay to (a) the operating room if immediate surgery was indicated, (b) the imaging suite if he or she was stable, (c) the intensive care unit if observation was the planned course of action, or (d) a regular gurney / stretcher in the emergency department for observation before discharge home . Chicago, il, usa) and microstat - w software (india, cnet download.com). Categorical data were presented as number and percentages whereas quantitative data were expressed as a mean standard deviation . Chi - square test, fisher's exact test, z - test, student's t - test, mann whitney u - test, and mcnemar's test were used as tests of significance . Receiver operating characteristic (roc) curve was used to determine cutoff values of the studied mortality scores with optimum sensitivity and specificity . Binary logistic regression model was used to detect the significant predictors of mortality and to design an equation for predicted mortality from big score . The accepted level of significance in this work table 1 shows that there was a significant decrease in the age, body weight, sbp, and gcs and a significant increase in both inr and base excess of nonsurvivor patients when compared to survivor patients . Demographic, clinical, and laboratory variables as regard patient outcomes table 2 and figure 1 show trauma characteristics of the studied children as regard systems injured by trauma, regarding airway, 11 patients were with patent airway, 28 patients with maintainable airway, and 11 patients unmaintainable airway . Trauma characteristics of the studied children as regard patient outcomes comparison between airway and central nervous system status among studied patients regarding level of consciousness, 17 patients were awake and 33 patients were comatosed . Table 2 also and figure 2 show that the most common mechanism of injury among studied patients was fall from height by 44%, and motorcycle was the least common mechanism of injury by 16% . They show also that the most favorable prognosis was in fall from height, and the poorest prognosis was in both pedestrian collision and motorcycle . Table 2 also shows that the most common diagnosis of cause of death among studied patients was central nervous system lesion (brain death) (20%), followed by hemorrhagic shock (6%) while respiratory failure and multiple system organ failure were the least common diagnosis of cause of death (2% for each). Mechanism of injury among studied patients figure 3 shows no significant difference between mean value of niss among died and survived patients, whereas there was a significant increase in mean value of pts among survived patients and a significant decrease in mean value of big score among survived patients . Comparing survivors and nonsurvivors regarding trauma scores figure 4 compares roc curve for validity of the three studied trauma scores (big, pts, and niss) in prediction of mortality, big score 12.7 has sensitivity 86.7%, specificity 71.4%, positive predictive value (ppv) 56.5%, negative predictive value (npv) 92.6%, and confidence index (ci) 0.770.97, whereas pts at value 3.5 has sensitivity 63.3%, specificity 68.6%, ppv 42.2%, npv 77.4%, and ci 0.570.85 and niss at value 39.5 has sensitivity 53.3%, specificity 54.3%, ppv 33.3%, npv 73.1%, and ci 0.480.79 . Hence, the highest sensitivity and specificity of the three studied scores was to big score at cutoff value of 12.7, while the lowest sensitivity and specificity was to niss at cutoff value niss 39.5, while pts showing moderate specificity and sensitivity at cutoff value 3.5 . Figure 4 shows also that areas under the roc curve of niss, pts, and big were 0.87, 0.71, and 0.63 respectively . Receiver operating characteristic curves for the validity of big, pediatric trauma score, and new injury severity score trauma scores in prediction of mortality table 3 and figure 5 show relation of big score to observed mortality rate, there was a significant positive correlation between big score values and mortality rates as the higher the value of big score, the higher the incidence of mortality . Comparing observed and predicted mortality by big score relation of big score to observed mortality rate easy - to - use trauma scoring systems inform physicians of the severity of trauma in patients and help them decide the course of trauma management . The use of trauma scoring systems can be used before the patient reaches the hospital, to decide whether to send the patient to a trauma center, and can also be used for clinical decision - making when the trauma patient has just arrived at the emergency department (ed). When the patient is in the ed, trauma scoring systems can be used to prepare the patient for surgery, to call on medical staff for trauma support, and to inform the family of the severity of the patient's condition in the early stage . In the present prospective comparative study, pediatric trauma big score, pts, and niss scale, has been conducted on fifty polytraumatized pediatric patients attended to tanta emergency university hospital . In this study, we choose to register the subjects from only one center to avoid the possible variability in the triage system in different settings . As regarding mechanism of injury, we found that fall from height was the most common cause of admission in polytraumatized pediatric patients (44%) and this coincide with the study done by fiorentino et al . In hospital de nio ricardo gutirrez in sao paolo (54%). Another study made by derakhshanfar et al . Found that pedestrian car accidents and falling down were the most common causes of injuries (23.3% and 21.3%), respectively . Letts et al . In their study on the children's hospital of eastern ontario, a major pediatric trauma center, found that fall from height was the third common cause of trauma in children was by 14% and the most common cause was motor car accidents by 38.9% . In this study as regarding the age of patients, there was a significant decrease in age of nonsurvivors when compared to survivors (p = 0.01). . Concluded that survival after childhood injury is independent on the age groups in their study after controlling injury severity . In the present study as regarding body weight, there was a significant decrease in body weight in nonsurvivors and when compared to survivors (p = 0.011). It was against the study made by derakhshanfar et al . Who found that there was no significant relation between body weight of patients and their outcome . As regarding sbp, we found a significant decrease in sbp in nonsurvivors when compared to survivors (p = 0.004). Conducted their cross - sectional study included injured children from 34 emergency services showed that when compared with children with normal relative risk (rr) and sbp, children with an abnormal sbp and rr had a 5.0 (95% ci: 3.96.4) and 2.2 (95% ci: 1.53.1) times higher odds of death, respectively . Derakhshanfar et al . Conducted their study in tehran, iran, on 151 pediatric trauma patients and showed that the mean sbp was 96 11 mmhg . There have been several previous studies on the importance of be in evaluating trauma patients, but only a few examined its importance in the pediatric population . Those studies suggested that be was a good indicator of injury severity in pediatric trauma patients . In this work, we found that there was a significant increase in be in nonsurvivors when compared to survivors (p = 0.039). Randolph et al . Found that admission be was a strong indicator of injury severity, and a be below 5 was predictive of severe injury and mortality . In severely injured children (all requiring mechanical ventilation), be <8 predicted a higher incidence of infectious complications and a less favorable outcome . Borgman et al . Reported that admission base deficit was also found to be an independent predictor of mortality in 707 children injured in iraq and afghanistan . Gcs is the most widely used scoring system for the evaluation of disorder of consciousness . The modified gcs is used for infants and young children to obtain the most accurate score . In this study, there was a significant decrease in gcs of nonsurvivor patients when compared to survivor patients . Most of the cases of death (66%) were due to head trauma, so gcs work better in these cases than other causes of death such as other, hemorrhagic shock, respiratory failure, or multiple system organ injury . That proved that gcs provides a prediction about morbidity and mortality after head injury . In this study, there was a significant increase in inr of nonsurvivor patients when compared to survivor patients . Verma and kole study showed that inr is indeed a good predictor of mortality in trauma patients, with a high diagnostic accuracy . In this study, pts showing moderate specificity and sensitivity at cutoff value 3.5 with sensitivity 63.3% and specificity 68.6% . There are conflicting reports on the effectiveness of the pts as a tool for assessing prognosis and in identifying those who will need a transfer to a pediatric trauma center . Studied the prognostic value of pts in pediatric trauma patients on 151 pediatric patients in tehran and found it to be an independent predictor of morbidity . Reported that the pts has no advantage in children, even in children younger than 14 years . Another study made by eichelberger et al . Has reported no difference between the predictive capabilities of the trauma score and the pts in identifying severely injured children . Further refinements of the pts include the age - specific pts and the triage age - specific pts . These scoring systems, however, have not yet been validated and are rarely used . In 1997, a simple modification of injury severity score (iss) was formulated by osler et al . And referred to as the niss, in the present study, we found that niss at cutoff value 39.5 has sensitivity 53.3% and specificity 54.3% for predicting mortality . Sullivan et al . Found that the niss performs as well as the iss in predicting mortality in pediatric trauma patients who are not severely injured (iss <24). It is not intended to reflect patient outcomes, but only to score an individual injury . An adult trauma study made by brockamp et al ., to compare the big score with other trauma scores revealed that big score is a good predictor of mortality in the adult trauma population, and they added that in a penetrating trauma population, the big score performed better than in a population with blunt trauma . The big score has the advantage of being available shortly after admission and may be used to predict clinical prognosis or as a research tool to risk stratify trauma patients into clinical trials . In the present study, we found that pediatric trauma big score was more sensitive and specific and easily applicable score in predicting mortality than pts and niss, and this coincides with the study done by borgman et al . Paediatric trauma big score may be applied as a mortality prediction tool in pediatric emergency for its ease and simplicity of application at the time of entry at the emergency department . This would also help in evaluating for early invasive monitoring and treatment decisions in the intensive care unit . We recommend that leaflets for big score be formed and calculated easily in the emergency department to help predicting mortality and decision - making regarding polytraumatized pediatric patients . We recommend that leaflets for big score be formed and calculated easily in the emergency department to help predicting mortality and decision - making regarding polytraumatized pediatric patients . We recommend that leaflets for big score be formed and calculated easily in the emergency department to help predicting mortality and decision - making regarding polytraumatized pediatric patients.
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The acute respiratory distress syndrome (ards), a clinically important complication of severe acute lung injury (ali) in humans, is a significant cause of morbidity and mortality in critically ill patients [15]. Infectious etiologies, such as sepsis and pneumonia, are leading causes of ali [1, 2, 5]. Histologically, ali in humans is characterized by a severe acute inflammatory response in the lungs and neutrophilic alveolitis [1, 5]. The physiological hallmark of ards is disruption of the alveolar - capillary membrane barrier, leading to development of noncardiogenic pulmonary edema, in which a proteinaceous exudate floods the alveolar spaces, impairs gas exchange, and precipitates respiratory failure [1, 57]. Ali can result in persistent respiratory failure and prolonged dependence on mechanical ventilation, increasing susceptibility to multiorgan dysfunction and mortality . Despite extensive investigation aimed at early diagnostic and pathogenetic factors of ali, current management is mainly supportive, as specific therapies have not been identified [5, 913]. Animal models focused on ali pathogenesis have yielded insights into mechanisms that initiate injury; however, little is known about potential determinants of resolution . Thus, new strategies are still required for achieving effective treatment of ali, which might ultimately aid the clinical therapy for ali patients . Progranulin (pgrn), also known as granulin epithelin precursor (gep), pc - cell - derived growth factor (pcdgf), proepithelin, and acrogranin, is an evolutionarily conserved, secreted glycoprotein with 7 granulin (grn) repeats [14, 15]. Pgrn played a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, host defense, and tumorigenesis [1520]. Of interest, recent findings suggested that pgrn was a key regulator of inflammation and that pgrn might mediate its anti - inflammatory effects, at least in part, by blocking tnf- binding to its receptors . However, whether pgrn could inhibit the lung inflammation and ultimately ameliorate the ali was still unclear . Recent evidence showed that elevated soluble tumor necrosis factor- receptor levels in bal fluid were found to be associated with poor patient outcome in ali, implying that blockade of pgrn by the soluble tumor necrosis factor- receptor might contribute to the development of ali . Thus, we hypothesized that pgrn might exert as a promising molecule for treatment of inflammation in ali . To address this issue, here we carefully evaluate the potential role of pgrn in treatment of ali using the murine model of lps - induced ali . We found that administration of pgrn significantly reduced lps - induced pulmonary inflammation and resulted in remarkable reversal of lps - induced increases in lung permeability, accompanied by a significant reduction of histopathology changes of lung . Our findings strongly demonstrated that pgrn could effectively ameliorate the lps - induced acute lung injury in mice, suggesting a potential role for pgrn - based therapy to treat clinical ards . Female balb / c mice at 6 weeks old were purchased from the center of experimental animals of tongji university . All mice were housed in the pathogen - free animal facilities of tongji university school of medicine . All animal experiments were performed according to the guide for the ethical guidelines of the shanghai medical laboratory animal care and use committee and the ethical guidelines of the tongji university laboratory animal care and use committee . Briefly, female balb / c mice (n = 6 per group) were anaesthetized and orally intubated with a sterile plastic catheter, and challenged with intratracheal instillation of 800 g of lps (e. coli 055:b5; sigma) dissolved in 50 l of normal pbs . Naive mice (without lps instillation) were injected with the same volume of pyrogen - free pbs to serve as controls . Mice were humanely killed at 3 d after lps challenge to collect tissues for analysis . Tnfr1 (cd120a) and tnfr2 (cd120b) antibodies for neutralizing studies were purchased from ebioscience . Our initial experiment showed that 200 g of tnfr2 antibody was effective to significantly inhibit the protective effect of pgrn on the lps - induced ali . Thus, 200 g of tnfr1 antibody or tnfr2 antibody was used for neutralization experiment in this study . Groups of mice were treated with pgrn via intratracheal instillation 30 min after their challenge with lps . The administration dose of pgrn was 2 g per mouse which was based on our initial experiments . The level of pgrn in bal fluid was determined by western blot using the murine pgrn affinity purified polyclonal antibody (r&d systems) or by elisa using the commercial murine pgrn elisa kit (r&d systems). According to previously described, bal was performed by instilling 0.9% nacl containing 0.6 mmol / l ethylenediaminetetraacetic acids in two separate 0.5 ml aliquots . An aliquot of the bal fluid was processed immediately for total and differential cell counts . The remainder of the lavage fluid was centrifuged and the supernatant was removed aseptically and stored in individual aliquots at 70c . Number of neutrophils was calculated as the percentage of neutrophils multiplied by the total number of cells in the bal fluid sample . All analyses were performed in a blinded fashion . In line with previously described, bal fluid collected was centrifuged at 800 g for 10 min, and supernatant was collected for analysis of total protein, albumin, igm, and cytokine / chemokine levels . Proinflammatory cytokine levels including tnf-, il-1, and il-6 in bal fluid were measured with murine cytokine - specific quantikine elisa kits (r&d systems). Chemokine levels including cxcl2, je (the murine homolog of human ccl2) and kc (the murine homolog of human il-8) in bal fluid were measured using cytokine - specific bead kits (r&d systems). Albumin and igm levels in bal fluid samples were measured using with a murine - specific albumin elisa kit (alpco diagnostics) and a murine - specific igm elisa kit (bethyl laboratories), respectively . Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 m thick sections . Sections were stained with hematoxylin and eosin, and images were taken with a nikon eclipse e800 microscope (200x). For the lung injury score, images were evaluated by an investigator who was blinded to the identity of the slides as previously described [5, 22]. In brief, the extent of the pathological lesions was graded from 0 to 3 as shown in table 1 . The score for each animal was calculated by dividing the total score for the number of sections observed . Differences between the treated groups versus the injured group were assessed using a one - way anova with statistic software (graphpad prism version 4.00). To assess the potential role of pgrn in lps - induced ali, we determined the level of pgrn protein in bronchoalveolar lavage (bal) fluid of lps - induced ali mice using western blot at day 3 after lps challenge . We found that the level of pgrn in bal fluid was significantly decreased on day 3 in mice challenged with lps compared with the control groups (figures 1(a) and 1(b), p <0.05). To further confirm this result, we further performed elisa assay to detect the level of pgrn in the bal fluid . Similarly, we revealed that the protein level of pgrn was downregulated in bal fluid on day 3 in lps - induced ali mice (figure 1(c), p <0.05). Further, we evaluated the time course of pgrn levels in bal fluid in lps - induced ali mice and the control mice . As shown in the figure 1(d), we found a substantial increase of pgrn protein on day 1 and then decreased since day 2, which indicated that pgrn might be subjected to proteolysis during inflammation in lung . Consistently, we indeed revealed an elevated expression of granulin, which were the units of pgrn, in bal fluid in lps - induced ali mice (figure 1(e)). Combing to access the potential role of pgrn in the development of ali, we evaluated the effect of pgrn administration in the maintenance of body weight and mortality of lps - induced ali mice . As shown in figure 2(a), we revealed that the loss of body weight was about 20% in lps - induced ali mice . Notably, we found that administration of pgrn effectively abrogated the loss of body weight of lps - induced ali mice (p <0.05). Interestingly, when pgrn was administered twice at 40 h intervals, it could further maintained the body weight of lps - induced ali mice to a level similar to the control mice (figure 2(a), p <0.05). Furthermore, we found that the mortality was approximately 40% in lps - induced ali mice, while administration of pgrn in lps - induced ali mice effectively maintained their survival, which was more apparent in ali mice received pgrn twice at 40 h intervals (figure 2(b), p <0.05). These findings suggested that pgrn was an effective candidate for preventing the development of ali . To investigate the possible mechanism underlying the protective effect of pgrn on lps - induced ali, we detected the total cell and neutrophil counts in bal fluid from mice treated with lps with or without pgen . As shown in figure 3(a), the total inflammatory cell count in the bal fluid was increased dramatically at day 3 after administration of lps (p <0.05). We revealed that neutrophils accounted for about 80% of the increased inflammatory cells and was significantly elevated in bal fluid (figure 3(b), p <0.05). Notably, we found that administration of pgrn could significantly reduce the total cell and neutrophil counts in bal fluid (figures 3(a) and 3(b), p <0.05). When pgrn was administered twice at 40 h intervals, it could further reduce the total cell and neutrophil counts in bal fluid to a significant lower level (figures 3(a) and 3(b), p <0.05). To further assess the anti - inflammatory effect of pgrn we found that proinflammatory cytokines, including tnf-, il-1, and il-6, as well as chemokines including cxcl2, je (the murine homolog of human ccl2), and kc (the murine homolog of human il-8), were all significantly elevated in bal fluid in response to lps challenge (figures 3(c) and 3(d), p <0.05). In contrast, administration of pgrn effectively decreased the levels of proinflammatory cytokines and chemokines (figures 3(c) and 3(d), p <0.05). Consistent to the above findings, administration of pgrn twice at 40 h intervals further reduced the proinflammatory cytokines and chemokines to a significant lower level in bal fluid (figures 3(c) and 3(d), p <0.05). We next determined the concentrations of total protein, albumin, and igm in bal fluid to evaluate the integrity of the alveolar - capillary membrane barrier and assess pulmonary vascular leakage as a marker for ali . As shown in figures 4(a)4(c), we found that the levels of total protein, albumin, and igm in bal fluid were all significantly increased in mice challenged with lps compared with that in the control mice (p <0.05). Whereas treatment with pgrn effectively reduced total protein, albumin, and igm levels (figures 4(a)4(c), p <0.05). Notably, administration of pgrn twice at 40 h intervals restored these lung injury indicators to levels similar to the control mice (figures 4(a)4(c)). To evaluate the potential role of pgrn in the histopathology changes of lung in lps - induced ali mice, histological assessment of lung sections 3 days after the administration of lps with or without treatment was performed . We revealed the marked inflammatory infiltrates, interalveolar septal thickening, and interstitial edema in lps - induced ali mice (figure 5(a)). Administration of pgrn effectively reduced the airspace inflammation, which was more apparent in mice treated with pgrn twice at 40 h intervals (figure 5(a)). Furthermore, severity of lung injury was also scored using a semiquantitative histopathology score system [5, 21], which evaluates lung injury in four categories: alveolar septae, alveolar hemorrhage, intra - alveolar fibrin, and intra - alveolar infiltrates . We found that treatment with pgrn could significantly reduce lung injury scores, which was more apparent in mice treated with pgrn twice at 40 h intervals (figure 5(b), p <0.05). Recent findings suggested that pgrn could bind to tnfr and thus mediate its anti - inflammatory effects in collagen antibody - induced arthritis and collagen - induced arthritis . Therefore, we next assessed the possible role of pgrn / tnfr interaction in the protective effect of pgrn on lps - induced ali . Groups of mice were pretreated with neutralizing antibodies to tnfr1 or tnfr2, respectively, and then challenged with lps with or without pgrn treatment . As shown in figures 6(a)6(d), we found that neutralization of tnfr1 had no significant influence on the protective effect of pgrn on the lps - induced ali as evidenced by similar levels of total inflammatory cell count, proinflammatory cytokines, albumin, and igm in bal fluid . In contrast, blockade of tnfr2 significantly abrogated the protective effect of pgrn on the lps - induced ali as evidenced by elevated levels of total inflammatory cell count, proinflammatory cytokines, albumin and igm in bal fluid (figures 6(a)6(d), p <0.05). Consistently, we found that blockade of tnfr2 but not tnfr1 could effectively inhibit the protective effect of pgrn on the histopathology changes of lung in lps - induced ali mice (figures 6(e) and 6(f), p <0.05). Similar results were also obtained in mice treated with pgrn twice at 40 h intervals (data not shown). These findings suggested that pgrn / tnfr2 interaction was crucial for the protective effect of pgrn on lps - induced ali . Ards is a complex clinical syndrome that is initiated by injury to the lung, often in the setting of pneumonia or sepsis . Here we carefully evaluated the potential role of pgrn in treatment of ali using the murine model of lps - induced ali . We found that administration of pgrn effectively maintained the body weight and survival of lps - induced ali mice . Furthermore, pgrn administration significantly reduced lps - induced pulmonary inflammation and resulted in remarkable reversal of lps - induced increases in lung permeability . Moreover, administration of pgrn contributed to a significant reduction of histopathology changes in lung of lps - induced ali mice . Our results provided clues for developing pgrn - based therapies to treat with ali . Accumulating data suggested that pgrn played an important role in inflammatory response [15, 23, 24]. Here we evaluated the expression of pgrn protein in bal fluid of lps - induced ali mice . We found that the level of pgrn protein in bal fluid was significantly downregulated 3 days after lps challenge in lps - induced ali mice . Previous study showed that during inflammation, neutrophils, and macrophages released proteases which digested pgrn into individual 6 kda granulin units, which were actually proinflammatory and could neutralize the anti - inflammatory effects of intact pgrn [23, 24], which might partly explain the decreased level of pgrn protein in bal fluid of lps - induced ali mice . Consistently, we indeed revealed an elevated expression of granulin, which was the units of pgrn, in bal fluid in lps - induced ali mice . However, the precise mechanism underlies the downregulation of pgrn in bal fluid of lps - induced ali still remains to be elucidated . In the present study our findings suggested that pgrn was a key regulator of inflammation and exerted an anti - inflammatory effect, which were in line with previous studies . As the half - life time for pgrn is about 40 hours, we further performed the second injection of ali mice with pgrn at 40 h intervals, and found that this strategy resulted in a more apparent reduction of the development of lps - induced ali . It should be pointed out that we did not observe any significant effect of pgrn alone on the lung injury of nave mice in this study (data not shown). Our data strongly suggested that pgrn was an optimistic candidate for the treatment of ali . However, the lps - induced model of ali cannot fully reproduce the complexity of clinical ali / ards in human patients . Therefore, it is necessary to reproduce these findings in more clinically relevant models . Besides, it is important to define the therapeutic window of pgrn intervention for ali at different dose and time points . In addition, it is also important to explore the possible effect of pgrn administration on host immune response in ali . The translation of our results into an effective new therapy for ards in patients will require, at the very least, that these issues be addressed . Tnf-/tnfr signaling has received great attention due to its position at the apex of the proinflammatory cytokine cascade and its dominance in the pathogenesis of various disease processes [2528]. Previous study showed that pgrn could bind to tnfr and then block the tnf- binding to its receptors . In this study, we evaluated the potential role of pgrn / tnfr interaction in the protective effect of pgrn on lps - induced ali . We demonstrated that blockade of tnfr2 but not tnfr1 could significantly inhibit the protective effect of pgrn on the lps - induced ali . In addition, we found that neutralization of tnfr1 or tnfr2 had no significant effect on the total cell response of ali mice (data not shown). Our findings were consistent with previous study which showed that tnfr2 seemed to play an important role in ards . One is that tnfr1 is expressed ubiquitously, whereas tnfr2 expression is tightly regulated and found predominantly in hematopoietic cells [30, 31]. However, the precise mechanism for the effect of pgrn on the development of lps - induced ali undoubtedly needed successive studies . In the present study, we demonstrated a murine model of ali that administration of pgrn effectively prevented the development of lps - induced ali, at least in part, through their interaction with tnfr2 . These findings might have potentially important implications for the treatment of ards, a clinical syndrome resulting from ali in human.
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Pityriasis rubra pilaris (prp) includes a spectrum of rare chronic, idiopathic inflammatory disorders with papulosquamous eruptions of unknown cause . Prp shows consistent clinical heterogeneity; consequently, it is hard to predict the outcome of treatment . A 29-year - old female presented for evaluation of a skin condition previously diagnosed as psoriasis vulgaris . The lesions first appeared at age six years with no preceding trauma or infection and were characterized by palmoplantar keratoderma and demarcated, hyperkeratotic plaques on the elbows and knees . The patient had been on many systemic treatments over the last two years without response, including cyclosporine (3 mg / kg / day for 3 months), methotrexate (15 mg / weekly for 4 months), adalimumab (two subcutaneous injection of 40 mg at day 0, a subcutaneous injection of 40 mg at day 7 and every 14 days thereafter for 16 weeks), etanercept (50 mg weekly subcutaneous injections for 12 weeks), infliximab (5 mg / kg given as an intravenous infusion at weeks 0, 2, 8). Physical examination showed a diffuse, orange - pink palmoplantar keratoderma [figure 1]. Well - defined keratotic follicular papules also involved the dorsal aspects of the hands and feet, elbows and knees [figures 2 and 3]. A skin biopsy revealed alternating parakeratosis and orthokeratosis, pronounced irregular acanthosis, focal hypergranulosis and mild focal spongiosis . Based on clinical and histologic findings, the patient was diagnosed having prp, type iv (circumscribed juvenile). After voluntary, informed consent, ustekinumab 45 mg subcutaneously at weeks 0 and 4, and quarterly thereafter (patient's weight = 55 kg) was then started, the same posology as in psoriasis . Keratoderma of the hands with a sharp demarcation of the borders diffuse transgrediens palmoplantar keratoderma on the dorsum of hands follicular hyperkeratosis and erythema on the elbows alternating parakeratosis and orthokeratosis, irregular acanthosis, focal hypergranulosis and a perivascular lymphocytic infiltration in the papillary dermis (h and e 10) pityriasis rubra pilaris occurs equally in male and female patients, with a bimodal age distribution, peaking during the first and then the sixth decade . Griffiths proposed a classification for prp in five subtypes, based upon age, duration, and type of cutaneous involvement . Type i, or classic prp, is the commonest type (50% of cases) and occurs in adults . The patient is usually erythrodermic with diffuse thickening of the palms and soles and possibly ectropion . 80% of patients experience clinical resolution within 3 years . On the basis of griffith's classification clinical manifestations occurred in her prepubertal age and relapsed at age 27, after a long - lasting remission . Type iv prp develops in prepubertal children presenting with sharply - demarcated areas of follicular hyperkeratosis and erythema on the elbows and knees . A waxy, orange - red, diffuse, palmoplantar keratoderma is also commonly observed . A standard therapeutic approach does not exist as cases are few and treatment is protracted . An increasing number of reports document the effectiveness of tumor necrosis factor- (tnf-) blockers in recalcitrant prp . Further, some case reports have documented favorable response of prp to ustekinumab, a fully human monoclonal antibody which binds to interleukin-12 (il-12) and il-23 with high specificity and affinity . An upregulation of tnf mrna in lesional compared with nonlesional skin in two patients with type i prp has been demonstrated . A recent retrospective revision investigating treatment options showed a marked clinical in more than 50% of patients with type, a systematic review of reports of prp responding positively to tnf- blockers does not recommend them due to possible reporting bias and spontaneous remissions . In the literature, the patients who achieved remission with tnf- blockers or ustekinumab were all consistent with classical type 1 prp . Our patient who presented a type iv prp was unresponsive to the tnf - blockers adalimumab, etanercept and infliximab . Our clinical experience suggests that in patients with type iv prp, tnf and il-12/il-23 blockade may not be useful targets . Since we observed a primary lack of response to all available tnf - blockers, we suggest that yet unmapped signaling pathways may be involved in type iv prp . There seems to be inadequate response of circumscribed variants of prp to standard therapies that are effective in classical prp.
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Elderly depressed people who experience cognitive impairment or dementia need to be assessed using special instruments . The cornell scale for depression in dementia (csdd) is considered one of the best tools to use with cognitively impaired residents, and is thus widely used in nursing home and long - term care (ltc) facilities.1 the validity of the csdd has been investigated and substantiated, including cross - culturally.15 one of the important features of the csdd is that informants other than the residents themselves can be used to provide the data necessary for diagnosis; having agreement between sources of information is important, though only a few studies have addressed this issue.6,7 the csdd comes in two formats: one for residents, and the other for caregivers, proxies, or nurses . . The greater the level of agreement between items, the greater the potential level of overall interrater reliability and in turn increased likelihood of validity . A number of studies have focused on the level of agreement between residents and caregivers; for example, schreiner and morimoto3 and amuk et al8 found little agreement between residents and caregivers for the items retardation and mood - congruent delusion in either depressed or nondepressed residents . With regard to the level of agreement as it might vary with the cognitive status of the individual, towsley et al9 found a significant score discrepancy between residents and nurse proxies in that nurses evaluated residents as less depressed than residents evaluated themselves . They did find that the overall level of agreement between the nurses and residents csdd scores was poor (r = 0.16). This is similar to the findings of burrows et al.7 these authors felt that the lack of strong agreement was, in part at least, a function of underreporting of symptoms in individuals with dementia; relying on nurse - proxy judgments may further worsen the problem of underdetection of depressive symptoms.7,9 the csdd s interitem correlation is also of interest . Csdd factors are drawn from consensus based on clinically relevant syndromes, rather than from statistical reasoning.1 harwood et al found that a four - factor solution accounted for 43.1% of the overall variance: general depression (lack of reactivity to pleasant events, poor self - esteem, pessimism, loss of interest, physical complaints, psychomotor retardation, and sadness), rhythm disturbances (difficulty falling asleep, multiple night awakenings, early morning awakenings, weight loss, and diurnal variations in mood), agitation / psychosis (agitation, mood - congruent delusions, and suicide), and negative symptoms (such as appetite loss, weight loss, a lack of energy, a loss of interest, and a minimal reaction to pleasant events).10 ownby et al found that the csdd revealed the same four - factor structure, but also found differences in factor content for several items across hispanic and anglo - saxon cultures.4 in asia, schreiner and morimoto examined use of the csdd with japanese poststroke residents and found a four - factor solution with some items loaded on to an undesignated factor, with the most prevalent symptoms being anxiety, irritability, and sadness, and the least prevalent being multiple physical complaints, appetite loss, and mood - congruent delusions.3 lin and wang, studying 147 residents from institutionalized care facilities in taiwan, found anxiety, sadness, and a subscale of ideational disturbance loaded onto the same factor, while a lack of some items linked to mood - related signs and behavioral disturbance were not loaded onto the expected factors.5 barca et al examined a large sample of 1159 residents and found a five - factor solution, as hypothesized; this is in contrast to the results reviewed above.11 it should be noted that several items did not load onto their designated factors.11 what does all of this lead to? As we know, the greater the csdd score, the more severe the depressive condition; if some items are not relevant to the scale s results (low interitem correlation), this may increase error and subsequently affect the score . This will in turn potentially adversely affect both the reliability of the scale and its accuracy (assessing by the area under the curve).12 since the csdd concept uses both interrater and interitem agreement to develop a final score, as given by a clinician, both procedures are deemed important . The aims of our study were: (1) to explore the level of interrater agreement between the csdd items, as rated by residents and caregivers, and (2) to compare the theoretical csdd model with alternative models using this sample . In this study, we carried out a secondary analysis of data derived from a cross - sectional study into the prevalence of major depressive disorder (mdd) in an ltc facility in chiang mai, thailand.13 data were collected between march and june of 2011 . The study was approved by the ethics committee of the faculty of medicine, chiang mai university . In the study, a trained research nurse performed a diagnostic interview in order to evaluate for psychiatric and cognitive function disorders using the thai version of the mini - international neuropsychiatric interview (mini) version 5.0, and the minimental state examination thai 2002 instrument (mmse - thai 2002).14,15 the interviews were conducted separately for each resident and caregiver; therefore, the interviewees were not aware of each other s responses . Eighty - four of the 111 residents in the ltc during the study period agreed to participate in the study, and all gave informed consent . Seven refused to participate, eight had active physical or psychiatric diseases that became an obstacle to interviewing, seven were unable to give intelligible answers, and five provided incomplete data . The final set of participants included 84 residents aged 60 years and over, and their five nonprofessional caregivers (ltc facility staff members). The mini instrument, as developed by sheehan et al,16 was used here as the standard for diagnosing diagnostic and statistical manual of mental disorders (fourth edition) major depression, while the thai version was translated and validated by kittiratanapaiboon and khamwongpin.17 it has kappa ranges of between 0.27 and 0.87 to suggest the presence of depressive disorders . The research nurse administered the mini across all participants and was not aware of the results of the csdd carried out by each patient . The csdd is divided into five subscales: (1) mood - related signs (anxiety, sadness, a minimal reaction to pleasant events, and irritability), (2) behavioral disturbance (agitation, psychomotor retardation, multiple physical complaints, and a loss of interest), (3) physical signs (a loss of appetite, weight loss, and a lack of energy), (4) cyclic functions (diurnal variation in mood, difficulty falling asleep, multiple awakenings during the night, and early morning awakening), and (5) ideational disturbance (suicidal tendencies, low self - esteem, pessimism, and mood - congruent delusions).1 it contains nineteen questions, each of which can be given a score ranging from 0 (absent) to 2 (severe), or a score the total score ranges from 0 to 38; a higher score denotes greater levels of depression . The csdd scale was carried out by a trained psychogeriatric research nurse, who interviewed each resident and each caregiver within the same day . The thai version of the csdd was developed following the translation and cultural adaptation method, a process that included an initial translation into thai by a geriatric psychiatrist (nw), followed by a backward translation into english by a bilingual translator (in this case, a university professor) who had no prior knowledge of the questionnaire.18 the two versions produced were then assessed and compared item by item by the authors and the bilingual translator . The resulting final draft was then field - tested with depressed elderly residents in a geriatric outpatient clinic (this group was not involved with the current study). The mmse - thai 2002 was modified from the original version by folstein et al to measure cognitive impairment in thai people.15,19 information regarding the participants level of education is required for the interpretation of cognitive impairment or dementia of this version of mmse . The cutoff score for cognitive impairment or dementia is 22 . For those who did not complete elementary schooling the total score for participants who are illiterate is 23, and for these participants the cutoff score is 14 . Means and standard deviations were used, and t - tests were employed when comparing the two groups . Intraclass correlation coefficients (iccs) were calculated in order to determine the level of absolute agreement between the scores for each item given by the residents and by the caregivers, using agreestat software version 2011.3 (advanced analytics, gaithersburg, md, usa). Confirmatory factor analysis (cfa) was used to compare the hypothesized five - factor model and an alternative model, as follows: (1) a five - factor model as per the original theoretical construct, (2) a four - factor model in which factor a (mood - related signs) and factor e (ideational disturbance) were collapsed into a single factor (as supported by prior studies, reviewed above). The fit statistics used included a comparative fit index score of 0.95, nonnormed fit index or tucker lewis index values of 0.9, a root - mean - square error of approximation of 0.06 with values as high as 0.08 indicating a reasonable fit, a standardized root - mean - square residual of 0.08, as well as the results of equation /degrees of freedom <3.2023 amos 18 (ibm, armonk, ny, usa) was used for the analysis.24 the level of reliability of the model s internal consistency was assessed using cronbach s alpha . Missing data were found in the response of only one of the caregivers, for whom three of the csdd items were missing . Eighty - four of the 111 residents in the ltc during the study period agreed to participate in the study, and all gave informed consent . Seven refused to participate, eight had active physical or psychiatric diseases that became an obstacle to interviewing, seven were unable to give intelligible answers, and five provided incomplete data . The final set of participants included 84 residents aged 60 years and over, and their five nonprofessional caregivers (ltc facility staff members). The mini instrument, as developed by sheehan et al,16 was used here as the standard for diagnosing diagnostic and statistical manual of mental disorders (fourth edition) major depression, while the thai version was translated and validated by kittiratanapaiboon and khamwongpin.17 it has kappa ranges of between 0.27 and 0.87 to suggest the presence of depressive disorders . The research nurse administered the mini across all participants and was not aware of the results of the csdd carried out by each patient . The csdd is divided into five subscales: (1) mood - related signs (anxiety, sadness, a minimal reaction to pleasant events, and irritability), (2) behavioral disturbance (agitation, psychomotor retardation, multiple physical complaints, and a loss of interest), (3) physical signs (a loss of appetite, weight loss, and a lack of energy), (4) cyclic functions (diurnal variation in mood, difficulty falling asleep, multiple awakenings during the night, and early morning awakening), and (5) ideational disturbance (suicidal tendencies, low self - esteem, pessimism, and mood - congruent delusions).1 it contains nineteen questions, each of which can be given a score ranging from 0 (absent) to 2 (severe), or a score (result) of symptoms not possible to evaluate . The total score ranges from 0 to 38; a higher score denotes greater levels of depression . The csdd scale was carried out by a trained psychogeriatric research nurse, who interviewed each resident and each caregiver within the same day . The thai version of the csdd was developed following the translation and cultural adaptation method, a process that included an initial translation into thai by a geriatric psychiatrist (nw), followed by a backward translation into english by a bilingual translator (in this case, a university professor) who had no prior knowledge of the questionnaire.18 the two versions produced were then assessed and compared item by item by the authors and the bilingual translator . The resulting final draft was then field - tested with depressed elderly residents in a geriatric outpatient clinic (this group was not involved with the current study). The mmse - thai 2002 was modified from the original version by folstein et al to measure cognitive impairment in thai people.15,19 information regarding the participants level of education is required for the interpretation of cognitive impairment or dementia of this version of mmse . The cutoff score for cognitive impairment or dementia is 22 . For those who did not complete elementary schooling the total score for participants who are illiterate is 23, and for these participants the cutoff score is 14 . The mini instrument, as developed by sheehan et al,16 was used here as the standard for diagnosing diagnostic and statistical manual of mental disorders (fourth edition) major depression, while the thai version was translated and validated by kittiratanapaiboon and khamwongpin.17 it has kappa ranges of between 0.27 and 0.87 to suggest the presence of depressive disorders . The research nurse administered the mini across all participants and was not aware of the results of the csdd carried out by each patient . The csdd is divided into five subscales: (1) mood - related signs (anxiety, sadness, a minimal reaction to pleasant events, and irritability), (2) behavioral disturbance (agitation, psychomotor retardation, multiple physical complaints, and a loss of interest), (3) physical signs (a loss of appetite, weight loss, and a lack of energy), (4) cyclic functions (diurnal variation in mood, difficulty falling asleep, multiple awakenings during the night, and early morning awakening), and (5) ideational disturbance (suicidal tendencies, low self - esteem, pessimism, and mood - congruent delusions).1 it contains nineteen questions, each of which can be given a score ranging from 0 (absent) to 2 (severe), or a score the total score ranges from 0 to 38; a higher score denotes greater levels of depression . The csdd scale was carried out by a trained psychogeriatric research nurse, who interviewed each resident and each caregiver within the same day . The thai version of the csdd was developed following the translation and cultural adaptation method, a process that included an initial translation into thai by a geriatric psychiatrist (nw), followed by a backward translation into english by a bilingual translator (in this case, a university professor) who had no prior knowledge of the questionnaire.18 the two versions produced were then assessed and compared item by item by the authors and the bilingual translator . The resulting final draft was then field - tested with depressed elderly residents in a geriatric outpatient clinic (this group was not involved with the current study). The mmse - thai 2002 was modified from the original version by folstein et al to measure cognitive impairment in thai people.15,19 information regarding the participants level of education is required for the interpretation of cognitive impairment or dementia of this version of mmse . The cutoff score for cognitive impairment or dementia is 22 . For those who did not complete elementary schooling the total score for participants who are illiterate is 23, and for these participants the cutoff score is 14 . Means and standard deviations were used, and t - tests were employed when comparing the two groups . Intraclass correlation coefficients (iccs) were calculated in order to determine the level of absolute agreement between the scores for each item given by the residents and by the caregivers, using agreestat software version 2011.3 (advanced analytics, gaithersburg, md, usa). Confirmatory factor analysis (cfa) was used to compare the hypothesized five - factor model and an alternative model, as follows: (1) a five - factor model as per the original theoretical construct, (2) a four - factor model in which factor a (mood - related signs) and factor e (ideational disturbance) were collapsed into a single factor (as supported by prior studies, reviewed above). The fit statistics used included a comparative fit index score of 0.95, nonnormed fit index or tucker lewis index values of 0.9, a root - mean - square error of approximation of 0.06 with values as high as 0.08 indicating a reasonable fit, a standardized root - mean - square residual of 0.08, as well as the results of equation /degrees of freedom <3.2023 amos 18 (ibm, armonk, ny, usa) was used for the analysis.24 the level of reliability of the model s internal consistency was assessed using cronbach s alpha . Missing data were found in the response of only one of the caregivers, for whom three of the csdd items were missing . Table 1 shows the demographic data, descriptive clinical data (eg, mdd diagnosis), and the csdd scores . Prior to admission to the facility, 34.5% were single, 8.3% were married, 2.4% were separated, 3.6% were divorced, and 63% were widowed . Most had an elementary school - level education, with a mean of 3.5 years in school . The mean and standard deviation of the mmse scores were 18.2 and 7.0, respectively, and 42% met the criteria for cognitive impairment, according to their level of education . Twenty - five of the 84 participants (29.8%) had mdd (see table 1), as diagnosed with the mini . It was found that the mean resident total csdd scores were not significantly different from those of the caregivers (mean [standard deviation] = 5.1 [4.51] vs. 4.46 [4.33], t = 1.51, p = 0.136). Alpha for the entire group was found to be 0.87, while for residents and caregivers only, the scores were 0.84 and 0.86, respectively . In order to compare the level of agreement for each csdd item response given by the residents and caregivers, icc was calculated for all participants, revealing a fair - to - moderate agreement for 14 of the items (between 0.3 and 0.6). In the cognitively impaired group, eleven items were found to have significant agreement (sadness, irritability, retardation, multiple physical complaints, loss of interest, lack of energy, difficulty falling asleep, multiple awakenings, suicidal ideation, self - deprecation, and mood - congruent delusions) compared to only five items in the cognitively intact group (irritability, retardation, multiple physical complaints, early morning awakening, and self - deprecation; table 3). In terms of the total csdd score, there was strong agreement (icc = 0.71) between caregivers and residents in the cognitively impaired group, but only a fair level of agreement in the cognitively intact group (icc = 0.32). Before comparing the models, the factor - analytic study results from the present study (using residents and caregivers combined) were compared with five previous findings.3,5,10,11,20 all studies used factor analysis with the principal - components method and a varimax rotation . Items 58 from factor b (behavioral disturbance) appear to be incorrectly loaded, especially item 7 . All the studies found that item 7 was not loaded onto factor b (behavioral disturbance), as expected, and four out of the six studies found item 7 was not loaded onto any factor (see the items and the factors in bold). Disagreement was also found in items 3, 4, 9, 12, 16, and 19 . All studies came up with a five - factor solution, except for harwood10 and kurlowicz26 (four - factor); however, these five - factor solutions were not the same as that produced by alexopoulos,1 since factors a (mood - related signs) and e (ideational disturbance) were loaded onto the same factor, while the fifth factor was comprised of the problematic items mentioned above . In fact, the fifth factor was loaded with different items, those that varied from study to study . It should be noted that item 4 (irritability) and item 5 (agitation) were loaded onto the same factor across all the studies (see items highlighted in grey). Item 19, mood - congruent delusions, generally loaded on factor a, while in the present study it did not load on any factor . Both the five - factor and four - factor models were found to be similar and displayed a poor fit to the data, as they were not able to reach threshold criteria (comparative fit index = 0.760.79, tucker lewis index = 0.720.75, root - mean - square error of approximation = 0.0890.094, and standardized root - mean - square residual = 0.0900.097). In order to compare the level of agreement for each csdd item response given by the residents and caregivers, icc was calculated for all participants, revealing a fair - to - moderate agreement for 14 of the items (between 0.3 and 0.6). In the cognitively impaired group, eleven items were found to have significant agreement (sadness, irritability, retardation, multiple physical complaints, loss of interest, lack of energy, difficulty falling asleep, multiple awakenings, suicidal ideation, self - deprecation, and mood - congruent delusions) compared to only five items in the cognitively intact group (irritability, retardation, multiple physical complaints, early morning awakening, and self - deprecation; table 3). In terms of the total csdd score, there was strong agreement (icc = 0.71) between caregivers and residents in the cognitively impaired group, but only a fair level of agreement in the cognitively intact group (icc = 0.32). Before comparing the models, the factor - analytic study results from the present study (using residents and caregivers combined) were compared with five previous findings.3,5,10,11,20 all studies used factor analysis with the principal - components method and a varimax rotation . Items 58 from factor b (behavioral disturbance) appear to be incorrectly loaded, especially item 7 . All the studies found that item 7 was not loaded onto factor b (behavioral disturbance), as expected, and four out of the six studies found item 7 was not loaded onto any factor (see the items and the factors in bold). Disagreement was also found in items 3, 4, 9, 12, 16, and 19 . All studies came up with a five - factor solution, except for harwood10 and kurlowicz26 (four - factor); however, these five - factor solutions were not the same as that produced by alexopoulos,1 since factors a (mood - related signs) and e (ideational disturbance) were loaded onto the same factor, while the fifth factor was comprised of the problematic items mentioned above . In fact, the fifth factor was loaded with different items, those that varied from study to study . It should be noted that item 4 (irritability) and item 5 (agitation) were loaded onto the same factor across all the studies (see items highlighted in grey). Item 19, mood - congruent delusions, generally loaded on factor a, while in the present study it did not load on any factor . Both the five - factor and four - factor models were found to be similar and displayed a poor fit to the data, as they were not able to reach threshold criteria (comparative fit index = 0.760.79, tucker lewis index = 0.720.75, root - mean - square error of approximation = 0.0890.094, and standardized root - mean - square residual = 0.0900.097). The csdd demonstrated a moderate level of interrater agreement between residents and caregivers, and was more reliable when used with cognitively impaired residents . These data support the use of proxy informants (eg, staff members) in the assessment of depression utilizing the csdd in elderly populations, particularly those with cognitive impairment . The cfa indicated a poorly fitting model, suggesting that the hypothesized model was not a good fit with the observed data . We found a few studies that compared caregiver and resident ratings using the csdd in a nursing home setting.3,9,27 compared to towsley et al,9 we found differences in three out of the five subscale scores and between the two groups of informants, even though there was no difference in the mean total scores . It may be that this is attributable to caregivers (or nurses) limitations in observing the residents signs and symptoms . Our findings yielded a fair level of agreement in the mean total scores (between the residents and caregivers), which were consistent with the results of schreiner and morimoto.3 the outstanding feature of the csdd was a strong level of agreement between the answers given by cognitively impaired residents and those given by caregivers, denoting that information given by the caregivers was similar to that given by the group of residents . This finding supports the use of a proxy, as suggested by leontjevas et al.6 this may be explained by the fact that in such settings, caregivers may pay more attention to those residents who are obviously vulnerable, such as those who have physical disabilities or cognitive problems (regardless of whether these conditions are caused by dementia or depression). Likewise, stroke residents suffering from cognitive disability may tend to be more closely observed, leading to a high number of significant correlations and a high level of agreement between the caregivers and the residents in terms of either their behavioral (eg, retardation) or psychological symptoms (eg, sadness, self - deprecation, and suicidal ideation).3 this clearly demonstrates the distinctive benefit of the csdd in detecting depression in residents or patients with cognitive impairment . The cfa also underscored how poor some factors are in relation to the whole set of items, and this confirms the previous findings of leontjevas et al6 and schreiner and morimoto.3 the item multiple physical complaints does not distinguish well between depressed and nondepressed residents; in fact, it was shown either not to load to any factor at all, or to load to an unintended factor.3,28 harwood et al, meanwhile, found it was loaded on to general depression, which comprises a mix of items: lack of reactivity to pleasant events, poor self - esteem, pessimism, loss of interest, physical complaints, psychomotor retardation, and sadness.10 this finding was also supported by a study carried out in asia by lin and wang, in which cross - loadings for the items of irritability, multiple physical complaint, and loss of interest were evident.5 even though multiple physical complaints are a common symptom among elderly depressed thais, this item may not be helpful in differentiating depression from nondepression, and in fact may contribute to poor construct validity within the csdd.29,30 even in a larger sample size, as studied by barca et al and kurlowicz et al, the same problem with this item (multiple physical complaints) was found.11,26 it is interesting to note that in addition to multiple physical complaints, both studies also found evidence of cross - loading for loss of interest, and for diurnal variation in mood . These latter findings were also found in studies using a smaller sample size.3,10 all studies found it difficult to differentiate between the items under factor e (ideational disturbance) and those under factor a (mood - related signs), as both these sets of symptoms reflect the presence of core depression, as found in ownby et al s and schreiner and morimoto s studies.3,4 mood - congruent delusion was found to be loaded in an inconsistent way depending on the nature of the sample . The level of depression in our sample may not have been so severe as to make this item successfully exhibit discriminatory performance, and it tends to perform better with a larger sample or with a sample with different characteristics, as found in other studies . The use of a small sample with low levels of depression impacts on the generalizability of the results of this study; however, there is some evidence from a number of findings (table 5) to suggest that the csdd may have better construct validity and model fit with minor revising, ie, grouping irritability and agitation together (regardless of what factor they are in), and taking out multiple physical complaints and diurnal variation in mood, since they are less likely to be able to measure actual depression . Multiple physical complaints, diurnal variation in mood, and mood - congruent delusion are important and relevant in specific clinical cases . If they are retained within the csdd scale for this reason, we suggest excluding these items from any calculation of the total csdd score during the screening or measuring for outcome of depression (due to the finding of lack of discriminatory power and potential for reduction in accuracy). The strength of this study is that we used only trained geriatric nurses to conduct the interviews with both the residents and caregivers . Using this method helped shed light on differences between informants when interviewed; however, this method may also be viewed as representing bias, as it may have inflated the level of agreement shown . First, the sample size was relatively small, which precluded precision in terms of the point estimates and limited the study s power, which may ultimately have impacted upon the factor structure . In addition, a test retest analysis was not performed to ensure this type of reliability . First, the sample size was relatively small, which precluded precision in terms of the point estimates and limited the study s power, which may ultimately have impacted upon the factor structure . In addition, a test retest analysis was not performed to ensure this type of reliability . The csdd demonstrated moderate interrater agreement between residents and caregivers, and was more reliable when used in cognitively impaired residents . Neither the five - factor model, as per the original theoretical construct, nor the four - factor model, in which factor a (mood - related signs) and factor e (ideational disturbance) were collapsed into a single factor, demonstrated a good fit
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Teratoma is a special type of mixed tumor that contains recognizable mature or immature cells or tissues representative of more than one germ cell layer and sometimes all three . Teratomas can develop in almost any area of the body, but usually occur in median sites . The most common sites are the sacrococcyx, anterior mediastinum, testicle, ovary, or retroperitoneum . 1 day old male baby was referred for respiratory distress secondary to the presence of an oropharyngeal mass . There no other abnormalities found in the head and neck region . Under general anesthesia the lesion was excised . On the gross pathological examination, the excisional material was a polypoid lesion of 3 2 1.5 cm in size whose surface was covered with skin (fig . Histologically, the mass consisted of a core of lobules of mature adipose tissue admixed with fascicles of striated skeletal muscle and covered by keratinized squamous epithelium containing many hair follicles associated with sebaceous and eccrine glands (fig . These findings confirmed the diagnosis of nasopharyngeal mature teratoma also known as congenital hairy polyp . . Teratomas generally occur in 1 of 4000 births, but only 10% are found in the head and neck . They most commonly arise from the midline or lateral nasopharyngeal wall and some reports have described a slight female predilection, but jiang et al . Found malignant teratoma to be more common in men . The majority of neonates with nasopharyngeal teratomas can be successfully intubated either via the oral or nasal routes, whereas the remainder usually require tracheostomy . In 6% of all cases, teratomas are associated with malformations such as cleft palate, bifid tongue, and bifid uvula . Most of the congenital nasopharyngeal lesions present as an emergency respiratory problem and require immediate surgery . Benign ones consist of mature tissue components, while those with malignant potential contain immature tissues; benign teratomas may undergo malignant change with age . Histopathological examination showed the mass in our case to be composed of multiple mature tissues with no juvenile cells, which confirmed a benign tumor . The main therapy of teratoma is complete surgical excision, which depends on the site of the tumor . Patients with teratomas are more likely to require intensive airway management prior to surgical excision of the lesion . Written informed consent was obtained from the patient's parent for publication of this case report and accompanying images . A copy of the written consent is available for the review of the editor - of - chief of this journal on request.
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Asthma is an enormous public health issue with 8.5% of people in the us diagnosed with asthma . Asthma disproportionately impacts the young . According to the 2011 national health interview survey,> 10 million children under age 18 years in the us or 14% have been diagnosed with asthma in their lifetime . This public health burden leads to missed school days for children and work days for adults, demands on families to manage asthma care for their children, and increased health care costs . The average annual health care cost for a child being treated for asthma exceeded $835 for prescription medications alone in 20072008, a large increase from the $350 spent in 19971998 . Others have quantified the costs associated with asthma, finding that annually> $50 billion in health care costs, almost $4 billion in lost work and school days, and> $2 billion from premature deaths is spent on a national basis in usa . The $56 billion total costs of asthma in that analysis in 2007 was almost $3 billion greater than the estimate from 2002 . Airborne allergen exposures and viral infections are indicated as the two major environmental contributors to the development and/or exacerbation of asthma . Reducing exposures to allergy and asthma triggers in residential settings is an important goal in treating asthma patients, since americans spend 70% of their time indoors at home . Asthma guidance for health care providers includes recommendations for controlling airborne allergens in the home . Most interventions focus on housekeeping activities, such as using high - efficiency particulate air (hepa) vacuum cleaners, improved bedding covers and laundering and even use of high - efficiency portable air cleaners . While many studies have been conducted on interventions to reduce asthma triggers, only a few evaluated the use of higher efficiency media filters in central ventilation systems . This gap in the literature is significant because central heating, ventilation and air conditioning (hvac) systems move a lot of air through and within homes, and 75% of homes in the us have ventilation systems with forced air for heating or cooling or both . The high prevalence of central ventilation systems presents an opportunity for improving mitigation of asthma triggers by filtration since high system airflow rates combined with the use of efficient filters are the two main requirements needed for this tactic to be effective . The goal of the current work was to assess whether readily available in - duct filtration in the form of better performing filters can have a measurable impact on reducing asthma triggers in the home . We compare modeling results to measurement studies and assess expected benefits of using filters with higher removal efficiencies . We conducted detailed modeling of aerosol concentrations typically found in residential indoor air to evaluate the expected in - use performance of six different types of filters . Four types of health - relevant aerosols in the indoor air of residences were modeled in this study: cat allergen, pm2.5 generated from indoor sources, pm2.5 of outdoor origin that infiltrates homes, and respiratory virus . A visual representation of the modeling analysis conducted in this report is depicted in figure 1 . Using contam, a widely accepted, peer - reviewed multi - zone indoor air quality model, we ran simulations over an entire year in representative homes in atlanta and chicago to evaluate changes in indoor concentrations of each aerosol under different filter intervention scenarios as well as varying indoor and outdoor conditions, including air exchange rates, using the energy plus model; [13; explained in supplementary material]. The performance of the contam model has been validated in many previous studies [1418].figure 1.flow diagram of modeling analysis . We conducted this modeling analysis on one attached and one detached sample home in each city . Characteristics are listed in supplementary table s3, and floor plans are provided in supplementary figures s3 and s4 . The overall results were similar between cities and for both home types, so only the results for the atlanta detached home are presented here . Supplementary table s5 lists the results comparing attached and detached dwellings in atlanta and chicago . The modeling simulations were conducted for a typical single - family residence in two specific geographic locations . One was representative of significant traffic - related pm2.5 and was located in an urban residential climate zone corresponding to chicago, illinois . The other was representative of an area with significant seasonal pollen and was representative of a suburban residential home corresponding to the atlanta, georgia, climate zone . Meteorological information is used by contam to simulate forced convection, radiant leakage and corresponding air exchange rates . As described previously, we used typical meteorological year (tmy3) data sets derived from 1991 to 2005 meteorological data . The meteorological data included hourly wind direction and speed, dry and wet bulb temperature, relative humidity and cloud cover data . Meteorological data for each of the locations was taken from the airport closest to that city . Jackson atlanta international airport . For each of the two cities modeled, two different contam building templates were used . The first was a template for a single family, detached home (dh72). The second was a template for a single family, attached home (ah41). Census bureau american housing survey and the doe residential energy consumption survey [2022] we modified the nist templates to allow for natural ventilation and leakage through and around windows sized to 11.5% of the area of each wall . To account for different types or eras of residential building constructions, three ventilation conditions low, medium and high annual median air exchange rate (aer) were modeled for each combination of climate zone and building type . The low, medium and high ventilation conditions correspond to annual median aer of 0.2, 0.5 and 1.5 per hour (h). Aer is an output of the contam model that is calculated minute - by - minute in the model based on building leakage area, ambient temperature, indoor temperature and ambient wind speed and direction . The building leakage rate was tuned to produce the desired annual median aer for each city and building template . These categories of ventilation rates are consistent with the range of residential air exchange rates recommended by the epa for use in exposure assessments . One - minute average whole house e1, e2 and e3 concentrations and ventilation rates from the individual rooms in a template for 365 days were output from the simulations . R statistical software version 2.15.1 (r development core team, vienna, austria) was used to aggregate the data into the aerosol concentrations and time scales of interest . The total concentration of each aerosol was calculated as the sum of the results for e1, e2 and e3 size bins . Similarly, the minute - average and spatially resolved data were reduced to hourly, daily and annual whole house averages for each combination of filter type, aerosol, building type and location . The main inputs to the contam model were particle sizes, aerosol generation rates, removal efficiencies of the various filters tested, and building factors, as shown in figure 1 . Figure 2 depicts the particle size ranges input for each of the aerosols modeled . For cat allergen, particle sizes were modeled as having the greatest fraction of particles in the 0.31.0 m range and smaller fractions in the 1.03.0 m and 3.09.0 m size ranges . For both pm2.5 source terms, the particle sizes were modeled as having> 90% of the particles in the 0.31.0 m size range with smaller fractions in the 1.03.0 m size range . Respiratory virus was modeled as an aerosol containing influenza and rhinovirus with most of the particles in the 1.03.0 m diameter size range and a small fraction in the 3.09.0 m diameter size range . Particle size distributions for viruses were determined from those measured in sneezes and coughs in previous work . A more detailed discussion of the particle size ranges input into the model is included in the supplementary material online.figure 2.particle size ranges for the modeled aerosols . Generation rates for the aerosols were obtained from several different sources and are presented in table 1 . Exposures in the home to cat allergen were modeled using the release of cat allergen on both a continuous basis as well as with burst emissions of cat allergen from periodic sudden releases in the home . Previous studies have shown upholstered furniture and carpets to serve as reservoirs for cat allergen, which can be released in bursts when someone sits on a couch or walks across a carpet . Indoor source pm2.5 was modeled from cooking activities generating particles for 10 min 3 times each day based on generation rates in particles / hour published previously . For outdoor pm2.5, we used actual measured outdoor pm2.5 concentrations from the geographic area and estimated the infiltration of pm2.5 from outdoors using contam . The monthly average outdoor pm2.5 concentration in atlanta during 2011 ranged from 8 g / m in january to 20 g / m in june . Respiratory virus was modeled as infectious dose in units of quant / hour, based on the literature related to outbreaks . We assumed the approximate mid - point of published quanta generation rates for influenza and rhinovirus, 67 and 5 q / h, respectively . The results for the virus model are representative of sick days in a home but were modeled all year round to evaluate the effectiveness of various filter media under varying home conditions, such as air exchange rate . The virus model assumed that coughing and sneezing were occurring on all days, since its intention was to understand effectiveness on virus particles produced on a regular, intermittent basis when a person with a virus was sick in the home . Virus generation was assumed to occur in the bedroom and living room, assuming an infected person would spend the most time in those two rooms . Because each day of a year was modeled and building factors, such as air exchange rate and air handler duty cycle, vary daily, the results provide a distribution of airborne virus levels for a wide range of scenarios that could reasonably be expected to occur in practice . Information in the supplementary material further explains the derivation of the generation rates for all aerosols.table 1.generation rates input into model by particle size category for each modeled aerosol.catindoor cooking pm2.5outdoor source pm2.5respiratory virusmodel inputsgeneration rate (g / h)% particles in size rangegeneration rate (particles / h)% particles in size range% particles in size rangegeneration rate (q / h)% particles in size rangee1 (0.31.0 m)0.138747.70e+109499e2 (1.03.0 m)0.688134.70e+096135.397e3 (3.09.0 m)20.51431.73 m, micrometer; g / h, micrograms per hour.abetween the hours of 7 a.m.10 p.m., the cat allergen concentration increases for 33% from the intermittent allergen release . Generation rates based on 15 . Percent (%) particles in each size range derived by converting g / h to particles per hour by dividing the generation rate by average particle volume, assuming unit density.bparticle generation rates in particles / hour for breakfast, lunch and dinner were obtained from howard - reed & emmerich .cthe percentage of outdoor pm2.5 particles that fall into the previously defined e1 and e2 particle ranges was known based on the diameter size distribution determined by wilson & suh .dgeneration rate of infectious doses (or quanta) per hour (q / h) based on liao et al . . M, micrometer; g / h, micrograms per hour . Between the hours of 7 a.m.10 p.m., the cat allergen concentration increases for 33% from the intermittent allergen release . Percent (%) particles in each size range derived by converting g / h to particles per hour by dividing the generation rate by average particle volume, assuming unit density . Particle generation rates in particles / hour for breakfast, lunch and dinner were obtained from howard - reed & emmerich . The percentage of outdoor pm2.5 particles that fall into the previously defined e1 and e2 particle ranges was known based on the diameter size distribution determined by wilson & suh . Generation rate of infectious doses (or quanta) per hour (q / h) based on liao et al . . We obtained removal efficiency data from the manufacturers for three size fractions most commonly tested by filter manufacturers: table 2 lists the reported particle removal efficiencies as well as the filter models we evaluated . Filter performance ratings tend to be based on removal efficiencies for specific size ranges of particles . The most widely used rating system for filters is the minimum efficiency removal value or merv rating system, which gives a single rating to a filter based on its performance removing particles in the e1, e2 and e3 size ranges . It does not readily translate as to how these filters can reduce exposures to health - relevant aerosols in the home . A limitation of focusing on removing a particular size fraction is that some common indoor allergens or other asthma triggers can be found on particles that dramatically differ in size . In our analysis, we used modeling techniques to apportion the particle sizes in these categories across common allergy triggers cat allergen, indoor sources of pm2.5, outdoor sources of pm2.5 and respiratory virus to evaluate the in - use effectiveness of the various filter grades on each asthma trigger . The merv scale remains the most readily available indicator of filter performance for a consumer . As a result, we use the term as an indicator of filter grades in this article to assist in translating our findings into consumer - relevant terminology.table 2.particle removal efficiencies by size fraction for each filter type evaluated.filter removal efficiencycalculated removal efficiencyfilter typetypical brand / modele1 (0.31 m)e2 (13 m)e3 (310 m)catindoor pm2.5outdoor pm2.5virusfiberglass (merv1)1 flanders e - z flow fiberglass filter1%10%15%4%2%1%10%basic pleated (merv7)flanders, naturalaire fpr 47%40%65%19%9%7%41%pleated a (merv8)3 m, filtrete 80020%55%70%31%22%20%55%pleated b (merv8)3 m, filtrete 100035%70%80%46%37%35%70%pleated c (merv12)3 m, filtrete 150050%80%90%59%52%50%80%pleated d (merv13)3 m, filtrete 190065%90%98%73%66%65%90%pleated 5 (merv16)lennox x6672, merv 16 carbon clean pleated air filter97%100%100%98%97%97%100%aremoval efficiencies using ashrae 52.2 test protocol reported by 3 m.bcalculated removal efficiencies derived using% particles in each size fraction from table 1 and the following formula for each aerosol and filter type:(% particles in e1 ree1) + (% particles in e2 ree2) + (% particles in e3% ree3)e.g . For removal of cat allergen by fiberglass filter: [0.74 0.01 + 0.13 0.10 + 0.14 0.15] 100 = 4% particle removal efficiencies by size fraction for each filter type evaluated . Removal efficiencies using ashrae 52.2 test protocol reported by 3 m. calculated removal efficiencies derived using% particles in each size fraction from table 1 and the following formula for each aerosol and filter type: (% particles in e1 ree1) + (% particles in e2 ree2) + (% particles in e3% ree3) e.g. For removal of cat allergen by fiberglass filter: [0.74 0.01 + 0.13 0.10 + 0.14 0.15] 100 = 4% the seven grades of filter tested were readily available for purchase and ranged in removal efficiencies according to their merv ratings from 1 to 16: a fiberglass filter (merv1), 1 thick basic pleated filter (pleated, merv7), four grades of 1 pleated filters (a, b, c, d ranging from merv 813) and a 5 thick pleated filter (merv16). Removal efficiencies for each asthma trigger were estimated from the particle size distributions for each of the triggers based on the literature combined with the measured removal efficiencies from the manufacturers . The published removal efficiencies using the ashrae method are based on particle counts, not particle mass . As a result, we present removal efficiencies and percent effectiveness based on particle counts by converting the mass concentrations output by the contam model using the average particle volume and an assumed particle density . All filters evaluated except the fiberglass were pleated, and pleated filters accounted for> 70% of filters offered for retail sale in a market survey conducted in california . Using the inputs described earlier, modeled indoor concentrations were compared across the six filter types . Effectiveness (%) was determined as the percent reduction in the median 24-h whole house concentration of a given pollutant for each filter grade compared to the baseline case, which was the fiberglass filter . The fiberglass filter was chosen as the referent filter because it is one of the two most common filters used in home hvac systems, and its removal efficiencies are very low, approximately equivalent to no filtration of health - relevant aerosols . This follows previous work to determine effectiveness measures for both in - duct and portable air cleaners in homes, with 50% effectiveness on a whole house basis considered a minimum level of meaningful performance . We compared annual operating costs among the filter - based and air cleaner interventions, including in - duct electronic particle removal systems and portable air cleaners . Annual operating costs for each of the filter types were determined from the manufacturer s retail price combined with the minimum recommended filter change - outs per year . The cost estimate for a generic in - duct electrostatic particle removal system was obtained from an online home repair calculator, and annualized costs were spread over 10 years . Electricity usage by a comparable unit (white rogers, sst2000) was estimated to be 40 w. similarly, a mid - priced portable hepa air cleaner (alen, a350) was selected for comparison . The frequency of filter changes as well as electricity usage were obtained from the owner s manual, and replacement filter costs were obtained from the manufacturer s website (model bf15a, alencorp.com). Electricity costs were added for the in - duct system and the portable hepa unit, using local electricity costs for the atlanta area . We conducted this modeling analysis on one attached and one detached sample home in each city . Characteristics are listed in supplementary table s3, and floor plans are provided in supplementary figures s3 and s4 . The overall results were similar between cities and for both home types, so only the results for the atlanta detached home are presented here . Supplementary table s5 lists the results comparing attached and detached dwellings in atlanta and chicago . The modeling simulations were conducted for a typical single - family residence in two specific geographic locations . One was representative of significant traffic - related pm2.5 and was located in an urban residential climate zone corresponding to chicago, illinois . The other was representative of an area with significant seasonal pollen and was representative of a suburban residential home corresponding to the atlanta, georgia, climate zone . Meteorological information is used by contam to simulate forced convection, radiant leakage and corresponding air exchange rates . As described previously, we used typical meteorological year (tmy3) data sets derived from 1991 to 2005 meteorological data . The meteorological data included hourly wind direction and speed, dry and wet bulb temperature, relative humidity and cloud cover data . Meteorological data for each of the locations was taken from the airport closest to that city . Jackson atlanta international airport . For each of the two cities modeled, two different contam building templates were used . The first was a template for a single family, detached home (dh72). The second was a template for a single family, attached home (ah41). Census bureau american housing survey and the doe residential energy consumption survey [2022] we modified the nist templates to allow for natural ventilation and leakage through and around windows sized to 11.5% of the area of each wall . To account for different types or eras of residential building constructions, three ventilation conditions low, medium and high annual median air exchange rate (aer) were modeled for each combination of climate zone and building type . The low, medium and high ventilation conditions correspond to annual median aer of 0.2, 0.5 and 1.5 per hour (h). Aer is an output of the contam model that is calculated minute - by - minute in the model based on building leakage area, ambient temperature, indoor temperature and ambient wind speed and direction . The building leakage rate was tuned to produce the desired annual median aer for each city and building template . These categories of ventilation rates are consistent with the range of residential air exchange rates recommended by the epa for use in exposure assessments . One - minute average whole house e1, e2 and e3 concentrations and ventilation rates from the individual rooms in a template for 365 days were output from the simulations . R statistical software version 2.15.1 (r development core team, vienna, austria) was used to aggregate the data into the aerosol concentrations and time scales of interest . The total concentration of each aerosol was calculated as the sum of the results for e1, e2 and e3 size bins . Similarly, the minute - average and spatially resolved data were reduced to hourly, daily and annual whole house averages for each combination of filter type, aerosol, building type and location . The main inputs to the contam model were particle sizes, aerosol generation rates, removal efficiencies of the various filters tested, and building factors, as shown in figure 1 . Figure 2 depicts the particle size ranges input for each of the aerosols modeled . For cat allergen, particle sizes were modeled as having the greatest fraction of particles in the 0.31.0 m range and smaller fractions in the 1.03.0 m and 3.09.0 m size ranges . For both pm2.5 source terms, the particle sizes were modeled as having> 90% of the particles in the 0.31.0 m size range with smaller fractions in the 1.03.0 m size range . Respiratory virus was modeled as an aerosol containing influenza and rhinovirus with most of the particles in the 1.03.0 m diameter size range and a small fraction in the 3.09.0 m diameter size range . Particle size distributions for viruses were determined from those measured in sneezes and coughs in previous work . A more detailed discussion of the particle size ranges input into the model is included in the supplementary material online.figure 2.particle size ranges for the modeled aerosols . Generation rates for the aerosols were obtained from several different sources and are presented in table 1 . Exposures in the home to cat allergen were modeled using the release of cat allergen on both a continuous basis as well as with burst emissions of cat allergen from periodic sudden releases in the home . Previous studies have shown upholstered furniture and carpets to serve as reservoirs for cat allergen, which can be released in bursts when someone sits on a couch or walks across a carpet . Indoor source pm2.5 was modeled from cooking activities generating particles for 10 min 3 times each day based on generation rates in particles / hour published previously . For outdoor pm2.5, we used actual measured outdoor pm2.5 concentrations from the geographic area and estimated the infiltration of pm2.5 from outdoors using contam . The monthly average outdoor pm2.5 concentration in atlanta during 2011 ranged from 8 g / m in january to 20 g / m in june . Respiratory virus was modeled as infectious dose in units of quant / hour, based on the literature related to outbreaks . We assumed the approximate mid - point of published quanta generation rates for influenza and rhinovirus, 67 and 5 q / h, respectively . The results for the virus model are representative of sick days in a home but were modeled all year round to evaluate the effectiveness of various filter media under varying home conditions, such as air exchange rate . The virus model assumed that coughing and sneezing were occurring on all days, since its intention was to understand effectiveness on virus particles produced on a regular, intermittent basis when a person with a virus was sick in the home . Virus generation was assumed to occur in the bedroom and living room, assuming an infected person would spend the most time in those two rooms . Because each day of a year was modeled and building factors, such as air exchange rate and air handler duty cycle, vary daily, the results provide a distribution of airborne virus levels for a wide range of scenarios that could reasonably be expected to occur in practice . Information in the supplementary material further explains the derivation of the generation rates for all aerosols.table 1.generation rates input into model by particle size category for each modeled aerosol.catindoor cooking pm2.5outdoor source pm2.5respiratory virusmodel inputsgeneration rate (g / h)% particles in size rangegeneration rate (particles / h)% particles in size range% particles in size rangegeneration rate (q / h)% particles in size rangee1 (0.31.0 m)0.138747.70e+109499e2 (1.03.0 m)0.688134.70e+096135.397e3 (3.09.0 m)20.51431.73 m, micrometer; g / h, micrograms per hour.abetween the hours of 7 a.m.10 p.m., the cat allergen concentration increases for 33% from the intermittent allergen release . Percent (%) particles in each size range derived by converting g / h to particles per hour by dividing the generation rate by average particle volume, assuming unit density.bparticle generation rates in particles / hour for breakfast, lunch and dinner were obtained from howard - reed & emmerich .cthe percentage of outdoor pm2.5 particles that fall into the previously defined e1 and e2 particle ranges was known based on the diameter size distribution determined by wilson & suh .dgeneration rate of infectious doses (or quanta) per hour (q / h) based on liao et al . . M, micrometer; g / h, micrograms per hour . Between the hours of 7 a.m.10 p.m., generation rates based on 15 . Percent (%) particles in each size range derived by converting g / h to particles per hour by dividing the generation rate by average particle volume, assuming unit density . Particle generation rates in particles / hour for breakfast, lunch and dinner were obtained from howard - reed & emmerich . The percentage of outdoor pm2.5 particles that fall into the previously defined e1 and e2 particle ranges was known based on the diameter size distribution determined by wilson & suh . Generation rate of infectious doses (or quanta) per hour (q / h) based on liao et al . . We obtained removal efficiency data from the manufacturers for three size fractions most commonly tested by filter manufacturers: table 2 lists the reported particle removal efficiencies as well as the filter models we evaluated . Filter performance ratings tend to be based on removal efficiencies for specific size ranges of particles . The most widely used rating system for filters is the minimum efficiency removal value or merv rating system, which gives a single rating to a filter based on its performance removing particles in the e1, e2 and e3 size ranges . It does not readily translate as to how these filters can reduce exposures to health - relevant aerosols in the home . A limitation of focusing on removing a particular size fraction is that some common indoor allergens or other asthma triggers can be found on particles that dramatically differ in size . In our analysis, we used modeling techniques to apportion the particle sizes in these categories across common allergy triggers cat allergen, indoor sources of pm2.5, outdoor sources of pm2.5 and respiratory virus to evaluate the in - use effectiveness of the various filter grades on each asthma trigger . The merv scale remains the most readily available indicator of filter performance for a consumer . As a result, we use the term as an indicator of filter grades in this article to assist in translating our findings into consumer - relevant terminology.table 2.particle removal efficiencies by size fraction for each filter type evaluated.filter removal efficiencycalculated removal efficiencyfilter typetypical brand / modele1 (0.31 m)e2 (13 m)e3 (310 m)catindoor pm2.5outdoor pm2.5virusfiberglass (merv1)1 flanders e - z flow fiberglass filter1%10%15%4%2%1%10%basic pleated (merv7)flanders, naturalaire fpr 47%40%65%19%9%7%41%pleated a (merv8)3 m, filtrete 80020%55%70%31%22%20%55%pleated b (merv8)3 m, filtrete 100035%70%80%46%37%35%70%pleated c (merv12)3 m, filtrete 150050%80%90%59%52%50%80%pleated d (merv13)3 m, filtrete 190065%90%98%73%66%65%90%pleated 5 (merv16)lennox x6672, merv 16 carbon clean pleated air filter97%100%100%98%97%97%100%aremoval efficiencies using ashrae 52.2 test protocol reported by 3 m.bcalculated removal efficiencies derived using% particles in each size fraction from table 1 and the following formula for each aerosol and filter type:(% particles in e1 ree1) + (% particles in e2 ree2) + (% particles in e3% ree3)e.g . For removal of cat allergen by fiberglass filter: [0.74 0.01 + 0.13 0.10 + 0.14 0.15] 100 = 4% particle removal efficiencies by size fraction for each filter type evaluated . Removal efficiencies using ashrae 52.2 test protocol reported by 3 m. calculated removal efficiencies derived using% particles in each size fraction from table 1 and the following formula for each aerosol and filter type: (% particles in e1 ree1) + (% particles in e2 ree2) + (% particles in e3% ree3) e.g. For removal of cat allergen by fiberglass filter: [0.74 0.01 + 0.13 0.10 + 0.14 0.15] 100 = 4% the seven grades of filter tested were readily available for purchase and ranged in removal efficiencies according to their merv ratings from 1 to 16: a fiberglass filter (merv1), 1 thick basic pleated filter (pleated, merv7), four grades of 1 pleated filters (a, b, c, d ranging from merv 813) and a 5 thick pleated filter (merv16). Removal efficiencies for each asthma trigger were estimated from the particle size distributions for each of the triggers based on the literature combined with the measured removal efficiencies from the manufacturers . The published removal efficiencies using the ashrae method are based on particle counts, not particle mass . As a result, we present removal efficiencies and percent effectiveness based on particle counts by converting the mass concentrations output by the contam model using the average particle volume and an assumed particle density . All filters evaluated except the fiberglass were pleated, and pleated filters accounted for> 70% of filters offered for retail sale in a market survey conducted in california . Using the inputs described earlier, modeled indoor concentrations were compared across the six filter types . Effectiveness (%) was determined as the percent reduction in the median 24-h whole house concentration of a given pollutant for each filter grade compared to the baseline case, which was the fiberglass filter . The fiberglass filter was chosen as the referent filter because it is one of the two most common filters used in home hvac systems, and its removal efficiencies are very low, approximately equivalent to no filtration of health - relevant aerosols . This follows previous work to determine effectiveness measures for both in - duct and portable air cleaners in homes, with 50% effectiveness on a whole house basis considered a minimum level of meaningful performance . We compared annual operating costs among the filter - based and air cleaner interventions, including in - duct electronic particle removal systems and portable air cleaners . Annual operating costs for each of the filter types were determined from the manufacturer s retail price combined with the minimum recommended filter change - outs per year . The cost estimate for a generic in - duct electrostatic particle removal system was obtained from an online home repair calculator, and annualized costs were spread over 10 years . Electricity usage by a comparable unit (white rogers, sst2000) was estimated to be 40 w. similarly, a mid - priced portable hepa air cleaner (alen, a350) was selected for comparison . The frequency of filter changes as well as electricity usage were obtained from the owner s manual, and replacement filter costs were obtained from the manufacturer s website (model bf15a, alencorp.com). Electricity costs were added for the in - duct system and the portable hepa unit, using local electricity costs for the atlanta area . Table 2 shows particle removal efficiencies for the major size fractions of particles (e1, e2 and e3) reported for each filter type as well as the calculated removal efficiencies for the four asthma triggers calculated from their measured removal efficiencies as well as the proportion of the particles commonly associated with each asthma trigger (cat allergen, indoor source pm2.5, outdoor source pm2.5 and virus). These results show the large variation in the fraction of particles removed by the various grades of filters . The lowest performing filter, a fiberglass filter, had a removal efficiency of only 4% for airborne cat allergen, while the highest efficiency filter tested, a 5-in pleated, had a removal efficiency of at least 97% for particles associated with the four categories of asthma triggers . The next highest efficiency filter tested, filter d, had a removal efficiency of> 70% for cat allergen and 65% or more for pm2.5 from indoor and outdoor sources . Since viruses have a greater fraction of particles associated with larger size ranges, the calculated removal efficiency would be> 50% for all filters equivalent to at least filter a, which removed> 50% of virus particles relative to the fiberglass filter . Modeled median home indoor concentrations of the four aerosols were highest for the fiberglass filter and substantially lower for all other filter types . For filters c and d, all pollutants were> 50% lower than the fiberglass filter, meeting our a priori criteria for effectiveness.table 3.modeled indoor concentration for detached home at median air exchange rate of 0.5 air changes per hour.contaminantfilter typemedian 24-h home indoor concentration% effectiveness (reductioncompared to fiberglass)cat allergen (ng / m)fiberglass (merv1)2.7basic pleated (merv7)1.932pleated a (merv8)1.739pleated b (merv8)1.545pleated c (merv12)1.450pleated d (merv13)1.3535 pleated (merv16)1.255indoor source pm2.5 (g / m)fiberglass (merv1)1.8basic pleated (merv7)1.324pleated a (merv8)1.044pleated b (merv8)0.855pleated c (merv12)0.762pleated d (merv13)0.6665 pleated (merv16)0.571pm2.5 infiltration (g / m)fiberglass (merv1)7.2basic pleated (merv7)5.720pleated a (merv8)4.241pleated b (merv8)3.355pleated c (merv12)2.663pleated d (merv13)2.2695 pleated (merv16)1.776respiratory virus (10 quanta / m)fiberglass (merv1)17.1basic pleated (merv7)9.544pleated a (merv8)7.755pleated b (merv8)6.363pleated c (merv12)5.568pleated d (merv13)4.8725 pleated (merv16)4.375 modeled indoor concentration for detached home at median air exchange rate of 0.5 air changes per hour . In figure 3, we plotted filter removal efficiencies and modeled effectiveness for each filter type compared to a fiberglass filter to assess the other filters performance in reducing home indoor levels of cat allergen (a), indoor source pm2.5 (b), outdoor source pm2.5 (c) and respiratory virus (d). In addition to the pleated 5 filter, filters c and d, which are merv12 and 13 standard size filters (1 thickness) achieved the recommended 50% effectiveness for all of the modeled aerosols, including cat allergen . These plots also show the diminishing returns on effectiveness for filters with high removal efficiencies . For outdoor pm2.5, the 70% removal efficiency of filter d achieves 70% effectiveness, while the 5 pleated filter has a removal efficiency of> 99% and achieves only 76% effectiveness on outdoor pm2.5 . A similar relationship is seen for pm2.5 generated indoors from cooking with maximum effectiveness of 72% for the 5 pleated filter, which had removal efficiency of> 97% for indoor pm2.5.figure 3.plots of filter removal efficiencies (from table 2) for each filter type versus effectiveness compared to the fiberglass filter in reducing indoor levels in homes for cat allergen, indoor source pm2.5, outdoor source pm2.5 and respiratory virus . Plots of filter removal efficiencies (from table 2) for each filter type versus effectiveness compared to the fiberglass filter in reducing indoor levels in homes for cat allergen, indoor source pm2.5, outdoor source pm2.5 and respiratory virus . Table 4 lists the annual operating costs of various air cleaning interventions for the home . The lowest priced interventions are the fiberglass and basic pleated filters with annual replacement costs of $20/year, but they also are the lowest performing filters . Higher grade filters can reduce common triggers by 50% for $40 to $80 per year . The tested filter d can achieve> 50% reductions in all four asthma triggers measured, at an annual operating cost of $80/year . Filters with removal efficiencies greater than those for filters c and d will not likely reduce in - home levels dramatically, and they come with additional costs . The portable hepa unit offered little effectiveness on a whole - home basis and had the greatest annual operating cost at $140/year, including replacement filters and electricity, but excluding the original purchase price.table 4.cost estimates for filtration interventions.area treatedintervention typemodelannual operating costsnoteswhole housefilterfiberglass (merv1)$18change monthly for high use periods . Assumed 6/year.basic pleated (merv7)$20change every 3090 days.pleated a (merv8)$40change at least every 90 days . Assumed 4/year.pleated b (merv8)$45change at least every 90 days . Assumed 4/year.5 pleated (merv16)$98change at least once per year.in-duct esp unitgeneric in - duct$120wash filter every 12 months.one roomportable hepaalen a350$140change filter every 48 months.aassumes 20 20 1 filter.bassumes 20 20 5 filter.cassumes $1100 installation cost spread over 10 years, and annual electricity cost of $10 .dassumes hepa filters replaced every 6 months at $50 each, and annual electricity cost of $40 . Annual cost does not take into account $400 purchase price of the air cleaner . Cost estimates for filtration interventions . Assumes 20 20 1 filter . Assumes 20 20 5 filter . Assumes $1100 installation cost spread over 10 years, and annual electricity cost of $10 . Assumes hepa filters replaced every 6 months at $50 each, and annual electricity cost of $40 . Annual cost does not take into account $400 purchase price of the air cleaner . This analysis showed that large reductions in indoor asthma and allergy triggers can be achieved using relatively inexpensive, high - efficiency in - duct air filters . We used a physical model that took into account factors, such as aerosol generation rates, how often the ventilation system ran, outdoor air temperature and air exchange rates in the modeled homes . To evaluate the reliability of our results, we compared the modeled concentrations to data from measurement studies, focusing on absolute levels and indoor / outdoor ratios for pm2.5 . Airborne levels of the pollutants modeled in our study had relatively low absolute concentrations (1.8 g / m attributed to indoor source pm2.5 and 7.2 g / m for infiltrated pm2.5), but not atypical of other studies of home indoor particle levels . Previous studies attributed 0.4 g / m to indoor pm2.5 levels for each instance of frying or cooking, and previous in - home monitoring studies we conducted measured average indoor pm2.5 concentrations of 10 g / m in winter and 12 g / m in summer for 25 homes in boston . We calculated the average ratio of pm2.5 that infiltrated indoors to outdoor pm2.5 and compared those results to values from the literature . The average indoor outdoor ratio for the atlanta detached model home was 0.60 for infiltrated pm2.5 in the current study . This value is comparable to values reported in the literature for outdoor source pm2.5, as indicated by sulfate which is used as a tracer for outdoor particulate air pollution . Compiled indoor outdoor sulfate ratios for a group of panel studies, including one in atlanta that measured indoor a theoretical modeling analysis conducted using a mass balance model found similar effectiveness on cat allergen (2060%) for a range of filter efficiencies similar to those tested in our study . The results we present coincide with this previous work, which also showed a plateau of effectiveness, even with a simulated hepa in - duct filter, which showed no greater effectiveness on cat allergen compared to filters with removal efficiency ratings close to our tested filter c or a merv12 . For infiltrated pm2.5, fisk and colleagues also showed 50% effectiveness for a filter (ashrae85) that would be comparable to filter d tested in our study; and only a full hepa filter was able to achieve 80% reduction for outdoor source pm2.5 . That paper did not evaluate indoor sources of pm2.5 or virus loads and took into account only continuous emissions of cat allergen; in addition, our model was able to incorporate air flow between rooms . The magnitude of exposure reduction obtained by the low cost, higher efficiency filters (e.g. Those with at least 50% removal efficiency for e1 size fraction) in our analyses has been associated with improvements in health . A recent review article suggests that improved health outcomes can result from asthma interventions employing filtration to reduce particle levels, particularly for those with asthma and/or allergies . Only one paper was identified that included high efficiency filters (merv12) into their asthma intervention program . Filters were combined with other efforts to improve ventilation system performance in the residences studied . These included servicing the air handling unit, if needed, or improving the housing for poorly fitting filters in the hvac system, so filter effectiveness alone was not assessed directly . Johnson and colleagues showed that the greatest reductions in coughing and wheeze were associated with the hvac intervention . Statistically significant (p <0.05) reductions in 0.01) were also found in the hvac intervention group . While the authors did not present results, they state that median non - viable mold spore counts were also reduced post - intervention for the hvac group . A recent exposure modeling study in multi - unit public housing estimated reductions in pm2.5 by> 50% for a combination of interventions that included use of kitchen and bathroom exhaust fans, replacement of gas with electric stoves, introduction of a no smoking policy and improved weatherization in the apartment . No other individual intervention in that modeling study was able to reduce pm2.5 levels by> 50%, and use of a portable hepa air cleaner alone was able to reduce modeled pm2.5 levels by only 25% . Our analysis focused on single family houses rather than apartments, but we showed> 50% reduction in pm2.5 levels using a relatively simple and inexpensive intervention . To achieve 50% reductions in indoor pm2.5, the combined intervention evaluated by fabian and colleagues involved replacing the stove, installing a kitchen exhaust fan and implementing extensive weatherization of the unit . The weatherization program alone was estimated to cost thousands of dollars, and it would take> 11 years to see a return on investment taking into account energy savings from that component of the intervention . Reducing indoor particulate levels use of two portable hepa air cleaners in apartments was shown to reduce indoor pm2.5 levels by> 50%, and the authors attribute increased symptom free days to the reductions in pm levels . This study included residences with smokers, but reducing indoor levels of pm may be an important factor for reducing exposures of the many asthmatic children living with smokers . Another study found 30% reductions in pm2.5 levels in the asthmatic children s bedrooms after introduction of a multi - faceted intervention program that included a portable hepa air cleaner in the bedroom . At 9-month post - intervention, the proportion of children experiencing asthma symptoms during the daytime was reduced 20%, but 3 months later returned to approximately baseline levels . While asthma intervention programs are widely recommended, most have been shown to be minimally to moderately effective at reducing triggers and even less effective at improving health outcomes . Most asthma intervention studies focus on activities in the home to reduce exposures to dust mite or cockroach allergen . In - duct filtration is unlikely to significantly affect dust mite and cockroach allergen, because the particles associated with those allergens tend to be too large to remain suspended in air for sufficient durations following release or resuspension to be able to enter the returns of the home ventilation system . One study showed limited reduction in dust mite allergen levels in house dust for homes when a mechanical ventilation system was installed in the homes, although the authors credited reduced humidity to the slightly reduced levels . Studies have also shown single interventions to be less effective than multi - pronged interventions at reducing asthma triggers in the home . Previous research we conducted, both modeling and experiments in a test home, indicated that in - duct particle removal systems are most effective at reducing particulate levels on a whole house basis and that portable hepa filters are effective only in a single room . In experiments in a test home, we found running five portable hepa air cleaners in the home yielded similar performance to using a lower grade (merv8) hvac filter in the duct . These studies also showed poor performance of the two lowest grades and most commonly used filters the fiberglass and basic 1 pleated filters . Another study used a theoretical modeling analysis to test grades of filters on two of the asthma triggers (cat allergen and outdoor pm2.5 infiltrating indoors), and found similarly poor results for the most commonly used filter media . Portable hepa filter units placed in the patient s bedroom may still be advised for sensitive patients, since source proximity is an important factor in reducing exposures and some studies have shown benefits from use of such units, particularly in a bedroom . The fiberglass filter is one of the two most commonly used filters in residences in the us, however, its poor performance is not widely understood by consumers . While the key performance characteristic for filters is particle removal efficiency, effectiveness in the real world is an important consideration . Our results show that when used in a typical home even filters with high removal efficiencies are unlikely to achieve 80% reductions for most airborne asthma and allergy triggers, which is the effectiveness recommended by the association of home appliance manufacturers (aham) for portable air cleaners . Another effectiveness measure, known as clean air delivery rate (cadr), pertains to portable air cleaners and is based on 80% effectiveness, given certain parameters regarding air flow and room size . Similar to the merv rating, cadr only measures removal efficiency of a device for certain particle size fractions under limited conditions, specifically for a single room . It does not show how effective a given portable unit would be in a whole house . In fact, most portable air cleaners do not have powerful enough fans to be effective on a whole house basis . It should be noted that the analysis presented here included periodic running of the home s fan system based on outdoor temperature and home conditions . It is likely that greater reductions would be achieved if the home s fan setting was set to remain on at all times . Removal efficiencies are not reported directly for filters; rather they are incorporated into rating scales with merv being the most widely recognized . In addition, the california energy commission is considering proposals to require labeling of removal efficiencies by particle size as well as a measure of the resistance to airflow of a filter . One limitation of some higher efficiency filters is that they can make it harder for air to pass through (often referred to as pressure drop). This can lead to greater energy use if the ventilation system must run more often to cool or heat the same home volume, if a filter is not designed for both low resistance to air flow and effective filtration . While this is driven mostly from an energy conservation perspective, labeling of filters with their removal efficiencies may provide additional information to consumers . However, as this analysis shows, there can be a plateau in effectiveness for filters above approximately a merv13 rating when used in a typical home . Rating systems, such as the merv, can be confusing to consumers and have some limitations . The merv is a 16-point scale based on filter removal efficiencies, however, only filters rated at or above merv14 are currently required to show any efficacy for removal of particles <1 m in size . This size fraction makes up a large proportion of particles associated with asthma triggers, particularly for cat allergen and both indoor and outdoor source pm2.5 . While we also modeled a merv16 filter in this analysis, removal efficiencies corresponding to this merv rating are only found in filters that are 35 thick, as compared to the standard 1 thick filters that are most common in the us homes . In addition, while the merv is a standard test it does have a number of limitations, the most important being that performance in the real world does not coincide directly with filter removal efficiencies measured using a laboratory testing method such as merv . However, our findings show that use of a filter that effectively removes smaller particles (<1 m), such as a merv12 or 13, will also be effective for asthma and allergy triggers that span size ranges . Proposed revisions to the merv guidelines may more effectively address the performance ratings for filters on submicron particles . The revised merv ratings would add removal efficiencies for the smallest size fraction (0.31 m) for merv11 - 14 filters and reduce slightly the removal efficiencies for the larger size fractions . Millions of people in the us, particularly children, have a current diagnosis of asthma, resulting in large health care costs as well as missed work and school days . A large and growing fraction of the us homes have forced air ventilation systems, providing potential low cost opportunities to reduce airborne asthma and allergy triggers . While not all asthma and allergy triggers are airborne, some key ones are, including cat allergen, pm2.5 and respiratory virus . We evaluated the performance of different grades of filters in a modeling analysis, and we identified filters performing at an approximate merv12 rating to be effective at reducing airborne asthma triggers by at least 50% . Despite the widespread and common use of media filters in forced air ventilation systems, an analysis of their efficacy that reflects a distribution of real - world emission and household scenarios has not previously appeared in the literature to our knowledge . The enormous burden of asthma in terms of costs and adverse health effects are well known by families and health care professionals . While some asthma interventions are quite costly, an extra $50 per year for well - performing ventilation filters is a small additional cost that may have significant impacts on indoor levels of many triggers in homes . Asthma interventions are and should be multi - factorial, and the evidence in our article suggests that in - duct filters could be an important, relatively low - cost component of efforts to reduce allergens in homes.
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Patients who present to the emergency department with chest pain require rapid triage, evaluation and management . Myocardial infarction with st segment elevation (ste) on electrocardiography (ecg) is a common presentation in emergency rooms across the world . Acute coronary syndrome (acs) marked by ste on the ecg warrants consideration for emergent cardiac catheterization and possible percutaneous coronary intervention (pci). In fact, there are now core measures and quality metrics in place that grade a hospital's efficiency at caring for such patients . However, the time pressure to optimize such quality metrics may lead to an unintended rush to treatment prior to adequate evaluation . In this report, we present a rare case where myocardial infarction was seen as a presenting feature of an underlying hematologic disease, thrombotic thrombocytopenic purpura (ttp). This case highlights the importance of a thorough, yet efficient, clinical evaluation in which the history, physical exam, ecg and laboratory data were needed to make the appropriate triage decision and not miss an unusual diagnosis . A 48-year - old woman with no known coronary risk factors was transferred from an outside facility to the cardiac catheterization laboratory of our hospital with the diagnosis of ste myocardial infarction for primary pci . On arrival to our catheterization laboratory, the ecg from the referring hospital showed sinus tachycardia with normal axis and intervals . There was ste in leads i, ii, avl, v46 and reciprocal st segment depression in lead iii [figure 1]. Laboratory data were not yet available . However, the patient's history of present illness was significant for malaise, fever, chills and lethargy that began 3 days prior to hospitalization . Further questioning established that she had mild generalized abdominal pain and one episode of non - bloody diarrhea . The family also noted that she had been intermittently confused and was talking gibberish . On the morning of admission, she had severe chest pain associated with nausea, vomiting and dyspnea on exertion, which led her to seek medical care . Sinus tachycardia at 121 beats per minute with st segment elevation in lead i, ii, avl, v46 on examination, she appeared toxic and in respiratory distress . Vital signs revealed a blood pressure of 126/70 mmhg with a heart rate of 121 beats per minute . In addition, mottling of her skin and livedo reticularis over the thighs was noted . There were a few purpuric skin lesions observed in her antecubital fossa and upper arms . Her jugular venous pressure was elevated up to the angle of the jaw . Cardiac exam revealed a normal first and second heart sound along with a fourth heart sound . Peripheral pulses were not palpable in the feet and were only faintly palpable in the arms . Neurologically, she was somewhat confused, but the sensory and motor exam was essentially normal . Given that the patient was not having active chest pain, the history was inconsistent with acs and she appeared more toxic than expected for a lateral wall myocardial infarction; cardiac catheterization was deferred and emergent laboratory studies were obtained . Initial laboratory studies revealed a white blood cell count of 13.5 10/mm; hematocrit of 24%; mean corpuscular volume of 88.4 fl and platelet count of 6 10/l . Her lactate dehydrogenase was elevated at 2820 units / l and haptoglobin was low at less than 10 mg / dl . Coagulation profile showed international normalized ratio of 1.2, prothrombin time of 12.6 seconds, fibrinogen 199 mg/ l and d - dimer 1.27 feu electrolytes were within normal limits; acute kidney injury was noted with blood urea nitrogen 51 mg/ dl and creatinine 1.9 mg / dl . Total bilirubin was markedly elevated at 32 mg% with an indirect bilirubin of 2.1 mg% . Cardiac biomarkers were elevated with creatinine kinase of 487 units / l and mb fraction of 28.8 ng / ml . Troponin - t was 0.86 ng / ml . Urinalysis showed ph of 6.0, 3 + albumin, 3 + hemoglobin, eight wbcs and greater than two rbcs with some amorphous crystals . Echocardiography demonstrated an ejection fraction of 4045% with severe hypokinesis of the inferior and basal anteroseptal wall . No significant valvular lesions were noted . In view of the acute onset of symptoms associated with microangiopathic hemolytic anemia, thrombocytopenia, acute kidney injury and waxing and waning mental status, the presumptive diagnosis of ttp was made . A disintegrin and metalloproteinase with thrombospondin motifs (adamst-13) activity was found to be low with presence of adamst-13 inhibitors in the plasma . She improved clinically with this treatment and normalized her platelets and lactate dehydrogenase, and ste in her ecg resolved [figure 2]. Outpatient cardiovascular follow - up demonstrated normalization of the left ventricular ejection fraction by echocardiography and a completely normal nuclear myocardial perfusion stress study . Further testing with invasive catheterization was not pursued as the entire episode was considered to be transient, secondary to metabolic derangement . Ttp is defined as a severe, thrombotic microangiopathy that is characterized primarily by systemic platelet - von willebrand factor aggregation, organ ischemia, profound thrombocytopenia and fragmentation of erythrocytes . Pathologically, focal areas of necrosis and hemorrhage may be seen in the pancreas, adrenals, heart, brain and kidneys. [35] although myocardial injury and necrosis are observed in a large number of patients with ttp, it is infrequently the initial presentation, and most likely thought to be due to microthrombi from massive platelet aggregation than plaque rupture thrombosis cascade . Various studies have determined the incidence of myocardial infarction in ttp to range from 1541%. [69] however, the heart is one of the most frequently involved organs at autopsy examination of patients with ttp . Mortality is considerably higher in patients with ttp who have positive cardiac biomarkers, necessitating closer monitoring in this subgroup . Early recognition of myocardial injury in a case of ttp is crucial as it identifies higher risk . However, invasive therapy in the form of cardiac catheterization and pci may be fraught with complications and is precluded by acute kidney injury and low platelet count . Thrombocytopenia also prevents the use of usual medical management in acs such as antiplatelet and anticoagulant therapy . Beta blockers and hmg coa reductase inhibitors may be used although their role is questionable . In acute bouts of ttp, such as this case, the treatment of choice is rapid initiation of plasmapheresis . In addition, immunosuppressive therapy including steroid therapy is helpful, especially in the setting of auto - antibodies against adamst-13 factor . Relapsing cases of ttp have been treated with rituximab, a monoclonal antibody against cd20 on memory b cells with good effect . Although cardiac involvement is common in ttp, as an index event it can be misleading . As swift protocols are activated in the emergency room and catheterization laboratories to ensure quality control, it is equally important to integrate all aspects of patient's clinical and objective data to detect unusual disease entities
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Cavernous sinus thrombosis (cst), which was first described by dease in 1778, is a rare disease associated with high mortality and morbidity rates if not treated immediately1 . Cst can result from infection of the paranasal sinus or any of the anatomic structures drained by the cavernous sinus, including the mid - face, orbit, and oral cavity2 . Deep neck infection containing lateral pharyngeal, pterygomandibular and infratemporal space that patient are presenting symptom of the limited mouth opening can cause infection of the cavernous sinus from the pterygoid plexus3,4 . In this study, we report a patient with cst who initially presented with limited mouth opening . A 55-year - old male visited our department with a chief complaint of limited mouth opening, which began suddenly on august 13, 2013 . On physical examination, mouth opening was limited to approximately 10 mm, with mild pain on opening . However, body temperature was normal and signs of infection including odynophagia, dyspnea, swelling and tenderness were absent . In the transcranial view of the temporomandibular joint, the translation of both temporomandibular joints was limited. (fig . 2) the provisional diagnosis was myospasm of the masticatory muscles; we prescribed a muscle relaxant and analgesic with physiotherapy . Three days later, on august 16, 2013, the patient was admitted to the department of ophthalmology complaining of orbital swelling and pain . Clinical examination revealed ptosis, proptosis, moderate chemosis, diplopia, and complete ophthalmoplegia of right side. (fig . 3) peripheral sensation around the right orbit was good and cranial nerves vii through xii were intact . 4) computed tomography (ct) and magnetic resonance imaging (mri) revealed proptosis and engorgement of the right superior ophthalmic vein that was accompanied by enhancement of retrobulbar tissues and swelling of the lateral pterygoid muscle with pus formation . Also, contrast enhancement and dilation indicative of inflammation of the cavernous sinus was noted. (fig . 5) the paranasal sinuses, including the ethmoid and maxillary sinus of the ipsilateral side, exhibited mucosal thickening and retention of purulent material. (fig . 6) initially, the patient was empirically treated with intravenous cephalosporin . On hospital day 3, he was prepared for incision and drainage under general anesthesia . A large amount of pus was drained from the lateral pharyngeal, pterygomandibular and infratemporal spaces . The wound was irrigated with copious saline through silastic drains placed in the abscess cavity . Repeated ct 10 days after surgery demonstrated improvement in the engorgement of the superior ophthalmic vein and dilation of the cavernous sinus. (fig . 7) over the following 11 months, his orbital symptoms resolved completely and his range of mouth opening returned to normal. (fig . The cavernous sinus, which is located to the rear of the optic canal and the superior orbital fissure, is an important structure containing the internal carotid artery and cranial nerve iii, iv, v, and vi . Thus, if infection or thrombosis occurs in the cavernous sinus, various symptoms referred to as' cavernous sinus syndrome' can appear, including ophthalmoplegia, diplopia and ptosis due to impairment of cranial nerve iii, iv, and vi . Involvement of the ophthalmic nerve and maxillary nerve can cause paresthesia around the orbital cavity5 . Thus, proptosis and chemosis can cause drain blockages of ophthalmic veins to the cavernous sinus6 . There are also connections between the contralateral cavernous sinus, the intercavernous sinus, facial veins that have not valve, and the pterygoid plexus, which is located very close to the cavernous sinus . Given the complex anastomosis of veins with the cavernous sinus, sometimes orbital symptoms appear on both sides, or on the opposite side7,8 . Childs and courville11 reported that an odontogenic source was responsible in up to 10% of cases . Pavlovich et al.2 presented two cases with different mechanisms: sinusitis and dental infection . In our case, chronic periodontitis of the maxillary posterior teeth and mucosal thickening of the right ethmoidal sinus were observed . Because the initial clinical symptom was the limitation of mouth opening, however, the infection was assumed to be from an odontogenic source . Mazzeo4 reported that mandibular infections may spread directly to the dural sinuses through the pterygoid plexus . In our case, the patient exhibited symptoms of the proptosis, ptosis, and ophthalmoplesia, accompanied by swelling of the face and neck due to what appeared to be a deep neck infection . The infection was thought to originate around the pterygoid muscle, since trismus preceded orbital symptoms . The route of infection to the cavernous sinus was deemed to be via the pterygoid plexus . Numerous causes of superior orbital fissure syndrome including trauma, infection of the cavernous sinus, neoplasm, aneurysm of the internal carotid artery, carotid cavernous fistula or idiopathic etiology have been reported in the literature12,13 . Imaging studies such as ct, mri, and magnetic resonance angiography should be employed in patients with findings consistent with a cst . If a vascular lesion of the cavernous sinus is suspected, cerebral angiography may be necessary5 . Typical radiographic signs of cst include expansion of the cavernous sinus, convexity of the normally concave lateral wall, irregular filling defects, and asymmetry; indirect signs include venous obstruction, dilation of the superior ophthalmic vein, proptosis, and thrombus in the veins14,15 . Our patients underwent angiography with simultaneous ct and mri to distinguish the clinical signs from the vascular lesion . On angiography, abnormal blood vessels were not observed, but direct and indirect radiographic signs of cst were demonstrated on ct and mri . The treatment of cst includes the use of antibiotics directed at the causative organism and surgery to remove the primary source of infection . The role of steroid and anticoagulant therapy for the treatment of cst remains controversial14 . In summary, cst is a rare disease that can cause life threatening complications . Infections of the pterygoid plexus from an odontogenic source can present as trismus, and clinicians should consider the potential for progression to cst . Prompt diagnosis and appropriate treatment are essential for preventing serious complications.
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Microalbuminuria (ma) was delineated many years ago to describe the appearance of small quantities of albumin, too little to detect by standard dipstick methods, in urine samples from subjects with diabetes [1, 2]. According to recent guidelines, ma may be defined as the urinary excretion of 30300 mg / d in a timed urine collection in adults . When spot urine samples are used, the recommended definitions of ma are the excretion of 17250 mg / g of creatinine in men and 25355 mg / g of creatinine in women . The gender - associated differences in definition result from variation in daily creatinine generation females have lower creatinine generation than males . In addition, variations in the quantification of albuminuria for these definitions of ma may need to be applied to the elderly (who have greatly decreased creatinine generation) or to excessively muscular individuals (who have high creatinine generation). Urinary albumin concentration in untimed urinary collections may also be used to define ma values of 330 mg / dl generally are regarded as fulfilling the definition . Albumin in urine is most often measured by immuno - turbidimetry using antibodies reactive with intact albumin . Alternatively, high - pressure liquid chromatography can be used, but this method also measures nonimmunologically reactive albumin and albumin fragments, which yields higher values than immuno - turbidimetry . In general, ma corresponds to a trace reading on dipstick testing of urine, but results depend on the degree of urine concentration . Dye - impregnated strips that have enhanced sensitivity for albumin are also available . Simple, portable, point - of - service instruments that measure small amounts of albumin make assessment of ma readily available, reliable, and inexpensive . The dominant viewpoint is that albumin is normally filtered by the glomeruli in small but significant amounts, about 1 to 2 mg / min (~ 2 g / d), but the great majority (99%) of filtered albumin is reabsorbed and degraded by the proximal tubule, leaving less than about 5 g / min (7 mg / d) to be excreted . A minority view is that larger amounts of albumin are filtered, as much as 200 g / d, but a retrieval pathway in the proximal tubule reclaims the filtered albumin intact and delivers it to the circulation, except for small amounts of intact and partially or fully degraded albumin . In pathologic states, the glomeruli may become increasingly permeable to circulating albumin by virtue of disturbances in endothelial cell function, basement membrane abnormalities, or podocyte (visceral epithelial cell) disorders . Currently, the focus is on disturbances of endothelial or podocyte function as causes of excessive albumin glomerular permeability, rather than disturbances of basement membrane physiology . Albumin is negatively charged at physiologic ph and may be impeded in transglomerular passage by the anionic residues in endothelial cell, glomerular basement membrane, or podocyte . However, the existence of a charge - selective permeability barrier has been challenged . A reduction in proximal tubule reclamation or reabsorption of filtered albumin . Finally, physicochemical alterations in the circulating albumin molecule can affect its permeation through the glomerular capillary wall, by altering the shape or possibly the electrical charge . Thus, the mechanisms underlying increased urinary albumin excretion are complex and it is often difficult to ascribe ma in specific patients to a unique pathophysiologic process; however, endothelial cell dysfunction, a podocytopathy, or both seem to underlie most instances . Nevertheless, it remains possible that alterations in proximal tubular function may contribute to albuminuria in some circumstances, such as during poorly controlled glycemia in diabetes mellitus . These considerations are important when one attempts to relate albumin excretion rates to the definition of kidney disease, because albuminuria arising from diffuse endothelial cell injury (eg, in atherosclerosis and hypertension) might best be regarded as a sign of a systemic disease process, affecting many organs and systems, rather than one originating in the kidneys . Alternatively, albuminuria arising from a podocyte dysfunction could legitimately be regarded as a manifestation of a kidney disease . Regardless of the underlying mechanisms and interpretation of the pathophysiologic meaning of albuminuria, important associations exist between the quantities of albumin excreted in the urine and progressive chronic kidney disease (ckd) and cardiovascular (cv) events . Microalbuminuria, as defined above, has been consistently associated with an increased likelihood of progression of generic ckd to more advanced stages or even to end - stage renal disease (esrd) in large epidemiologic studies [12, 13, 14, 15]. These observations have been used to justify the inclusion of ma in the definition of generic ckd in the original classification schema of the kidney disease outcome quality initiative (kdoqi) of the national kidney foundation (nkf) in 2002 . Indeed, the presence of ma (defined as uacr of 30299 mg / g creatinine without gender or age adjustments) as the sole reason for diagnosing generic ckd accounted for 90% of those with stage 1 ckd and 87% of those with stage 2 ckd in the us national health and nutrition examination survey (nhanes) conducted from 1999 to 2004 . In this cohort (n = 13,233), 6.8% had self - reported diabetes and 27% had diagnosed hypertension . Thus, the great majority of subjects designated as having stage 1 or 2 ckd in nhanes had isolated ma (ma without a clear reduction in estimated glomerular filtration rate [egfr], urine sediment changes, or structural kidney disease) as the defining abnormality . It is natural to ask whether such isolated ma is sufficient reason to designate a person as having generic ckd . As shown by a recent, very large, collaborative meta - analysis (chronic kidney disease prognosis consortium; n = 105,872 subjects), the hazard ratio (hr) for all - cause mortality and cardiovascular mortality (adjusted for the effects of age, ancestry, history of cv disease, systolic blood pressure, diabetes, smoking, and total cholesterol concentration) rises progressively above a uacr of about 10 mg / g [17]. In subjects with normal renal function (egfr = 90104 ml / min/1.73 m), the hr for cv mortality is 1.63 at a uacr of 1029 mg / g, 1.82 at 30299 mg / g, and 4.77 at greater than 300 mg / g . A decrease in egfr magnifies the effect of an increased uacr on both all - cause and cv mortality, especially at an egfr of 45 ml / min/1.73 m or lower . Thus, there is little or no doubt that increased albumin exertion, even below the conventional ma range, is associated with an increased likelihood for mortality, both all - cause and cv - related . Is elevated albumin excretion a biologic marker of underlying systemic disease (eg, cancer, atherosclerosis), or is it somehow involved in the causal pathway for fatal events, including cv disease? Similarly, the risk of developing a progressive form of ckd is linked somehow to the presence of elevated albumin exertion . A large, community - based study from alberta, canada, (n = 920,875) showed that subjects with normal or nearly normal renal function (egfr 60 ml / min/1.73 m, average age 46 years) and ma had event rates of esrd of 1.5 and doubling of serum creatinine of 2.8 times those of subjects with absolute events rates were low: 0.06 per 1000 person - years of follow - up for esrd in the normo - albuminuric group versus 0.09 per 1000 patient - years for the ma group . A lowered egfr magnified the effect of ma on the occurrence of esrd and doubling of the serum creatinine, especially at levels below 45 ml / min/1.73 m. in another community - based study (n = 65,589 adults; 3.3% diabetic; average age, 50.1 years), researchers noted that the likelihood of developing (and surviving long enough) to receive treatment for esrd among subjects with ma and egfr 60 ml / min/1.73 m was 27 times that of subjects with normo - albuminuria and similar egfr levels [13]. Subjects with an egfr less than 60 ml / min/1.73 m and ma had a 5.4-fold to 81-fold increased risk of esrd compared to those with an egfr 60 ml / min/1.73 m, depending on the degree of decrease in egfr . Thus, the addition of ma to egfr greatly enhances the ability to detect and quantify risk of progressive ckd, particularly when egfr is less than 60 ml / min/1.73 m. these observations call for a revision of the 2002 version of the kdoqi - nkf classification schema for ckd that does not include albuminuria for diagnosis of stage 3 or above ckd taken together, these findings from epidemiologic studies using large databases strongly support the view that ma needs to be considered as a biomarker of adverse outcomes, even among those subjects with normal or nearly normal renal function . However, the strength of this association between ma and outcomes may vary by subject age and underlying disease (eg, diabetic or nondiabetic) or by concomitant illness known to influence albumin excretion rates (eg, obesity). For example, in the aforementioned meta - analysis conducted by the chronic kidney disease prognosis consortium [17], the pooled estimates of fully adjusted hr for cv mortality tended to be higher in those younger than 65 years of age compared to those older than 65 years with apparently equivalent degrees of ma . An analysis of the impact of ma on outcomes in the old - old (> 75 years of age) has not been conducted; however, as stated above, uacr has a tendency to increase with normal aging, predominantly because of the effects of sarcopenia on creatinine generation, not necessarily because of an absolute increase in albumin excretion with aging . Furthermore, the association of ma with adverse outcomes in subjects with normal or nearly normal renal function (egfr 60 ml / min/1.73 m), after adjustment for the effects of aging may simply reflect the overall burden of atherosclerotic vascular disease in this group of subjects . As recently pointed out by kalaitzidis and bakris, untangling the influences of metabolic disturbances and hypertension in diabetic and nondiabetic subjects with concomitant ma can be problematical . It is still uncertain if ma is an integral part of the pathophysiologic pathways for cv disease or simply a bystander . The sine qua non of specific forms of ckd in which ma is present is that of diabetes mellitus . Indeed, the concept of ma arose from studies of the natural history of diabetic nephropathy during the period 1981 to 1982 [1, 2]. Shortly after its initial description in type 1 diabetes, ma we now recognize that ma in type 1 diabetes is a dynamic process with frequent spontaneous regression to normo - albuminuria [19, 20]. We also recognize the wide array of extra - renal factors that can influence albumin excretion, such as obesity, age, gender, distant inflammation, and certain drugs (eg, rosuvastatin) [21, 22, 23]. Furthermore, studies done at the joslin clinic over many years indicate that more advanced stages of ckd are not uniformly associated with progression of ma to overt microalbuminuria [19, 24]. Pathologic abnormalities indicative of diabetic nephropathy (eg, increased mesangial fractional volume) precede the development of ma, and very clearly renal functional decline can occur in the absence of ma, at least in type 1 diabetes . Type 2 diabetes may be another story, because the relationship of ma and renal functional decline is not as well studied or understood as in type 1 diabetes, and this relationship is confounded by concomitant accelerated atherosclerotic macrovascular disease . Nevertheless, several studies showed that progression of type 2 diabetes can occur in the absence of ma [21, 2226]. Interventions designed specifically to reduce ma have not consistently shown a reduction of later progression to advanced ckd or esrd . Some recent interventional trials (eg, the avoiding cardiovascular events through combination therapy in patients living with systolic hypertension [accomplish] trial) involving type 2 diabetic subjects showed that the change in albumin excretion rates can be disassociated from risk of progression to esrd under the influence of combined renin - angiotensin system blockade and calcium channel antagonism [21, 27]. Taken together, these observations in type 1 and type 2 diabetes suggest, but do not prove, that ma may not be a reliable marker of kidney disease or its progression . Among patients with well - established kidney disease (ie, egfr <4550 ml / min/1.73 m), the magnitude of albuminuria can be viewed as a marker of a more progressive course and a more rapid loss of renal function (a risk marker rather than a risk factor) for both diabetic and nondiabetic renal disease [28, 29]. This finding may well be a manifestation of lead - time bias, in that those with lower levels of albumin excretion have disease in its earlier stages and thus a greater reserve of functioning nephrons, whereas those with higher levels of albuminuria have no reserves and with each loss of nephrons, a corresponding decline in renal function becomes evident . In this sense, the level of albumin excretion is a relevant marker of kidney disease and risk of its progression . A conundrum exists when renal function is entirely or nearly normal: no overt structural renal disease is evident, yet albumin excretion is elevated into the ma range . No doubt this circumstance somehow predisposes to, or is a marker of a predisposition to, cv disease . For example, albuminuria correlates with parameters of aortic stiffness and carotid plaque formation but not with carotid intima - media thickness as a surrogate for widespread atherosclerosis . On the other hand, ma does correlate with signs of altered endothelial function, such as endothelium - dependent vascular dilatation [3133]; however, it is often difficult to disentangle the effects of alterations in renal function and concomitant metabolic and dyslipidemia states in identifying direct relationships between ma and endothelial dysfunction . Isolated ma may well be a biomarker of widespread vascular injury and atherosclerotic burden . In this sense, it is not measuring a kidney disease per se, but only a secondary and indirect effect of a distant disease process on kidney physiology . Although this conclusion may appear to be semantic rather than reality - based, it has immediate relevance for the diagnosis of ckd using current classification schema (ie, kdoqi - ckd). Stage 1 and 2 ckd in this schema cannot be reliably distinguished on the basis of egfr alone because current creatinine - based egfr formulas are imprecise . Whether newer cystatin c based formulas will correct this deficiency remains to be seen; preliminary findings are encouraging, but not uniformly so [34, 35]. Most cases of stage 1 and 2 ckd are currently defined on the basis of ma, typically in an isolated form . This definition is useful to stratify the eventual risk of cv events and all - cause mortality; however, does it have the same utility for defining the presence of and risk for progressive ckd and eventual esrd at all levels of egfr and in all ages? Current evidence suggests that the utility of ma in predicting renal outcomes is most pronounced in those with well - established kidney disease (eg, egfr <4550 ml / min/1.73 m) and in younger subjects (eg, <65 years of age). In my opinion, whether it is appropriate to regard isolated ma as equivalent to kidney disease is still an open question . A compromise position would be to regard this laboratory finding as an indication of kidney damage rather than giving it the more ominous label of disease . This issue is not trivial about 30% of those diagnosed as having ckd in epidemiologic studies have their kidney disease identified by the finding of ma and egfr 60 ml / min/1.73 m in the absence of a self - reported diagnosis of diabetes.
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Diabetes mellitus (dm) is a metabolic disorder characterised by an increase in plasma glucose level due to insulin deficiency and/or resistance that can lead to damage to multiple organs . Currently, approximate 347 million people are suffering from dm worldwide and the number will continue to increase . There are two types of dm: type 1 diabetes mellitus (t1 dm) and type 2 diabetes mellitus (t2 dm). T1 dm is caused by the destruction of the pancreatic -cells due to an autoimmune reaction, leading to absolute insulin deficiency, whilst t2 dm is characterised by insulin resistance, where the body fails to produce an appropriate physiological response to circulating insulin . T2 dm accounts for approximately 90% of all dm cases with the prevalence increasing with age . Tremendous effort has been invested to understand the complications of dm and its impact on vision loss, neuropathy, and cardiovascular diseases; however, dm - induced cognitive dysfunction is seldom addressed and is not as well understood . Since life expectancy has been markedly prolonged with advances in medicine, it has been suggested that the incidence of t2 dm and dementia would increase as the population ages [1, 2]. Indeed, several epidemiological studies have shown that people with t2 dm have a significantly higher risk of developing cognitive impairments and dementia when compared to those with normal blood glucose levels [35]. A recent population - based longitudinal study has shown that the relative risk of alzheimer's diseases (ad) and vascular dementia (vad) in the dm population was 1.46 (95% ci: 1.201.77) and 2.5 (95% ci: 2.13.0), respectively, when compared to people without dm . Moreover, dm has been suggested to be an individual risk factor for dementia [7, 8], independent of other established risk factors, such as hypertension and atherosclerosis [8, 9]. Traditionally, ad is considered as the major diagnosis of dementia; however numerous clinical - pathological studies have suggested a significant contribution of cerebrovascular diseases to cognitive decline [11, 12]. Although the exact pathophysiology of dm - mediated dementia has not been fully elucidated, existing evidence has shown that both cerebrovascular changes and neurodegeneration are implicated in the development and progression of dm - mediated cognitive dysfunction . To date nevertheless, numerous reports have highlighted the therapeutic potential of antidiabetic therapies in the treatment and prevention of cognitive dysfunction [1315]. In the largest randomized controlled trial to date, the accord - mind study, it was shown that the decline in total brain volume was significantly reduced in the intensive glycemic control group, compared to the standard glycemic control group . Although the cognitive outcomes were not different, the effect of glycemic control in preserving cerebral structure cannot be denied . Findings from these studies have clearly indicated that treatments targeting dm could be a novel strategy to prevent dementia development and potentially to slow down the progression of cognitive dysfunction . Through the advances in pharmacological therapy, many oral antidiabetic agents have become available . Interestingly, oral antidiabetic drugs such as thiazolidinedione and metformin have been shown to have beneficial effects to slow the progression of dementia in both clinical and animal studies [17, 18]. However, many of these pharmaceutical agents are associated with various undesirable side - effects, such as weight gain, fluid retention, and increased risk for heart failure, limiting their compliance and utility in clinical practice . Traditional chinese medicine (tcm), including chinese herbal medicines (chm) and acupuncture, has been used for thousands of years for the management of disease, maintenance of health, and prolongation of life expectancy . Accumulated evidence suggests that many chms and their active ingredients possess hypoglycemic properties and that some tcm interventions have beneficial effects in the treatment and prevention of dm and its complications, with minimal toxicity and fewer adverse reactions . In addition, existing evidence has demonstrated the therapeutic potential of tcms in dm - mediated cognitive dysfunction . In this paper, we present a comprehensive review of current understanding of dm - mediated dementia and the scientific research on the use of tcms for the management of cognitive dysfunction in dm . An increasing number of studies have reported an acceleration of cognitive decline in patients with dm, independent of common cardiovascular risk factors [8, 22]. To date, dm is recognised as an independent risk factor for the development of cognitive dysfunction . In a meta - analysis based on twenty - five studies, it was estimated that t2 dm patients have 1.5-fold greater risk of cognitive dysfunction and 1.6-fold increased risk of dementia, when compared to people without diabetes . Similarly, a recent report has shown a 1.5-fold higher risk of ad in people with diabetes than those without diabetes . Most reports so far have suggested an increased risk of global cognitive dysfunction in diabetes [6, 23, 24], while some reports showed more selective cognitive impairment, mainly affecting learning, mental speed, and visuospatial process [2527]. It is important to point out that the discrepancy between these studies may simply be due to the variation of neurocognitive testing, such as age, education, sex, history of other illnesses, and the duration / severity of diabetes [28, 29]. Many complications of diabetes, such as retinopathy, lower limb ulcers, and atherosclerosis, usually take years to develop before becoming clinically apparent . However, cognitive function decrement has been observed in the early stage of t2 dm . In children with t1 dm, deficits in cognitive development, including vocabulary, block design, general intelligence, speed of processing, and learning, have been observed as early as 2 years after the onset of t1 dm . These findings suggested that deficits in regulation of blood sugar level, even at an early stage, would have detrimental effects on cognitive function . Interestingly, recent studies have demonstrated that elevated blood glucose levels may be a risk factor for impaired cognitive function leading to dementia, even among people without diabetes [32, 33] highlighting the relationship between high blood glucose level and dementia outcome . There is ample evidence from neuropsychological studies reporting that people who have dm also suffer from mild cognitive impairment (mci). Longitudinal studies have shown that approximate 55% of patients with mci developed probable alzheimer's dementia over 3 years [34, 35] and the progression rate reached 100% after 9.5 years . It has been suggested that dm patients have 50% higher chance of developing alzheimer's disease than those without dm . Progression of mci to dementia has been shown to be markedly accelerated by diabetes in elderly subjects who were either cognitively intact or diagnosed with mci at baseline . Brain imaging studies have provided direct evidence to support dm - mediated mci and dementia [39, 40]. Resting - state functional magnetic resonance imaging (rs - fmri) studies have revealed abnormalities in amplitude of low - frequency fluctuations (alff) in t2 dm patients in multiple brain regions . These present as decreased alff in the bilateral middle temporal gyrus and left fusiform gyrus and increased alff in bilateral cerebellum posterior lobe and right cerebellum culmen . Moreover, recent studies have shown that alternation of alff and reduced connectivity of the hippocampus are associated with the presence of diabetic vascular disease and poor cognitive performance in t2 dm patients [42, 43]. Although the mechanism between mci and the increased risk of dementia under dm is not fully understood, it has been suggested that the dm - mediated mci and dementia are not likely to form a continuum, given the difference in etiologies and risk factors between mci and dementia [44, 45]. People suffering from dm over a long period have been shown to express an elevated level of dementia [46, 47]. There are an extensive number of studies examining the effect of dm on cognitive functions in elder population [4850]. It has been shown that the prevalence of dementia in t2 dm patients increased over age, from 2.4% in the age group of 6576 and 5% in 7685 to 8.3% for patients over 85 years of age . Several studies have also reported an increased incidence of dementia in individuals who were diagnosed with dm in midlife after an extended follow - up of 2535 years . However, the exact effect of midlife against late - life dm onset on cognitive impairment and dementia remains to be clarified . The mechanisms underlying the development of cognitive dysfunction in diabetes have not been fully elucidated . Many hypotheses have been suggested based on the pathophysiological mechanisms through which diabetes might affect the initiation and progression of the pathology of dementia . These proposed mechanisms include various diabetic - specific factors or signalling pathways that may influence cognitive functioning, such as hyperglycemia, insulin deficiency, microvascular complications, and inflammation . In this section, we will highlight some of the risk factors and possible mechanisms related to cognitive dysfunction in diabetes (figure 1). Vascular complications, including atherosclerosis, hypertension, stroke, and vascular comorbidity, are closely associated with dm . Recent studies have reported that vascular complications are likely to be an important determinant of cognitive dysfunction and dementia . Increased cerebral infarcts and reduction of amyloid - beta load a meta - analysis of longitudinal studies suggested that there is a stronger association of vascular - related cognitive impairment than ad with dm patients . Interestingly, less alzheimer's - like pathology has been observed, but more ischemic lesions in t2 dm patients with a clinical diagnosis of dementia have been observed . Indeed, an increasing number of studies are suggesting that the reduction of cerebral perfusion plays a significant role in the development of ad, supporting the hypothesis that cerebrovascular pathology such as stroke predisposes cognitive decline and dementia development . The detrimental effects of dm on cognitive function in vascular dementia have been demonstrated in a recent preclinical study . Showed that exacerbated cognitive functions caused by diabetes were mediated via augmentation of neuronal cell death in the hippocampus through creb / bdnf signalling pathway in an animal model of vascular dementia . Hypertension has been shown to be a significant risk factor for poor cognitive performance in both t1 dm and t2 dm patients [57, 58]. In addition to hypertension, atherogenic dyslipidemia is another common vascular risk factor in dm . Dyslipidemia contributes to atherosclerosis development and has been found to increase risk of dementia in diabetes . Moreover, reduced cerebral blood flow, upregulation of inflammatory cytokines [63, 64], endothelial dysfunction, and abnormalities in cerebral capillaries have been demonstrated in patients with diabetes . Changes in cerebral vasculature by these factors are closely associated with stroke and brain damage, including brain infarct and white matter lesions, contributing to cognition deterioration in diabetes . Given the complex pathophysiology of vascular complications in diabetes, more research is required to explore the exact mechanisms around how these dm - related vascular risks contribute to cognitive decline and dementia . However, majority of current findings have confirmed the association of vascular risk factors and cognitive decrements in diabetes and support the belief that predominant cerebrovascular pathology in diabetes could aggravate cognitive functioning . Blood glucose levels are regulated by the endocrine system involving multiple organs and signalling molecules and pathways . Upset of this precisely regulated process could lead to imbalance of blood glucose level, resulting in organ damage . Although the exact mechanisms behind the association between dm and cognitive impairment or dementia are unclear, studies have shown that it is a multifactorial process where metabolic condition plays a significant role . Chronic high blood glucose levels have been shown to have negative effects on cognitive functions and brain structure . Numerous studies have demonstrated a close relationship between glucose intolerance and cognitive decrements and dementia [24, 69, 70]. It has been shown that people with poor glycemic control, with glycosylated hemoglobin (hba1c) higher than 7.0%, have a 4-fold higher risk of developing cognitive impairment . Similarly, an inverse association of hba1c and cognitive function such as working memory, learning, and executive functioning has been observed in t2 dm patients . The results of these studies highlight the contribution of poor glycemic control in cognitive function deterioration process . Multiple toxic effects of hyperglycemia on the brain, such as formation of advanced glycated end products (ages), generation of reactive oxygen species (ros), and activation of polyol, diacylglycerol, and hexosamine pathways, have been suggested . Ages have been shown to contribute to microvascular complications, accelerated amyloid - beta deposition and senile plaque formation [9, 71]. A preclinical study has demonstrated that increased cerebral ages expression is associated with cognitive dysfunction in diabetic mice . Similarly, increased ages levels have been observed in ad patients with t2 dm, when compared to nondiabetic ad patients . Moreover, ages lead to ros generation via activation of a rage cell surface receptor for ages, which in turns leads to neuronal injury [76, 77]. It is well established that oxidative stress is implicated in both the onset and progression of diabetes and its complications . It has been shown that cognitive deficit caused by hyperglycemia in diabetic rat is associated with an increase in ros levels and reduction of antioxidant levels [78, 79]. In addition, increased ros generation has been shown to activate various cellular signalling pathways, such as the polypol pathway, protein kinase c activation, and increase of glucose shunting via the hexosamine pathway, all of which are related to neuronal injury and cerebral damage . Interestingly, it was shown that administration of antioxidants could reverse the cognitive dysfunction in the diabetic rats [78, 79], suggesting a potential therapeutic target for dm - mediated cognitive impairment . Sufficient glucose supply is vital for normal brain function and it is well established that hypoglycemia has detrimental effects on the brain [81, 82]. Repeated hypoglycemic episodes are a common side - effect in patients who receive intensive insulin therapy for diabetes . In animal studies, it has been shown that exposure to low blood glucose levels can cause cerebral energy failure, neuronal necrosis, and brain damage leading to a flat electroencephalograph and cognition dysfunction . In human autopsy studies, multifocal or diffuse necrosis of the cerebral cortex, basal ganglia, and hippocampus was observed in patients who died of hypoglycemia . A dose - response relationship between the occurrence of severe hypoglycemic episodes and risk of dementia development has been reported in a retrospective study involving 16,667 t2 dm patients . Although contradicting results have been reported by some studies [85, 86], arguing that tolerance to a hypoglycemic state can be developed in patients exposed to hypoglycemia chronically, the effect of hypoglycemia on some high - risk groups cannot be ignored . For example, it has been shown that impairment of memory functioning is strongly correlated with severe hypoglycemia in t1 dm patients . The blood glucose level is regulated by insulin, a hormone generated by the beta cells in the pancreas . Traditionally, it was believed that the brain is an insulin independent organ; however, recent studies have suggested otherwise . It has been shown that insulin is actively transported across the blood brain barrier and is also produced locally in the brain . Furthermore, insulin receptors are expressed in the hippocampus and the cortex, indicating its functional role in the brain . In addition, being a regulator of food intake and energy homeostasis, insulin also plays a role in memory and learning . Changes in insulin levels and receptor sensitivity could lead to deficits in cognitive function . In ad, impairments of cerebral insulin receptors activation and elevated insulin level in the csf have been reported, indicating the contribution of insulin in cognitive decline and dementia development . Hyperinsulinemia is a common characteristic of t2 dm and has been identified as a risk factor for cognitive dysfunction and dementia progression [95, 96]. It has been suggested that hyperinsulinemia is associated with reduction of amyloid metabolism, due to downregulation of insulin - degrading enzyme (ide) levels in the brain . Therefore reduced ide levels would lead to a accumulation in the brain, contributing to ad and cognitive impairment . It has been suggested that inhibition of insulin - mediated pathways can lead to hyperphosphorylation of tau and a production, via activation of the glycogen synthase kinase 3 (gsk3) signalling [99, 100]. It has been suggested that people suffering from dm are under a state of subclinical chronic inflammation [101, 102]. Numerous proinflammatory markers and cytokines, such as c - reactive protein (crp), tumour necrosis factor- (tnf-), interleukin- (il-) 1, and il-6, have been shown to be upregulated in both t1 dm and t2 dm . Given the fact that many of these inflammatory markers found in dm patients are closely associated with the pathogenesis of ad, there is an increasing interest in the link between dm and dementia . Yaffe et al . Reported that impaired cognitive functions were observed in dm patients with elevated crp and il-6 levels, but not in patients with normal levels of these markers . Firstly, it has been shown that chronic inflammation in dm can induce changes in blood brain barrier (bbb) permeability . Moreover, increased bbb permeability can also allow access of toxic substances and metabolites into the brain, leading to cerebral damage . Secondly, neuroinflammation is a well - established factor in the development of cognitive decline, dementia, and other neurodegenerative diseases [109, 110]. It has been demonstrated that inflammatory cytokines can cause activation of glia cells leading to neuronal damage . For example, tnf- has been shown to induce hippocampal dysfunction, via activation of the jnk and the ib kinase / nfb signalling pathway [111113]. Finally, inflammation plays a central role in the development of complications in vasculature, including stroke, contributing to cognitive impairment and dementia development as discussed in section 3.1 . Given the fact that dm is now an established risk factor for cognitive dysfunction and dementia, there is an increasing interest in targeting dm for the treatment of cognitive decline and dementia . Several studies suggested that oral antidiabetic drugs such as thiazolidinedione and metformin could offer therapeutic benefits to reduce dm - related cognitive dysfunction in both patients and animal models [17, 18]. A recent study demonstrated that glycemic control using empagliflozin significantly prevented cognitive impairment via attenuation of cerebral oxidative stress and increase in cerebral brain - derived neurotropic factor in a t2 dm mouse model . This highlighted the potential of antihyperglycemic agents in the treatment of t2dm - related cognitive dysfunction . However, some clinical studies have demonstrated rather limited and inconsistent benefits of these glucose lowering agents in limiting cognitive decline and dementia development [1416]. Furthermore, the risks associated with the use of antiglycemic / insulin therapies have raised the concerns regarding the long - term safety and effectiveness of these interventions for the management of dm - induced cognitive dysfunction and dementia [84, 85]. For example, hypoglycemia is commonly observed in t1 dm patients with tight glycemic control and in advanced t2 dm patients [116, 117]. Although intensive insulin therapy has shown to successfully control glycemia and reduce vascular complications in dm patients, several reports have highlighted possible neuronal damage and cognitive impairment due to the incidence of hypoglycemia associated with the insulin therapy . Traditional chinese medicine (tcm) has been used to treat dm for over thousands of years . A large number of tcm interventions belonging to several key modalities (such as herbal medicine, acupuncture, and taichi) have been used for the management of dm and its complications (table 1). According to traditional chinese medical system, chinese practitioners, who often adopt a holistic approach in treating their patients, manage diabetes through integrated care: nourishing and strengthening the body's functions rather than focusing solely on blood glucose control [119, 120]. Despite a lack of scientific validation of the tcm interventions for diabetes, the accumulated evidence has demonstrated some promising results in relieving the symptoms and complications of diabetes [121, 122]. In this section, we will firstly review the current clinical findings of tcms used for dm - related cognitive dysfunction and then highlight some of the potential mechanisms underlying the tcm effects on the condition . Numerous clinical studies have demonstrated the beneficial effects of tcm interventions on cognitive dysfunction and dementia . Data from a meta - analysis suggest that tcm interventions appear to be a safer and more effective treatment for vascular dementia, based on 31 randomised clinical trials comparing 1605 patients on tcm treatments with 1263 patients on western medicine or placebo . It has been suggested that sleep apnea hypopnea syndrome (sahs) patients with t2 dm have a higher risk of cognitive decline than the nondiabetic sahs patients . Interestingly, in a clinical study, a 2-week treatment with danhong injection, consisting of extracts of salvia miltiorrhiza and carthamus tinctorius l., significantly improved the montreal cognitive assessment (moca) score, especially in the executive function and memory domains, in 86 sahs patients with t2 dm . Although blood glucose levels were not examined in this study, the results indicate that danhong injection could improve cognitive function in t2 dm patients . Another clinical study in 36 t2 dm patients has demonstrated that combined huang qi (radix astragali) and chuanxiong (ligusticum wallichii) injections over 30 days significantly reduced blood glucose levels and improved cognitive function, whilst the standard pharmaceutical care with antidiabetic agents in the control group only reduced the blood glucose levels, indicating that the herbal formula may provide therapeutic benefits to dm patients beyond its blood glucose lowering property . In a clinical trial involving 164 diabetic patients with complicated coronary heart disease (chd), it was shown that while both isosorbide mononitrate (20 mg, twice a day) and fufangdanshendiwan (consisting of salvia miltiorrhiza, panax pseudoginseng var . F.) (270 mg, once daily) treatments for 16 weeks significantly improved the cardiac ischemia burden (as measured by 24-hour ambulatory ecg monitoring), improvements in insulin sensitivity and lipid metabolism indexes were only observed in patients received fufangdanshendiwan . Moreover, the herbal formula was also shown to reduce a formation and improve cognitive function in the dm patients . As mentioned in section 3, increased oxidative stress and inflammation li and yeung have demonstrated that an 8-week treatment with zhi nao capsule consisting of extracts of codonopsis pilosula, polygonatum sibiricum, ligusticum wallichii, and acorus tatarinowii significantly increased serum superoxide dismutase (sod), reduced crp level, and limited cognitive decline and dementia development in t2 dm patients . In china, it is not an uncommon practice to use integrative strategies, combining tcm and western medicine interventions, in the treatment of dm and its complications . Numerous studies have assessed the efficacy of the combined therapies to treat cognitive dysfunction in dm patients . Nao xin tong, a complex herbal formula (consisting of radix astragali, salvia miltiorrhizae, angelicae sinensis, ligusticum wallichii, paeoniae rubra, flos carthami tinctorii, gummi olibanum, resina commiphorae myrrhae, ramulus cinnamomi cassiae, buthus martensi, lumbricus, and hirudo seu whitmaniae) has been shown to improve cognitive function in 32 stroke patients with t2 dm . In patients who received a combined therapy of alprostadil with lao xin tong, a greater cognitive enhancing effect was observed when compared to the alprostadil only group . In a clinical trial, 48 dm patients with vad were randomised to receive piracetam or piracetam plus oral shengmaidingzhi decoction (consisting of pseudostellaria heterophylla (miq .) Pax, ophiopogon japonicus, schisandra chinensis, wolfiporia extensa, polygala tenuifolia willd ., acorus tatarinowii, pinellia ternata (thunb .) Breit, semen persicae, panax pseudoginseng var . The combined therapy group demonstrated greater improvements in the activities of daily living scale (adls) and the scale of elderly cognitive functions (secf) when compared to that of the piracetam only group . Several studies have demonstrated that a combined therapy of bushenquyuyizhi decoction (consisting of cistanche deserticola, acorus tatarinowii, and panax pseudoginseng var . Notoginseng) with nimodipine produced a significantly greater effect than nimodipine alone on cognitive function [155, 156]. In a more recent study conducted by zhao et al ., 85 patients with dm - mediated vascular cognitive dysfunction were allocated to receive a 6-month treatment of aspirin (100 mg, once daily) or aspirin (100 mg, once daily) plus bushenquyuyizhi decoction (2.5 g per day, orally), over 6 months . At the completion of the 6-month treatment, cognitive dysfunction was improved in both treatment groups . However, the cognitive improvement was only maintained in the combined therapy group 12 months after the treatment, indicating a potential long - term effect of the herbal intervention, possibly via enhancement of general health and limiting disease progression in the patients . Electroacupuncture is a form of acupuncture by way of applying a small electric current between pairs of fine needles that are inserted into acupoints selected according to the tcm theory . Several recent studies have also demonstrated that electroacupuncture improved cognitive function and quality of life in diabetic patients [159, 160]. Although the sample sizes were small (ranging from 25 to 32 patients) in these studies, the therapeutic potential of electroacupuncture should not be ignored and further investigations with a rigorous design are warranted . A large number of in vitro and in vivo preclinical studies have been conducted to assess the underlying mechanisms of tcm interventions in diabetes - related cognitive dysfunctions . Reduced antioxidative levels and increased ros generation are closely associated with the pathogenesis of diabetes and its complications . Antioxidant properties of chms have been demonstrated in numerous studies [161, 162]. For example, green tea, which is commonly consumed in eastern and asian countries, contains of a mixture of plant polyphenols that possess antioxidative and radical - scavenging activities . In obese kk - ay mice, green tea catechins reduced blood glucose levels and insulin resistance via inhibition of the tnf--induced ros generation . It has also been shown that green tea catechins markedly suppressed memory regression in samp10 mice, a mouse model of brain senescence with cerebral atrophy and cognitive dysfunctions . Daily consumption of green tea catechins significantly reduced brain atrophy and suppressed dna oxidative damage . These effects were associated with the improvement of plasma antioxidative activity caused by daily green tea consumption . A recent study showed that green tea catechins remarkably ameliorated learning and memory impairments in a diabetic rat model, via the reduction of oxidative stress and nitric oxide modulation . In this study, green tea catechins also significantly reduced the blood glucose levels, indicating that green tea can suppress diabetes - mediated cognitive dysfunction via both hypoglycemic and antioxidative effects . Indeed, numerous other chms, such as berberine and ginsenoside, have been shown to reduce diabetes - mediated cognitive decline via reduction of oxidative stress [167, 168]. Tanshinol (tsl), a bioactive component of danshen (salvia miltiorrhiza), widely used for vascular disease, was shown to improve spatial working memory and attenuated vascular dementia in rats, via an increase in acetylcholine levels and reduction of acetylcholinesterase activity . Tanshinone iia (tan iia), another bioactive component of danshen, was also shown to restore diabetes - induced nerve deficiency . The regulation of the cholinergic neurotransmission in the brain plays a vital role in memory and cognitive function . Similar to tsl, several other herbal constituents, including lycopene, berberine, and curcumin, have been shown to ameliorate diabetes - related cognitive dysfunction, via protection of the cholinergic neurotransmission [167, 172, 173]. In addition, several other mechanisms have been proposed as underlying the effect of chms on dm - mediated cognitive dysfunction, such as through reduction of ages - mediated neuroinflammation and the downregulation of cerebral amyloid - beta (a). For example, danshensu, a bioactive component of salvia miltiorrhiza, has been shown to improve learning and memory in diabetic mice via suppression of age - mediated neuroinflammation . These effects were independent of blood glucose, insulin, and glycosylated hemoglobin levels, indicating a direct neuroprotective and anti - inflammatory effect of danshensu . Liuwei dihuang decoction (lwdhd), a well - established tcm formulation, consisting of six herbs (rehmannia glutinosa libosch ., cornus officinalis sieb ., dioscorea oppositifolia l., paeonia ostii, alisma orientale (g. samuelsson) juz ., and poria cocos (schw .) Wolf), has been shown to attenuate neural apoptosis and a deposition in the hippocampus and cerebral cortex in a streptozotocin - induced diabetic rat model . In addition, lwdhd also reduced blood glucose levels, decreased oxidative stress, and suppressed inflammation in hippocampus of the animals . Interestingly, chen et al . Showed that administration of zibupiyin recipe (zbpyr), a modification of the zicheng decoction (consisting of 12 herbs: panax ginseng c. a. meyer, dioscorea opposita thunb . Tomentosa, and glycyrrhiza uralensis fisch . ), over a 6-week treatment, prevented dm - associated cognitive decline in db / db mice . The observed effect was possibly due to improving dendritic spin density and attenuating brain leptin and insulin signalling pathway injury . Although these findings need to be confirmed in humans, it provides important preclinical data to support the potential benefits of tcm in preventing and slowing the development and progression of dm - associated cognitive dysfunction . Data from the above studies have clearly indicated that the positive effects of chms could be mediated by multiple pathways and mechanisms . Combination therapy underpins the philosophy of chms, where patients are generally treated with multiherb formulations . Complex chemical mixtures of chms enhance therapeutic efficacy by facilitating synergistic action and/or ameliorating / preventing potential side - effects . The multicomponent and multitarget approach of chms makes them ideal therapies for disorders such as dm - mediated cognitive dysfunction and dementia, which have multifactorial / multisystem pathophysiological components . Substantial effort has been invested to understand the effects of diabetes on cognitive decline and dementia in the past decade . Recent studies have identified the risk factors and possible mechanisms underlying the pathogenesis of the dm - mediated cognitive dysfunction . By taking advantage of recent advancements in these processes, it is possible to develop better therapies for dm - related cognitive complications, including ad, vascular dementia, and cognitive decline . Numerous clinical studies have highlighted the potential of tcms in the treatment of dm - related cognitive decline . Despite the fact that most of these trials have shown positive outcomes, significant methodological issues such as small sample sizes and poor randomization exist in many of these studies . Therefore, more rigorously designed randomized controlled trials are required to further validate these findings . Finally, most of the preclinical studies assessing the effects of chms on dm - mediated cognitive dysfunction were performed using single herbs or isolated active ingredients . Given that the traditional use of chms is based on complex formulation, more research on the synergic effects of herbal combinations is required for a more comprehensive understanding of the mechanisms underlying their effect on the diseases.
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Metastatic disease is rare and usually occurs within the first two years after initial diagnosis, and is extremely rare after that time period . However, metastases to forearm bones are very rare with only very few reports published till date . Here we present a case of bilateral retinoblastoma with metastases to right forearm bones four years after the initial treatment . An 8-year - old boy treated four years back for bilateral retinoblastoma with enucleation of left eye along with adjuvant chemotherapy and radiotherapy presented with a right forearm swelling . An x - ray revealed a mass lesion in right forearm with destruction of lower one third of right ulna . Bone scan was performed three hours after intravenous injection of 5 mci of 99tc mdp . Whole body images showed increased tracer uptake in right forearm bones [figure 1]. Static images revealed increased tracer uptake in middle and lower one - third of right ulna consistent with x - ray findings . In addition, bone scan showed involvement of lower one - third of left radius . However, special stains excluded ewing's sarcoma and other primitive neuroectodermal tumors (pnets). Involvement of radius picked up by bone scan had important implication in deciding the nature of disease as ewings tumor with multi - focal disease as second primary is very unlikely . Tc99m - mdp bone scan; (a) whole body anterior view; (b) whole body posterior view showing increased tracer uptake in right forearm bones . Static images of bilateral forearms; (c) anterior; (d) posterior revealing involvement of right ulna and radius osseous metastases from retinoblastoma are reported most commonly in the skull and long bones . The prognosis for most children diagnosed with retinoblastoma is good, with a 5-year survival of rate of more than 90% . Complications include local recurrence, metastases, and the development of a second primary tumor . Focal and diffuse involvement of the bones and bone marrow is known to occur, but metastases are usually seen within two years of initial diagnosis and later metastases are said to be extremely rare . These patients are also at risk of increased second primaries like osteosarcoma, leiomyosarcoma, spindle cell sarcoma, malignant fibrous histiocytoma, rhabdomyosarcoma, angiosarcoma, ewing sarcoma, and pnet . These typically develop after a latent period of 815 years. [68] involvement of multiple bones would point the diagnosis towards metastases rather than multifocal second primary . Imaging modalities available for skeletal metastatic work up include x - ray, ct, mri and functional imaging modalities like bone scan . X - rays are in general less sensitive to pick up the metastases, whereas whole body mri is not widely available, so conventional wb bone scan is essential in the metastatic work up of these patients . In our case, x - ray picked up ulnar involvement but radius involvement was missed . However, bone scan revealed radius involvement suggesting a metastatic disease rather than second primary . Later, histopathology revealed round blue cell tumour involving both radius and ulna and ihc was negative for cd99 excluding diagnosis of ewings sarcoma . Skeletal scintigraphy remains an essential diagnostic procedure in evaluation of these patients and can have important implications in management.
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Parosteal lipoma is an extremely rare benign tumor composed mainly of mature adipose tissue with a bony component . It is among the rarest neoplasias of skeleton, accounting for less than 0.1% of primary bone tumors and 0.3% of all lipomas . The most common locations for this tumor are the femur, proximal radius, humerus, tibia, clavicle and pelvis . Thought to arise from mesenchymal cells in the periosteum, parosteal lipomas share histopathologic features with the commonly occurring soft - tissue lipomas, and cytogenetic evidence suggests a common histopathogenesis . Depending on the degree of chondroid modulation and enchondral ossification, parosteal lipomas may rest directly on the cortex without cartilage or bone elements; may have a narrow bony stalk with a lucent lipomatous cap, mimicking a pedunculated exostosis; may have a densely ossified broad - based osteochondromatous element beneath a thin lipomatous cap, simulating a sessile exostosis; or may have patches of chondroid and bone scattered throughout the lipomatous mass . This study reports the case of a 65-year old woman with a big mass on the posteromedial aspect of the right upper arm since 1 year (admitted on 05/07/2013). The swelling was slow growing, painless, soft, nontender, immobile, with well defined margins and no fixity to skin . On roentgenogram of right humerus there was an evidence of ill defined soft tissue swelling in the upper arm posteromedially with radiopacity in continuation with the surface of humerus suggestive of bony excrescences (fig . Mri right shoulder joint (plain + contrast) revealed a large 13 cm 5 cm 8 cm well defined, nonenhancing, lobulated, heterointense, predominantly fat intensity lesion with a small area of chondroid component measuring 2 cm 1.6 cm in posteromedial aspect of the proximal right humerus, seen completely separate from the adjacent muscles (fig . Vertical elliptical incision was taken over the posterior border of the right upper arm over the tumor . The tumor was under the lower part of deltoid which formed the roof near the upper end of humerus, between the long and medial heads of triceps muscles (fig . 6 cm 5 cm 5 cm sized part of the tumor was under the long head of triceps displacing it medially along with radial nerve and vessels . 7 cm 6 cm 3 cm sized part of tumor was under the medial head of triceps displacing it laterally . After hemostasis, wound was closed with a suction drain in situ . The specimen (fig . 5a) weighed 250 g. postoperatively a shoulder sling was given for 3 weeks to prevent any inadvertent fracture following the use of osteotome over the humerus during the surgery . 1b) of right humerus was normal and showed no bony excrescences . On gross pathology, the lesion was multilobulated, well circumscribed and irregular mass of size 12 cm 5 cm 8 cm with a bony part measuring 2.5 cm 2 cm 1 cm . 5b), the lesion was composed of mature lipocytes that had an intimate relationship with the periosteum, consistent with parosteal lipoma . Parosteal lipomas, benign adipose tissue tumors situated directly on bone cortex, are unusual neoplasms that appear to emerge from multidirectional mesenchymal modulation within the periosteum . Periosteal lipomas, chondrolipomas of soft tissue and lipomas of nerves but they are most commonly believed to originate from the periosteum . These tumors are frequently associated with chondroid and/or osseous modulation, which permit subclassification into 4 distinct variants: (i) no ossification; (ii) pedunculated exostosis; (iii) sessile exostosis; and (iv) patchy chondro - osseous modulation . Parosteal lipoma presents as an immobile, nontender, slow growing mass over bones that is not fixed to skin . On radiographs, a parosteal lipoma is a well - defined area of lucency located adjacent to a long bone . In one series of parosteal lipomas, 60% had definite bony alterations, mostly hyperostotic reactive changes (fine linear densities, calcification, cortical thickening or undulation, or frank excrescences of bone), but these lipomas also have cortical bowing and smooth cortical erosions . Parosteal lipomas have a homogeneous lobulated appearance and are adherent to the surface of the adjacent bone . When present, osseous excrescences may mimic osteochondromas, but the former lack the contiguity of the marrow space with the underlying bone that is characteristic of the latter . Parosteal lipomas that gain clinical attention are those that compress neurovascular bundles and cause motor and sensory function deficits . The tumor is identified on mr images as a juxtacortical mass with signal intensity identical to that of subcutaneous fat, regardless of pulse sequence . Heterogeneity in these lesions is invariably present and corresponds to the pathologic components in the lesion . Areas with intermediate signal intensity on t1-weighted images that are high signal intensity on t2-weighted images represent the cartilaginous components in parosteal lipoma . Fibrovascular septa may cause a lobulated appearance of the fat component, with low - signal - intensity strands on t1-weighted images that become higher in signal intensity on the long tr images (particularly with fat suppression). Larger areas of bone production surrounded by the lipomatous components are also well demonstrated with mr imaging . Adjacent muscle atrophy, poorly demonstrated by ct, is identified on mr images as increased striations of fat in the affected muscle and is caused by associated nerve entrapment . This finding is best appreciated on t2-weighted images because of the decreased signal intensity of normal muscle relative to fat . Finally, mr imaging best demonstrates the relationship of the tumor to the underlying native bone and muscle, and this information is important for surgical planning because parosteal lipoma is usually firmly adherent to the underlying cortex at the site of surface bone production . Pathologically the lesion is usually a multi - lobulated yellowish mass composed of mature adipocytes, and it is well encapsulated with a broad base of attachment to the underlying bone . Microscopically, the fat cells of a parosteal lipoma appear histochemically identical to the adipocytes that are found in the subcutaneous tissues . There has been no indication to date that this tumor undergoes malignant degeneration, although minimal cellular pleomorphism may occasionally occur . The treatment of parosteal lipoma is complete surgical resection . In the case with nerve entrapment, the tumor must be removed before irreversible muscle atrophy occurs so as to maintain function . The nerve must also be separated from the parosteal lipoma and care must be taken to spare it during surgical excision . In our case, adequate surgical removal of a parosteal lipoma requires either subperiosteal dissection, which is the separation of the lesion from the underlying bone using an osteotome or segmental resection of bone; this is in contrast to the relatively easy dissection for a soft tissue lipoma lying adjacent to bone . Several cases have shown a malignant transformation having radiological and histological features of benign lipomas together with histologic fields of either malignant fibrous histiocytoma or liposarcoma . Malignant transformation of a lipoma should be suspected when rapid bone destruction is seen in a radiolucent lipoma . Parosteal osteosarcomas and well - differentiated liposarcomas (wdlps) of soft tissue share several features: they are slowly progressive, locally aggressive tumors, tend to recur locally, and rarely or never metastasize if not dedifferentiated . Microscopically, both are well differentiated tumors, very like their normal tissue counterpart . A biopsy is required from its two components, i.e. Bony base and peripheral fat . Written informed consent was obtained from the patient for publication of this case report and accompanying images . Dr . Rohan chaudhary performed the study design, data collections, data analysis, writing and he was the operating surgeon.
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We sampled ants in three regions of morocco: first in the north - west, in the region of tangiers - tetouan where we focused on several areas: (1) mediona and cap spartel within the province of tangiers, (2) al hamra and balota within the province of tetouan, and (3) dardara, fifi, talassemtane national park, and bouhachem natural park within the province of chefchaouen . Second, we surveyed the eastern region where we focused on two areas: (1) site of bni snassen within the province of berkan and (2) temsamane within the province of driouch . The third region of interest was marrakech - tensift - el haouz situated in the south - west of morocco, where the okaimden mountain in the province of al - haouz, was surveyed (fig . 1.the provinces and localities where sampling was undertaken for this study . The provinces and localities where sampling was undertaken for this study . For collections of ants from nests, specimens were collected under direct vision using an aspirator and forceps . A berlese funnel technique was also utilized for extracting ants from soil and leaf litter and at a few sites we used pitfall traps . Specimens were studied under a leica s4d stereomicroscope and identified using available keys (cagniant 1996, 2009) taxonomic nomenclature follows bolton (2014). Below, our collections are organized as follows: a2808: (3w, 1q); where a2808 refers to the collection code of reyes - lpez, and where (3w, 1q) refers to 3 workers and 1 queen . This information is followed by the date, locality, gps coordinates, and altitude in meters above sea level . Plagiolepis pygmaea, formicinae material and data: a2816: nest; 11 june 2011; talassemtane national park, tazaout; 35 7.95 n, 05 06.76 w; 1,680 m; a1627: (14w); 15 july 2010; talassemtane national park, jbel bouslimame, spanish fir, abies pinsapo, forest; pitfall trap; 35 07.05 n, 05 08.16 w; 1,500 m. this species was found in rabat and casablanca by cagniant (1962). Although it was described as a rare exotic of european origin, introduced to morocco through human activities, cagniant has never mentioned this species again . The workers that we examined, correspond to the latest description of this species (sharaf et al . 2011), in having funicular segments 2 and 3 of equal length, and distinctly broader than long, and 9 ommatidia in the longest row of the eyes . As jbel bouslimame is in a protected area, very remote from human settlements, we suspect p. pygmaea is indigenous to morocco . Ponera testacea, ponerinae material and data: a2808: (3w, 1q); 16 may 2011; talassemtane national park, akchour; 35 15.88 n, 05 08.83 w; 566 m; a2807: (18w); 05 june 2011; talassemtane national park, tazaout; 35 15.90 n, 05 08.19 w; 1,475 m; a2743: (1w); 11 september 2011; bouhachem natural park; 34 59.51 n, 04 49.21 w; 1,390 m; a2676: (4w, 3q); 5 june 2011; talassemtane national park, tazaout; 35 15.90 n, 05 08.19 w; 1,475 m; a2673: (2w); 29 may 2011; talassemtane national park; 35 04.78 n, 05 10.10 w; 890 m. a2618: (10w); 12 july 2011; bouhachem natural park; 35 16.33 n, 05 29.47 w; 1,152 m. it seems this species is widespread in the north of morocco . We found a multitude of them, including whole nests, in leaf litter and soil samples, and under rocks and moss . The main place where we found this species is the talassemtane national park (chefchaouen). X. espadaler (unpublished data) previously collected it in morocco at: (1) bab - bou - idir, 25 may 1986, 1,500 m, q. canariensis, q. ilex; espadaler leg . ; and, (2) tazzeka, taza, 29 march 1997, 1,400 m, c. hernando leg . Strumigenys tenuipilis, myrmicinae material and data: a2596: (1w); 13 july 2011; chefchaouen, quercus suber forest; 35 06.10 n, 05 18.20 w; 488 m; a2598: (4w); 14 june 2011; dardara, quercus suber forest; 35 07.24 n, 05 17.60 w; 374 m. extracted from leaf litter and soil samples using the berlese funnel method . At dardara, it was given species status by brown (1953, p. 132) as s. tenuipilis . After further changes in generic placement, however, baroni urbani and de andrade (2007) have argued that it, along with many dacetines, should revert to the original strumigenys . In morocco, s. baudueri, p. argiola (cagniant 2006) and recently p. membranifera (taheri andreyes - lpez 2011), all of which are now placed in strumigenys, have been recorded from morocco . Temnothorax pardoi, myrmicinae material and data: a2822: (14w); 04 september 2011; tangers, mediona, cap spartel; 35 46.57 n, 05 54.42 w; 214 m; a2821: (1w); 13 september 2011; chefchaouen, fifi; 35 01.39 n, 05 12.25 w; 1,190 m. extracted from samples of soil and leaf litter using the berlese funnel method . This species was described from workers captured in the south of the iberian peninsula (tinaut 1987). There are other records of this species being found in different areas of the iberian peninsula, including portugal (tinaut 1987). The present finding is important because up to now this species has been considered as endemic to the iberian peninsula . We intend to survey the coastal areas of the tingitane peninsula (north of the rif) in order to precisely delimit the area of its distribution . Tetramorium parvioculum, myrmicinae material and data: a2599: (19w); 14 june 2011; chefchoauen, dardara, cork oak forest; 35 07.24 n, 05 17.60 w; 374 m; a2597: (6w); 13 july 2011; bouhachem, quercus suber woodland; 35 06.10 n, 05 18.20 w; 488 m; a2605: (2w); 11 july 2011; chefchaouen, dardara, akerat, cork oak forest; 35 06.10 n, 05 18.20 w; 488 m. t. parvioculum was described only recently from gibraltar, on the southern tip of the iberian peninsula (guillem and bensusan 2009). It has since been found on both sides of gibraltar, namely in spain and in morocco (guillem et al . Aenictus vaucheri, dorylinae material and data: a2602: (10 w) nest; 13 july 2011; chefchaouen, akerrat forest, cork oak forest; 35 06.10 n, 05 18.20 w; 488 m; a2197: nest; tetouan, al hamra; 35 23.83 n, 05 22.27 w; 139 m; a2866: nest; 30 april 2011; near balota; mosaic of scrub and open woodland with pistacia lentiscus, quercus suber, calicotome villosa, cistus spp . ; 34 57.13 n, 05 31.66 w, 140 m. this species is common in three separate locations in morocco . Two are from the atlantic coast area of the tingitane peninsula and the other from the atlas of beni mellal (cagniant 2006). Despite its hypogaeic habitat, cagniant considered this species to be very common in koumch (the region of beni mellal). Aphaenogaster pallida group, myrmicinae in morocco, the pallida group (see schulz 1994) is represented by two species given below: foreli and leveillei . According to cagniant (1996), however, due to their subterranean existence, records are scarce and those which have been cited (from elsewhere in morocco) are not georeferenced . Material and data: a2721: (6w); 03 april 2011; berkan, sibe of bni snassen; 34 48.91 n, 02 24.30 w; 729 m. aphaenogaster leveillei material and data: a2084: (11w); 12 february 2011; driouch, temsanane, abelkhache, open area surrounded by fields of crops; 35 12.86 n, 003 41.23 w; 385 m; a2656: (1w); 2 april 2011; pitfall traps; 34 48.90 n, 02 24.30 w; 732 m. material and data: a2802: nest; 22 september 2011; chefchaouen, dardara, cork oak forest; 35 06.24 n, 05 17.65 w; 433 m; a2848: nests; 26 february 2012; chefchaouen, akarat, forest of hayouna; 35 06.64 n, 07 19.43 w; 722 m. a2850: nest; 20 february 2012; marrakech, jbel okaimdem; 31 11.75 n, 07 51.35 w; 2,170 m. this species has been found in the tingitane peninsula and in the middle atlas (cagniant 1964, de haro and collingwood 1994), as well as in tunisia and algeria . Our record from marrakech indictes that this species exists at much higher altitude (2,170 m) than previously recorded (200800 m). Linepithema humile, dolichoderinae material and data: a2867: 4 january 2012; chefchaouen city; 35 10.07 n, 05 16.13 w; 591 m; 15 september 2012; rabat city, hassan; 34 01.045 n, 6 49.522 w; 15 september 2012; temara, harhoura (road to skhirat); 33 54.520 n, 6 58.672 w. l. humile is an invasive species that has been recorded many times in morocco, principally in some coastal areas of the tingitane peninsula (tangiers and cape spartel, cagniant 1964; martil and cabo negro, de haro and collingwood 1994). We can now confirm the widespread presence of this species in rabat and temara (especially in public gardens). . We have found it in various places in the town of chefchaouen, especially in gardens and in the walls of buildings . Proceratium algiricum, proceratiinae material and data: a2395: (2w); 14 june 2011; chefchaouen, dardara, quercus suber forest; 35 07.24 n, 05 17.60 w; 374 m. this species has been recorded in morocco from the central zone, the middle atlas, and chefchaouen (cagniant, 2006). The ant fauna of morocco is particularly diverse with 233 species, when compared to only 180 species in both algeria and tunisia (cagniant 2006), but it remains less than the recorded 295 species in the iberian peninsula (roig and espadaler 2010). The rate at which ant species are added to the list of the moroccan fauna would suggest that there remain more to be discovered . Our investigations also suggest that several species previously thought to be restricted in distribution or in choice of habitat are in fact more widespread and less selective (such as t. pardoi or t. parvioculum). It is difficult to speculate how climate change and an array of anthropogenic factors including farming practice will influence the ant fauna . For this reason, frequent and regular field work is required, with accurate data particularly of the macro and micro habitats where ant species are thriving, if we are to understand ant ecology in morocco.
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More than 10 million patients with new cases of cancer are diagnosed every year, and about 27 million new cases of cancer will have been recorded by 2030.1,2 some traditional cancer therapies, such as radiotherapy and chemotherapy, have enhanced the 5-year survival rates of cancer patients . For improving the therapeutic efficiency against cancer, increasing amounts have been used to develop more new approaches, with the aims of fewer side effects, enhanced safety, and decreased invasiveness . Hyperthermia is known to induce apoptotic cell death in many tissues, in which the local temperature is raised more than 40c . The heat generation sources, radiofrequency waves, microwaves, or ultrasound, have been used to produce moderate heating in a specific target region.3 heat energy can cause irreversible cell damage by denaturing proteins and the local cells or tissues are selectively destroyed . However, a lack of specificity for tumor tissues would induce unavoidable cell damage in the surrounding healthy tissues, which has limited use in cancer treatment.3 while still in a relatively immature stage, gold nanoparticle - mediated photothermal therapy has contributed to great advances in cancer therapy . Gold nanostructures, as highly biocompatible materials, are widely used for biological application and medical purposes including imaging, drug delivery, and hyperthermia therapy.46 gold nanostructures provide precise control of sizes, shapes, and flexible surface chemistry for bioconjugation of biological molecules, which can offer molecular - level specificity for particular biocoupling in cancer cells . Due to unique and highly tunable optical properties, when gold nanostructures are exposed to light at their resonance wavelength, the conduction band electrons at the gold surface generate a collective coherent oscillation, resulting in strong light absorption or light scattering of gold . The absorbed light can be converted into localized heat, which can be readily employed for therapy based on photothermal destruction of cancer cells.710 pitsillides et al first reported the photothermal therapy in lymphocytes with a short pulsed laser in the presence of gold nanoparticle immunoconjugates in 2003.11 zharov et al reported gold - induced thermal destruction of cancer cells using a nanosecond laser.12,13 research on the use of gold in cancer treatment has also been carried out by el - sayed et al.10,14 several studies have reported on the feasibility and efficiency of tumor - specific targeting of gold nanostructures for photothermal cancer therapy, such as gold nanorods,15 nanoshells,5,16 and nanocages.17 in this study, on the basis of successfully synthesizing gold nanoparticle - antibody conjugates, l-428 hodgkin s cell - killing experiments induced by the photothermal effect of gold nanoparticles were implemented . Under laser irradiation, through specific targeting of ligands to receptors, light strongly absorbed by gold is transferred to the antibody molecules and the cell environment, so that the very high killing efficiency of cancer cells can be achieved . The irradiation laser was a frequency doubled q - switched neodymium (nd):yag laser (surelite i; continuum, santa clara, ca), with nonlinear crystals to enable conversion of the fundamental wavelength frequency from 1064 nm to 532 nm (2.5 mm spot size, 6 ns pulse width, 10 hz repetition rate), which was used for matching the gold surface plasmon resonance peak for photothermal cancer treatment . The output laser power, which is measured with a power meter, was adjusted by using an attenuator placed between the laser and the first mirror . Then, the laser was irradiated on a sample micro - cuvette with 18 wells with a diameter of 2 mm, which was custom - made in a 25 75 mm optical glass slide . The two antibodies, anti - cd30 monoclonal antibody (mab) berh2 and anti - cd25 mab act1, were provided by the research center borstel (borstel, germany). Gold nanoparticles 15 nm in size were purchased from british biocell international ltd (cardiff, uk). The stable gold antibody conjugates were prepared by passive absorption of proteins to the surface of the gold . For steady conjugation of antibodies and gold, the ph of the gold solution must be adjusted to be just above (~0.5) the isoelectric point (pi) of the antibody.18 an important parameter to consider is the amount of antibody bound to the gold . To find the amount of antibody needed to saturate and stabilize the gold, a titration procedure was used to fix the antibody concentration using a 96-well microtiter plate . First, 50 l solution with the same concentration of gold per well was placed into ten plates . The pi of berh2 and act1 antibodies was about 7.5, the ph of the gold in every plate was adjusted by 1% k2co3 solution to 8.0 to match the pi of the protein . Then, various amounts of antibodies with the same concentration were added . After stewing for 5 minutes, 10 l of nacl solution (10%) per well was added and mixed homogenously, while observing the solution color change . The amount that maintained the red color before the blue envelope was used for conjugation . For gold - berh2 and gold - act1 conjugates, 70 l 1% k2co3 solution was added into 5 ml gold to make the ph = 8.0, and the best conjugation amount of berh2 (9.5 mg / ml) was 7.5 l, while the best conjugation amount of act1 (7.4 mg / ml) was 12.5 l . To take the preparation of gold - berh2 conjugates as an example, 5 ml gold nanoparticles with 15 nm were transferred into a 15 ml tube, and 70 l 1% k2co3 solution was added into the tube to adjust the ph . Then, 7.5 l berh2 mabs (9.5 mg / ml) were mixed into the gold solution . After incubating for 30 minutes at room temperature while stirring, the bottom scarlet solution was extracted and centrifuged for 45 minutes at 40,000 rpm to wash the unbound antibodies . Finally, the pellet was resuspended in phosphate - buffered saline (pbs) supplemented with 0.1% bovine serum albumin (bsa), and stored at 4c until use . Cells were maintained in rpmi1640 medium supplemented with 10% heat - inactivated fetal calf serum, 2 mm l - glutamine, and antibacterial antibiotic solution . The cell line was maintained at 37c in an incubator in 5% co2 and 95% air and passaged twice a week . For the experiments, l-428 cells at the logarithmic growth phase were centrifuged at 1400 rpm for 5 minutes at 20c and then resuspended in pbs with cell densities of 10 ml . For testing the stability of gold antibody conjugates and the staining ability of different conjugates to the l-428 cells, either gold - berh2 or gold - act1 conjugates were added to the cell suspension at certain ratios between the conjugates and the cells . After incubation for 20 minutes at 37c, the cells were centrifuged and washed twice and resuspended in pbs . Then, a secondary antibody, goat anti - mouse alexa 488 (am - a488) was added into the cell suspension to couple with the berh2 or act1 antibody . After incubation for 20 minutes and centrifugation at 1400 rpm for 5 minutes, the cell samples were resuspended in pbs and analyzed using a flow cytometer (facscan; becton dickinson, franklin lakes, nj). Equipped with a 488 nm air - cooled argon - ion laser . Data analysis was based on the collection of 10,00050,000 events . To implement the gold nanoparticle - induced l-428 cell - killing experiments, the gold antibody conjugates, gold - berh2 and gold - act1, and unbound gold with the same optical density, were added into the cell suspension, respectively . After incubation for 20 minutes at 37c, the cells were centrifuged at 1400 rpm for 5 minutes, and resuspended in pbs for the next photothermal treatment . In the l428 cell killing experiments, there were four experimental groups for comparing the killing efficiency with or without gold, the control cell group, cells with unbound gold, cells with gold - berh2 conjugates, and cells with gold - act1 conjugates . Four groups of prepared cell samples was seeded into sample micro - cuvettes per well . The cells were treated in the scanning mode of laser irradiation with 050 mw, 5 pulses . After photothermal treatment, all of the cells were washed, resuspended in pbs, and incubated for 15 minutes, followed by the cell viability analysis . Cell viability was determined using calcein - am / propidium iodide (pi) double - staining solution . Briefly, after photothermal treatment, 2 g / ml of calcein - am solution was added into the irradiated resuspended cell sample and incubated at 37c for 20 minutes . The samples were analyzed using flow cytometry . To determine the percentage of necrotic cells relative to the total number of cells, 10,00050,000 events were acquired per sample using cellquest software (bd biosciences). All the data were processed with winmdi 2.8 software (microsoft corporation, redmond, wa). The results were confirmed by fluorescence microscopy (bh2-rfl - t2; olympus corporation, tokyo, japan), and the fluorescent images were recorded using a digital camera . The irradiation laser was a frequency doubled q - switched neodymium (nd):yag laser (surelite i; continuum, santa clara, ca), with nonlinear crystals to enable conversion of the fundamental wavelength frequency from 1064 nm to 532 nm (2.5 mm spot size, 6 ns pulse width, 10 hz repetition rate), which was used for matching the gold surface plasmon resonance peak for photothermal cancer treatment . The output laser power, which is measured with a power meter, was adjusted by using an attenuator placed between the laser and the first mirror . Then, the laser was irradiated on a sample micro - cuvette with 18 wells with a diameter of 2 mm, which was custom - made in a 25 75 mm optical glass slide . The two antibodies, anti - cd30 monoclonal antibody (mab) berh2 and anti - cd25 mab act1, were provided by the research center borstel (borstel, germany). Gold nanoparticles 15 nm in size were purchased from british biocell international ltd (cardiff, uk). The stable gold antibody conjugates were prepared by passive absorption of proteins to the surface of the gold . For steady conjugation of antibodies and gold, the ph of the gold solution must be adjusted to be just above (~0.5) the isoelectric point (pi) of the antibody.18 an important parameter to consider is the amount of antibody bound to the gold . To find the amount of antibody needed to saturate and stabilize the gold, a titration procedure was used to fix the antibody concentration using a 96-well microtiter plate . First, 50 l solution with the same concentration of gold per well was placed into ten plates . The pi of berh2 and act1 antibodies was about 7.5, the ph of the gold in every plate was adjusted by 1% k2co3 solution to 8.0 to match the pi of the protein . Then, various amounts of antibodies with the same concentration were added . After stewing for 5 minutes, 10 l of nacl solution (10%) per well was added and mixed homogenously, while observing the solution color change . The amount that maintained the red color before the blue envelope was used for conjugation . For gold - berh2 and gold - act1 conjugates, 70 l 1% k2co3 solution was added into 5 ml gold to make the ph = 8.0, and the best conjugation amount of berh2 (9.5 mg / ml) was 7.5 l, while the best conjugation amount of act1 (7.4 mg / ml) was 12.5 l . To take the preparation of gold - berh2 conjugates as an example, 5 ml gold nanoparticles with 15 nm were transferred into a 15 ml tube, and 70 l 1% k2co3 solution was added into the tube to adjust the ph . Then, 7.5 l berh2 mabs (9.5 mg / ml) were mixed into the gold solution . After incubating for 30 minutes at room temperature while stirring, the bottom scarlet solution was extracted and centrifuged for 45 minutes at 40,000 rpm to wash the unbound antibodies . Finally, the pellet was resuspended in phosphate - buffered saline (pbs) supplemented with 0.1% bovine serum albumin (bsa), and stored at 4c until use . Cells were maintained in rpmi1640 medium supplemented with 10% heat - inactivated fetal calf serum, 2 mm l - glutamine, and antibacterial antibiotic solution . The cell line was maintained at 37c in an incubator in 5% co2 and 95% air and passaged twice a week . For the experiments, l-428 cells at the logarithmic growth phase were centrifuged at 1400 rpm for 5 minutes at 20c and then resuspended in pbs with cell densities of 10 ml . For testing the stability of gold antibody conjugates and the staining ability of different conjugates to the l-428 cells, either gold - berh2 or gold - act1 conjugates were added to the cell suspension at certain ratios between the conjugates and the cells . After incubation for 20 minutes at 37c, the cells were centrifuged and washed twice and resuspended in pbs . Then, a secondary antibody, goat anti - mouse alexa 488 (am - a488) was added into the cell suspension to couple with the berh2 or act1 antibody . After incubation for 20 minutes and centrifugation at 1400 rpm for 5 minutes, the cell samples were resuspended in pbs and analyzed using a flow cytometer (facscan; becton dickinson, franklin lakes, nj). Equipped with a 488 nm air - cooled argon - ion laser . Data analysis was based on the collection of 10,00050,000 events . To implement the gold nanoparticle - induced l-428 cell - killing experiments, the gold antibody conjugates, gold - berh2 and gold - act1, and unbound gold with the same optical density, were added into the cell suspension, respectively . After incubation for 20 minutes at 37c, the cells were centrifuged at 1400 rpm for 5 minutes, and resuspended in pbs for the next photothermal treatment . In the l428 cell killing experiments, there were four experimental groups for comparing the killing efficiency with or without gold, the control cell group, cells with unbound gold, cells with gold - berh2 conjugates, and cells with gold - act1 conjugates . Four groups of prepared cell samples was seeded into sample micro - cuvettes per well . The cells were treated in the scanning mode of laser irradiation with 050 mw, 5 pulses . After photothermal treatment, all of the cells were washed, resuspended in pbs, and incubated for 15 minutes, followed by the cell viability analysis . Cell viability was determined using calcein - am / propidium iodide (pi) double - staining solution . Briefly, after photothermal treatment, 2 g / ml of calcein - am solution was added into the irradiated resuspended cell sample and incubated at 37c for 20 minutes . The samples were analyzed using flow cytometry . To determine the percentage of necrotic cells relative to the total number of cells, 10,00050,000 events were acquired per sample using cellquest software (bd biosciences). All the data were processed with winmdi 2.8 software (microsoft corporation, redmond, wa). The results were confirmed by fluorescence microscopy (bh2-rfl - t2; olympus corporation, tokyo, japan), and the fluorescent images were recorded using a digital camera . To evaluate the conjugation stability of gold and antibodies and the binding specificity of different conjugates to l-428 cells, the two conjugates, gold - berh2 (anti - cd30 receptor) and gold - act1(anti - cd25 receptor), and the goat anti - mouse alexa 488 were adopted in this experiment . Both cd30 and cd25 are cell membrane proteins of the tumor necrosis factor receptors, but only cd30 has a high overexpression on the surface of l-428 . Through the specific coupling of goat anti - mouse immunoglobulin g antibody to monoclonal antibody, stable coupling was achieved between am - a488 and berh2/act1 . Whether or not am - a488 attaches to l-428 depends on the expression of protein on l-428 . The results indicate that only for the cells in the gold - berh2 group, the detection of the fluorescence signal was positive . The fluorescence signal on the cells in the gold - act1 group was the same as that for the control cell group (negative). The gold - berh2 conjugates bound specifically to cd30 receptors on the surface of l428 cells, which were also stained with am - a488 . A conceptual diagram of this binding process is shown in the inset box in figure 2 . The conjugation stability was confirmed on one hand, but the different binding abilities of different conjugate - bound cells would bring a different performance in the cell - killing experiment . Figure 3 shows cell viability of l428 cells after some hours of exposure to a certain concentration of gold, or gold - berh2 conjugates . The ratio of pure gold to cells was 10:1, and the ratios of gold conjugates to cells were 10:1 and 10:1, respectively . After incubation, the number of viable cells were stained with calcein - am (2 g / ml) and then measured using flow cytometry . For all samples, 20,000 events were acquired . For the groups of unbounded gold and gold - berh2 conjugates incubated with l-428 cells at a ratio of 10, the cell viability was greater than 90% after 24 hours of incubation, which had no significant difference compared with the control cell group . At the ratio of 10:1, a bigger decrease in cell viability was observed after incubation for 24 hours and l-428 cell viability dropped to 84% . Therefore, for a short incubation period (less than 12 hours), there was no significant increase in the number of dead cells incubated with gold at a relatively low concentration . To avoid the cytotoxicity induced by gold with high doses and a long incubation period, the highest ratio of gold to cells used for the photothermal killing experiment was not more than 10:1, and the incubation period was less than 6 hours . To monitor the necrotic effect of laser irradiation on living cells in the presence of gold, we performed cell damage experiments on l-428 cells, which were incubated with gold - berh2 conjugates at a 10:1 ratio for 20 minutes, and then irradiated with a laser at 50 mw, 5 pulses . After irradiation, cell viability was assessed using calcein - am (2 g / ml) and pi (1 g / ml) staining, and tested using flow cytometry and optical fluorescence microscopy . The events for facs were 20,000 events . The cells positively stained with pi presumably represented the later stages of cell death, when membrane integrity was lost . The percentage of death was calculated as the number of pi positive cells divided by the total number of cells . Figure 4 shows the photothermal treatment results, where severe destruction of l-428 cells was observed when the cells were exposed to laser irradiation (50 mw, 5 pulses) with gold - berh2 conjugates . The flow cytometry results of l-428 cells incubated with gold - berh2 conjugates treated with or without laser irradiation are shown on the left side . The corresponding fluorescent images are shown on the right side . Without laser irradiation, about 93% of the cell population was alive, which exhibited bright green fluorescence after calcein - am staining . More than 95% of the cell population was dead after laser irradiation at 50 mw, 5 pulses, and the dead targets exhibited red fluorescence after pi staining . Furthermore, the effect of laser influence on laser - induced cell damage has been considered in this section . To test whether the increased cell death rate is linked to laser irradiated power, we treated cell samples with different laser power settings . L-428 cells incubated with gold - berh2 conjugates and the control cell group were seeded into the sample micro - cuvette respectively . The cell concentration was 4 10/ml, and the gold - berh2 conjugate to cell ratio was 10:1 . The cell samples were irradiated with 5 pulses and different laser power settings, and then tested using the flow cytometry by calcein - am / pi double staining . Figure 5a and b show a representative flow cytometry dot plot illustrating the change in cell viability induced by different power laser irradiation . Right lower quadrants expressed the calcein - am positive cells as a percentage of the total cell population . Left upper quadrants expressed the pi positive cells as a percentage of the total cell population . As shown in figure 5a, for the control cell group, there was no remarkable change in cell viability when increasing the laser - irradiated power from 0 mw to 50 mw . But for the cells in the gold - berh2 conjugates group as shown in figure 5b, cell damage efficiency was affected significantly by the laser - irradiated power . Most of the cells died when the laser power was increased to 50 mw . In the presence of gold for the control cell group incubated without gold, the cell viability curve did not change much with the variation of laser - irradiated power; however, the curve of the cells with gold - berh2 conjugates showed a rapid decrease in cell viability as the laser power increased . Excessive irradiated laser power caused devastating damage or complete necrosis of l-428 cells incubated with gold antibody conjugates . Due to the specific aggregation of the cd30 receptor and berh2 antibodies, efficient conversion of light strongly absorbed by the gold to heat energy induced a significant increase in cell death . The effect of the photothermal treatment of l-428 cells was evaluated in the above experiments by combining laser irradiation and gold nanoparticles . In this section, we compared the efficiency of different binding modes of gold to cells and cell viability . There were four experimental groups for comparing the killing efficiency with or without gold nanoparticles: the control cell group, cells with gold, cells with gold - berh2 conjugates, and cells with gold - act1 conjugates . From the cell - binding specificity tests, it was known that berh2 antibodies could bind specifically on the surface of l-428 cells, but act1 antibodies could not . Thus, the above four experimental groups represented four combination conditions between gold and cells: pure cells, cells with unbound gold, cells targeted by few nonspecific gold antibody conjugates, and cells targeted by many specific gold antibody conjugates . The cell samples were treated by laser irradiation with 40 mw, 5 pulses, which would be compared with the nonirradiated part . Then, 10,000 events were tested using flow cytometry by calcein - am / pi double - staining . The experimental analysis results are shown in figure 6, and were divided into two parts: irradiated and non - irradiated . In the non - irradiated part, there was no difference of cell viability between the four cell samples without laser irradiation . In the irradiated part, there was no significant difference from the control group in the cell viability . For the cells in the unbound gold group and in the gold - act1 conjugates group, a slight increase in the death rate can be found . Due to the small volume of the sample micro - cuvettes, gold or gold - act1 conjugates in the sample volume might distribute in the surrounding cells, which could induce some heat energy shift to the cells . Compared with the other three groups, due to the specific coupling of cd30 antigen and berh2 antibody, gold - berh2 conjugates have a tight binding on the surface of l428 cells . Under laser irradiation with 40 mw, 5 pulses, a large damage rate of about 96% was observed in the l-428 cells . To evaluate the conjugation stability of gold and antibodies and the binding specificity of different conjugates to l-428 cells, the two conjugates, gold - berh2 (anti - cd30 receptor) and gold - act1(anti - cd25 receptor), and the goat anti - mouse alexa 488 were adopted in this experiment . Both cd30 and cd25 are cell membrane proteins of the tumor necrosis factor receptors, but only cd30 has a high overexpression on the surface of l-428 . Through the specific coupling of goat anti - mouse immunoglobulin g antibody to monoclonal antibody, stable coupling was achieved between am - a488 and berh2/act1 . Whether or not am - a488 attaches to l-428 depends on the expression of protein on l-428 . The results indicate that only for the cells in the gold - berh2 group, the detection of the fluorescence signal was positive . The fluorescence signal on the cells in the gold - act1 group was the same as that for the control cell group (negative). The gold - berh2 conjugates bound specifically to cd30 receptors on the surface of l428 cells, which were also stained with am - a488 . A conceptual diagram of this binding process is shown in the inset box in figure 2 . The conjugation stability was confirmed on one hand, but the different binding abilities of different conjugate - bound cells would bring a different performance in the cell - killing experiment . Figure 3 shows cell viability of l428 cells after some hours of exposure to a certain concentration of gold, or gold - berh2 conjugates . The ratio of pure gold to cells was 10:1, and the ratios of gold conjugates to cells were 10:1 and 10:1, respectively . After incubation, the number of viable cells were stained with calcein - am (2 g / ml) and then measured using flow cytometry . For all samples, 20,000 events were acquired . For the groups of unbounded gold and gold - berh2 conjugates incubated with l-428 cells at a ratio of 10, the cell viability was greater than 90% after 24 hours of incubation, which had no significant difference compared with the control cell group . At the ratio of 10:1, a bigger decrease in cell viability was observed after incubation for 24 hours and l-428 cell viability dropped to 84% . Therefore, for a short incubation period (less than 12 hours), there was no significant increase in the number of dead cells incubated with gold at a relatively low concentration . To avoid the cytotoxicity induced by gold with high doses and a long incubation period, the highest ratio of gold to cells used for the photothermal killing experiment was not more than 10:1, and the incubation period was less than 6 hours . To monitor the necrotic effect of laser irradiation on living cells in the presence of gold, we performed cell damage experiments on l-428 cells, which were incubated with gold - berh2 conjugates at a 10:1 ratio for 20 minutes, and then irradiated with a laser at 50 mw, 5 pulses . After irradiation, cell viability was assessed using calcein - am (2 g / ml) and pi (1 g / ml) staining, and tested using flow cytometry and optical fluorescence microscopy . The cells positively stained with pi presumably represented the later stages of cell death, when membrane integrity was lost . The percentage of death was calculated as the number of pi positive cells divided by the total number of cells . Figure 4 shows the photothermal treatment results, where severe destruction of l-428 cells was observed when the cells were exposed to laser irradiation (50 mw, 5 pulses) with gold - berh2 conjugates . The flow cytometry results of l-428 cells incubated with gold - berh2 conjugates treated with or without laser irradiation are shown on the left side . About 93% of the cell population was alive, which exhibited bright green fluorescence after calcein - am staining . More than 95% of the cell population was dead after laser irradiation at 50 mw, 5 pulses, and the dead targets exhibited red fluorescence after pi staining . Furthermore, the effect of laser influence on laser - induced cell damage has been considered in this section . To test whether the increased cell death rate is linked to laser irradiated power, we treated cell samples with different laser power settings . L-428 cells incubated with gold - berh2 conjugates and the control cell group were seeded into the sample micro - cuvette respectively . The cell samples were irradiated with 5 pulses and different laser power settings, and then tested using the flow cytometry by calcein - am / pi double staining . Figure 5a and b show a representative flow cytometry dot plot illustrating the change in cell viability induced by different power laser irradiation . Right lower quadrants expressed the calcein - am positive cells as a percentage of the total cell population . Left upper quadrants expressed the pi positive cells as a percentage of the total cell population . As shown in figure 5a, for the control cell group, there was no remarkable change in cell viability when increasing the laser - irradiated power from 0 mw to 50 mw . But for the cells in the gold - berh2 conjugates group as shown in figure 5b, cell damage efficiency was affected significantly by the laser - irradiated power . The cell death rate was about 40% when using 15 mw laser irradiation; most of the cells died when the laser power was increased to 50 mw . In the presence of gold antibody conjugates, the cell death rate mostly depends on the laser irradiated power . The statistical analysis of the repeated experiments is shown in figure 5c . For the control cell group incubated without gold, the cell viability curve did not change much with the variation of laser - irradiated power; however, the curve of the cells with gold - berh2 conjugates showed a rapid decrease in cell viability as the laser power increased . Excessive irradiated laser power caused devastating damage or complete necrosis of l-428 cells incubated with gold antibody conjugates . Due to the specific aggregation of the cd30 receptor and berh2 antibodies, efficient conversion of light strongly absorbed by the gold to heat energy induced a significant increase in cell death . The effect of the photothermal treatment of l-428 cells was evaluated in the above experiments by combining laser irradiation and gold nanoparticles . In this section, we compared the efficiency of different binding modes of gold to cells and cell viability . There were four experimental groups for comparing the killing efficiency with or without gold nanoparticles: the control cell group, cells with gold, cells with gold - berh2 conjugates, and cells with gold - act1 conjugates . From the cell - binding specificity tests, it was known that berh2 antibodies could bind specifically on the surface of l-428 cells, but act1 antibodies could not . Thus, the above four experimental groups represented four combination conditions between gold and cells: pure cells, cells with unbound gold, cells targeted by few nonspecific gold antibody conjugates, and cells targeted by many specific gold antibody conjugates . The cell samples were treated by laser irradiation with 40 mw, 5 pulses, which would be compared with the nonirradiated part . Then, 10,000 events were tested using flow cytometry by calcein - am / pi double - staining . The experimental analysis results are shown in figure 6, and were divided into two parts: irradiated and non - irradiated . In the non - irradiated part, there was no difference of cell viability between the four cell samples without laser irradiation ., there was no significant difference from the control group in the cell viability . For the cells in the unbound gold group and in the gold - act1 conjugates group, a slight increase in the death rate can be found . Due to the small volume of the sample micro - cuvettes, gold or gold - act1 conjugates in the sample volume might distribute in the surrounding cells, which could induce some heat energy shift to the cells . Compared with the other three groups, due to the specific coupling of cd30 antigen and berh2 antibody, gold - berh2 conjugates have a tight binding on the surface of l428 cells . Under laser irradiation with 40 mw, 5 pulses, a large damage rate of about 96% was observed in the l-428 cells . With the preliminary successes of photothermal therapy in cancer studies, gold nanoparticle - mediated thermal therapy became a new and minimally invasive tool for cancer treatment.19,20 in the present study, to evaluate the effectiveness of gold in the destruction of cancer cells or tumor tissue, three main aspects were considered: the photothermal therapy system, specific gold nanoparticle we set up a photothermal therapy system (figure 1). A pulsed light with 532 nm wavelength was chosen to match the surface plasmon resonance absorption peak of 15 nm gold nanoparticles used in the experiment for photothermal cancer treatment . Specificity of targeting to achieve high efficiency of photothermal conversion of gold nanoparticles was a key problem . Specific targeting should produce a high photothermal effect on the targeted cancer, and only minimal heat should be applied to the surrounding normal cells and tissues . After synthesizing two different gold nanoparticle antibody conjugates, gold - berh2 and gold - act1 conjugates, their conjugation stability and binding specificity for l-428 cells were evaluated (figure 2). L-428 cells are positive for cd30, but negative for cd25 . Through flow cytometry measurements using alexa 488, the results indicate that the gold - berh2 conjugates bound specifically to the surface of l-428 cells, but that gold - act1 conjugates did not, which seems to agree with our predictions . Prior to considering gold nanoparticles for photothermal therapy application, it is important to understand their characterization of biocompatibility.21 the toxicity and side effects of nanoparticles are determined by the shape, dose, surface chemistry, incubation duration, etc.2224 we compared the toxicity of pure gold nanoparticles and gold antibody conjugates to l-428 cells under different incubation time periods and different concentrations . After incubation for 24 hours, pure gold and gold antibody conjugates incubated with l-428 cells did not induce a decrease in cell viability compared with control cells under a relatively low concentration (the ratio of gold to cells was not more than 10:1); but, with an increase in the gold concentration, a decrease in cell viability was observed . A relatively low concentration of gold and a short incubation time were adopted in the photothermal experiment to exclude the influence of cytotoxicity of gold on cell viability . During the photothermal treatment, the temperature distribution on the surrounding cell surface was the dominant factor leading to cell death.2526 it is important to note that the temperature distribution is determined by the gold s shape and concentration and the wavelength and power of laser irradiation . At the molecular level, hyperthermic effects can induce protein denaturation, membrane rupture, and cell shrinkage.27 cytotoxic effects have occurred in cells maintained for 1 hour at 42c, but this duration could be shortened to 34 minutes by using a higher temperature such as 80c.14,2729 numerical analysis found that cells with higher gold - loading required a lower laser - irradiated power to achieve the temperature rise for cell destruction.14 photothermal therapy tests on living l-428 cells were initially performed to confirm the gold nanoparticle - mediated photothermal effect on cell viability . When cells were treated with gold or laser alone, after laser irradiation in the presence of the gold, the cell death rate had a severe increase (figure 4), and the results were also confirmed by fluorescence microscopy . With the combination of gold nanoparticles and laser irradiation, few calcein - am positive cells were observed and most l428 cells were positive for pi staining . The effect of laser irradiation on cell damage has been evaluated (figure 5). For the control cells without gold, there was no remarkable change in cell viability when increasing the power of laser irradiation . But in the presence of gold - berh2 conjugates, cell damage efficiency was affected significantly by the laser - irradiated power, and the cell death rate mostly depended on the power of laser irradiation . For successful cancer killing, the cells must be heated to a minimum temperature by proper laser irradiation to cause cancer cell death . The different binding mode of gold to cells can also induce different therapy efficiency on cell viability (figure 6). Under the same laser irradiation, the specific binding of gold - berh2 conjugates to l-428 induced extensive cell damage, but there was a slight increase in the cell death rate for the nonspecific binding groups, cells with the unbound gold group, and cells with the gold - act1 conjugates group . The results again confirmed the advantage of specific targeting of gold to cells and high therapy efficiency for gold - targeted cancer cells and slight damage to untargeted cells . In vivo photothermal therapy of gold nanostructures in preclinical and, potentially, clinical use is a crucial direction for our future research . Due to the low absorption by tissues in the 700850 nm near infrared (nir) region, gold nanostructure - mediated photothermal therapy is predominantly designed to operate in this window of wavelengths to enhance light penetration capability, and to prevent undesirable damage to healthy tissue . The absorption peak of gold nanorods, nanoshells, and nanocages are all in the nir region . Some recent studies have focused on in vitro and in vivo photothermal cancer treatments;3036 however, the optimization of gold nanoparticle - based therapy techniques to physiological environments needs further study, to determine the clinical success of gold nanostructure - based nanomedicine.19,37,38 many experimental parameters, including the absence of gold or laser irradiation, the laser - irradiated power, and the binding mode of gold to cells, were all tested for their influence on cell viability . When conjugated with berh2 (anti - cd30 receptor) antibodies, these gold nanoparticles specifically bind l-428 cells that overexpress the cd30 receptor; and, with laser irradiation at suitable wavelength and power, very high effectiveness in killing l-428 cells is achieved . Many optimization problems, including the compatibility of gold nanostructures, the stability of gold ligand conjugates, and laser irradiation modes, need further modification to make them more suitable for in vivo tumor model studies . Gold nanoparticle - mediated photothermal cancer therapy provides a relatively safe microsurgery system for cancer therapy development.
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Systemic lupus erythematosus (sle) is a multifactorial autoimmune disease characterized by several clinical manifestations and the appearance of multiple autoantibodies.14 analysis of sera obtained from the same sle patient over a long period of time has demonstrated that different autoantibody specificities appear at different time intervals with a general trend towards increasing the number of antigens recognized by the sera.3 these observations of increasing complexity of this autoimmune response over a period of time are very similar to those reported by lehmann et al.4 in their mode of epitope spreading in experimental autoimmune encephalomyelitis . Thus, it was generally accepted that intermolecular epitope spreading was one of the mechanisms for the amplification and diversification of autoantibody responses in sle.5,6 in short, sle is a multisystem autoimmune disorder of unknown etiology6 or the agent (or agents) triggering this autoimmune response remains to be identified, but it is thought that a combination of genetic and environmental factors are required.68 reactive oxygen species (ros) has the potential to initiate damage to proteins, dna and other cell biomolecules under pathological conditions.9 protein oxidation, which results in functional disruption, is not random but appears to be associated with increased oxidation in specific proteins.1014 we previously reported that many serum proteins were found to be oxidatively modified leading to the formation of neoantigens which could in turn initiate autoimmunity in various diseases1520 including sle.21 oxidative stress and formation of oxidatively - modified protein are associated with sle,8,21,22 however, the potential role of oxidative stress, especially the consequences of oxidative modification of proteins, in the pathogenesis and progression of sle remains unresolved . Immunoglobulin g (igg) is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids, constituting 75% of serum immunoglobulins in human . It is well known that igg is quite vulnerable to ros.2329 many studies showed the presence of elevated levels of oxidized igg in patients with rheumatic diseases.2628 in patients with sle, igg dysfunction has been reported.26 now, it is well documented that igg behaves not only as an antibody, but also as a putative antigen for rheumatoid factor.20,26 therefore, igg is continuously exposed to oxidative stress, as the alterations in its conformation and function could occur, resulting in modification of its biological properties . In the present study, we demonstrated that after modification with ros, human igg became highly immunogenic in experimental animals and the induced antibodies against ros - modified igg showed cross - reactions with native and ros - modified nucleic acid conformers . Therefore, we hypothesized that oxidative by - products, such as hydroxyl radicals - damaged human igg, help to initiate autoimmunity in sle . To test this hypothesis, we investigated the binding characteristics of naturally occurring sle autoantibodies to native and hydroxyl radical - modified igg igg from normal human sera was purified using protein a - sepharose cl-4b affinity column (sigma - aldrich st . Louis, mo, usa) as previously described.20 briefly, serum diluted with equal volume of pbs, ph 7.4 and applied to the column equilibrated with the same buffer . The bound igg was eluted with 0.58% acetic acid in 0.85% sodium chloride and neutralized with 1.0 ml of 1.0 mol / l tris hcl, ph 8.5 . The purified igg was dialyzed against pbs, ph 7.4 and stored at 20 c . Human dna was purified from the blood of healthy human subjects, which was free of protein, rna and single - stranded regions as previously described.30 chromatin was purified from goat liver as described previously.31 human igg, hsa (sigma - aldrich co.), human hemoglobin (sigma - aldrich co.), human transferring (sigma - aldrich co.), human dna and chromatin was modified in pbs (10 mm sodium phosphate buffer containing 150 mm nacl, ph 7.4) by our published procedure.18 proteins or nucleic acid conformers were modified by hydroxyl radicals, generated with uv irradiation (30 min) of hydrogen peroxide (15.1 mm) at 254 nm . Ages was prepared by independent reactions of different plasma proteins (hsa, human igg, human transferrin, or hemoglobin) with glycoaldehyde (sigma - aldrich, co.) according to our published procedure.3234 the reaction was terminated by removing non - reacted glycoaldehyde using dialyzing extensively against pbs, ph 7.2 . The immunization of random bred, female new zealand white rabbits was performed as described previously18,20 briefly, rabbits (n = 4; two each for native and ros - human igg antigens) were immunized intramuscularly at multiple sites with 25 g of antigen, emulsified with an equal volume of freund s complete adjuvant (sigma - aldrich, co.). The animals were boosted in freund s incomplete adjuvant (sigma - aldrich, co.) at weekly intervals for 6 weeks with the same amount of antigen . Test bleeds were performed 7 days post boost, which gave appropriate titer of the antibody . The animals were bled and the serum separated from the blood (pre - immune and immune) was heated at 56 c for 30 min to inactivate complement proteins and stored at 80 c . The study group included 72 patients (63 females and 9 males; aged (sd) 43.6 13.3 years) with sle, as defined by the american college of rheumatology 1997 revised criteria34 and the age range was 2969 years (43.6 13.3 years). The control group comprised 39 healthy volunteers (36 females and 3 males; aged (sd) 45.7 13.8 years). Venous blood samples from the control subjects and from sle patients were collected and serum was separated . Full informed consent was obtained prior to the blood extraction from sle patients and healthy subjects . All serum samples were decomplemented at 56 c for 30 min and stored in small aliquots at 80 c . Protein carbonyl contents in sle patients sera and in healthy human sera were analyzed as described previously35 with slight modifications . The reaction mixture containing 0.5 ml of 10 mm 2,4-dinitrophenylhydrazine (dnph)/2.5 mhcl and 1:100 diluted sera from study subjects and was thoroughly mixed . After addition of 250 mm tca (20%) and centrifugation, the pellet was collected and washed three times with 1 ml ethanol: ethylacetate (1:1) mixture . The pellet was then dissolved in 1 ml of 6 m guanidine solution and incubated at 30 c for 15 min . After centrifugation, the supernatant was collected and carbonyl contents were estimated using molar absorption coefficient of 22,000 m cm . Samples were spectrophotometrically analyzed against a blank of 1 ml of guanidine solution (6 m). Protein concentration was determined in the samples by the method of lowry et al.36 antibodies against native and ros - modified human igg were detected and quantified using elisa assays performed on flat bottom 96 wells, polystyrene immunoplates as previously described.15,16 polystyrene polysorp / maxisorp immunoplates (thermo fisher scientific, fremont, ca, usa) were coated with 100 ml of native or modified igg (5 g / ml) or dna (2.5 g / ml) in carbonate bicarbonate buffer (0.05 m, ph 9.6). The plates were coated for 2 h at 37 c and overnight at 4 c . Each sample was coated in duplicate and half of the plates served as control devoid of only antigen coating . Unbound antigen was washed thrice with tbs - t (20 mm tris, 150 mm, nacl, ph 7.4 containing 0.05% tween-20) and unoccupied sites were blocked with 2% fat free milk in tbs (10 mm tris, 150 mm nacl, ph 7.4) for 46 h at 37 c . The test serum serially diluted in tbs - t in tbs (100 ml / well) was adsorbed for 2 h at 37 c and overnight at 4 c . Bound antibodies were assayed with human alkaline phosphatase conjugate using p - nitrophenyl phosphate as substrate . The absorbance (a) of each well was monitored at 410 nm on an automatic microplate reader . The antigenic specificity of the antibodies was determined by competition elisa.17,18 varying amounts of inhibitors (020 mg / ml) were mixed with a constant amount of serum samples . The mixture was incubated at room temperature for 2 h and overnight at 4 c . Percent inhibition was calculated using the formula: percent inhibition=[1(ainhibition / auninhibition)]100 all measurements were performed in duplicates and repeated at least 3 times using age- and sex - matched sle or control samples . Comparisons were performed using origin 6.1 software package (northampton, ma, usa) (one paired two tailed t - test with one way anova analysis). P values less than 0.05 were considered significant, and p values less than 0.001 were considered highly significant . Igg from normal human sera was purified using protein a - sepharose cl-4b affinity column (sigma - aldrich st . Louis, mo, usa) as previously described.20 briefly, serum diluted with equal volume of pbs, ph 7.4 and applied to the column equilibrated with the same buffer . The bound igg was eluted with 0.58% acetic acid in 0.85% sodium chloride and neutralized with 1.0 ml of 1.0 mol / l tris hcl, ph 8.5 . The purified igg was dialyzed against pbs, ph 7.4 and stored at 20 c . Human dna was purified from the blood of healthy human subjects, which was free of protein, rna and single - stranded regions as previously described.30 chromatin was purified from goat liver as described previously.31 human igg, hsa (sigma - aldrich co.), human hemoglobin (sigma - aldrich co.), human transferring (sigma - aldrich co.), human dna and chromatin was modified in pbs (10 mm sodium phosphate buffer containing 150 mm nacl, ph 7.4) by our published procedure.18 proteins or nucleic acid conformers were modified by hydroxyl radicals, generated with uv irradiation (30 min) of hydrogen peroxide (15.1 mm) at 254 nm . Ages was prepared by independent reactions of different plasma proteins (hsa, human igg, human transferrin, or hemoglobin) with glycoaldehyde (sigma - aldrich, co.) according to our published procedure.3234 the reaction was terminated by removing non - reacted glycoaldehyde using dialyzing extensively against pbs, ph 7.2 . The immunization of random bred, female new zealand white rabbits was performed as described previously18,20 briefly, rabbits (n = 4; two each for native and ros - human igg antigens) were immunized intramuscularly at multiple sites with 25 g of antigen, emulsified with an equal volume of freund s complete adjuvant (sigma - aldrich, co.). The animals were boosted in freund s incomplete adjuvant (sigma - aldrich, co.) at weekly intervals for 6 weeks with the same amount of antigen . Test bleeds were performed 7 days post boost, which gave appropriate titer of the antibody . The animals were bled and the serum separated from the blood (pre - immune and immune) was heated at 56 c for 30 min to inactivate complement proteins and stored at 80 c . The study group included 72 patients (63 females and 9 males; aged (sd) 43.6 13.3 years) with sle, as defined by the american college of rheumatology 1997 revised criteria34 and the age range was 2969 years (43.6 13.3 years). The control group comprised 39 healthy volunteers (36 females and 3 males; aged (sd) 45.7 13.8 years). Venous blood samples from the control subjects and from sle patients were collected and serum was separated . Full informed consent was obtained prior to the blood extraction from sle patients and healthy subjects . All serum samples were decomplemented at 56 c for 30 min and stored in small aliquots at 80 c . Protein carbonyl contents in sle patients sera and in healthy human sera were analyzed as described previously35 with slight modifications . The reaction mixture containing 0.5 ml of 10 mm 2,4-dinitrophenylhydrazine (dnph)/2.5 mhcl and 1:100 diluted sera from study subjects and was thoroughly mixed . After addition of 250 mm tca (20%) and centrifugation, the pellet was collected and washed three times with 1 ml ethanol: ethylacetate (1:1) mixture . The pellet was then dissolved in 1 ml of 6 m guanidine solution and incubated at 30 c for 15 min . After centrifugation, the supernatant was collected and carbonyl contents were estimated using molar absorption coefficient of 22,000 m cm . Samples were spectrophotometrically analyzed against a blank of 1 ml of guanidine solution (6 m). Antibodies against native and ros - modified human igg were detected and quantified using elisa assays performed on flat bottom 96 wells, polystyrene immunoplates as previously described.15,16 polystyrene polysorp / maxisorp immunoplates (thermo fisher scientific, fremont, ca, usa) were coated with 100 ml of native or modified igg (5 g / ml) or dna (2.5 g / ml) in carbonate bicarbonate buffer (0.05 m, ph 9.6). The plates were coated for 2 h at 37 c and overnight at 4 c . Each sample was coated in duplicate and half of the plates served as control devoid of only antigen coating . Unbound antigen was washed thrice with tbs - t (20 mm tris, 150 mm, nacl, ph 7.4 containing 0.05% tween-20) and unoccupied sites were blocked with 2% fat free milk in tbs (10 mm tris, 150 mm nacl, ph 7.4) for 46 h at 37 c . The test serum serially diluted in tbs - t in tbs (100 ml / well) was adsorbed for 2 h at 37 c and overnight at 4 c . Bound antibodies were assayed with human alkaline phosphatase conjugate using p - nitrophenyl phosphate as substrate . The absorbance (a) of each well was monitored at 410 nm on an automatic microplate reader . The antigenic specificity of the antibodies was determined by competition elisa.17,18 varying amounts of inhibitors (020 mg / ml) were mixed with a constant amount of serum samples . The mixture was incubated at room temperature for 2 h and overnight at 4 c . All measurements were performed in duplicates and repeated at least 3 times using age- and sex - matched sle or control samples . Comparisons were performed using origin 6.1 software package (northampton, ma, usa) (one paired two tailed t - test with one way anova analysis). P values less than 0.05 were considered significant, and p values less than 0.001 were considered highly significant . Human igg was purified from normal human sera by affinity chromatography using protein - a sepharose cl-4b affinity column . The purified igg was found to elute as a single symmetrical peak and gave a single band on sds - page (data not shown). Our earlier report20 showed alterations in human igg following exposure to the hydroxyl radicals, generated by the uv - irradiation of hydrogen peroxide . Loss of secondary structures, hypochromicity at 280 nm, loss of tryptophan fluorescence intensity, increase in protein carbonyl contents were observed in hydroxyl treated human igg . We also showed previously that immunization of ros - modified human igg in rabbits induced high titre antibodies (> 1:12,800), whereas with native human igg the titre was low (1:6400).20 in the present study, we studied the antigenic specificity of the experimentally induced antibodies against native and ros - modified human igg by competitive inhibition assay . A maximum of 97% inhibition of the affinity purified anti - ros - human igg antibodies with the immunogen as inhibitor, was observed (table 1). Competition experiments with native human igg used as inhibitor showed 51.2% inhibition at 20 g / ml . The affinity purified anti - ros - human igg antibodies exhibited a variable recognition of chromatin, dna, ros - modified - chromatin and ros - modified dna (table 1). Native chromatin and native dna showed maximum inhibition of 48.2% and 56.3%, respectively, and 18.2 g / ml of native dna was required for 50% inhibition, whereas the ros - modified conformers of chromatin and dna showed maximum inhibition of 63.2% and 65.4%, respectively at 20 g / ml of inhibitor concentration . 50% inhibition of anti - ros - igg antibodies was achieved at 7.4 and 10.2 g / ml of ros - modified dna and ros - modified chromatin, respectively . Percentage of relative affinity of anti - ros - igg antibodies in respect with inhibitors was also estimated which further confirmed that anti - ros - human igg antibodies exhibited diverse antigen binding characteristic with native and ros - modified nucleic acid conformers (table 1). Glycated igg showed negligible inhibitions of 16.5%, whereas native hsa and glycated hsa showed inhibition of 18.0% and 14.1%, respectively . Native human hemoglobin and glycated hemoglobin showed inhibition of 11.0% and 17.0%, respectively, whereas ros - modified conformers of hsa and hemoglobin showed moderate inhibition of 28.0% and 27.0%, respectively . The complete antigen binding specificity of affinity purified anti - ros - human igg antibodies has been summarized in table 1 . Competitive inhibition elisa results of antigen binding characteristics of affinity purified antihuman igg antibodies were shown in table 2 . Our data with anti - native human igg antibodies showed a maximum inhibition of 89.0% with the immunogen as inhibitor . Only 9.7 g / ml of native igg was required to inhibit 50% its activity . The induced antibodies partially recognized ros - modified human igg as it showed a maximum inhibition of 56.1% . Inhibitor 17.2 g / ml was required to inhibit 50% antibody binding activity to native igg . Native chromatin and native dna showed maximum inhibition of 32.2% and 22.2%, respectively, whereas ros - chromatin and ros - dna showed maximum inhibition of 41.1% and 34.1%, respectively (table 2). To probe the possible role of the ros in the pathogenesis of sle, 72 sle sera were selected for binding to native and ros - modified human igg . Majority of sle sera (35/72) showed strong binding to ros - modified igg over native igg (p <0.01) (fig . Normal human sera (nhs) showed negligible binding with native or modified igg (p> 0.05). Native dna was used as an immunochemical marker for sle, showed strong binding to sle serum antibodies; whereas antibodies from nhs showed negligible binding to native dna (fig . The average absorbance at 410 nm (sd) of 35 sle sera binding to native and ros - damaged igg was 0.51 0.10 and 1.3 0.11, respectively . Whereas, 39 nhs binding to native and ros - human igg antigens was 0.19 0.12 and 0.22 0.10, respectively . The binding specificity of antibodies from 35 selected sle sera was evaluated by competition elisa using native and ros - modified igg as inhibitors . Figure 2 illustrates inhibition of sle autoantibodies (in sera 1 to 6) binding to native dna by native or ros - modified igg . Our data showed strong reactivity of autoantibodies in patient s sera towards ros - igg over native igg (p <0.01). Similarly, the rest of the sera showed high percent inhibitions with modified igg over native igg (data summarized in table 3). The average percent inhibition (sd) in the binding of 35 sle sera to ros - modified and unmodified igg was 54.4 8.2 and 27.8 7.9, respectively . The data reveals striking differences in the recognition of native and oxidized igg by sle autoantibodies (p <0.001) (table 3). Elevated level of protein carbonyl contents in the serum is considered to be the most reliable biomarker of oxidative stress . The data showed significantly increase in serum protein carbonyl contents (p <0.05) in sle patients, compared with normal subjects of the same age group . The average carbonyl contents (sd) of 12 independent assays of sle serum proteins and normal human serum (nhs) proteins were 3.9 0.42 and 2.4 0.21 nmol / mg protein, respectively (table 3). A p - value of <0.01 indicates significant difference in the carbonyl contents of sle - serum and nh - serum . Reactive oxygen species (ros) play a key role in both normal biological functions and in the pathogenesis of certain human diseases . These species are continuously generated in cells by cellular metabolism and by exogenous agents but increase in their steady states are thought to be responsible for a variety of pathological conditions, including sle, cancer and aging.7,22,37 ros in excessive amount have the ability, either directly or indirectly, to damage proteins, dna and other cell biomolecules.722 among the ros, hydroxyl radicals are the most potent damaging ros which can react with almost all biological macromolecules.1521 proteins are the major targets for free radical attack mainly by hydroxyl radicals.17 protein oxidation, results in cellular dysfunctions, functional disruption and structural changes and contributes to the etiology of many human diseases.9,1114 these oxidative modifications on protein may lead to the formation of neoantigens which could in turn initiate autoimmunity . It is well established that igg is an abundant protein in the circulatory system, whose redox modifications modulate its physiologic functions,23 as well as may serve as a biomarker of oxidative stress.2325,2729 igg is continuously exposed to oxidative stress2325 bringing about alterations in conformation and functions of igg, resulting in modification of its biological properties . We previously demonstrated that ros caused extensive damaged to human igg and damaged igg was found to be a potent antigenic stimulus inducing high titre antibodies in rabbits, whereas with native human igg, antibodies titre was low.20 the antigenic specificity of affinity purified anti - ros - human igg and anti - native igg antibodies reiterated that induced antibodies were immunogen specific . The substantially enhanced immunogenicity of ros - human igg in comparison to native analogue could possibly be due to the generation of potential neo - epitopes against which antibodies are raised . In the present study, we demonstrated for the very first time that experimentally induced antibodies against ros - damaged human igg exhibited diverse antigen binding characteristics . Native dna, native chromatin, and their ros - modified conformers were found to be effective inhibitors of induced antibody - immunogen interaction . Whereas, induced antibodies against native igg showed negligible binding to the above mentioned nucleic acid conformers . This notable feature of induced antibodies against ros - modified igg resembled the diverse antigen - binding characteristics of naturally occurring sle anti - dna autoantibodies . Sle is a chronic autoimmune disease that is characterized by increased production of autoantibodies, but the initial immunizing antigens that drive the development of sle are largely unknown . Autoantibody production in sle has been attributed to either selective stimulation of autoreactive b cells by self antigens or antigens cross - reactive with self.1,2,7 numerous modified forms of dna have been found to be immunogenic and are recognized by sle anti - dna antibodies.7,31,38 despite the power of modern molecular approaches and persistent investigative efforts, lupus remains an enigmatic disorder6 and the agent (or agents) triggering this autoimmune response remains to be identified . Patients with sle have a diverse array of anti - nuclear autoantibodies, but the cellular and molecular mechanisms that are responsible for the production of anti - nuclear antibodies in sle and the way by which these antibodies participate in tissue destruction remain highly controversial.39 it was thought worthwhile to investigate the binding characteristics of natural sle autoantibodies to ros - damaged human igg as that the possible involvement of ros - modified igg in sle could be ascertained . Sera from 72 sle patients having high titre anti - dna antibodies and 39 normal human subjects were collected for the present study . Of these, 48.6% sle sera showed preferentially high binding to ros - igg as compared to its native analogue (p <0.01) as determined by direct binding elisa assays . Native or modified igg showed no appreciable binding with normal subjects (p> 0.05). We also noticed that unmodified igg showed some binding to sle serum antibodies as compared with the antibodies from nhs (p <0.05), this may be due to the partial structural similarities of native and modified igg, which may shared common paratope of sle autoantibodies . Our data clearly indicated that sle autoantibodies showed substantial difference in the recognition of modified igg over native igg (p <0.01). This increase is due to the oxidation of lys, arg, pro or other amino acid residues . In short, protein carbonyl groups are the biomarker of oxidative stress.35 in human plasma, all amino acids in the protein are susceptible to oxidative modification by oxidants such as hydroxyl radicals and hypochlorous acid.35 in view of these, carbonyl contents present in the total serum protein of sle were investigated . Our results showed that total serum protein carbonyl contents were significantly increased in sle patients, when compared with the carbonyl contents present in total serum protein of healthy human subjects (p <0.05). These results indicated that in sle patients with increased oxidative stress, the oxidative modification of plasma proteins has been greatly enhanced . Since the abundant protein of plasma is igg, it is likely to be extensively damaged and might be responsible for the pathological conditions associated with sle . These results suggest that igg is continuously exposed to oxidative stress, so much so that alterations in its biological properties could result in the conformational changes of igg . Our results demonstrate the presence of ros - induced human igg damage in sle patients, which might play an active part in the progression of disease . The present study further proposed that, in addition to igg in serum concentration, the quality of igg molecules may be not only a crucial factor affecting its protective effects, but also a risk factor as a pro - oxidant in sle patients . Ours is the first report to show the role of hydroxyl radical damaged immunoglobulin g in sle . Our results provide new suggestions that hydroxyl radical modification of igg causes perturbations, resulting in the generation of neo - epitopes, and making it a potential immunogen . The induced antibodies against hydroxyl radical - modified igg resembled the diverse antigen - binding characteristics of naturally occurring sle anti - dna autoantibodies . The igg modified with the hydroxyl radicals may be one of the factors for the induction of circulating sle autoantibodies.
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The emergence of primordial metabolism has been postulated to play a central role in the origins of life . Many of the investigations so far have centered on the reductive citric acid cycle . However, in his glyoxylate scenario, eschenmoser theorized that glyoxylate 1 may have played an important role in the development of a primordial metabolism, acting as a primal source for biogenic molecules such as sugars, amino acids, and nucleobases . Crucial to the scenario was the hypothetical formation of dihydroxyfumarate (dhf) 5, by dimerization of glyoxylic acid under the influence of an umpolung catalyst such as cyanide . Dhf was proposed to undergo reaction with glyoxylate and aldoses, ultimately yielding biologically relevant -keto acids and sugars, respectively . Subsequently it was shown by sagi et al . That the reaction of dhf with glyoxylate, glycolaldehyde, and glyceraldehyde led, via -keto acid intermediates, to the formation of triose, tetrulose, and pentuloses, with remarkable efficiency . This success emphasized the need to demonstrate the formation of dhf from glyoxylate . To this end, we set out to investigate the cyanide - mediated dimerization of glyoxylate (glyoxoin reaction) with the expectation of chemistry similar to the benzoin reaction . Herein we report the results of this investigation: the unanticipated formation of meso- and dl - tartrates (the formal reduction products of dhf) rapidly and in high yield (scheme 1). We also present evidence that the glyoxoin reaction proceeds via dhf as a putative intermediate, thus strengthening the proposals made in the glyoxylate scenario . This facile production of tartrate combined with the known dehydration of tartrate to give oxaloacetate, and its decarboxylation to give pyruvate, potentially, provides an alternate entry into the citric acid cycle based on the glyoxylate scenario . The (homogeneous) glyoxoin reaction of glyoxylate (1.0 m) with a catalytic amount of nacn (0.1 m) in aqueous medium at room temperature in 2.0 m naoh (ph 14) showed complete consumption of glyoxylate (by c nmr after 28 h); no signals corresponding to the expected dhf were observed . (high ph was required to ensure cyanide was present only as its anion; however ph 14 was obtained when 2.0 m naoh was prepared rather than 0.5 m.) rather, the c nmr spectrum of the crude reaction mixture (figure 1) suggested the formation of meso- and dl - tartrates 8 in high yields (in some cases exceeding 80%, see table s1 for example calculations) along with carbonate, oxalate 9, formate 11, tartronate 12, and glycolate 13 . The identities of products were confirmed by comparison to and by spiking with authentic materials (figure s1). In addition, the tartrates were isolated from the reaction mixture as calcium salts according to literature procedure and confirmed by c nmr spectroscopy and mass spectrometry (figure s2). The meso - tartrate is produced in equal or greater quantity than the combined dl - tartrates (table s1). No interconversion of meso - tartrate to and from dl - tartrates (nor conversion to any other reaction product) was observed under reaction conditions (figure s3). Reaction of 1.0 m sodium glyoxylate (182/94 ppm, completely consumed) with 0.1 m cyanide (167 ppm) in aqueous 2.0 m naoh (room temperature, 1 h) produces meso - tartrate 8 (178.9/76.1 ppm) and dl - tartrates 8 (179.8/75.0 ppm). Signals for carbonate (169.1 ppm), oxalate 9 (173.6 ppm), formate 11 (172 ppm), tartronate 12 (179.7/76.2 ppm), and glycolate 13 (trace, 181.4/62.4 ppm) were observed . The glyoxoin reaction also produced tartrates at lower concentrations (0.01 m of 1 and 0.002 m cn) and at lower temperatures (4 c). The reaction is, in fact, so resilient that even under heterogeneous conditions (when insoluble lithium glyoxylate was substituted in place of sodium glyoxylate), tartrates were still produced (figure s4). However, when the ph was lowered (ph 9), tartrates were not observed, indicating that high ph was crucial . When a control reaction (at ph 14) was conducted by omitting the cyanide ion, no tartrates were formed; only the disproportionation of glyoxylate to glycolate 13 and oxalate 9 was observed, proceeding through the well - known cannizzaro reaction (figure s5c). The production of tartrates (as opposed to dhf) from the glyoxoin reaction in basic media was unanticipated but did implicate dhf as an intermediate, since tartrate is formally the reduction product of dhf . Therefore, a reaction of dhf 5 (5 mmol, insoluble), nacn (1 mmol/0.1 m), and glyoxylic acid (5 mmol/0.5 m) in aqueous naoh (2.0 m/10 ml) at room temperature was investigated . A vigorous bubbling was observed in this heterogeneous reaction mixture for 2 h, at which point a c nmr spectrum of the supernatant showed the production of tartrates 8 along with carbonate, oxalate 9, formate 11, tartronate 12, and glycolate 13 . More revealingly, when the dhf / glyoxylate reaction was repeated, omitting the cyanide, tartrates 8 were still formed . When the reaction of 0.25 m of dhf 5 with 0.25 m glyoxylic acid (in the absence of cyanide) was repeated in a mixture of 1.0 m naoh and 1.0 m lioh, the reaction mixture became homogeneous (similar to the glyoxoin reaction). C nmr spectrum, after 1 h, showed production of tartrates 8, (in 69% yield by quantitative c nmr) with complete glyoxylate conversion and little carbonate formation (figure 2); the ratios of the accompanying product peaks were similar to the glyoxoin reaction (figure s5a, b) with higher proportion of meso - tartrate (table s1). Interestingly, in the heterogeneous case, the c nmr spectrum of the reaction was also nearly identical to the glyoxoin reaction with the exception of a much more intense carbonate peak (figure s6b). In both heterogeneous and homogeneous cases, these results demonstrated that dhf could, by itself, mediate the transformation of glyoxylate to tartrates (figure s6). Typical c nmr of the homogeneous dhf / glyoxylate (without cyanide) reaction . Reaction of 0.25 m glyoxylate with 0.25 m dhf in aqueous 1.0 m naoh and 1 m lioh (room temperature, 1 h). For nmr details while these reactions showed that dhf might be an intermediate, it may not be the only path in the cyanide - mediated glyoxoin reaction . Tartrates could have been additionally produced from the base catalyzed condensation reaction of glycolate 13 (a product of the cannizzaro reaction) with glyoxylate 1 . This pathway was ruled out, since tartrates were observed neither in the glyoxylate control reaction discussed above nor when glyoxylate and glycolate were combined under identical reaction conditions . This leaves dhf formation and its reaction with glyoxylate as the most likely route to tartrates . Two possible mechanisms were considered for this formal reduction of dhf 5 (scheme 2). The first is a simple cross - cannizzaro reaction in which the hydroxide adduct of glyoxylate transfers a hydride to the carbonyl of the keto form of dhf leading directly to tartrates 8 and oxalate 9 . The second pathway involves an aldol reaction between dhf and glyoxylate resulting in a six - carbon tricarboxylate 6 . This six - carbon compound 6 can undergo a hydroxide promoted fragmentation to form tartrates 8 and oxalate 9 (scheme 2). Cross - cannizzaro reaction involving a hydride transfer (top) versus an aldol - reaction followed by hydroxide - mediated fragmentation (bottom). To differentiate between these two possible pathways, a reaction of dhf and glyoxylate was conducted using c - dilabeled glyoxylate . If the cross - cannizzaro hydride transfer was the sole pathway, only the formation of c labeled oxalate 9 * would be expected . If the fragmentation of the six - carbon tricarboxylate 6 * was the only pathway, only signals corresponding to labeled tartrates 8 * would be observed . However, when c - dilabeled glyoxylic acid (0.1 m) was reacted with dhf (0.1 m) in 1.0 m naoh and 1.0 m lioh (homogeneous reaction), thec nmr spectrum showed that both labeled tartrates 8 * and oxalate 9 * were formed . While the presence of labeled tartrates 8 * indicates that an aldol reaction fragmentation reaction must have occurred, formation of labeled oxalate 9 * could be explained via the competing self - cannizzaro (as opposed to the cross - cannizzaro) reaction of glyoxylate . However, integration of the c signals corresponding to the carboxylate peaks of oxalate 9 * and glycolate 13 * revealed that there is 50% less labeled oxalate than labeled glycolate in the reaction mixture (figure s7c). This excess of glycolate indicated that (a) there is likely some side reaction that results in the production of labeled glycolate 13 *; and (b) if the cross - cannizzaro hydride transfer reaction is taking place, it is doing so to a lesser extent than this glycolate producing side reaction . Therefore, although the cross - cannizzaro reaction cannot be ruled out entirely, this experiment supports the aldol reaction a separate reaction using unlabeled glyoxylate (1.0 m) and c - labeled nacn (0.01 m), clearly revealed the glyoxylate cyanohydrin 2 at 125 ppm an otherwise empty spectral region; as the reaction progressed, a second peak appeared at 126 ppm (attributed to the dhf cyanohydrin 4) persisting for 4 h before disappearing (figure s8). In the heterogeneous reaction (in 2.0 m aq naoh) of labeled glyoxylate with dhf, a second pathway was suggested by the formation of significant amount (10%) of singly labeled tartrates 8 (figure s7b), along with singly labeled tartronate 12 . Interestingly, the (single) labeling occurs only at the carboxylate moiety of tartrates 8 and tartronate 12 as evidenced by a lack of splitting of the carboxylate signal . Some of this could be explained by incomplete labeling of the starting material; however, the starting material contains <1% of singly labeled material (by c nmr, figure s7a). This suggested that the carbon carbon bond in glyoxylate is being broken during the course of the reaction . Based on the above observations, we propose an overall mechanism (scheme 3), which accounts for the bulk of the tartrates 8 and oxalate 9 . In this mechanism the deprotonated glyoxylate cyanohydrin 3 then reacts with an additional molecule of glyoxylate to form the cyanohydrin adduct of dhf 4, which is then converted to the keto form of dhf 5 (which tautomerizes to the typical enol form). Dhf, thus formed, can react via an aldol reaction with an additional molecule of glyoxylate (c labeling shown in red) to yield a six - carbon tricarboxylate intermediate 6 *, which can (under high ph) rearrange to 7 * via a cyanide - mediated retro - aldol pathway . This mechanism accounts for the primary pathway to tartrates, in which oxalate and tartrates are formed in a 1:1 ratio . These intermediates 6*/7 * can be attacked at the carbonyl by a free hydroxide anion (under high ph) and undergo fragmentation to yield tartrates 8/8 * and oxalate 9/9*. This mechanism accounts for the primary pathway to tartrates, in which oxalate and tartrates are formed in a 1:1 ratio . Additional side products are formed by pathways (scheme 4) that include a series of benzoin - type rearrangements, which are possible under these high alkaline conditions and which also account for the formation of singly labeled products . In these pathways the tricarboxylate intermediate 6(6*)/7(7 *) can undergo benzoin - type rearrangements to a six - carbon aldehyde intermediate which fragments to yield singly labeled tartrates and formate . Alternatively 6(6*)/7(7 *) can undergo a retro - aldol reaction generating dhf 5/5 *, which under these high - alkaline conditions undergoes a benzoin - type rearrangement to an aldehyde intermediate 10/10*. This intermediate can then react with hydroxide and fragment to form bicarbonate, formate 11/11 *, tartronate 12/12 *, and glycolate 13/13*. This second reaction pathway was identified from the experiments dealing with the heterogeneous decomposition of dhf alone to give formate and tartronate in 2.0 m naoh . This also accounts for the presence of formate in these samples; glycolate and oxalate are also produced by the hydroxide promoted fragmentation reaction of the keto form of dhf . In addition, there is a side reaction that occurs when glyoxylate 1 is in a far higher concentration than dhf 5, where tartrates 8 and tartronate 12 are formed in a 1:1 ratio . This side reaction (scheme 4, bottom) begins at intermediate 6/7, which decarboxylates to intermediate 14, which then undergoes an aldol reaction with an additional molecule of glyoxylate 1 to form a seven carbon tricarboxylate 15 . Further benzoin - type rearrangements followed by fragmentation gives rise to tartrates 8 and tartronate 12 . The three pathways shown above account for all observed side products, including isotopically labeled side products . The results of the dhf glyoxylate reaction presented here are in sharp contrast to the work of sagi et al . ; there, dhf was reacted with glyoxylate in lithium hydroxide at ph 78 to produce dihydroxyacetone and pentulosonic acid by way of six- and seven - carbon tricarboxylate intermediates (6 and 15) via several decarboxylations . However, in our work, 6 and 15 undergo attack by free hydroxide at the carbonyl, yielding oxalate 9, tartronate 12, and tartrates 8 . While the carbonate produced in the glyoxoin reaction is indicative of decarboxylations occurring, no evidence was seen for any of the intermediates or products observed in the work of sagi et al . (6) this contrasting result demonstrates how even in consistently basic environments, ph may be used to alter the product suite of this reaction, from selective production of pentulosonic acid to selective production of tartrate . This dependence on ph underscores the need to investigate a wide range of conditions for potentially prebiotic reactions to fully explore the possibilities for producing these biologically relevant molecules . The products of this simple and robust glyoxoin reaction are stable in the high - ph environment, illustrating their potential as feedstock for further reactions . However two questions must be raised to determine the relevance to prebiotic chemistry; specifically, what are the abiotic availabilities of glyoxylate and of a high - hydroxide concentration? For the former there are many possibilities: (a) glyoxylate has been shown to be readily produced by photo - oxidation of acetylene under anoxic conditions; (b) reductive conversion of carbon dioxide and carbon monoxide to glyoxylate has been experimentally demonstrated; some of these could be reasonably extrapolated to early earth scenarios; and (c) glyoxylate was shown by weber to form reliably in the reaction of glycolaldehyde and formaldehyde, catalyzed by various primary amines, though it was not the primary product . The results of this investigation underscore the need and the opportunity to further explore these options and fully investigate formation of glyoxylate under prebiotic conditions . High - hydroxide concentrations are considered to be extreme (and unusual) in the conventional prebiotic line of thought . This usually renders any high - ph reaction problematic in this context . However, there are at least three plausible prebiotic scenarios one can consider . The first possible scenario is the widely investigated alkaline hydrothermal vent system; however, this system is not without drawbacks . Related reservoirs of natural hydroxide are lakes fed by alkali springs, which could act as a natural reactor for this type of chemistry . While these lakes may have the advantage of sidestepping the magnitude of both dilution and thermolysis faced by oceanic vents, they too have their limitations . Another possible high - alkaline environment can be found in the interlayer framework of double - layered hydroxides (e.g., hydrotalcites), which are especially conducive for uptake and concentration of anions, such as glyoxylate and cyanide, from dilute solutions . While each of these possibilities appear to be highly localized with their attendant weaknesses in a prebiotic context, they do offer potential for highly alkaline environments on early earth, that need to be validated from a prebiotic perspective, for the chemistry described here . Notwithstanding the arguments presented above, it remains that the cyanide - mediated dimerization of glyoxylate at high ph to produce tartrates proceeds with remarkable consistency and speed, even at low temperature, low concentration, and low solubility . The fact that these reactions occur reliably and that the products are stable at high hydroxide concentrations demonstrates that a high ph environment need not be antithetical to production of such organics in a primordial setting . Moreover, the use of cyanide as a catalyst is to be contrasted with many other conventional prebiotic scenarios that start with cyanide as a source molecule . In the context of primordial metabolism, the robust production of tartrates from glyoxylate opens up new venues for the origins of biologically relevant small molecules . The simple and known dehydration of tartrates results in oxaloacetate which is known to decarboxylate to give pyruvate, thus providing an entry into a prebiotic citric acid cycle . In extant biology, oxaloacetate is the entry point to the citric acid cycle, reacting with pyruvate to form citrate . There are examples in extant biology where tartrate is utilized and metabolized for production of oxaloacetate to be used in the citric acid cycle . Thus, it is reasonable to posit that tartrates could have acted as a source of small molecules, which could become part of an emerging proto - metabolic process (e.g., reductive citric acid cycle). Thus, the glyoxoin reaction (and the glyoxylate scenario) could serve as a plausible alternative start for rudimentary chemical evolution (scheme 5). Glyoxylic acid monohydrate (0.110 mmol, 10100 equiv) and sodium cyanide (0.011 mmol, 1 equiv) were each weighed in individual 6 dram vials . Ten ml of 2.0 m naoh or 2.0 m lioh was then added to the vial containing the sodium cyanide . The solution was then transferred by syringe to the vial containing the glyoxylic acid . The cyanide solution was added down the side of the vial to the glyoxylic acid over the course of 20 s and then closed, and the vial was cooled (by running cool water over the vial) as necessary to avoid with excess heating . Dihydroxyfumaric acid dihydrate (0.15 mmol, 5 equiv), glyoxylic acid monohydrate (0.15 mmol, 5 equiv), and sodium cyanide (0.011 mmol, 1 equiv) were each weighed in individual 6 dram vials . Five ml of 2.0 m naoh was then added to the vial containing the sodium cyanide . In a separate vial an additional 5 ml of 2.0 m aq naoh was then added to the vial containing the glyoxylic acid . The glyoxylate solution was added down the side of the vial to the dihydroxyfumaric acid over the course of 20 s and then closed, and the vial was cooled (by running cool water over the vial) as necessary to avoid with excess heating . Dihydroxyfumaric acid dihydrate (0.15 mmol, 1 equiv) and glyoxylic acid monohydrate (0.15 mmol, 1 equiv) were each weighed in individual 6 dram vials . Five ml of 2.0 m naoh as then added to the vial containing the glyoxylic acid monohydrate . This vial was mixed until no solid remained . In a separate vial, 5 ml of 2.0 m aq lioh the glyoxylate solution was then transferred into the vial containing the dhf solution, and the vial was closed and shaken . Alternately, adding dhf to glyoxylate resulted in no noticeable differences.
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Prostate cancer is the second most common cancer and second cause of cancer death in chilean men . Prostate specific antigen (psa) is the most accurate serum marker for prostate and the only biomarker routinely used for the early detection of prostate cancer . Although psa is highly specific for prostate, an elevated level is not specific for prostate cancer, being increased in benign pathologies [2, 3]. Consequently, approximately 70% of men with an increased serum psa, defined as> 4.0 ng / ml, do not have prostate cancer and thus undergo unnecessary prostate biopsies . A psa cutoff of 4.0 ng / ml is currently used to select men for prostate biopsy; however, this misses many cancers and it has been suggested that lowering the cutoff to 2.6 ng / ml will detect small but clinically significant cancers . The prostate cancer prevention trial reported that 39.2% of men with a psa 2.13.0 ng / ml, 27.7% of men with a psa 1.12.0 ng / ml, and 1.3% of men with a psa <1.0 ng / ml had end of trial prostate biopsies with foci of adenocarcinoma . In other words 38% of men with prostate cancer have a psa <4.0 ng / ml and 70% of men with a psa> 4.0 ng / ml do not have cancer . The subject is further complicated by the high prevalence of prostate cancer detected at autopsy, the high frequency of positive prostate biopsies in men with a normal digital rectal examination (dre) and psa <4.0 ng / ml, the contrast between the incidence and mortality rates for prostate cancer, and the need to treat an estimated 37 men with screened detected prostate cancer to prevent one prostate cancer death [9, 10]. To achieve a relative mortality reduction of 40% by screening for prostate cancer, 50% of screened detected prostate cancers may be overtreated . This is especially so as the risks of prostate biopsy are not insignificant; rietbergen et al ., in a study of 5,802 patients undergoing transrectal prostate biopsy, reported an incidence of complications of 0.5% hospitalizations, 2.1% rectal hemorrhage, 2.3% fever, and 7.2% persistent hematuria . The use of primary malignant cpc (mcpc) detection as a sequential test for deciding the need for prostate biopsy may resolve in part some of these problems . In the study protect of 228 patients undergoing first biopsy where 28.5% of patients had cancer diagnosed, the detection of primary mcpcs and the association with a positive biopsy had a sensitivity of 86.2% and specificity of 90.8%, with a positive predictive value of 78.9% and negative predictive value of 97.1% . The use of the detection of primary mcpcs as a sequential test to select men, with suspicion of prostate cancer for an elevated psa, to determine the need for prostate biopsy raised concern of the possibility of missing clinically significant prostate cancer . We present the results of 328 men diagnosed with prostate cancer as a result of a screening program and compare the results of primary mcpc detection with the biopsy gleason score, percent of infiltration of the samples by cancer, and the number of positive cores . After ethical committee approval of the study for the use of primary mcpc detection, a prospective study was carried out . All patients attended in the carabineros de chile health system and had a serum psa> 4.0 ng / ml and/or a digital rectal examination (dre) suspicious of prostate cancer and were referred for prostate biopsy . Immediately before the biopsy 8 ml of venous blood was collected in edta (beckinson - vacutainer) and sent to a central laboratory . Patients were coded and clinical details of serum psa, age, and biopsy results were collected . The prostate biopsy and primary cpc detection were independently analyzed, with the evaluators being blinded to the clinical details and results of the biopsy or primary mcpc test . Mononuclear cells were obtained by differential centrifugation using histopaque 1,077 (sigma - aldrich), washed, and resuspended in 100 l of autologous plasma . 25 l aliquots were used to make slides (silanized, dako, usa), dried in air for 24 hours, and fixed in a solution of 70% ethanol, 5% formaldehyde, and 25% phosphate buffered saline, ph 7.4 . Primary mcpcs were detected using a monoclonal antibody directed against psa and clone 28a4 (novocastro laboratory, uk) and identified using an alkaline phosphatase - anti - alkaline phosphatase based system (lsab2, dako, usa), with new .- fushcin as the chromogen . Positive samples underwent a second process with anti - p504s clone 13h4 (dako, usa) and were identified with a peroxidase based system (lsab2, dako, usa) with dab (3,3-diaminobenzidine tetrahydrochloride) as the chromogen . A primary mcpc was defined according to the criteria of ishage (international society of hematotherapy and genetic engineering) and the expression of p504s according to the consensus of the american association of pathologists . A malignant primary cpc (mcpc) was defined as a cell that expressed psa and p504s; a benign primary cpc (bcpc) expressed psa but not p504s and leucocytes could be p504s positive or negative but did not express psa (figures 1(a)1(c)). A test was considered positive when at least 1 cell was detected/4 ml blood . The test was classified as positive or negative for mcpc; the number of mcpcs/4 ml of blood was not used as a parameter in order to simplify the result of the test . Immunocytochemical staining of the slides was analyzed by one observer blinded to clinical and pathological details . An ultrasound guided 12-core biopsy was taken according to standard recommendations; samples were fixed in formaldehyde and sent to the pathology service . 3 m paraffin embedded sections were cut, deparaffinized, and stained with h&e as per standard procedure . If cancer was detected, gleason score, number of cores positive for cancer, and percent of infiltration were recorded . The prostate biopsy was analyzed by a single pathologist blinded to clinical details and results of the mcpc test . The ultrasound was used to guide the biopsy; not all reports had the prostate volume calculated or whether the prostate was homogeneous or heterogeneous . As it is not routine in chile the use of prostatic ultrasound prebiopsy, we did not include this parameter in the analysis . Both the nccn guidelines and nice (uk) guidelines recommend as a treatment option active surveillance in men with the following criteria: clinical stage t1c or less, gleason score 6, less than 3 cores positive for cancer, and less than 50% or 10 mm infiltration in any one core . The frequency of patients who were primary mcpc positive and negative was compared using these guidelines to evaluate the number of patients complying with the criteria for active surveillance . We used the 1994 criteria for as and not those of tosoian et al . Descriptive statistics were used for demographic variables, expressed as mean and standard deviation in the case of continuous variables with a normal distribution . In case of an asymmetrical distribution the median and interquartile range (iqr) values were used . The student t - test was used to compare continuous variables with a normal distribution, the mann - whitney test for ordinate and continuous variables with a nonnormal distribution, and chi - squared for the differences in frequency . Statistical significance was defined as a p value less than 0.05 to all tests were two tailed . Analysis was performed using the stata 11.0 program (statacorp lp, college station, texas, usa). The study was directed with complete conformity with the principles of the declaration of helsinki and approval of the local ethical committees . A total of 328 men of a cohort of 1123 patients had a prostate biopsy positive for cancer, with an overall incidence of 29.2% of all biopsies . 42/328 (12.8%) of these men were negative for the detection of primary mcpcs . Men negative for primary mcpcs were significantly older and had lower serum psa levels, lower gleason scores, lower number of cores positive for prostate cancer, and cores less infiltrated with cancer . Of the 1123 patients, 90 (8%) were positive for mcpcs but had an initial prostate biopsy negative for cancer . Comparing men mcpc negative with those mcpc positive using the epstein criteria for active surveillance, 38/42 (91%) of mcpc negative men compared with 34/286 (12%) (p <0.0001) of mcpc positive men complied with the criteria for active surveillance of their prostate cancer (table 2). Four men in the cpc negative group did not comply with the criteria for active surveillance; their details are shown in table 3 . In patient number 3 patient number 1 had a cancer which needed treating and underwent radical prostatectomy as did patients 2 and 4 . Models of prostate cancer detection and estimates of progression suggest that 2342% of screen detected prostate cancers are overtreated . The introduction by epstein et al . Of criteria to predict pathologically insignificant prostate cancer has been useful but there is caution about using it as the sole reference for making clinical decisions as many as 8% of these cancers were not organ confined based on postsurgical findings . Active surveillance is considered the best option for patients with low risk cancers or a short life expectancy . Thus if there is a significant percent of men overtreated for their prostate cancer and active surveillance is an accepted treatment option, then the aim of the prostate biopsy in men with an elevated psa is not to detect each and every prostate cancer but to detect those prostate cancers with the potential for causing harm . Men with clinically insignificant prostate cancers that were never destined to have symptoms or to affect their life expectancy may not benefit from knowing that they have the disease . The detection of clinically insignificant prostate cancer could be considered as an adverse effect of the prostate biopsy . As such, there is considerable anxiety and distress found in men undergoing active surveillance . There are no directly relevant studies comparing immediate and delay biopsy in men with a raised psa level . A number of observational studies have reported risk factors for high grade prostate cancer in men referred for biopsy, related to age, psa, dre result, prior prostate biopsy, black ethnicity, and prostate volume [2325]. However, there are concerns over delaying a prostate biopsy because of the uncertainties of the natural history of untreated prostate cancer, the missed opportunity to detect and treat a curable cancer, or that due to delay in performing a biopsy the treatment of a larger or more aggressive cancer may lead to a more complex surgery with greater side effects . The use of primary cpc detection to select men for prostate biopsy fails to detect men with cpc negative cancer . This represents approximately 5% of all primary cpc negative men and an elevated psa of between 4.0 and 10.0 ng / ml . This study suggests that, of these 5% of primary cpc negative men, 9% would have a prostate cancer complying with the guidelines for treatment or that approximately 99.5% of all primary cpc negative men would not have a prostate cancer needing treatment or in the majority of cases primary cpc negative having benign prostatic hyperplasia . Thus from these results the concern of missing significant prostate cancer is minimal, much less than the 38% of prostate cancers missed when using a psa level of 4.0 ng / ml as a cutoff point for recommending prostate biopsy . An ongoing study of the followup of all primary cpc negative men with an elevated psa is currently in progress . We used the epstein criteria to define active surveillance rather than those of tosoian et al . ; men do not routinely undergo prostatic ultrasound as part of the prostate cancer screening program; thus the decision to refer the patient for prostate biopsy based on psa and mcpc determinations would not include prostate volume determinations . Previously we reported the use of the number of cpcs / ml blood detected; however, the increase in specificity using a cutoff point of 4 cells / ml blood was minimal, 8%, with an important decrease in sensitivity . There are two questions not answered by the study, which are currently part of an ongoing investigation . Firstly, mcpc positive men with a negative biopsy are these men at increased risk of prostate cancer, in that they have cancer but the biopsy failed to detect it; studies have shown that approximately 20% of men have cancer detected on repeated biopsy . If follow - up studies show that these men do indeed have cancer, it may be advisable to repeat the biopsy earlier or a biopsy for saturation . Secondly, in mcpc negative men, we are currently repeating total and free psa with mcpc testing on a 6-month basis; men who become positive, with an abnormal dre or significant change in total psa and/or free psa, are referred for biopsy . It would be important to determine if a change from mcpc negative to positive was associated with a change in the clinic - pathological parameters to indicate active treatment . In chile, few men choose to undergo active surveillance, preferring active treatment, as we have little data on this type of patient . It is important to emphasize that these are primary mcpcs and are not associated with the prognosis; most of these cells disappear after radical treatment; men who remain positive, secondary mcpc positive, have a higher frequency of biochemical failure . One concern over the use of circulating tumor cell technologies is the discordant results achieved using different methods of detection . Using a dual psa / prostate specific membrane antigen rt - pcr method eschwge et al davis et al . Found no association between cpc detection using the cellsearch system and the clinical parameters prior to radical prostatectomy or between men with local pc or controls . However, stott et al . Found primary cpcs in 42% of patients with localized cancer; fizazi et al ., using anti - berep-4 epithelial antigen combined with telomerase activity, detected primary cpcs in 79% of patients with localized cancer, a similar figure to that reported using this same methodology . We believe that part of the difference documented is caused by the relatively high detection in control patients; one explication is that cpc can be found in men with prostatitis and benign hyperplasia; however, these cpcs are p504s negative . We also designed the test using cpcs to produce a result considered as positive or negative; the fundamental question was is there cancer and will it harm the patient? ; consequently we considered that the presence of single cell is sufficient to classify patients as positive or negative for cancer . We also need to emphasize that this is a single institution study; thus the test needs to be used in other centers to determine the reproducibility of the test . Within our institution preanalytical variables are controlled and are limited; immunocytochemical staining can be automatically performed and thus the main variable is the observer . Observer variation could be minimized with adequate training but is a variable that needs to be considered . The inter - observer variation in reported gleason scores has been a problem; recently published data on the clinical implications of interobserver variation showed an overall agreement of 80.789%, but this would lead to up to 10% of patients recommended for active observation would have received different treatments based on inter - observer variation . The objective of this study was to address the concern of not detecting potentially harmful prostate cancer using the detection of p504s expressing primary cpcs . The results suggest that the majority of cancers that the test failed to detect, when used as a sequential screening test in men with a psa level of between 4.0 and 10.0 ng / ml, are low grade small volume tumors which would comply with the criteria for active observation . Thus in primary cpc negative men the possibility of a harmful cancer being missed is minimal; in these men prostate biopsy could be avoided or delayed, thus not exposing the patient to the adverse effects of biopsy or the anxiety or distress of active observation.
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Aging is a gradual process, proportional to time, that causes structural and functional changes due to internal degeneration . Aging can be divided into intrinsic aging, which is natural with the progression of time, and environmental aging caused by the external environment . Uv - induced photoaging is usually observed on skin that is exposed to the sun . Its clinical characteristics include earlier observation than intrinsic aging, deep and wide wrinkles, inconsistent pigmentation and solar lentigo on the exposed skin surface2 . Along with recent socio - financial improvements, improvements in medical technology, hygiene and nutrition have increased life expectancy . As the elderly population increases, many treatment methods to reduce skin aging and improve wrinkles and pores are currently being researched4, 5 . However, ablative laser treatment causes a financial burden and side effects, such as erythema, edema, and hyperpigmentation . In addition, the damage on the epidermis caused by ablative laser treatment delays the time between returning to daily activities6,7,8 . Recently, non - ablative skin rejuvenation, which improves skin aging and photoaging without damaging the epidermis, has been the preferred treatment9 . Previous studies suggest that radiofrequency (rf) effectively reduces abdominal obesity by transferring heat to the dermis, increases blood volume in the capillaries and hence increases blood flow, induces collagen formation, and increases the thickness of the epidermis10,11,12 . Electroacupunture (ea) has been reported to reduce inflammation, induce healing of damaged tissues, reduce pain, and improve facial rejuvenation13,14,15 . Low - level laser therapy (lllt) has been used in various research studies and has been reported to have anti - inflammatory effects, reduce pain, reduce edema, induce healing of damaged tissue or fracture, produce collagen, induce skin rejuvenation and to be effective for acne and vitiligo16,17,18,19,20,21 . In this study, the effects of rf, ea, and lllt on wrinkles and moisture content were investigated in 30 female subjects aged 3055 years . The results will be used as a reference for the further development of skin physical therapy . The subjects of this study were 30 adult women aged between 30 and 55 years who presented at m dermatology hospital in yongin, gyeonggi - do, south korea and who voluntarily agreed to participate after listening to an explanation of the purpose of the study . The subjects were assigned to rf, (n=10), ea (n=10) or lllt (n=10) groups . Inter - group homogeneity testing for general characteristics prior to treatment revealed no significant differences . The ethics committee of namseoul university, south korea approved the study (irb approval number: 104147 - 201504-hr-003). The characteristics of the subjects are shown in table 1table 1.general characteristicsvariableslllt(n=10)ea(n=10)rf(n=10)age (years)42.9 3.743.4 7.142.8 4.9height (cm)159.8 4.3158.0 4.3163.0 7.9weight (kg)52.4 6.158.8 7.256.7 lllt: low - level laser therapy group; rf: radiofrequency group; ea: electroacupuncture group . Lllt: low - level laser therapy group; rf: radiofrequency group; ea: electroacupuncture group for the rf group, prix iii (shenb co., ltd ., korea) was used at 1 mhz (rf energy, maximum 450w / cm) in two 15-minute sessions per week for six weeks22 . For the ea group, disposable stainless steel acupuncture (0.20 15 mm) was used . For ea stimulation, an ots h-306 (hanil tm co., ltd ., korea) was used at a frequency of 3 hz, with 5 seconds of on time and 2 seconds of off time . The intensity of the ea was increased until the subject felt simulation and a small muscular contraction and was then maintained . For the lllt group, redpulsar (sometech inc ., korea), which is a diode laser, was used in two 15-minute sessions per week for six weeks at 635 nm, with 10mw of the laser class iiib23 . A janus skin measurement device (psi co., ltd . The faces of the subjects were fixed in a specific spot and the facial skin conditions were meticulously photographed using a 10 megapixel canon dslr camera lens . Wrinkles under both eyes were measured through front - view photos, whereas wrinkles on the eye rims were measured through profile - view photos . To minimize the measurement errors, the same rater performed measurements from the beginning to the end in similar indoor environments (room temperature 2021 c and humidity 5060%). A moisture checker (psi co., ltd ., korea) was used to measure the skin moisture levels on the forehead and right cheek . In order to reduce error, three measurements were taken and the average was calculated (table 2table 2.moisture ratevariablesforeheadcheekvery dry (au)124124dry (au)25342534moisturized (au)35443544sufficiently moisturized (au)45654565au: arbitrary unit). All data were coded and analyzed using the spss statistical analysis program (ver.18). To determine the post - intervention changes in the number of wrinkles and moisture content in each group, a test of normality was conducted . If subjects passed the test, a paired samples t - test was conducted; otherwise, the wilcoxon rank - sum test was conducted . To determine the inter - group differences in wrinkles and moisture content, one - way analysis of variance was used when they satisfied normality . Treatment of under eyes showed that wrinkles have significantly decreased on both sides after rf, ea and lllt (p<0.05). Treatment of eye rims showed that wrinkles have significantly decreased on the right side after rf, ea and lllt (p<0.05) (table 3table 3.changes in the number of wrinkles after rf, ea or lllt and a comparison of wrinkle improvements between groupswrinklesinterventionprepostunder eyesrightrf*11.2 2.88.3 3.6ea*14.7 9.510.7 7.3lllt*14.4 6.211.3 4.4leftrf*12.9 4.58.0 3.3ea*16.5 8.913.0 7.3lllt*12.7 5.810.1 4.6eye rimsrightrf*8.1 4.16.0 3.1ea*12.3 8.19.8 5.9lllt*7.6 3.76.6 2.9leftrf7.9 5.26.8 4.3ea13.0 8.211.2 6.7lllt7.7 4.06.6 3.8values are shown as the mean standard deviation, * significant difference between the pre- and post - tests within each group, p<0.05 . Paired samples t - test; wilcoxon rank - sum test; rf: radiofrequency; ea: electroacupuncture; lllt: low - level laser therapy). Values are shown as the mean standard deviation, * significant difference between the pre- and post - tests within each group, p<0.05 . Paired samples t - test; wilcoxon rank - sum test; rf: radiofrequency; ea: electroacupuncture; lllt: low - level laser therapy treatment of forehead showed that skin moisture has significantly increased after rf, ea and lllt (p<0.05). Treatment of cheek showed that skin moisture has significantly increased after rf, ea and lllt (p<0.05). Significant differences have been observed on skin moistures of the cheek between the two groups (p<0.05) (table 4table 4.changes in skin moisture after rf, ea or lllt and comparison of skin moisture improvements between groupsmoisture contentinterventionprepostforeheadrf*35.3 2.537.1 3.0ea*33.7 3.838.7 3.2lllt32.9 4.836.9 4.9cheekrf*36.3 2.938.9 2.5ea*33.7 3.641.5 3.5lllt*34.5 2.939.9 3.3values are shown as the mean standard deviation, * significant difference between the pre- and post - tests within each group, p<0.05 . Paired samples t - test; wilcoxon rank - sum test; lllt: low - level laser therapy; ea: electroacupuncture; rf: radiofrequency; 1: rf; 2: ea; 3: lllt). Values are shown as the mean standard deviation, * significant difference between the pre- and post - tests within each group, p<0.05 . Paired samples t - test; wilcoxon rank - sum test; lllt: low - level laser therapy; ea: electroacupuncture; rf: radiofrequency; 1: rf; 2: ea; 3: lllt desire for anti - aging and antioxidants has increased with economic growth and life expectancy . Active participation of females in all aspects of society has increased the attention paid to skin management3, 21 . Therefore, this study aimed to investigate the effects of rf, ea and lllt on wrinkles and skin moisture content . A previous study by el - domyati et al.10, which conducted a quantitative evaluation of dermal collagen after rf treatment, reported an increase in type i and type ii collagens and a decrease in the number of wrinkles under the eyes and on the eye rims (p<0.01). A study by abraham et al.24 also reported improvements in wrinkles, pore size, and the laxity of facial skin . The present study reported similar findings in that wrinkles under the eyes and on the eye rims were significantly decreased (p<0.05). Based on previous studies that reported that rf induces the formation of new collagen by transferring heat to the dermis25, 26, we hypothesize that the formation of new collagen contributed to the improvement of wrinkles under the eyes and on the eye rims in this study . Barrett14 reported that the number of wrinkles on eye rims decreased by 50% and wrinkles under the eyes decreased by 20% after 10 sessions of ea . Kwon et al.27 reported that miso facial rejuvenation acupuncture significantly decreased the width of wrinkles on eye rims from 4.87 3.63 to 2.43 2.04 . Using jae - seng acupuncture, cho et al.28 reported significant improvements in the number of eye wrinkles, consistent with the present study (p<0.001). A previous study by barolet et al.29, in which 12 led treatments at 660 nm, 50 mw / cm and 4 j / cm were conducted on a 3d model of tissue - engineered human reconstructed skin, reported improvements in wrinkle depth, surface roughness and fitzpatrick wrinkling severity scores . Russell et al.30 incorporated combination led light therapy (633 nm and 830 nm) and also reported improvements in periorbital wrinkles . In the present study, wrinkles under the eyes on both sides and wrinkles on the left eye rims were significantly decreased after lllt treatment (p<0.05). Based on previous studies, which reported that lllt treatment induced collagen up regulation and down - regulation of matrix metalloproteinase, it can be inferred that the increase in collagen on the epidermis contributed to the improvement of wrinkles . Kwon et al.31 reported that the moisture content of the right side of the face increased from 49.10 10.95au to 63.20 12.42au after miso facial rejuvenation acupuncture (p<0.001). In the present study, the moisture content improved significantly on the forehead and the cheek in the ea group (p<0.05). A previous study by oh et al.32 reported that the relaxation of the skin and the wrinkles increased as aging progresses since the moisture content decreases . Therefore, it can be inferred that the increase in the moisture content observed in the present study was caused by the decrease in wrinkles . Firstly, since the study was not conducted on a large number of participants and it was only conducted on female adults between the ages of 3055 years, generalization of the results is limited . Secondly, since the study only confirmed the effects in a span of 6 weeks, evaluation of long - term effects was not possible . Therefore, further studies should be conducted on a larger sample pool composed of female participants from a larger age range, and long - term effects should be confirmed during a span of at least 6 months.
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The phantom consisted of 5 components; the driver, the control, the support, the balloon phantom and the ecg . The phantom's end - diastolic phase, with the longest motion - free periods, was designed at 85% of the r - r interval despite of the changes of the hr . Different sized acrylic tubes (3, 4 and 5 mm) were used to simulate coronary arteries . Inside them, artificial plaques (+ 100 hounsfield unit [hu]) simulated different degrees of stenosis (0, 25, 50 and 75%). These tubes were attached to the surface of a pulsating cardiac phantom (fuyo co., tokyo, japan) to simulate real heart motion . We diluted iodinated contrast medium (angiografin, 306 mg i / ml, schering ag, berlin, germany) with distilled water to a density of about 330 hu, and then we filled the cardiac phantom and the simulated coronary arteries with it (fig . The pulsating cardiac phantom equipped with the simulated coronary arteries was scanned with a 64-slice mdct (lightspeed vct; ge medical systems, milwaukee, wi). First, we scanned the cardiac phantom with static scanning to confirm the lumen size and the degree of stenosis in the simulate coronary arteries . The scan protocol was 120 kv, 600 ma, 0.35 second / rotation, a 0.625 mm slice - thickness, a 0.625 mm slice interval, a 0.16 fixed pitch factor and a 50 cm field of view (fov) for both the static and cardiac helical scanning . The phantom and the simulated coronary arteries were scanned at 9 different hrs: 40, 50, 60, 70, 80, 90, 100, 110 and 120 beats per minute (bpm). Both reconstruction algorithms, the halfscan reconstruction (single sector reconstruction, snapshot segment) and the multisector reconstruction (two sector reconstruction, snapshot burst), were applied to the 9 different hrs with the reconstructed phase at 85% of the r - r interval of the diastolic cardiac cycle of the phantom . In this study, we choose a two - sector reconstruction algorithm instead of a four - sector reconstruction algorithm because the number of sectors used for image reconstruction in the multisector reconstruction is fluctuant (5). The ct scanner will automatically adjust the number of sector used according to the hr . If the four - sector reconstruction algorithm was chosen, then the number of sectors used may oscillate between one to four sectors depending on the hr . A two - sector reconstruction algorithm was used in this study to ensure that the number of sectors used at higher hrs was constant . The effective temporal resolution (175 msec) is half of the gantry rotation time (grt, 350 msec) in the halfscan reconstruction, but actually the time required for one image acquisition includes the time gantry rotation for 180 plus a beam fan angle (approximately 30 - 60). So, the temporal window is approximately around 60 - 66% of the grt for the halfscan reconstruction . In the multisector reconstruction with the m segment, the effective temporal resolution varies between grt/2 and grt/2 m (6 - 9). There was a total of 324 segments of the simulated coronary arteries to be analyzed ([3 segments with different degrees of stenosis + 1 non - stenotic segment] 3 different sizes of acrylic tubes 9 different hrs 3 observers). Analysis was performed on an aw workstation (aw 4.3, ge medical systems, milwaukee, wi) by three experienced radiologists who were kept blinded to the luminal diameters, the degree of stenosis, the hr and the reconstruction algorithm . The radiologists measured the diameter of the patent lumen from each segmented image, instead of the stenotic rate . By doing this, the radiologist could not know the actual diameter of each segment since the stenotic and nonstenotic segments were separately presented . The images were reviewed with a fixed viewing window of 800/200 (ww / wl) and a viewing fov of 3.2 cm . The degree of stenosis (%) was calculated as: (the luminal diameter at the nonstenotic segment - the luminal diameter at the stenotic segment) 100%/the luminal diameter at the nonstenotic segment the ct scans were interpreted independently by three board - certified radiologists . Grade 1 equals a good quality image without artifacts, grade 2 equals an acceptable quality image with mild motion artifacts that do not interfere with the diagnosis and grade 3 equals a nondiagnostic, poor quality image with significant motion artifacts (fig . Paired t tests were used to analyze the differences in image quality . P value less than 0.05 were considered to indicate a significant difference . Segments with a quality score of 3 were defined as nonassessable segments due to the nondiagnostic image quality, and the segments with a quality score of 1 or 2 were defined as assessable segments . Both the numbers of nonassessable segments and the measurement error, defined as the relative error, of all the segments were recorded . The correlations between the number of nonassessable segments, the relative error and the reconstruction algorithm, hr, luminal diameter, stenotic severity and observers were analyzed . The phantom consisted of 5 components; the driver, the control, the support, the balloon phantom and the ecg . The phantom's end - diastolic phase, with the longest motion - free periods, was designed at 85% of the r - r interval despite of the changes of the hr . Different sized acrylic tubes (3, 4 and 5 mm) were used to simulate coronary arteries . Inside them, artificial plaques (+ 100 hounsfield unit [hu]) simulated different degrees of stenosis (0, 25, 50 and 75%). These tubes were attached to the surface of a pulsating cardiac phantom (fuyo co., tokyo, japan) to simulate real heart motion . We diluted iodinated contrast medium (angiografin, 306 mg i / ml, schering ag, berlin, germany) with distilled water to a density of about 330 hu, and then we filled the cardiac phantom and the simulated coronary arteries with it (fig . The pulsating cardiac phantom equipped with the simulated coronary arteries was scanned with a 64-slice mdct (lightspeed vct; ge medical systems, milwaukee, wi). First, we scanned the cardiac phantom with static scanning to confirm the lumen size and the degree of stenosis in the simulate coronary arteries . The scan protocol was 120 kv, 600 ma, 0.35 second / rotation, a 0.625 mm slice - thickness, a 0.625 mm slice interval, a 0.16 fixed pitch factor and a 50 cm field of view (fov) for both the static and cardiac helical scanning . The phantom and the simulated coronary arteries were scanned at 9 different hrs: 40, 50, 60, 70, 80, 90, 100, 110 and 120 beats per minute (bpm). Both reconstruction algorithms, the halfscan reconstruction (single sector reconstruction, snapshot segment) and the multisector reconstruction (two sector reconstruction, snapshot burst), were applied to the 9 different hrs with the reconstructed phase at 85% of the r - r interval of the diastolic cardiac cycle of the phantom . In this study, we choose a two - sector reconstruction algorithm instead of a four - sector reconstruction algorithm because the number of sectors used for image reconstruction in the multisector reconstruction is fluctuant (5). The ct scanner will automatically adjust the number of sector used according to the hr . If the four - sector reconstruction algorithm was chosen, then the number of sectors used may oscillate between one to four sectors depending on the hr . A two - sector reconstruction algorithm was used in this study to ensure that the number of sectors used at higher hrs was constant . The effective temporal resolution (175 msec) is half of the gantry rotation time (grt, 350 msec) in the halfscan reconstruction, but actually the time required for one image acquisition includes the time gantry rotation for 180 plus a beam fan angle (approximately 30 - 60). So, the temporal window is approximately around 60 - 66% of the grt for the halfscan reconstruction . In the multisector reconstruction with the m segment, the effective temporal resolution varies between grt/2 and grt/2 m (6 - 9). There was a total of 324 segments of the simulated coronary arteries to be analyzed ([3 segments with different degrees of stenosis + 1 non - stenotic segment] 3 different sizes of acrylic tubes 9 different hrs 3 observers). Analysis was performed on an aw workstation (aw 4.3, ge medical systems, milwaukee, wi) by three experienced radiologists who were kept blinded to the luminal diameters, the degree of stenosis, the hr and the reconstruction algorithm . The radiologists measured the diameter of the patent lumen from each segmented image, instead of the stenotic rate . By doing this, the radiologist could not know the actual diameter of each segment since the stenotic and nonstenotic segments were separately presented . The images were reviewed with a fixed viewing window of 800/200 (ww / wl) and a viewing fov of 3.2 cm . The degree of stenosis (%) was calculated as: (the luminal diameter at the nonstenotic segment - the luminal diameter at the stenotic segment) 100%/the luminal diameter at the nonstenotic segment the ct scans were interpreted independently by three board - certified radiologists . Grade 1 equals a good quality image without artifacts, grade 2 equals an acceptable quality image with mild motion artifacts that do not interfere with the diagnosis and grade 3 equals a nondiagnostic, poor quality image with significant motion artifacts (fig . Segments with a quality score of 3 were defined as nonassessable segments due to the nondiagnostic image quality, and the segments with a quality score of 1 or 2 were defined as assessable segments . Both the numbers of nonassessable segments and the measurement error, defined as the relative error, of all the segments were recorded . The correlations between the number of nonassessable segments, the relative error and the reconstruction algorithm, hr, luminal diameter, stenotic severity and observers were analyzed . At an hr of 40 and 50 bpm, the temporal window was 227 ms for both the halfscan and multisector reconstructions, so the fan angle in our study was about 53.5. with an increasing hr (60 - 120 bpm), the temporal window improved in the multisector reconstruction with the best temporal window being 127 ms, while the temporal window in the halfscan reconstruction remained unchanged at 227 ms (fig . In contrast, at hrs above 70 bpm (70 - 120 bpm), the multisector reconstruction achieved better image quality than the halfscan reconstruction did, except for a paradoxical reverse phenomenon at an hr of 90 bpm (fig . The halfscan reconstruction performed better than the multisector reconstruction despite of the tendency for a better performance of multisector reconstruction at higher hrs . Even so, at the intersection hr of 60 - 80 bpm, the two reconstruction algorithms provided equally good images . In summary, multisector reconstruction achieved better or equally good image quality at an hr higher than 60 bpm . The trend for an increasing number of nonassessable segments with an increasing hr was more obvious for the halfscan than for the multisector reconstruction algorithm . For the 324 segments, 79 (24.4%) halfscan segments were nonassessable and 49 (15.1%) multisector segments were nonassessable . This advantage of multisector reconstruction was conspicuous above an hr of 80 bpm (fig . The number of nonassessable segments was correlated with the reconstruction algorithm (p = 0.004) and hr (p <0.001), but not with the luminal diameter, the degree of stenosis and the observer . With excluding the nonassessable segments, we found no significant difference between the reconstruction algorithms for the accuracy of measuring the degree of stenosis (fig . We also used the relative error to analyze the accuracy of measuring the degree of stenosis according to the following equation: relative error (%) = (the measured degree of stenosis - the true degree of stenosis)100%/true degree of stenosis so less relative error represented better accuracy . When analyzing the accuracy of stenosis detection, also referred to as the relative error, both the assessable and nonassessable segments should be included . Because the relative error of the nonassessable segments is measureless, we have to make an assumption for the relative error of the nonassessable segments . The relative error of the nonassessable segments could be presumed to be a variable degree of error (100, 150 and 200%) because the relative error of the assessable segments varies from 0 to 196% and the relative error of the nonassessable segments should be presumed to be greater than that of the assessable ones . On the linear regression analysis, better accuracy of stenosis detection (a smaller relative error) was correlated with multisector reconstruction, slower hrs and a larger luminal diameter, and it was not correlated with the degree of stenosis and the observer (table 1). For example, at presumed 150% error of the nonassessable segment, better accuracy was correlated with multisector reconstruction (p = 0.005), slower hrs (p <0.001) and larger luminal diameter (p = 0.014), and it was not correlated with the degree of stenosis and the observer . Despite of the variable degree of the assumptive relative error of the nonassessable segments, the correlation remained the same . At hr of 40 - 70 bpm; both reconstruction algorithms achieved a similar relative error . However, multisector reconstruction achieved a better performance at an hr above 80 bpm, except at an hr of 90 bpm (fig . 7). The volume rendering images of the two reconstruction algorithms at different hrs showed the better performance of the multisector reconstruction algorithm at higher hrs (fig . There was less cardiac motion related to banding artifacts for the multisector reconstruction algorithm, and especially at higher hrs . At an hr of 40 and 50 bpm, the temporal window was 227 ms for both the halfscan and multisector reconstructions, so the fan angle in our study was about 53.5. with an increasing hr (60 - 120 bpm), the temporal window improved in the multisector reconstruction with the best temporal window being 127 ms, while the temporal window in the halfscan reconstruction remained unchanged at 227 ms (fig . In our study, a lower score equates with better image quality . At hrs below 60 bpm in contrast, at hrs above 70 bpm (70 - 120 bpm), the multisector reconstruction achieved better image quality than the halfscan reconstruction did, except for a paradoxical reverse phenomenon at an hr of 90 bpm (fig . The halfscan reconstruction performed better than the multisector reconstruction despite of the tendency for a better performance of multisector reconstruction at higher hrs . Even so, at the intersection hr of 60 - 80 bpm, the two reconstruction algorithms provided equally good images . In summary, multisector reconstruction achieved better or equally good image quality at an hr higher than 60 bpm . The number of nonassessable segments increased with an increasing hr for both algorithms . The trend for an increasing number of nonassessable segments with an increasing hr was more obvious for the halfscan than for the multisector reconstruction algorithm . For the 324 segments, 79 (24.4%) halfscan segments were nonassessable and 49 (15.1%) multisector segments were nonassessable . This advantage of multisector reconstruction was conspicuous above an hr of 80 bpm (fig . The number of nonassessable segments was correlated with the reconstruction algorithm (p = 0.004) and hr (p <0.001), but not with the luminal diameter, the degree of stenosis and the observer . With excluding the nonassessable segments, we found no significant difference between the reconstruction algorithms for the accuracy of measuring the degree of stenosis (fig . We also used the relative error to analyze the accuracy of measuring the degree of stenosis according to the following equation: relative error (%) = (the measured degree of stenosis - the true degree of stenosis)100%/true degree of stenosis so less relative error represented better accuracy . When analyzing the accuracy of stenosis detection, also referred to as the relative error, both the assessable and nonassessable segments should be included . Because the relative error of the nonassessable segments is measureless, we have to make an assumption for the relative error of the nonassessable segments . The relative error of the nonassessable segments could be presumed to be a variable degree of error (100, 150 and 200%) because the relative error of the assessable segments varies from 0 to 196% and the relative error of the nonassessable segments should be presumed to be greater than that of the assessable ones . On the linear regression analysis, better accuracy of stenosis detection (a smaller relative error) was correlated with multisector reconstruction, slower hrs and a larger luminal diameter, and it was not correlated with the degree of stenosis and the observer (table 1). For example, at presumed 150% error of the nonassessable segment, better accuracy was correlated with multisector reconstruction (p = 0.005), slower hrs (p <0.001) and larger luminal diameter (p = 0.014), and it was not correlated with the degree of stenosis and the observer . Despite of the variable degree of the assumptive relative error of the nonassessable segments, the correlation remained the same . At hr of 40 - 70 bpm; both reconstruction algorithms achieved a similar relative error . However, multisector reconstruction achieved a better performance at an hr above 80 bpm, except at an hr of 90 bpm (fig . 7). The volume rendering images of the two reconstruction algorithms at different hrs showed the better performance of the multisector reconstruction algorithm at higher hrs (fig . There was less cardiac motion related to banding artifacts for the multisector reconstruction algorithm, and especially at higher hrs . The temporal resolution of mdct with using the halfscan reconstruction algorithm has improved (6 - 9) and this permits reliable assessment of the main coronary branches in patients with hrs below 65 bpm (10, 11). However, the temporal resolution of the gated reconstruction images is of major concern when considering the heart because the motion of the coronary arteries can reach up to 69.5 mm / s during the cardiac cycle (10). So we confirmed that even for an hr below 65, the halfscan algorithm provided better image quality than the multisector reconstruction algorithm did . When hrs are faster, the temporal resolution of the halfscan reconstruction algorithm will be too long for a motion - free image . The multisector reconstruction algorithm retrospectively composes images from different cardiac cycles (two to five), which theoretically could significantly reduce the effective temporal resolution (12). Thus, marked improvement of the effective temporal resolution theoretically achieves better image quality at faster hrs . But the maximum benefit for temporal resolution occurs when the gantry rotation and cardiac motion are fully asynchronous, which depends on the relationship between the hr and the pitch (13 - 15), so the image quality of both reconstruction algorithms will degrade with heart rates higher than 90 bpm, as was our result . A successful multisector reconstruction also requires no misregistration due to arrhythmia or a changing hr (16). Coronary artery visualization and analysis requires high resolution . However, it cannot be expected that the coronary artery returns to exactly the same position from one cardiac cycle to the next . One disadvantage of multisector reconstruction is the nonconstant improvement of the temporal resolution because of synchronous gantry rotation and the cardiac cycle; this also explains the paradoxical reverse, at an hr of 90 bpm, of both the image quality (fig . Similar results were reported in a 32-patient study that found better image quality with an hr> 75 bpm than with an hr of 65 - 75 bpm (17), though the temporal resolution, another disadvantage of multisector reconstruction is the higher radiation exposure required at a lower pitch for obtaining high spatial resolution, and this radiation exposure is estimated to be 30% higher than that of the halfscan reconstruction (18). There might be a certain degree of ethical concern for the unnecessary radiation dose if a lower pitch is routinely used for low hr patients for both the halfscan and multisector reconstructions . Using ecg - pulsed tube current modulation should be considered to decrease the radiation dose (19). Two studies (4, 20) have reported a decreased image quality with using halfscan reconstruction in mdct units when the hr exceeds 65 bpm in phantoms and patients . One of these studies reported that an increase in image quality by multisector reconstruction when the hr exceeds 65 bpm (variable gantry rotational speed and variable pitch values) leads to a significant increase in imaging time with using 4-detector mdct (4). Another study reported better sensitivity, specificity, accuracy and fewer nonassessable segments with multisegment reconstruction versus halfscan reconstruction with using a 16-slice ct scanner in 34 patients with normal hrs (21). The same group latter reported better diagnostic accuracy and longer vessel lengths that were free of motion artifacts with multisector reconstruction in all hr groups (<65 bpm, 65 - 74 bpm and> 74 bpm) on 16-slice mdct in a study of 126 patients (22). In a 32-patient study (17), there was no significant improvement in image quality in any of the hr groups with employing dual - segment reconstruction versus halfscan reconstruction and using 64-detector mdct . They proposed that the temporal resolution (165 ms) using 64-detector mdct and halfscan reconstruction was similar to the temporal resolution (approximately 160 ms) using 16-detector mdct and multisector reconstruction (17). In another similar 40-patient study with hrs that ranged from 61 to 87 bpm (23), there was no significant difference of the single- and two - segment reconstruction algorithms in the number of visible segments and the quality scores . Better image quality was observed for two - segment reconstruction only at a certain hr range in that study . However, our phantom study on a 64-detector mdct demonstrated that multisector reconstruction achieved better or equally good image quality at an hr higher than 60 bpm (fig . 4) and better or equally good diagnostic accuracy for all the hr groups (fig . The diagnostic accuracy was significantly better for the multisector reconstruction than that for the halfscan reconstruction at hrs of 80, 100, 110 and 120 bpm . This differences could have resulted from the lack of inter - heart beat variability in our study or the limited number of patients with a high hr in their studies (17, 23). One advantage of our study was that the true degree of stenosis in the simulated coronary arteries was assured and this was also confirmed by static scanning . This method should be more accurate than 2-plane coronary angiography, and more objective than expensive intracoronary ultrasound (24). Another advantage was that the hrs in our study were well controlled by the pulsating cardiac phantom, and so we avoided the confounders of unexpected arrhythmia or changing hrs due to contrast medium injection and the vagal tone during breath - hold or that was due to anxiety . However, this was also a limitation, since it means our results cannot reflect the reality of clinical settings . Since our phantom had rigid vessel walls, it cannot be a good emulation of a real heart in terms of the rate of wall motion, as well as the actual distortion of the epicardial surface . In addition, a simulated coronary stenosis excludes artifacts from calcium plaque, making it less close to the real pathology seen in clinical settings . Finally, the diameters of our simulated coronary arteries (3 - 5 mm) do not fully represent the range of diameters of the true main coronary arteries (1.46 - 6.09 mm) (25), nor do they represent the scenario of eccentric stenoses . According to our study, multisector reconstruction achieved equally good or better diagnostic accuracy for all the hr groups and at least equally good image quality at an hr higher than 60 bpm . The application of multisector reconstruction for hrs between 60 and 80 bpm is acceptable, and the application of multisector reconstruction for the group with higher hrs would be beneficial . However, the reproducibility of our results should be confirmed in the setting of inter - heart beat variability . In conclusion, our study results showed that at higher hrs, multisector reconstruction in a 64-detector mdct achieved better temporal resolution, fewer nonassessable segments, better image quality, less cardiac motion related banding artifacts and less error in measuring the degree of stenosis.
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Most studies about laparoscopic skills acquisition are focused on the validation of simulators, i.e., devices that recreate operative conditions . However, other important issues concerning the learning process, like feedback, are seldom discussed . This often leads to the absence of validated feedback during training . In the historical apprenticeship system of see one, do one, teach one, feedback was directly provided during surgery in the operating room (or). Due to ethical and financial constraints and because of limitation of resident work hours, simulators were introduced in the last decade . This enables repeated skills training in a stress - free environment without causing harm to patients . Simulators can roughly be subdivided into video box trainers and computerized virtual reality (vr) trainers . Box trainers need the assistance of expert tutors during training sessions and courses to provide feedback . Experts can provide feedback on basic laparoscopic skills (bls) or can guide the trainee step by step through difficult tasks, e.g., intracorporeal knot tying, while giving tips and tricks . They can also increase motivation, which enhances the learning process, though objective assessment of the laparoscopic skills is not directly present in a traditional box trainer . On the other hand, vr trainers are programmed to give objective feedback about time, errors, and movement parameters . This implicates that several aspects of bls, for example the position of the hands and arms, will not be learned without the presence of a tutor . However, due to scarce time and relative high costs, the expert should be exploited optimally . Therefore, we conducted an observational study of the role of expert feedback in laparoscopic skills acquisition during a mandatory course in residency training . In 2007, an observational study was performed during a basic laparoscopic skills course in the netherlands . This course, named the cobra - alpha, is a basic laparoscopic skills course, which is mandatory for dutch obstetrics and gynecology (ob / gyn) residents since 1997, and should be attended in the first or second postgraduate year (pgy). The goal of this course is to train the participants in bls and provide knowledge necessary for continued training . In summary, the bls include camera navigation, body positioning, pointing and grasping tasks, and the use of the scissors . Although it is an advanced task, laparoscopic knot tying is also trained as it incorporates all other bls . For the laparoscopic skills training, five validated laparoscopic box trainer exercises were used . These exercises were placing a pipe cleaner through four small circles, placing beads in the form of a b on a board, stretching out a rubber band around 16 nails on a wooden board, cutting a circle from a rubber glove and intracorporeal knot tying . The level of performance on each task was established by adding the time to completion of a task with penalty points, consequently rewarding precision and speed . Additionally, the resulting score could be compared to a pre - established expert level, used as performance standard . Three hours of hands - on training started after a 10-min introduction video in which the exercises were explained . Consequently, one resident could hold the camera, while the other performed the exercises . Although not specifically trained to teach basic laparoscopic skills, they work as consultants in teaching hospitals and have at least 5 years of experience in these skills courses . They had been instructed to guide the participants through the exercises and to provide feedback and instructions on the bls . A questionnaire was developed by the authors on the role of expert feedback in laparoscopic skills acquisition . The questionnaire consisted of a part that was handed out prior to and a part immediately after the course . In the first part, residents characteristics were collected, as well as their self - perceived level of competence on their bls . The second part concerned the expert feedback and instructions; additionally, the self - perceived level of competence was asked again . As primary outcome measure, the expert feedback was studied . Participants were asked to which extent they had perceived feedback on 13 items (1 = no feedback, 2 = little feedback, or 3 = extensive feedback). For analysis, these items were categorized in five topics: body position, camera navigation, use of instruments, laparoscopic knot tying, and other tips and tricks . Furthermore, participants were asked what feedback they judged to be helpful in acquiring bls and on which topic they had desired more feedback (multiple options were possible). Finally, ideas or comment on the course could be noted . Secondarily, the self - perceived competence was determined before and after the course in order to obtain a measure of a participant s learning effect . The self - perceived level of competence had to be rated on a visual analog scale (vas). The vas ranked from unskilled to very skilled and was interpreted in the range 1 to 5 . For data analysis, a vas of 3 was chosen as cutoff point, and a score of 3 or higher was considered to be skilled . The self - perceived skills levels prior to and after the course were compared, using wilcoxon signed - rank test . The correlation coefficient between resident s experience (expressed in number of postgraduate years) and self - perceived skill level prior to the course was analyzed by a nonparametric spearman s rank correlation test . These exercises were placing a pipe cleaner through four small circles, placing beads in the form of a b on a board, stretching out a rubber band around 16 nails on a wooden board, cutting a circle from a rubber glove and intracorporeal knot tying . The level of performance on each task was established by adding the time to completion of a task with penalty points, consequently rewarding precision and speed . Additionally, the resulting score could be compared to a pre - established expert level, used as performance standard . Three hours of hands - on training started after a 10-min introduction video in which the exercises were explained . Consequently, one resident could hold the camera, while the other performed the exercises . Although not specifically trained to teach basic laparoscopic skills, they work as consultants in teaching hospitals and have at least 5 years of experience in these skills courses . They had been instructed to guide the participants through the exercises and to provide feedback and instructions on the bls . A questionnaire was developed by the authors on the role of expert feedback in laparoscopic skills acquisition . The questionnaire consisted of a part that was handed out prior to and a part immediately after the course . In the first part, residents characteristics were collected, as well as their self - perceived level of competence on their bls . The second part concerned the expert feedback and instructions; additionally, the self - perceived level of competence was asked again . As primary outcome measure, participants were asked to which extent they had perceived feedback on 13 items (1 = no feedback, 2 = little feedback, or 3 = extensive feedback). For analysis, these items were categorized in five topics: body position, camera navigation, use of instruments, laparoscopic knot tying, and other tips and tricks . Furthermore, participants were asked what feedback they judged to be helpful in acquiring bls and on which topic they had desired more feedback (multiple options were possible). Finally, ideas or comment on the course could be noted . Secondarily, the self - perceived competence was determined before and after the course in order to obtain a measure of a participant s learning effect . The self - perceived level of competence had to be rated on a visual analog scale (vas). The vas ranked from unskilled to very skilled and was interpreted in the range 1 to 5 . For data analysis, a vas of 3 was chosen as cutoff point, and a score of 3 or higher was considered to be skilled . The self - perceived skills levels prior to and after the course were compared, using wilcoxon signed - rank test . The correlation coefficient between resident s experience (expressed in number of postgraduate years) and self - perceived skill level prior to the course was analyzed by a nonparametric spearman s rank correlation test . Out of the 28 participants who attended the course, 24 (86%) completed the entire questionnaire . The remaining four participants had incompletely filled out the questionnaire and were excluded from further analysis . Residents ranged between their first and fifth pgy (median: pgy 3). Among the participants, 83% reported prior experience on a box trainer or vr trainer . The highest amount of experienced feedback was focused on laparoscopic knot tying and in some lesser extent on instrument handling (table 1). Additionally, participants appraised feedback on these two topics as most valuable . Less than 30% of the participants indicated to have received extensive feedback on the other topics . Least attention was paid to body positioning; 43% claimed to have received no feedback on that topic . In general, 66% of the participants thought that more feedback would have resulted in more improvement of their skills, except from feedback on camera navigation . Table 1amount of experienced feedbacktopicfeedback (%) nolittleextensivebody positioning433621camera navigation403228instrument handling313931intracorporeal knot tying114247other tips and skills462529 amount of experienced feedback prior to the course, the median self - perceived skills assessment varied between 1.2 and 3.4 (table 2). Participants already rated themselves to be skilled in camera navigation and pointing and grasping tasks prior to the course . Residents who were in an earlier phase of residency, expressed in smaller number of pgy, rated their skillfulness prior to the course lower than the ones who were more experienced (spearman s rank correlation test, = 0.19, p = 0.04). In general, the self - perceived competence improved significant for laparoscopic knot tying and the use of the scissors (wilcoxon signed - rank test, p <0.001). Table 2self - perceived skill improvementskillmedian vas scoredifference before and afterbeforeafterp valuebody positioning2.82.90.67 n.s.camera navigation3.43.20.18 n.spointing en grasping3.03.10.13 n.suse of the scissors2.73.1<0.001intracorporeal knot tying1.22.4<0.001vas: self - perceived level of competence, range 1 (unskilled) to 5 (very skilled)vas visual analog scale, n.s not significantwilcoxon s signed - rank test self - perceived skill improvement vas: self - perceived level of competence, range 1 (unskilled) to 5 (very skilled) vas visual analog scale, n.s not significant wilcoxon s signed - rank test the improvement in relationship to the skill level prior to the course is shown in fig . 1 . This figure shows that participants, who rated themselves not as skilled, improved more than the already skilled participants, except for body positioning . However, six of the eight residents that scored less than 3.0 prior to the course showed improvement (wilcoxon signed - rank test, p = 0.06). X - axis represents vas score of participant s self - perceived skills level prior to the course; y - axis represents the skills improvement (vas score after minus vas score before the course), n = 24 participants . Vas visual analog scale, in which 1 = very unskilled, 5 = very skilled skills improvement during a basic laparoscopic skills course . X - axis represents vas score of participant s self - perceived skills level prior to the course; y - axis represents the skills improvement (vas score after minus vas score before the course), n = 24 participants . Vas visual analog scale, in which 1 = very unskilled, 5 = very skilled this study revealed that residents judge feedback as a factor that positively influences their skills acquisition during a basic laparoscopic skills course . Alternatively, body positioning seems to have got the least feedback, and this skill did not show improvement . Admittedly, the self - perceived competence is not the most objective and accurate measure for assessment of skills level . However, in one study, residents showed to be capable to rate their own laparoscopic skills with good reliability and validity . Of course, no firm conclusions can be drawn from this observational study . However, a possible relation between feedback and learning can be hypothesized . This fits the results of the study of mahmood et al ., who showed that without feedback there can be no learning . Albeit, the objectives of a course need to be predefined in order to facilitate expert feedback on all training goals . Though it is worrisome that in the presence of six tutors, who were informed about the learning objectives, more than 70% of the participants denied to have received extensive feedback on some basic skills (e.g., camera navigation and body positioning), a possible explanation is that the more complex tasks distract the experts attention from the more basic tasks . Furthermore, the experts in the current study might be more eager to teach knot tying and cutting because they are more used to practicing and training these skills in clinical practice . This information may provide new leads for improving of the course . In the first place, tutors have to ascertain that their feedback has actually reached the trainee . Secondly, it may be helpful to make the learning objectives clear to the trainees as well, to enable them to ask for more specific feedback on their skills . The improvement of self - perceived competence due to the bls course was the highest for complex tasks, i.e., the use of scissors and intracorporeal knot tying, while residents indicated no or little improvement in the more basic skills . That reciprocates the finding that participants rated themselves better skilled in the basic than in the more complex skills . This phenomenon can be explained by the influence of feedback on a learning curve . In the beginning, the learning curve is steep and trainees experience most benefit from cognitive feedback . The cognitive feedback, which explains how and why something is performed as it should, can best be provided by experts . The positive effect that experts can generate in the start of the acquiring of motor skills was already revealed by rogers et al . . This group showed that novices have a significantly better performance on a knot tying task if expert feedback is added to computer - assisted learning . Particularly, in the beginning of the process of acquiring of new skills, training courses in presence of an expert are useful . Unfortunately, the residents who attended this course were not all in the beginning of residency . Most participants had already trained laparoscopic skills in dry laboratories or in the or during residency . Because participants obtain most benefit from the bls course in the beginning of their learning curve, residents should attend the training course early in residency . After progressing along a learning curve consequently, trainees are able to pay more attention to new elements, for example the anatomical structures during an operation . During this stage, learning relies more on intrinsic feedback, provided by sense organs and previous cognitive knowledge [3, 10]. Additionally, trainees have to become independent from external feedback . In this learning stage, feedback in the form of knowledge of results is important, instead of cognitive feedback . It can be argued whether basic skills like camera navigation, instrument handling, and body positioning require the same attention as more advanced skills like cutting and knot tying . In the aforementioned theory, every skill should be taught in an equivalent way, keeping the competence level of a trainee in mind to tailor the training to individual needs . Additionally, an expert can draw parallels to or practice and, last but not least, strongly enhances the motivation of a trainee . Further research needs to be done on the effect of expert feedback, in which more objective outcome measures might be used, though, with current knowledge, we can conclude that feedback plays an important role in skills acquisition and therefore the effectiveness of a training course . Training objectives need to be predefined and to be clear to both tutor and trainee, in order to guide and structure the feedback . In a bls course, most benefit can be obtained at the start of the learning curve.
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Clinically significant mrsa and mssa isolates, identified during january 2006march 2008, within 3 select communities (sites a site b also included 1 adjoining community, and sites a and b also included additional first nations reserves serviced by the community . A total of 1,280 isolates, obtained from skin and soft tissue infections (sstis), urinary tract infections, upper respiratory tract infections, and lower respiratory tract infections, were identified as s. aureus . A high proportion of these isolates, 692 (54.1%) of 1,280, were mrsa . Over the 2-year study period, rates of mrsa and mssa infections in the 3 communities ranged from 146482/10,000 and 112329/10,000 population, respectively . Trends of seasonality were apparent for mrsa infections, with the highest rates being observed in the third and fourth quarters of the year (figure 1). Overall, the highest quarterly rates of mrsa and mssa infections were observed at site c, with 738/10,000 and 610/10,000 population, respectively . Crude rates of community - acquired methicillin - resistant staphylococcus aureus (a) and methicillin - susceptible s. aureus (b) infections per 10,000 population in 3 select communities (sites a, b, and c) of northern saskatchewan, canada . The highest proportion of mrsa (30.4%) and mssa (32.1%) infections were identified in children <10 years of age (figure 2). Compared to mssa infections, mrsa infections were statistically more likely to be causing infections in persons <30 years of age (odds ratio [or] 1.46, 95% confidence interval [ci] 1.141.86, p = 0.002) and less likely to be causing infections in patients> 60 years of age (or 0.33, 95% ci 0.200.567), p<0.001) (figure 2). No significant difference was found in gender between those who acquired mrsa (46.7% male) and mssa (53.3% female, 49.4% male) infections . Age distribution of methicillin - resistant staphylococcus aureus (mrsa) and methicillin - susceptible s. aureus (mssa) infections in 3 select communities of northern saskatchewan, canada . Most mrsa (98.6%) and mssa (91%) isolates were obtained from sstis . Further analysis of sstis, comparing where on the body the infections were seen, showed significantly more mrsa infections in the axillae (or 3.04, 95% ci 1.396.89, p = 0.004), buttocks (or 2.1, 95% ci 1.273.49, p = 0.003), and trunk (or 2.25, 95% ci 1.543.31, p = <0.001) than mssa infections . Mrsa infections were significantly less likely to be found in feet (or 0.29, 95% ci 0.180.45, p<0.001), hands (or 0.45, 95% ci 0.30.68, p<0.001), and face or head (or 0.66, 95% ci 0.480.90, p = 0.009). Of the additional infection sites included in this study, mssa infections were statistically more likely to be identified in lower respiratory tract infections (or 5.6, 95% ci 1.524.62, p<0.05) and urinary tract infections (or 6.76, 95% ci 2.8716.71, p<0.001). A subset of 665 isolates were further characterized by antimicrobial drug susceptibility testing (table 1). In comparison to mssa, mrsa were significantly more likely to be susceptible to clindamycin, erythromycin, fusidic acid, and gentamicin, but were more likely to be resistant to mupirocin (table 1). In regards to the clindamycin - resistant isolates, 3 (18.8%) of the 16 mrsa isolates and 73 (93.6%) of the 78 mssa isolates all 328 of the mrsa isolates, but only 54 (70.1%) of the 77 mssa isolates, displayed high level resistance (> 128 g / ml). Mrsa, methicilllin - resistant staphylococcus aureus; mssa, methicillin - susceptible s. aureus; r, resistant; or, odds ratio; ci, confidence interval; mic50, 50% minimum inhibitory concentration; mic90, 90% inhibitory concentration; sxt, sulfamethoxazole / trimethoprim; ns, not significant . Pulsed - field gel electrophoresis (pfge) showed that most mrsa isolates (372/379, 98.2%) were usa400 . The remaining 7 mrsa isolates were identified as cmrsa10 (usa300, sequence type (st) 8) (n = 5), cmrsa2 (usa100/800, st5) (n = 1), and cmrsa8 (emrsa15, st22) (n = 1). As anticipated, pfge revealed much greater genetic diversity among the mssa strains circulating in these regions than in mrsa strains . Notably, however, most mssa pfge fingerprints (79.2%) were related to highly successful canadian epidemic mrsa strains, a finding that was further confirmed by using spa typing (5) (table 2). * mssa, methicilin - susceptible staphylococcus aureus; mrsa, methicillin - resistant s. aureus; pvl, panton - valentine leukocidin; pfge, pulsed - field gel electrophoresis; mlst, multilocus sequence typing; st, sequence type . Mrsa isolates were more likely to harbor the genes encoding panton - valentine leukocidin than were mssa isolates, 95.5% versus 5.2%, respectively . The pfge and spa types of the 15 panton - valentine leukocidin positive mssa isolates were associated with the ca - mrsa epidemic strain types usa400, usa300, and usa1000 (table 2). Rates of mssa and mrsa infections in these 3 northern saskatchewan communities (112482 cases/10,000 population) far exceed mrsa rates reported in the neighboring provinces of manitoba (16/10,000 population) (6) and alberta (10.7/10,000 population) (7), as well as benchmark hospital rates provided by the canadian nosocomial infection surveillance program (3.43 cases/10,000 patient days) (1). The high rates of s. aureus infections in remote northern saskatchewan communities has been attributed to a combination of risk factors, including overcrowding and poor housing conditions, inadequate hygiene, preexisting skin conditions, and previous high usage of antimicrobial drugs (8). Usa400 was by far the predominant strain type in all 3 communities, accounting for> 98% of the mrsa isolates . Usa400 was first reported in manitoba as an outbreak in the southern region in the late 1990s, but has since spread to the northern regions of the province from 2000 to 2004 (9). Usa400 was thereafter seen in a central eastern saskatchewan community adjacent to the manitoba border (2) and has since disseminated as far north as nunavut (10) and southwestern alaska (11). Because mrsa and mssa sstis tended to be identified more frequently from different body sites, it is appealing to speculate that ca - mrsa strains, such as usa400, might also colonize different body sites (e.g., axillae or intestines) more efficiently than other strains of s. aureus . This hypothesis coincides with a recent report in which nasal colonization was less likely in patients with ca - mrsa sstis than in those with hospital - acquired mrsa sstis (12). Intestinal carriage of s. aureus has been implicated as a risk factor for infection (13) and could be a strong contributor to environmental dissemination and transmission (14). This possibility was recently further supported by the results of a study in which the rectal carriage, but not nasal carriage, of usa300 was strongly associated with sstis in children (15). Further study is required to determine whether specific lineages of s. aureus are more proficient colonizers at non - nasal colonization sites, what host / bacteria genetic factors are involved, and whether this colonization plays a role in the high success of these ca - mrsa strain types . To address the high rates of s. aureus infections in northern saskatchewan, physician treatment algorithms and educational materials these materials are all freely available (www.narp.ca) and are intended to promote proper antimicrobial drug usage and hygiene to diminish the spread of s. aureus disease.
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A total of 2.78 million tickets were sold to the london paralympic games, making the paralympic games the world s third biggest sporting event, after the olympic games and the fifa world cup . The movement is genuinely global174 countries have national paralympic committees and participation is increasing, with> 6000 internationally registered athletes in the sport of athletics alone . Classification systems are integral to paralympic sport, being used to determine eligibility and control for the impact of eligible impairment types on the outcome of competition . Valid classification systems facilitate competition in which the athletes who succeed are not simply those who have less severe impairments than their competitors, but those who have the most favorable combination of athletic attributes and have enhanced them to best effect . Classification that is not valid or that is not perceived to be valid poses a significant threat to paralympic sport . At the elite level, the legitimacy of an individual s competitive success or athletic achievement can be significantly diminished by the perception that they are in the wrong class, with the potential for considerable personal and financial costs, as well as for discrediting the movement . At the grass - roots level, a classification system that is perceived to be unfair will discourage participation among people with disabilities, rather than achieve the goal of increasing it . Evidence - based decision making in classification is an essential means of enhancing classification validity, but evidence underpinning current methods of classification is weak . In 2007 the international paralympic committee (ipc) mandated the development of evidence - based methods of classification for all paralympic sports and the ipc position stand on classification outlines key requirements for the development of evidence - based systems, including valid, reliable methods for assessing impairment . Research in this area is limited; however, researchers are beginning to investigate the methods that may contribute to evidence - based classification in paralympic sport . Impairments classified in paralympic sport include impaired muscle strength, impaired range of movement, and limb deficiency . The focus of this article is impaired muscle strength, which is required in 16 of the 23 summer paralympic sports, and is a key component of classification in athletics (ie, track and field), the largest paralympic sport . Methods for assessing strength have remained essentially unchanged since the first classification system was described by sir ludwig guttman . They are based on manual muscle testing (mmt) methods in which the strength of individual muscle actions (eg, elbow flexion, knee extension) are assigned a grade from 0 (no voluntary muscle contraction) to 5 (normal strength through normal anatomical range of movement) according to their capacity to overcome gravity and/or manual resistance . For the purposes of classification, mmt has a number of advantages: it is widely understood and utilized in clinical practice and, because it does not require any instrumentation, it is inexpensive and space - efficient . One final advantage of using mmt for classification is that the ability to overcome manual resistance is assessed using the break test, an isometric contraction against manual resistance . Isometric assessments are advantageous for classification purposes because they are known to be relatively unresponsive to the high - speed, dynamic strength training regimes required for performance enhancement in athletics . As a consequence, athletes who have optimized sports performance through high - speed, dynamic resistance training are less likely to change their classification strength measures and be placed into a class for athletes with less severe impairments . Unfortunately mmt also has several important disadvantages that make it unsuitable as a method of strength assessment for classification . First, acceptable interrater reliability is difficult to achieve, a problem exacerbated by the wide range of mmt techniques that are described in published literature and used by classifiers from different countries in their clinical practice . For example, an athlete with full passive range of motion but only 15 degrees of active elbow extension against gravity is likely to experience much more activity limitation in the shot put than an athlete with 100 degrees active range, and yet the correct muscle grade for both actions is 2 . Ordinal measurement scales are unsuitable for research that aims to develop evidence - based methods of classification, as mandated by the international paralympic committee . Specifically, evidence - based methods of classification require quantification of the relative importance of different muscle actions in a given sporting movement, and therefore a ratio - scale measure of strength is necessary . When the relative importance of key muscle actions has been quantified using a ratio - scale measure, it will be possible to validly aggregate strength measures of contributing muscle groups in order to obtain an evidence - based estimate of how much activity limitation different strength impairments will cause, regardless of their distribution and severity . In order to permit the development of evidence - based methods of classification, our research group developed a battery of novel strength tests . Key features of the battery were that measures were isometric and therefore, according to theory, less training responsive than other dynamic strength tests; instrumented, yielding an outcome measure in newtons (a ratio scale); comprehensive, assessing all muscle actions of importance in the key disciplines of paralympic athletics (wheelchair racing, running, throwing, and jumping); and parsimonious, by assessing compound (or multijoint actions) and thereby minimizing the number of tests required and ensuring that individual tests accounted for the greatest possible variance in performance . The aims of this study were 3-fold: to establish normal performance ranges for each of the novel tests in nondisabled participants, to evaluate the reliability of each of the novel tests, and to assess the strength of association between individual test outcomes and body mass . In relation to the final aim, a sufficiently strong relationship with body size would indicate that, prior to applying these methods in classification, it may be necessary to develop body - size scaling methods that can be validly applied to measures obtained from athletes with neuromusculoskeletal impairments . Participants were 118 nondisabled, males (n = 63) and females (n = 55), ages 18 to 37 (mean sd 23.2 3.7) recruited from the university of queensland and local sports clubs . All were regularly active in competitive sport or engaged in 3 or more vigorous training sessions per week . The study was approved by the ethics committee of the school of human movement studies, university of queensland (number hms07/0406) and all participants provided written informed consent prior to participation . Participants completed testing in a single session, with the exception of 17 who returned in a minimum of 2 days and a maximum of 14 days from the initial session, so that test the sample size calculation was based on the requirement for calculation of allometric scaling exponents and a minimum number of 54 participants were required in each group (males and females) at an effect size of 0.15 with power set at 0.8 and probability at 0.05 . All participants completed a battery of 7 isometric strength tests grip strength and 6 novel tests which are presented in table 1 . The battery aimed to be both parsimonious and comprehensive, using the smallest possible number of tests to evaluate the strength of those movements considered most important in the key athletic disciplines (ie, running, jumping, throwing, and wheelchair propulsion). Strength and body size test protocols grip strength was assessed using a handheld dynamometer (smedlay s dynamometer, fabrication enterprises, white plains, ny, usa, 100 kg), with values entered directly to excel 2007 spreadsheet (microsoft, redmond, wa, usa) and then converted to newtons (n). The 6 novel tests were completed with the participant seated in a customized strength rig (figure 1). The rig comprised a rigid, aluminum rectangular frame with an s - type load cell (scale components, slacks creek, qld, australia) rated to 394 kg (1000 lb) mounted at one end, opposite a rigid seat . An aluminium plate (250 mm 196 mm 12 mm) was secured to the load cell and, once seated, participants applied force to the load cell from a seated position by either pushing or pulling on the plate . Three features of the rig permitted positioning of participants so that force was applied to the load cell from anatomically standardized positions: the load cell was adjustable vertically and horizontally to account for individual differences in sitting height and breadth (see figure 1, panel b); the seat position was adjustable in the fore - aft direction to account for individual differences in arm and leg length (see figure 1, panel a); backrest height was adjustable to permit positioning at the c7 vertebra regardless of the participant s sitting height . The arrow indicates the ability of the chair to move in the fore - aft direction . Panel b shows a magnified view of the load cell set up with arrows indicating the ability to move the load cell vertically and horizontally . The joint angles selected for each testing position aimed to position prime movers so that, as far as possible, length / tension relationships were optimized . To achieve the precise leg angles described in table 1, an anthropometric marking pen was used to clearly identify the following landmarks: acromion, greater trochanter, mid - point of lateral knee joint line, and the lateral malleolus . A sony camera, positioned 1.5 m from the rig, in line with the mid - point of the seat surface at 1 m high, fed a live, sagittal view video image of the participant seated in the strength rig into a personal computer, on which dartfish (version 4.0.9.0; dartfish, lausanne, switzerland) was installed . The dartfish angle tool was used to draw the required joint angles and the strength rig could then be adjusted until the participant was positioned so that their video image was matched with image created by the angle tool . This method could not be used for the arm angles which are only observable from an overhead view, and these angles were set using a handheld goniometer (sunshine diagnostic and measuring instruments, new taipei city, taiwan) and monitored visually by a member of the testing team . Figure 2 shows athletes positioned for an upper body strength measure (panel a) and a lower body strength measure (panel b). Panel a illustrates the position that participants were placed into for the bilateral supported arm push . Panel b illustrates the position that participants were placed into for the leg extensor strength test . Once positioned, participants performed 3 maximal isometric contractions of 5 seconds duration, each separated by 30 seconds of rest . Valid trials required peak force to be achieved slowly (> 2 seconds and <3 seconds) followed by a 3-second hold at peak . To assist participants achieve peak force slowly, 2 submaximal practice trials were performed with real - time visual feedback of the force time curve so that participants could easily identify when maximum force was achieved either too quickly or too slowly . Participants applied force with their hand(s) for upper limb tests and with their foot / feet for lower limb tests, with the rigid backrest permitting exertion of maximal voluntary force . Hands / feet were positioned so that force was directed through the long axis of the load cell and torque was minimized . Load cell output was captured by musclelab v4020e (ergotest, porsgunn, norway) at a sampling rate of 100 hz . Raw isometric strength scores were exported directly into an excel 2007 spreadsheet from the load cell data acquisition program (musclelab), and all trials were then processed in scilab using a custom - written algorithm (scilab enterprises, versailles, france) to acquire the peak isometric force from each trial . For each trial, the isometric force used for further analysis was calculated as the mean force over the 2-second period with the least variability . To ensure a plateau was achieved, a trial was deemed acceptable if the calculated force was a minimum of 95% of the peak force registered during the trial . Body mass was measured and recorded on each participant to the nearest 0.1 kg using scales (seca 760 mechanical scales, seca, hamburg, germany). All data were analyzed using spss v16 (spss inc, chicago, il). Independent t - tests were performed on the strength data of males and females to determine whether strength tests were significantly affected by sex and should be analyzed separately . Test retest reliability was assessed using dependant t - tests, 2-tailed intraclass correlations (icc) method 3,1, standard error of the mean (sem) and bland the relationship between body mass and each of the strength measures was determined by calculating pearson correlations and allometric scaling exponents . To calculate the latter, a log - linear regression analysis was conducted using strength as the dependent variable and body mass as the independent variable . The slope of the regression line was used as the allometric scaling exponent, and residual plots were assessed to check the fit of each model . Exponents were calculated on the dominant side for the upper body and the right side for the lower body . Participants were 118 nondisabled, males (n = 63) and females (n = 55), ages 18 to 37 (mean sd 23.2 3.7) recruited from the university of queensland and local sports clubs . All were regularly active in competitive sport or engaged in 3 or more vigorous training sessions per week . The study was approved by the ethics committee of the school of human movement studies, university of queensland (number hms07/0406) and all participants provided written informed consent prior to participation . Participants completed testing in a single session, with the exception of 17 who returned in a minimum of 2 days and a maximum of 14 days from the initial session, so that test the sample size calculation was based on the requirement for calculation of allometric scaling exponents and a minimum number of 54 participants were required in each group (males and females) at an effect size of 0.15 with power set at 0.8 and probability at 0.05 . All participants completed a battery of 7 isometric strength tests grip strength and 6 novel tests which are presented in table 1 . The battery aimed to be both parsimonious and comprehensive, using the smallest possible number of tests to evaluate the strength of those movements considered most important in the key athletic disciplines (ie, running, jumping, throwing, and wheelchair propulsion). Strength and body size test protocols grip strength was assessed using a handheld dynamometer (smedlay s dynamometer, fabrication enterprises, white plains, ny, usa, 100 kg), with values entered directly to excel 2007 spreadsheet (microsoft, redmond, wa, usa) and then converted to newtons (n). The 6 novel tests were completed with the participant seated in a customized strength rig (figure 1). The rig comprised a rigid, aluminum rectangular frame with an s - type load cell (scale components, slacks creek, qld, australia) rated to 394 kg (1000 lb) mounted at one end, opposite a rigid seat . An aluminium plate (250 mm 196 mm 12 mm) was secured to the load cell and, once seated, participants applied force to the load cell from a seated position by either pushing or pulling on the plate . Three features of the rig permitted positioning of participants so that force was applied to the load cell from anatomically standardized positions: the load cell was adjustable vertically and horizontally to account for individual differences in sitting height and breadth (see figure 1, panel b); the seat position was adjustable in the fore - aft direction to account for individual differences in arm and leg length (see figure 1, panel a); backrest height was adjustable to permit positioning at the c7 vertebra regardless of the participant s sitting height . The arrow indicates the ability of the chair to move in the fore - aft direction . Panel b shows a magnified view of the load cell set up with arrows indicating the ability to move the load cell vertically and horizontally . The joint angles selected for each testing position aimed to position prime movers so that, as far as possible, length / tension relationships were optimized . To achieve the precise leg angles described in table 1, an anthropometric marking pen was used to clearly identify the following landmarks: acromion, greater trochanter, mid - point of lateral knee joint line, and the lateral malleolus . A sony camera, positioned 1.5 m from the rig, in line with the mid - point of the seat surface at 1 m high, fed a live, sagittal view video image of the participant seated in the strength rig into a personal computer, on which dartfish (version 4.0.9.0; dartfish, lausanne, switzerland) was installed . The dartfish angle tool was used to draw the required joint angles and the strength rig could then be adjusted until the participant was positioned so that their video image was matched with image created by the angle tool . This method could not be used for the arm angles which are only observable from an overhead view, and these angles were set using a handheld goniometer (sunshine diagnostic and measuring instruments, new taipei city, taiwan) and monitored visually by a member of the testing team . Figure 2 shows athletes positioned for an upper body strength measure (panel a) and a lower body strength measure (panel b). Panel a illustrates the position that participants were placed into for the bilateral supported arm push . Panel b illustrates the position that participants were placed into for the leg extensor strength test . Once positioned, participants performed 3 maximal isometric contractions of 5 seconds duration, each separated by 30 seconds of rest . Valid trials required peak force to be achieved slowly (> 2 seconds and <3 seconds) followed by a 3-second hold at peak . To assist participants achieve peak force slowly, 2 submaximal practice trials were performed with real - time visual feedback of the force time curve so that participants could easily identify when maximum force was achieved either too quickly or too slowly . Participants applied force with their hand(s) for upper limb tests and with their foot / feet for lower limb tests, with the rigid backrest permitting exertion of maximal voluntary force . Hands / feet were positioned so that force was directed through the long axis of the load cell and torque was minimized . Load cell output was captured by musclelab v4020e (ergotest, porsgunn, norway) at a sampling rate of 100 hz . Raw isometric strength scores were exported directly into an excel 2007 spreadsheet from the load cell data acquisition program (musclelab), and all trials were then processed in scilab using a custom - written algorithm (scilab enterprises, versailles, france) to acquire the peak isometric force from each trial . For each trial, the isometric force used for further analysis was calculated as the mean force over the 2-second period with the least variability . To ensure a plateau was achieved, a trial was deemed acceptable if the calculated force was a minimum of 95% of the peak force registered during the trial . The best trial was then employed for statistical analysis . Body mass was measured and recorded on each participant to the nearest 0.1 kg using scales (seca 760 mechanical scales, seca, hamburg, germany). All data were analyzed using spss v16 (spss inc, chicago, il). Independent t - tests were performed on the strength data of males and females to determine whether strength tests were significantly affected by sex and should be analyzed separately . Test retest reliability was assessed using dependant t - tests, 2-tailed intraclass correlations (icc) method 3,1, standard error of the mean (sem) and bland the relationship between body mass and each of the strength measures was determined by calculating pearson correlations and allometric scaling exponents . To calculate the latter, a log - linear regression analysis was conducted using strength as the dependent variable and body mass as the independent variable . The slope of the regression line was used as the allometric scaling exponent, and residual plots were assessed to check the fit of each model . Exponents were calculated on the dominant side for the upper body and the right side for the lower body . Independent t - tests showed significant differences between males and females in all strength measures, and therefore these data were analyzed separately . Overall, both male and female participants were symmetrical, with no significant difference in mean force production between dominant and nondominant arms, left and right leg flexors, and left and right leg extensors . In the lower limbs, extensor strength (male mean right leg: 1822.4n 454.6n; female mean right leg: 1193.5n 302.1n) was approximately 5 times greater than flexor strength (male mean right leg: 352.9n 78.3n, female mean right leg: 237.4n 53.6n). Descriptive statistics for body mass and strength measure the results of analyses for test retest reliability are presented in table 3 . The mean difference between the test and retest was consistently low for all tests absolute difference range was 3.7 to 51.0 n, a relative difference of 0.2% to 7.3% . Retest reliability of novel strength measures (n = 17) pearson correlations showing the relationships between individual strength tests and body mass are presented in table 4 for males, females, and the total sample . Correlations were moderate and significant for 6 of the 7 tests in males (r = 0.430.61, p = 0.000) and 2 of the 7 tests in females (r = 0.300.33, p = 0.0140.03). Pearson correlations and allometric scaling for body mass and strength measures allometric exponents were calculated separately for males and females; exponents were higher for males (range = 0.581.27) than for females (range = 0.060.67). Currently, the method of strength assessment used in paralympic athletics classification is based on mmt, an ordinal - scale measure . This is problematic because ordinal - scale measures do not permit quantitative evaluation of the impact of impairment on athletic performance, a fundamental requirement for the development of evidence - based methods of classification . The results from this study indicate that the ratio - scale strength assessment battery described could be validly applied to address this important methodological shortcoming and facilitate the development of evidence - based methods of classification . In addition to furnishing a ratio - scale measure of strength, the battery has a number of features that would be advantageous for paralympic classification, including the measures are isometric and therefore training resistant; the battery is both comprehensive (assessing resultant forces produced by the muscle groups of principal importance in paralympic athletics) and parsimonious; and reliability is good to excellent . Furthermore, the sex - specific normal performance ranges that are reported will permit meaningful interpretation of results in athletes with impairments in future studies . The key advantages of the battery evaluated in this study are expanded in the following paragraphs . The following example illustrates why ratio - scale measurement is of fundamental importance to the development of evidence - based methods of classification . In wheelchair racing, the current class t51 is based on the strength impairment profile of a person with a complete spinal cord injury at neurological level c56, including impaired shoulder flexion strength (up to grade 4) and triceps strength grade 03 . The current class t52 profile is based on the strength impairment profile of a person with complete spinal cord injury at neurological level c78, including normal shoulder flexion and triceps strength (ie, grade 5). If an athlete with polio presents with normal shoulder flexion strength but grade 3 triceps, the classifier must decide whether the athlete is best in class t51 (even though they will have more shoulder flexion strength than other athletes in that class) or in class t52 (even though they will have less triceps strength than other athletes in class). In this instance, decision - making should be based on evidence regarding the relative importance of shoulder flexion and triceps extension to wheelchair propulsion . Current, ordinal - scale methods of strength assessment are not suitable for investigating this question . Two additional advantages conferred by the proposed test battery are that it is both parsimonious and comprehensive . These advantages occur because, rather than assessing individual muscle actions acting over a single joint, a number of the proposed tests assess the resultant force of a number of key muscle groups acting over more than one joint . For example, the leg extension test assesses the combined strength of hip and knee extensors simultaneously, while the single supported arm push assesses shoulder horizontal flexion and elbow extension simultaneously . Consequently, the test battery described in this study comprises 7 tests, rather than the 20 individual muscle grade tests required to assess the same muscle groups in the current system . In addition to saving time, the proposed test battery would considerably reduce the number of maximum contractions required from each athlete during classification, helping to ensure that fatigue did not the confound outcomes . Importantly, the significantly reduced number of tests does not result in a less comprehensive battery all the principal muscle actions required for the activities that are central in paralympic athletics are evaluated (ie, wheelchair racing, running, jumping, and throws both standing and seated). More specifically, the tests capture muscle synergies that are required for performance of the activities of interest . For example, the push pull test for the upper body uses the same prime movers as used for seated throwing with a pole: shoulder extension and elbow flexion of the nonthrowing arm simultaneously assessed at the same time as shoulder flexion and elbow extension on the throwing or dominant arm . The sex - specific isometric strength ranges reported in this paper on nondisabled individuals will allow the interpretation of results obtained from athletes with impairments . In general, the overall pattern of results was consistent with what is known about strength males were significantly stronger than females on all strength tests; the lower body was stronger than the upper body and the lower limb extensor strength was greater than the flexor strength . More specifically, grip strength means (males = 526 n; females = 344 n) were comparable with means previously reported for this protocol (males = 523 n; females = 319 n) and the mean bilateral supported arm push for males (n) was similar to that reported by hortobagyi (9976 n) for a similar protocol performed in supine . However, the battery also extends what is known about the relative strength of different movements for example, combined hip and knee flexion was only 19.4% of extension in males and 19.9% in females . These percentages are considerably less than those reported in studies of isolated lower limb movements, which indicate that knee flexion strength is between 43% and 90% of knee extension strength . One final feature of the test battery described in this study which is important for the purposes of classification is that the reliability of all strength tests was excellent (icc> 0.8) for all but one test . The icc for the leg flexor test was good with an icc of 0.71, which is acceptable given the low sem (314 n) and the small mean difference between test one and test 2 (66 n). As mentioned in the introduction, one advantage that current methods of strength assessment have this is an important feature because classification aims to control for the impact of strength impairment on athletic performance without controlling for other advantages conferred by body size . For example, 2 throwers with complete spinal cord injuries at t2 should compete in the same class for the discus, regardless of whether one is 2 m tall and weighs 100 kg and the other is 1.5 m tall and 65 kg . It will be important that any new measure of strength used for the purposes of classification is, as far as possible, independent of body size . Results from this study indicate that a number of measures1 in males (leg flexor strength) and 5 measures in females (single supported arm push, bilateral supported arm push, unsupported push pull, leg flexor and plantar flexor strength)were not strongly or significantly related to body mass . This indicates that it is likely that these measures are sufficiently independent of body size to be validly applied in classification . However, 6 tests in males were moderately and significantly related to body mass (r = 0.430.61) and 2 tests in females showed a weaker but still significant relationship (0.300.33). Allometric exponents were also calculated to give an indication of the slope of the regression line as this would indicate how much change in strength would be expected for a given change in body mass . The allometric exponents that were calculated were much larger for males (mean of 1.06) than for females (mean of 0.3). This indicates that a given change in body size is associated with a larger change in strength in males than in females . It has been suggested by previous research that this may be due to a decrease in range of strength scores seen when males and females are analyzed separately, or a result of higher relative percentages of lean body mass in males when compared with females . These results indicate that further research is required to determine whether raw measures require normalization in order to be validly applied in classification . Unfortunately the scaling exponents developed in this study on nondisabled participants based on body mass will not be able to be applied directly to athletes with disabilities . Body measurements such as body mass, height, or limb circumference are inappropriate in athletes with disabilities as neuromusculoskeletal impairment changes the relationship between these measures of body size and strength in ways that are unpredictable . This is because impairments to the central and peripheral nervous system and to the muscle fiber itself, which commonly affect paralympic athletes, will disrupt the fundamental premise for scaling that force is primarily determined by muscle cross - sectional area . For example, people affected by spastic paraplegia resulting from umn injury will retain muscle bulk better than those affected by lower motor neuron injuries due to the presence of intact spinal - level reflexes, although the impairment of structures may result in comparable impairments of strength function . It is posited that local bony dimensions for example, humerus length or biacromial width may be the most appropriate type of anthropometric measure by which to scale strength measures in athletes with neuromusculoskeletal impairment because, compared to body mass or muscle cross - sectional area, they more commonly remain unaffected by neuromusculoskeletal impairments . Research is required that identifies the most appropriate local bony dimensions by which to scale and subsequently evaluates whether normalized or raw scores are more valid for classification purposes . Ordinal - scale strength assessment methods are currently used in paralympic athletics classification, preventing the development of evidence - based classification systems . The results from this study indicate that the ratio - scale strength assessment battery described could be validly applied to address this important methodological shortcoming and facilitate the development of evidence - based methods of classification by allowing a valid and reliable assessment of muscle strength in athletes with neuromusculoskeletal impairments . The battery has a number of other features that are advantageous for classification, including the measures are isometric and therefore training resistant; the battery is both comprehensive (assessing resultant forces produced by the key muscle groups) and parsimonious; and reliability is good to excellent . Furthermore, the sex - specific normal performance ranges that are reported will permit meaningful interpretation of results in athletes with impairments in future studies . The results from this study have implications for the 16 paralympic sports that assess strength in the classification process.
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The incidence of esophageal cancer has increased in recent years, reflecting a shift in histologic type and primary tumor location in the west . Although adenocarcinoma of the esophagus is more prevalent in western countries, squamous cell carcinoma is still the most prominent histologic type worldwide . Esophageal cancer is a highly aggressive disease with a 3-year survival rate of less than 1% . Approximately 50% of patients have distant metastasis at the time of diagnosis, and of the remaining patients who initially present with only locoregional disease, 25% develop distant metastases . Compared to other solid tumors, randomized phase iii trials to test the efficacy of cytotoxic chemotherapy are lacking for metastatic esophageal squamous cell carcinoma (mescc). One randomized phase ii study published in europe more than 20 years ago showed that a combination of cisplatin / fluorouracil (5-fu) (fp) was superior to cisplatin alone (response rate, 35% vs. 19%, respectively), although no clear improvement in overall survival (os) was observed (33 weeks vs. 28 weeks, respectively). Hematological and non - hematological toxicities were more prominent in the combination treatment arm, with aplasia and septicemia being the most frequent toxicities observed . Treatment - related deaths were observed in seven patients (16%) in the combination treatment arm, whereas no treatment - related deaths were observed in the cisplatin alone treatment arm . In contrast, another phase ii study investigating capecitabine / cisplatin combination therapy in a different ethnic population showed promising anti - tumor activity, with a 57.8% response rate and more tolerable toxicity levels . The combination of cisplatin and 5-fu is the most commonly used regimen as first - line chemotherapy for mescc . However, the differences in treatment responses and toxicity levels in these two different clinical trial study populations highlight the difficulty in determining the most effective treatment regimens for mescc patients . These studies further illustrate the importance of identifying patients who would benefit from cytotoxic chemotherapy . Here, we developed and validated a prognostic nomogram to predict survival that will aid physicians and patients in making an appropriate clinical decision regarding treatment . This study was approved by the institutional review board (irb) of the samsung medical center, seoul, south korea and is registered on clinicaltrials.gov (nct#nct 01472419). The informed consent form was waived by the irb . Between january 2000 to december 2010, 239 patients were diagnosed with recurrent mescc and received either fp or capecitabine / cisplatin (xp) as first - line chemotherapy . The following clinicopathologic variables and treatment outcomes were collected: patient demographics, laboratory data at the time of first - line palliative chemotherapy, surgical record, pathologic report, tnm stage, treatment record, and vital status . Fp and xp regimens used to treat the patients are described below . In the fp group, 5-fu and 5-fu (1,000 mg / m) was infused over 24 hours during the four days . Cisplatin was given in one dose of 60 mg / m at the beginning of each cycle . In the xp group, capecitabine (1,250 mg / m bid) was administered for 14 days and cisplatin was given in one dose of 60 mg / m at the beginning of each cycle . The clinical tumor response was assessed according to world health organization (who) criteria or response evaluation criteria in solid tumors criteria (recist 1.0). As an external validation data set, 61 patients with mescc who received palliative fluoropyrimidine / platinum chemotherapy (xp or fp) at northern france cancer center were included . The primary endpoints of the study were os and progression - free survival (pfs). Os was measured from the date of first chemotherapy to the date of death or the last follow - up visit, and pfs was calculated from the date of first chemotherapy to the date of progression . A prognostic model was established by identifying all variables that significantly influenced os or pfs at a p - value less than or equal to 0.05 in univariate analysis . Factors included in the multivariate analyses were the following: age, sex, performance status, previous esophagectomy, weight loss (10% of weight lost for 3 months) at the time of chemotherapy, stent or percutaneous endoscopic gastrostomy, tracheoesophageal fistula, primary tumor location, histologic tumor grade, and metastatic site (lung, liver, bone, and adrenal gland). In addition, laboratory findings at the time of chemotherapy were included as categorical variables based on the normal ranges and consisted of the following: hemoglobin (> 11.2 g / dl vs. 11.2 g / dl), white blood cell count (> 8,630/l vs. 8,630/l), platelet count (> 13810/l vs. 13810/l), serum albumin (> 3.5 g / dl vs. 3.5 g / dl), total bilirubin (> 1.5 mg / dl vs. 1.5 mg / dl), aspartate aminotransferase (> 40 u / l vs. 40 u / l), and serum creatinine (> 1.1 mg / dl vs. 1.1 mg / dl). A nomogram was developed as a prognostic scoring system incorporating significant clinical and laboratory variables based on a multivariate cox proportional hazards regression model . For example, the predicted 12-month os hazard rate was calculated as follows: 12 months os hazard rate=0.06501exp (0.6755ecog 0 - 1 [if the ecog grade was 0 - 1, the score could be 1, if not, the score is 0])+1.0682ecog 2 (if the ecog grade1 was 2, the score could be 1, if not, the score is 0)+0.3784weight loss (absence of weight loss=0, presence of weight loss=1)-0.6562albumin (if serum albumin> 3.5 g / dl, the score is 1, if serum albumin 3.5 g / dl, the score is 0)-0.2848previous esophagectomy (absence=0, presence=1). The nomogram was then constructed using coefficients from the cox proportional hazard model for convenience . On the basis of the nomogram, patients were categorized into three groups according to their risk score percentile: favorable (0 - 50 percentile), intermediate (50 - 75 percentile), and poor (75 - 100 percentile) risk score . Discrimination was examined using the area under the receiver operating characteristic (roc) curve (auc). For internal validation, random sampling was repeated with replacement using the bootstrapping method over 1,000 times, and a new bias - corrected 95% confidence interval (ci) for auc was calculated using r 2.13.2 (r foundation for statistical computing, vienna, austria; isbn 3 - 900051 - 07 - 0; http://www.r-project.org). This study was approved by the institutional review board (irb) of the samsung medical center, seoul, south korea and is registered on clinicaltrials.gov (nct#nct 01472419). The informed consent form was waived by the irb . Between january 2000 to december 2010, 239 patients were diagnosed with recurrent mescc and received either fp or capecitabine / cisplatin (xp) as first - line chemotherapy . The following clinicopathologic variables and treatment outcomes were collected: patient demographics, laboratory data at the time of first - line palliative chemotherapy, surgical record, pathologic report, tnm stage, treatment record, and vital status . Fp and xp regimens used to treat the patients are described below . In the fp group, 5-fu and 5-fu (1,000 mg / m) was infused over 24 hours during the four days . Cisplatin was given in one dose of 60 mg / m at the beginning of each cycle . In the xp group, capecitabine (1,250 mg / m bid) was administered for 14 days and cisplatin was given in one dose of 60 mg / m at the beginning of each cycle . The clinical tumor response was assessed according to world health organization (who) criteria or response evaluation criteria in solid tumors criteria (recist 1.0). As an external validation data set, 61 patients with mescc who received palliative fluoropyrimidine / platinum chemotherapy (xp or fp) at northern france cancer center were included . The primary endpoints of the study were os and progression - free survival (pfs). Os was measured from the date of first chemotherapy to the date of death or the last follow - up visit, and pfs was calculated from the date of first chemotherapy to the date of progression . Os and pfs were estimated using the kaplan - meier product - limit method . A prognostic model was established by identifying all variables that significantly influenced os or pfs at a p - value less than or equal to 0.05 in univariate analysis . Factors included in the multivariate analyses were the following: age, sex, performance status, previous esophagectomy, weight loss (10% of weight lost for 3 months) at the time of chemotherapy, stent or percutaneous endoscopic gastrostomy, tracheoesophageal fistula, primary tumor location, histologic tumor grade, and metastatic site (lung, liver, bone, and adrenal gland). In addition, laboratory findings at the time of chemotherapy were included as categorical variables based on the normal ranges and consisted of the following: hemoglobin (> 11.2 g / dl vs. 11.2 g / dl), white blood cell count (> 8,630/l vs. 8,630/l), platelet count (> 13810/l vs. 13810/l), serum albumin (> 3.5 g / dl vs. 3.5 g / dl), total bilirubin (> 1.5 mg / dl vs. 1.5 mg / dl), aspartate aminotransferase (> 40 u / l), and serum creatinine (> 1.1 mg / dl vs. 1.1 mg / dl). A nomogram was developed as a prognostic scoring system incorporating significant clinical and laboratory variables based on a multivariate cox proportional hazards regression model . For example, the predicted 12-month os hazard rate was calculated as follows: 12 months os hazard rate=0.06501exp (0.6755ecog 0 - 1 [if the ecog grade was 0 - 1, the score could be 1, if not, the score is 0])+1.0682ecog 2 (if the ecog grade1 was 2, the score could be 1, if not, the score is 0)+0.3784weight loss (absence of weight loss=0, presence of weight loss=1)-0.6562albumin (if serum albumin> 3.5 g / dl, the score is 1, if serum albumin 3.5 g / dl, the score is 0)-0.2848previous esophagectomy (absence=0, presence=1). The nomogram was then constructed using coefficients from the cox proportional hazard model for convenience . On the basis of the nomogram, patients were categorized into three groups according to their risk score percentile: favorable (0 - 50 percentile), intermediate (50 - 75 percentile), and poor (75 - 100 percentile) risk score . Discrimination was examined using the area under the receiver operating characteristic (roc) curve (auc). For internal validation, random sampling was repeated with replacement using the bootstrapping method over 1,000 times, and a new bias - corrected 95% confidence interval (ci) for auc was calculated using r 2.13.2 (r foundation for statistical computing, vienna, austria; isbn 3 - 900051 - 07 - 0; http://www.r-project.org). Between january 2000 to december 2010, 239 mescc patients were treated with either fp or xp chemotherapy . The median age of the patients was 62 with a range of 40 to 79 . Two hundred and twenty - five patients (94.1%) had an eastern cooperative oncology group (ecog) performance score of 0 to 1 at the time of chemotherapy . Ninety - three patients developed recurrence even after curative resection (n=93), and 11 patients showed recurrence after definitive concurrent chemo and radiotherapy . The other 135 patients (56.5%) were diagnosed with metastatic disease at initial presentation . The most common metastatic site was the lung (n=97) followed by the liver (n=36). Sixty percent (n=143) of patients received fp and 40% received xp treatment as first - line chemotherapy . No significant differences in baseline characteristics were observed between the xp and fp groups, except for the proportion of patients who received esophagectomy (data not shown). The median age of this group of patients was 56, with a range of 42 to 77, and all patients received palliative fluoropyrimidine / platinum chemotherapy . Again, no significant differences in baseline characteristics were observed between the fp and xp groups . The patients in the validation set had fewer previous esophagectomies, more lung and liver metastases, and poorer performance status scores as a group than the patients in the training set . Univariate analyses revealed that poor ecog score, weight loss (loss of at least 10% of weight for 3 months), absence of previous esophagectomy, low hemoglobin level (11.2 g / dl), and low albumin level (3.5 g / dl) were significantly associated with low os . Moreover, poor ecog score, weight loss, absence of previous esophagectomy, low albumin level, leukocytosis (> 8,630/l), elevated alanine aminotransferase level, and the presence of liver metastasis predicted short pfs in univariate analysis (table 2). Multivariate analysis showed that poor performance status (ecog2) (p=0.01), low albumin level (p<0.01), previous esophagectomy (p<0.01), and weight loss (p=0.04) at the time of chemotherapy retained statistical significance to predict poor survival (table 3). Previous esophagectomy (p=0.03) and low albumin level (p<0.01) were also significant for predicting pfs in multivariate analysis . To build a nomogram to predict survival following frontline palliative chemotherapy in mescc, a scoring system was constructed that incorporates significant variables such as performance status scores, weight loss, low albumin levels, and previous esophagectomy (fig ., patients were categorized into ecog 0, 1, or 2 and scored at 0, 63, or 100 points, respectively, based on the cox regression model . For other variables, the scoring system was as follows: weight loss (+) vs. (-) (35 points vs. 0 points), low albumin vs. normal albumin (61 points vs. 0 points), and esophagectomy (+) vs. (-) (27 points vs. 0 points). Patients were then categorized into three groups by risk scores based on the nomogram: favorable (score 0 - 90, n=119), intermediate risk (score 91 - 135, n=58), and poor (score> 135, n=61) risk score (table 4). The nomogram could discriminate between risk groups very well with the following median os for each risk group: favorable risk group, 13.8 months (95% confidence interval [ci], 10.82 to 18.58 months); intermediate risk group, 11.2 months (95% ci, 8.7 to 11.89 months); and poor risk group (7.0 months; 95% ci, 3.56 to 10.04 months; p<0.001) (fig . Similar to the os findings, the nomogram significantly identified the pfs of the three groups as follows: favorable risk group, 5.9 months (95% ci, 4.6 to 7.2 months); intermediate risk group, 4.3 months (95% ci, 3.3 to 5.3 months); and poor risk group, 3.0 months (95% ci, 2.3 to 3.8 months) (p<0.001) (fig . The calibration curve showed the robustness of the nomogram to predict os probability at 12 months (fig . The nomogram was internally validated by repeated random sampling with replacement by bootstrapping over 1,000 times, yielding a bias - corrected 95% ci for auc of 0.59 to 0.74, in accord with the 95% ci for auc (0.58 - 0.74). The nomogram was also assessed on an external validation data set and discriminated the three risk groups with survival curves significantly similar to those obtained for the training data set (fig . The median os according to risk groups were: favorable risk group, 15.5 months; intermediate risk group, 7.8 months; and poor risk group, 5.1 months (p<0.001) (fig . Between january 2000 to december 2010, 239 mescc patients were treated with either fp or xp chemotherapy . The median age of the patients was 62 with a range of 40 to 79 . Two hundred and twenty - five patients (94.1%) had an eastern cooperative oncology group (ecog) performance score of 0 to 1 at the time of chemotherapy . Ninety - three patients developed recurrence even after curative resection (n=93), and 11 patients showed recurrence after definitive concurrent chemo and radiotherapy . The other 135 patients (56.5%) were diagnosed with metastatic disease at initial presentation . The most common metastatic site was the lung (n=97) followed by the liver (n=36). Sixty percent (n=143) of patients received fp and 40% received xp treatment as first - line chemotherapy . No significant differences in baseline characteristics were observed between the xp and fp groups, except for the proportion of patients who received esophagectomy (data not shown). The median age of this group of patients was 56, with a range of 42 to 77, and all patients received palliative fluoropyrimidine / platinum chemotherapy . Again, no significant differences in baseline characteristics were observed between the fp and xp groups . The patients in the validation set had fewer previous esophagectomies, more lung and liver metastases, and poorer performance status scores as a group than the patients in the training set . Univariate analyses revealed that poor ecog score, weight loss (loss of at least 10% of weight for 3 months), absence of previous esophagectomy, low hemoglobin level (11.2 g / dl), and low albumin level (3.5 g / dl) were significantly associated with low os . Moreover, poor ecog score, weight loss, absence of previous esophagectomy, low albumin level, leukocytosis (> 8,630/l), elevated alanine aminotransferase level, and the presence of liver metastasis predicted short pfs in univariate analysis (table 2). Multivariate analysis showed that poor performance status (ecog2) (p=0.01), low albumin level (p<0.01), previous esophagectomy (p<0.01), and weight loss (p=0.04) at the time of chemotherapy retained statistical significance to predict poor survival (table 3). Previous esophagectomy (p=0.03) and low albumin level (p<0.01) were also significant for predicting pfs in multivariate analysis . To build a nomogram to predict survival following frontline palliative chemotherapy in mescc, a scoring system was constructed that incorporates significant variables such as performance status scores, weight loss, low albumin levels, and previous esophagectomy (fig ., patients were categorized into ecog 0, 1, or 2 and scored at 0, 63, or 100 points, respectively, based on the cox regression model . For other variables, the scoring system was as follows: weight loss (+) vs. (-) (35 points vs. 0 points), low albumin vs. normal albumin (61 points vs. 0 points), and esophagectomy (+) vs. (-) (27 points vs. 0 points). Patients were then categorized into three groups by risk scores based on the nomogram: favorable (score 0 - 90, n=119), intermediate risk (score 91 - 135, n=58), and poor (score> 135, n=61) risk score (table 4). The nomogram could discriminate between risk groups very well with the following median os for each risk group: favorable risk group, 13.8 months (95% confidence interval [ci], 10.82 to 18.58 months); intermediate risk group, 11.2 months (95% ci, 8.7 to 11.89 months); and poor risk group (7.0 months; 95% ci, 3.56 to 10.04 months; p<0.001) (fig . 2). The nomogram was applied in the same patient cohort to predict pfs . Similar to the os findings, the nomogram significantly identified the pfs of the three groups as follows: favorable risk group, 5.9 months (95% ci, 4.6 to 7.2 months); intermediate risk group, 4.3 months (95% ci, 3.3 to 5.3 months); and poor risk group, 3.0 months (95% ci, 2.3 to 3.8 months) (p<0.001) (fig . The calibration curve showed the robustness of the nomogram to predict os probability at 12 months (fig . The nomogram was internally validated by repeated random sampling with replacement by bootstrapping over 1,000 times, yielding a bias - corrected 95% ci for auc of 0.59 to 0.74, in accord with the 95% ci for auc (0.58 - 0.74). The nomogram was also assessed on an external validation data set and discriminated the three risk groups with survival curves significantly similar to those obtained for the training data set (fig . The median os according to risk groups were: favorable risk group, 15.5 months; intermediate risk group, 7.8 months; and poor risk group, 5.1 months (p<0.001) (fig . This is largely because of the absence of largescale phase iii randomized clinical trials, since mescc is included as part of esophageal adenocarcinoma / gastric cancer in most trials . Despite the lack of clear evidence that palliative chemotherapy confers a survival benefit in mescc, many physicians are firmly convinced that chemotherapy has a favorable impact on outcome because some drugs (or combination of cytotoxic agents) have shown antitumor activity in phase ii trials [6,8 - 11]. These discrepancies between what is known and what is done mean that practice patterns differ greatly between continents, countries, and institutions . Moreover, no guidelines exist to select patients who might benefit the most from chemotherapy . Only a few studies to date have focused on prognostication of mescc with palliative chemotherapy . One study analyzed 351 patients either with adenocarcinoma or with squamous cell carcinoma and found that performance status and the extent of disease predict treatment outcome . The major limitation of this study was the heterogeneous patient population and the variety of chemotherapeutic regimens used . Another large - scaled prognostication paper included 1,080 patients from three randomized trials although only 50 patients had mescc . The largest study was reported by a french group who analyzed 284 patients, of whom 80% had mescc . This study identified dysphagia and the occurrence of a second primary cancer as independent predictors of poor outcome in a first set of 171 patients and validated these findings in a second set of 113 patients . However, when survival was adjusted with these prognostic factors, they found that chemotherapy did not substantially alter the natural course of mescc . In our study, we found that poor performance status (ecog2), low albumin level (3.5g / dl), absence of previous esophagectomy, and weight loss (at least 10% for 3 months) significantly predicted poor survival and short pfs following first - line fluoropyrimidine / cisplatin chemotherapy . It is worth noting that all of these factors, although independent prognosticators, are more or less related to nutritional matters in esophageal cancer . Hence, the actual benefit from chemotherapy in mescc patients with poor nutritional status as indicated by low albumin, poor performance or weight loss needs to be taken into consideration during decision making . The greatest advantage of our nomogram is that it is based on clinical parameters that are prospectively collected before chemotherapy . The robustness of the model was demonstrated using internal validation via the bootstrapping method as well as external validation on a series of french patients with mescc who received an fp regimen . Our study is also the largest study analyzing the prognostic factors of outcome in mescc treated with a fluoropyrimidine / cisplatin regimen, the most commonly used and most efficient regimen according to a review of 96 different trials performed by grunberger et al . ., the nomogram developed here predicts survival in mescc and should aid physicians and patients in decision - making regarding the use of fp chemotherapy . This nomogram may also be useful in stratifying patients in future clinical trials, similar to what is currently being done in an ongoing randomized phase ii trial for mescc (clinicaltrials.gov nct #nct00816634). The nomogram including the variables, ecog performance, albumin, weight loss and previous esophagectomy predicts survival in mescc patients and could serve as a guide for the use of fp / xp chemotherapy in mescc patients.
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In the industrialised nations, food allergy is a growing epidemic affecting all age groups and appearing at any time in life . A marked increase in the incidence of food allergy in young children is of particular concern, with a reported 68% of young children and 3 - 4% of adults having some type of food allergy [13]. Comparable to the trends first seen with asthma, countries such as the united states (us), united kingdom (uk), and australia have the highest rates of food allergy . In the past decade alone, prevalence rates in the us have increased by at least 18% [4, 5]. Similarly, a recent study in australia found that more than 10% of a cohort of infants had challenge - proven ige - mediated food allergy to one of the common allergenic foods (peanut, raw egg, and sesame). This escalation in the prevalence of food allergies underlies the importance of further research to improve prevention and treatment strategies . These aberrant allergic reactions are principally driven by a t helper type 2 (th2) immune pathway, as evidenced by high levels of allergen - specific immunoglobulin e (ige), th2 polarisation involving inflammatory cells, and cytokines / mediators, and the reported efficacy of therapies that inhibit th2 immune responses in human subjects [912]. There is now also recognition of the innate properties of allergens and their role in th2 polarisation of dendritic cells (dcs) and the process of allergen sensitisation [9, 13]. The most common foods that trigger food allergy are cow's milk, hen's egg, and peanuts and tree nuts, while less common food allergens include soy, wheat, fish, and shellfish [31, 32]. Food allergy is known to be most common in the first 3 years of life; however, studies have shown that most food allergies that begin early in life, such as milk, egg, soy, and wheat, are generally outgrown . Conversely, allergies to peanut, tree nuts, fish, and shellfish usually persist, becoming a lifelong burden [12, 31, 33]. Animal models hold great potential as powerful tools to help answer some of the difficult questions still surrounding the food allergy epidemic . Research in humans is limited by ethical concerns and the chance of fatal anaphylactic reactions . This has stimulated great interest in the use of relevant animal models to predict possible triggers for allergy, identify possible mechanisms involved in setting up the allergic pathway, as well as the testing of novel therapeutic treatments [12, 35, 36]. The purpose of this review is to discuss the application of animal models for the study of the three main food allergens: cow's milk, hen's egg, and peanuts / tree nuts and to provide an overview of the contribution of animal models to our understanding of these allergens and food allergy in general . Mice are the predominant laboratory animal used to study the development of many diseases, generally favoured for their size, short breeding cycles and manageable housekeeping, and the relative ease of genetic manipulation compared to larger models [35, 37]. The use of the murine species in research over several decades has led to the continued development of cellular and molecular tools to allow extensive investigation of mechanisms and pathways of interest . Today, mice have the most comprehensive characterisation of their biology, immunology, and genetic makeup . This background has led to mice providing the foundation for the development of numerous food allergy models . Murine models of food allergy have been investigated in several strains including c3h / hej, balb / c, c57/bl6, and dba/2 (table 1). These animals have the capacity to produce ige and/or igg1 anaphylactic antibodies, and strains can be divided into either high or low ige responders . One of the most challenging obstacles involved in developing murine models of food allergy is the tendency for the immune system to develop oral tolerance to ingested antigens [16, 17]. To avoid this, researchers have focussed on certain strains of mice, such as c3h / hej [14, 15, 23] and balb / c [1517, 27], which more readily display th2 responses than other common murine strains . The use of adjuvants such as cholera toxin (ct) to help stimulate a th2 response is also frequent in food allergy models [1417, 22, 23, 25, 29]. The rat is another common small animal model used in studies of food allergy, with the brown norway (bn) strain being the most suitable for inducing specific ige after oral sensitisation [18, 19, 40, 41]. Other rat strains such as the wistar, hooded lister, and piebald virol glaxo (pvg) rats, have also been examined; however, these strains fail to produce quantifiable levels of antigen - specific ige . The bn rat, as well as other murine strains, has also been used to predict the allergenicity of novel proteins, such as those used in agricultural biotechnology, as reviewed by ladics and colleagues . The guinea pig has also been used as a model to investigate the allergenicity of food proteins, specifically cow's milk (cm). . Demonstrated that within 13 days of drinking cm, guinea pigs could display fatal anaphylaxis if animals were subsequently challenged . There are obstacles in translating findings from guinea pigs to the human setting, including differences in immune physiology and having to estimate ige production in guinea pigs indirectly (e.g., through pca). The limited knowledge and tools available to study their immune system have also led to fewer studies on this model for food allergy research, though it has been a successful model for infectious diseases . Pigs, dogs, and sheep are the main examples of large animal models that have been investigated for food allergy (table 1). Like humans, dogs are one of the few species that can develop allergies to naturally occurring allergens including pollen, grass, house dust mite, and food . Dogs have previously been used to examine other food allergens including wheat, egg, and meat proteins and displayed positive oral challenges in addition to specific ige production, traits similar to those seen in human patients . The university of california developed an atopic dog colony specifically for use in allergy research; these high ige producing canines were first used for asthma studies involving environmental allergens such as grass pollen and ragweed, which were shown to elicit prominent airway hyperresponsiveness (ahr) in these animals . Pigs (swine) represent another large animal model that presents many advantages as a comparative model for food allergy . The intestinal physiology of swine is anatomically and histologically similar to humans, with a microflora more diverse than that seen in rodent models [12, 29, 49]. Pigs also represent an outbred population, with notable variation in the quality of immune responses raised by individuals . These traits are extremely important when examining the pathogenesis and immune responses to food allergens . Swine models have previously been used to investigate other allergic disorders such as asthma; in these studies, animals displayed airway obstruction, eosinophilia, and a late - phase asthmatic response following airway allergen challenge, as typically observed in human asthmatics . Sheep have the advantage of being similar in size and physiology to humans, are placid in nature, and their use poses fewer ethical constraints compared to the use of other large animal models [37, 50]. Sheep have previously been used for allergy studies involving house dust mite (hdm) allergen with a focus on human allergic asthma [37, 51], and more recently as a model for peanut food allergy . Some key advantages of using large animal models include their outbred nature, allowing studies that are more comparable to humans, the ability to conduct serial experiments within the same cohort of animals, and their relative longevity, allowing more relevant investigations into chronic disease as well as the long - term evaluation of specific therapies [37, 50]. There are multiple physiological routes that can induce allergic sensitisation including oral, nasal, intraperitoneal, intragastric, and cutaneous . Despite oral sensitisation being classified as one of the major routes for the sensitisation to food proteins, alternative pathways such as the skin and/or the respiratory tract may also play a role in allergic sensitisation . For example, in a human study it was found that peanut sensitisation arose from environmental exposure, primarily through cutaneous or inhalation routes, rather than from maternal or infant allergen consumption . Further, in a mouse model strid et al . Reported that an aqueous solution of either peanut or ova, when applied to the abraded skin induced the production of antigen - specific ige . It is worthy to note that the most effective route of food allergen sensitisation has also been shown to vary significantly between mouse strains [15, 56]. The route of allergen sensitisation is, therefore, an important and necessary consideration for the use of any relevant animal model of food allergy and will be the topic of further discussion throughout this review . Cow's milk (cm) allergy is one of the most prevalent food allergies that occurs in infants and young children, with the incidence estimated at 2.5% of the general population [31, 57]. Cm can be divided into two main classes, whole casein (bos d 8) which accounts for 80% of the total protein content and whey proteins that make up the remaining 20% . The casein fraction can be subdivided further into four main proteins (s1-, s2-, -, and -casein), whilst whey contains -lactoglobulin (lg or bos d 5), -lactalbumin (ala or bos d 4), immunoglobulins (bos d 7), bovine serum albumin (bsa or bos d 6), and traces of lactoferrin . Although each protein has the potential to act as an allergen, casein, blg, and ala are believed to be the most allergenic [5860]. Reactions to milk proteins can be either ige mediated (usually occurring immediately or within 2 hours of ingestion) or non - ige mediated (generally having a delayed onset). Clinical features of ige - mediated hypersensitivities can include reactions involving the skin, respiratory tract, gastrointestinal tract, or in extreme cases, systemic anaphylactic shock . The chance of developing ige - mediated food allergy is greater in atopic humans that have the genetic predisposition towards mounting an immediate hypersensitivity reaction to food proteins . Though research has shown that most patients outgrow cm allergy by the age of 3, those that suffer from ige - mediated hypersensitivities have a much poorer rate of outgrowing the disorder and are also at a greater risk of developing other atopic conditions [57, 61, 62]. Mice and rats have been employed in numerous studies on cm allergy (table 1) and were first used in this field to help define the immunopathogenic mechanisms responsible for eliciting allergen - specific ige production and other cell - mediated reactions [14, 18, 20, 23]. The brown norway (bn) rat is a high - immunoglobulin (specifically ige) responder, allowing some level of comparison to atopic humans [18, 41]. In a study by atkinson and miller, sensitised bn rats displayed reaginic antibody responses to a range of milk proteins, similar to those recognised in allergic patients with cm allergy . Milk proteins were also found to be less allergenic than ova, with the dose of antigen required to induce sensitisation being 20-fold higher . It has since been demonstrated that bn rats can also be sensitised orally and without the use of adjuvants through gavage dosing . Rats sensitised in this way produced significant antigen - specific ige responses, comparable to those seen in allergic patients . Li et al . Used several techniques to induce ige - mediated cm hypersensitivity in three - week - old c3h / hej mice . Sensitised via the intragastric (ig) route with milk proteins and ct as adjuvant, animals were boosted once a week for a five - week period . This study was one of the first murine models of cm allergy to generate systemic hypersensitivity by oral sensitisation and challenge . Six weeks after the initial sensitisation, cm - specific ige antibody levels were significantly increased and ig challenge with allergen provoked systemic anaphylaxis, with immediate reactions regularly accompanied by respiratory symptoms . Plasma histamine levels increased significantly in cm - sensitised mice after challenge, compared with ct - sham - sensitised mice and nave mice, suggesting histamine to be a major mediator involved in this anaphylaxis model . Heat - treated sera did not produce anaphylaxis in contrast to untreated sera, thus confirming the presence of ige . Furthermore, cytokine production in spleen cells of allergic mice was examined, and a significant increase in the type 2 cytokines interleukin- (il-) 4 and il-5, but not interferon- (ifn-) was detected . This finding provided strong support that th2 responses contribute to the development of cm allergy . Genetic susceptibility is known to be a contributing factor towards developing food allergies; however, trying to observe the expected development of allergy in humans is practically impossible . Morafo et al . Aimed to investigate the susceptibility of different strains of mice to food hypersensitivity, following the sensitisation protocol li et al . Their study focussed on c3h / hej and balb / c mice and involved sensitising animals to both cm and peanut (pn) allergen, via the ig route with ct . Interestingly, though balb / c mice are routinely used as models for food allergy (usually induced by systemic antigen sensitization, e.g., ip), ig sensitisation in this study failed to induce hypersensitivity reactions to either of the food allergens, whilst c3h / hej mice were shown to display reactions to both . Cm - specific ige levels at the time of challenge (week 6) were markedly increased in c3h / hej mice; however, ige levels in balb / c mice were only slightly higher than those of nave mice . Furthermore, anaphylactic reactions were observed in 87% of c3h / hej mice, whilst none were observed in balb / c mice or nave mice of either strain . Anaphylactic reactions were associated with increased plasma histamine levels found only in c3h / hej mice . The comparison of splenocyte cytokine profiles between the two strains illustrated that in balb / c mice ifn- production was significantly increased, whilst il-4 and il-10 were not . Conversely, il-4 and il-10, but not ifn-, levels were considerably higher in c3h / hej mice . Furthermore, a study by berin et al . Investigated the role of tlr4 in the development of allergic sensitisation to either cm or pn proteins, in both c3h / hej and balb / c mice . T - cell responses were th2 skewed in tlr4-deficient c3h / hej mice but not tlr4-sufficient c3h / hej mice; however, this th2 skewing was not observed in tlr4-deficient balb / c mice . Moreover, c3h / hej mice were susceptible to pn - induced anaphylaxis, whilst balb / c mice were completely resistant . This study concluded that though tlr4 status may impact t - cell responses and the severity of anaphylactic reactions to food proteins, the nature of the effect was highly dependent on the genetic background of the mouse . Together, these findings suggested that genetic background plays a major role in the development of food allergies . Other studies have demonstrated successful sensitisation to milk proteins via a systemic (ip) route [16, 17]. The most effective route of sensitisation has been shown to vary significantly between mouse strains and should be taken into consideration when developing a relevant food allergy model . A recent study by dunkin et al . Assessed the impact of milk allergens via different sensitisation routes, with and without adjuvant (ct). Three - week - old c3h / hej mice were exposed to ala, through ig, cutaneous, intranasal, or sublingual routes . Although sensitisation was successful via each route, cutaneous exposure was shown to induce the maximal ige response . Interestingly, the presence of the adjuvant ct was a more significant factor for sensitisation than the actual route . Hen's egg (he) allergy is the second most common food allergy in children, with the dominant egg allergens found in egg white . Egg yolk still holds some allergenic properties; however, these are considerably lower than the four major egg white proteins, ovomucoid (ovm or gal d 1), ovalbumin (ova or gal d 2), ovotransferrin (ovt or gal d 3), and lysozyme (lys or gal d 4). Though ova is the major protein in egg white (54% of total protein), ovm (gal d 1) has been reported as the immunodominant allergen . Similar to its use as a model for the study of cm allergy, the bn rat has been one of the most studied animal models for he allergy (table 1). Atkinson and colleagues [18, 19] effectively dosed rats orally (0.5 ml/100 g body weight) with solutions of 1.012.5 mg / ml ova in distilled water twice a week, for a six - week period . Oral sensitisation of ova was shown to induce both antigen - specific igg and ige antibodies (assessed by pca). Levels of igg antibody were detected in sera from day 21 onwards from animals dosed with 5 mg / ml and above, with levels peaking at day 28 . Interestingly, ige could be detected from as early as day 14 onwards, in animals given the higher doses (10.0 and 12.5 mg / ml), while the lower dose (5 mg / ml) only induced antibody from day 28 onwards . Levels of igg were also absent at lower doses, further illustrating the impact of allergen dose on sensitisation in this model and providing potential for its use in testing factors that could either enhance or inhibit sensitisation to food proteins . Knippels et al . In subsequent studies used bn rats to further characterise the rat he allergy model by investigating parental sensitisation to ova without the use of adjuvants . In this study, three main factors were examined including dose (0.00220 mg / ml), method of dose application (ad libitum via the drinking water or gavage), and frequency of dosing (daily, twice a week, once a week, or once every two weeks) over a period of 6 weeks . Rats were tested for anti - ova antibodies and delayed - type hypersensitivity (dth) responses on days 28 and 42 (separate groups). Daily administration of ova by gavage (1 mg / rat) induced ova - specific igg and ige responses in nearly all animals tested . In the same group of animals, no significant dth response was detected at day 28 but by day 42, dth responsiveness had developed . In comparison, upon ad libitum exposure to 0.002, 0.02, or 0.2 mg / ml ova via the drinking water, no ova - specific antibodies were produced . However, exposure to 2 or 20 mg / ml ova caused ova - specific igg responses . No ova - specific ige was detected for either of the time - points investigated . Interestingly, the most pronounced dth reactions were seen in rats exposed to ova via the drinking water at day 28, with weaker responses seen by day 42 . Results from this study clearly demonstrate how the method of dose application may impact on the quality and magnitude of the immune response . In further work with this model, knippels et al . Examined the effects of oral challenge with ova in previously sensitised bn rats, reporting a minor, transient effect on breathing frequency or systolic blood pressure, similar to that observed in food allergy patients . Another study by akiyama et al . Also investigated oral sensitisation in bn rats and three murine strains (balb / c, b10a, and ask) and found that both bn rats and b10a mice had the highest sensitisation to ova from the models examined; this confirmed that bn rats were a suitable model for assessing the allergenicity of food proteins . This study also found that age was a contributing factor to sensitisation in balb / c mice, with 20-week - old mice showing the highest ova - specific ige and igg1 responses among the three different age groups examined (7 weeks, 20 weeks, and 1 year). Though many studies have used ova to examine egg allergy, ovm (or gal d 1) has been reported as being the dominant allergen in hen's egg to cause allergic reactions in children . One study conducted by rupa et al . Aimed to induce allergy to ovm using a neonatal swine model . Three outbred litters of yorkshire piglets were used for this study, where animals were sensitised ip on days 14, 21, and 35 with 100 g of crude ovm, with ct as adjuvant (10, 25, or 50 g). Pigs were fasted overnight before oral challenge on day 46 with a mixture of egg white and yoghurt . The majority of animals sensitised to ovm displayed strong skin reactivity to direct skin testing on day 35, in contrast to control pigs that did not respond . Additionally, after oral challenge, only sensitised animals showed symptoms of allergic hypersensitivity . Sera analysed from these sensitised pigs revealed ovm - specific igg, whilst pca reactions confirmed ige - mediated antibody responses to ovm . Sera that were heat treated or collected from control animals failed to induce a positive pca response . These results confirm that ovm can successfully be used to sensitise and elicit allergy in pigs, and due to their outbred nature, these animals may also provide the opportunity to investigate some of the mechanisms that underlie allergic predisposition . Despite their appearance and name, peanuts (arachis hypogaea) are not actually a nut; they are a species in the legume or bean family . However, though peanuts and tree nuts originate from different families, they have both been known to contain potent allergens, with a us study reporting peanut and tree nut allergy to specifically account for 90% of fatal anaphylactic reactions . Unlike other food allergies such as cow's milk and egg, the ubiquitous use of pn proteins, together with the apparent increase in the prevalence of pn allergy over the last few decades, has generated great attention towards research in this field [68, 69]. As many as eleven pn allergenic proteins have been categorised (ara h 111), with ara h 1 and ara h 2 classified as the major pn allergens [70, 71]. Following from their work with a murine cm allergy model, li et al . Developed a murine model of pn - induced anaphylaxis using c3h / hej mice - sensitised ig with a low (5 mg / mouse) or high (25 mg / mouse) pn dose together with ct as adjuvant . In this study, both doses of the allergen induced pn - specific ige; however, the level of sensitisation was more effective with the lower dose . Systemic anaphylactic reactions after oral challenge were also more sever in mice sensitised with the low pn dose, with 12.5% of mice exhibiting fatal or near - fatal anaphylaxis . Furthermore, plasma histamine levels and mast cell degranulation from ear tissue were significantly increased in sensitised mice, suggesting that histamine and other mediators released from mast cells attributed significantly to the severe reactions (including anaphylaxis) seen in the pn - sensitised mice . Investigations into t- and b - cell responses in these mice showed similarities to human patients, with allergic mice exhibiting significant in vitro t - cell proliferative responses to crude pn and the major allergens ara h 1 and ara h 2 . More importantly, this study demonstrated that pn proteins are more allergenic than cm proteins, both with respect to a shorter sensitisation period (fewer doses) and the induction of hypersensitivity in adult (5 - 6-week - old) as well as 3-week - old mice [14, 23]. A comparison between oral and nasal routes of allergen sensitisation in a mouse model of pn allergy was performed by fischer et al ., using female c57bl/6 mice, sensitised with whole pn protein extract (ppe) and ct as adjuvant . Oral sensitisation was shown to induce higher pn - specific plasma ige antibody responses and lung eosinophilia following allergen challenge . This was in contrast to nasal sensitisation, which induced greater levels of pn - specific plasma igg and increased airway inflammation (recruitment of macrophages) after challenge . Furthermore, only nasal sensitisation was found to favour an inflammatory response to nasal challenge with unrelated antigens . Cytokine - specific mrna responses on whole - lung tissues were also analysed and compared for both groups before and after challenge . Before challenge, nasally and orally after nasal challenge, however, orally sensitised mice displayed a greater increase for two th2-associated cytokines, il-4 and ccl-11, whereas nasally sensitised mice expressed a greater increase in the th1 cytokine il-17 . Overall data from this study proposed that mice sensitised orally were more prone to allergic - type responses whilst nasal sensitisation was shown to promote nonallergic inflammation . Although peanuts are not actually classified as nuts, patients allergic to peanuts also regularly develop hypersensitivity to tree nuts including almonds, brazil nuts, cashew, and hazelnuts to name a few . [72, 73] found that the major pn allergen, ara h 2, shared similar ige binding epitopes with allergens from almond and brazil nuts, which may contribute to the increased rates of cosensitisation to peanuts and tree nuts in peanut - allergic individuals . Peanuts and tree nuts are frequently associated with life - threatening anaphylaxis, with both forms of allergy rarely outgrown with age . A cashew nut mouse model of allergy showed robust induction of specific ige following transdermal sensitisation, as well as th2 cytokines (il-4, il-5, and il-13) production by cultured splenocytes from sensitised animals . More recently, a long - term mouse model for hazelnut (hn) allergy was investigated to determine whether sensitivity would persist over time . Findings from this study in adult balb / c mice revealed that circulating hn - specific ige antibodies persist for long periods (up to 8 months) despite allergen withdrawal . These long - term memory ige responses were found to be associated with memory spleen cell il-4 responses . This data, therefore, illustrated possible mechanisms that could be involved with persistent nut allergies, even when the allergen is withdrawn for long periods of time . Dogs, pigs, and sheep have reportedly been used as large animal experimental models for pn allergy (table 1). While dogs have previously been employed for the testing of numerous allergens, teuber et al . Used atopic dogs for the first time to develop a canine model of pn / tree nut allergies . This study used inbred high ige - producing spaniel / basenji dogs, subcutaneously (sc) sensitising them with commercial extracts of either 1 g of pn, english walnut or brazil nut proteins, together with aluminium hydroxide (alum) as adjuvant . To test allergenicity, intradermal skin tests, ige immunoblotting and oral challenges were carried out with ground nut preparations . All animals skin tested at 6 months of age displayed positive wheal responses to the commercial extracts used for sensitisation, with pn generating the largest response and barley the least . Ige immunoblotting revealed specific recognition of nut proteins, with pn - sensitised dogs displaying specific ige binding to ara h 1, one of the major pn allergens recognised by pn - allergic subjects . Within 10 minutes of oral challenge, all pn - sensitised dogs vomited and showed signs of fatigue, further demonstrating the high allergenicity to pn proteins . Overall, this study demonstrated the successful use of the dog model to display symptoms seen in food allergy sufferers, as well as a model for concurrent sensitisation to a number of food allergens . Set out to develop a neonatal swine model of pn allergy that would not only display allergic reactions, but also immune and histological profiles similar to those seen in allergic patients . Optimal sensitisation was achieved in piglets sensitised ip with 500 g of crude pn extract with ct as adjuvant on days 9, 10, and 11 after birth, then boosted on days 18 and 25 . Pigs were later challenged by either ig challenge or skin tests at 1-week intervals . Following oral challenge, physical symptoms displayed by the animals were comparable to those seen in humans, including the appearance of rashes and distress associated with the gastrointestinal (diarrhoea) and respiratory (gasping / panting) systems . Serum igg antibodies were analysed and correlations between allergen - specific igg levels and clinical symptom scores, suggesting that high igg levels afford greater protection against food challenge . While ige levels were not directly assessed in this study, repeated positive skin tests and passive cutaneous anaphylaxis indicated the presence of ige . Finally, histological assessment of the small intestine revealed denudation of the villi, oedema, and infiltration of immune cells . Collectively, these findings demonstrate clinical, immunological, and pathological features of pn allergy as seen in humans, supporting the neonatal pig as a suitable model for investigating mechanisms of pn allergy . More recent studies report the use of sheep as a model for pn allergy involving animals sensitised via sc injections of crude pn extract (100 g) with alum as adjuvant . Sensitisation was achieved following 3 sc injections at 2-week intervals, followed by a boost injection after a 1-month rest period . Concurrent sc injections of ova (100 g) and hdm (50 g) were also given to compare allergenicity . Elevated pn - specific ige responses were detected in 4050% of immunised sheep, while only 10% (1 of 10 sheep) displayed detectable ova - specific ige . This level of sensitisation to pn allergen was similar to that seen in response to hdm allergen sensitisation here and elsewhere [51, 74] in sheep, and it likely reflects the outbred nature of this species . Though ova was shown to have a low capacity for specific ige induction, total ova - specific ig levels were shown to increase significantly . Conversely, the elevated pn - specific ige levels were not accompanied by a notable change in pn - specific total ig . Significantly, pn - allergic sheep showed strong ige reactivity to two of the major peanut allergens: ara h 1 and ara h 2 . Furthermore, 80% of sheep that responded to pn allergen with high ige levels also displayed an immediate hypersensitivity reaction following intradermal pn challenge . The sheep model of pn allergy displays a robust systemic ige - responsiveness to pn proteins, providing a new large animal experimental system for studies of allergen - associated immune mechanisms . Despite our improved understanding of food allergy in recent years, there is still no specific therapeutic option available . Currently, strict avoidance and the use of adrenaline in the event of an accidental exposure are the only approved treatments, although several forms of immunotherapy are presently under investigation including oral (oit), sublingual (slit), epicutaneous (epit), and subcutaneous allergen - specific immunotherapy (scit) [75, 76]. A recent study by srivastava et al . Demonstrated that anaphylaxis in a murine model can be prevented following treatment with the chinese herbal medicine formula fahf-2 . Findings from this study and other work from this group suggests that fahf-2 may have the potential to treat multiple food allergies, including peanut and egg [7779]. The high risk of anaphylaxis is a major factor limiting the development of food allergy immunotherapy in humans [75, 80]. In this context, animal models may play an important role in providing a platform for refining these treatments and ensuring thorough preclinical evaluation of their safety, prior to therapeutic human application.
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The first study of this dissertation (chapter 2) concentrated on trauma exposure in childhood . Early in life, the hpa - axis is in development and trauma exposure during childhood is thought to have profound effects on hpa - axis regulation . These effects may be evident even throughout adulthood and in the absence of psychiatric morbidity . In this chapter, we explored the influence of exposure to childhood trauma on hpa - axis regulation in a group of women without present and lifetime psychiatric disorders, and compared them with women without a history of childhood trauma . The results of this study support the hypothesis that hpa - axis regulation is durably changed by exposure to sustained childhood trauma, as we found a blunted cortisol and adreno - corticotropic hormone (acth) response to the dexamethasone / corticotropin - releasing hormone (dex / crh) challenge test . These findings are largely consistent with several lines of evidence, which comprise animal studies (de kloet, sibug, helmerhorst, & schmidt, 2005; sanchez, ladd, & plotsky, 2001; sapolsky & meaney, 1986; suchecki, rosenfeld, & levine, 1993). However, causal inference cannot be asserted based on our cross - sectional analysis . Therefore, an alternative interpretation of the results is that higher resilience against psychopathology explains why the women in our study had not developed psychiatric illnesses following childhood trauma exposure . The salivary cortisol responses to awakening, as well as the cortisol levels over the rest of the day, were not (lastingly) altered by childhood trauma exposure (fig . No effect of childhood trauma exposure on basal hpa - axis regulation was found; a blunted cortisol and acth response to the dex / crh challenge test were found in trauma - exposed (te) women . In chapter 3, we described the mental health of a large group of dutch peacekeeping veterans 1025 years after deployment to lebanon and former yugoslavia, and its association with deployment - related trauma exposure . We found that 1025 years post - deployment, dutch peacekeeping veterans did not show more psychological distress than the general dutch population as assessed with the brief symptom inventory (bsi). In addition, we did not find a significant association between past trauma exposure (1025 years ago) and current psychological distress . Moreover, trauma exposure explained only 9% of the variance in psychological distress . From these findings, we concluded that even though military peacekeeping operations may have a strong impact on the lives of soldiers, trauma exposure in this group of veterans did not seem to be related to current psychological distress . In other words, exposure to traumatic events often occurs during deployment, but this did not cause sustained psychological distress in the majority of the dutch peacekeepers . In chapter 4 the relationship between past trauma exposure (as described above) and current hpa - axis regulation in a group of peacekeeping veterans without current or lifetime dsm - iv axis i psychiatric disorders was explored . The hpa - axis was tested through basal salivary cortisol over the day as well as with the dex / crh challenge test . In contrast to our hypothesis that trauma exposure during adulthood would be associated with altered hpa - axis functioning, we did not find evidence of hpa - axis alterations . However, as hpa - axis regulation in military personnel may well be different than that of non - military controls due to e.g., chronic stress during deployment, military training, or personality structure, we also compared peacekeeping veterans with civilian non - trauma - exposed controls (ne) without current and lifetime psychiatric morbidity . Again, no differences were found . From these results we concluded that deployment - related trauma exposure in male peacekeeping veterans is unrelated to long term hpa - axis alterations (fig . 3). Established relationship between deployment - related trauma exposure and hpa - axis regulation . No effect of deployment - related trauma exposure on hpa - axis regulation was found . In chapter 5, we studied the effect of work - related trauma exposure on hpa - axis regulation in dutch railway personnel . Train drivers and conductors in the netherlands are frequently exposed to severely stressful and traumatic events during the course of their daily work . Verbal and physical aggression as well as person - under - train accidents and near - accidents occur on a regular basis for many of the men and women . In this study, we compared te male train drivers and conductors without current and lifetime dsm - iv axis i psychiatric disorders with ne controls, also without psychiatric disorders . In support of our hypothesis that trauma exposure during adulthood is associated with hpa - axis dysregulation, we found a blunted salivary cortisol response to awakening in the te subjects . However, no effect was found in hpa - axis reactivity during the dex / crh test . These results support the idea that trauma exposure during adulthood is associated with subtle basal hpa - axis alterations, even in the absence of psychiatric morbidity (fig . 4). Established relationship between civilian trauma exposure during adulthood and hpa - axis regulation . No differences were found on the cortisol and acth response to the dex / crh . The results from the studies on the effect of adulthood trauma on hpa - axis regulation in this dissertation are mixed . In order to describe and summarize all current evidence on the association between trauma exposure during adulthood and hpa - axis regulation in our and other studies that included te healthy individuals as well as ne healthy controls and ptsd patients, we performed two meta - analyses on 37 eligible studies published between 1995 and 2010 (chapter 6). In the first meta - analysis, the te subjects were compared with ne control subjects (including 21 studies) and, in the second meta - analysis, te subjects were compared with ptsd patients (including 34 studies). The results clearly show that trauma exposure during adulthood is not associated with basal hpa - axis dysregulation . In addition, no evidence was found for an association of ptsd with basal hpa - axis regulation . In subgroup analyses, however, we found increased cortisol suppression after dexamethasone in te subjects . Mechanisms that have been proposed to explain the enhanced cortisol suppression in response to dexamethasone are an up - regulation of pituitary glucocorticoid receptors or glucocorticoid receptor sensitivity, resulting in an increased negative feedback sensitivity in te individuals . As the results of the dst in te subjects and ne controls were based on only five publications, more studies on this topic are needed . The findings of the studies presented in this dissertation add to the literature on the association of trauma exposure and hpa - axis functioning in several ways . First, in the design of our studies, we focused on te individuals without current or lifetime psychiatric disorders and compared them with a non - psychiatric ne control group . In most studies on the association between trauma exposure, ptsd, and hpa - axis regulation, ptsd patients were compared with individuals without psychiatric disorders . This precludes the possibility to distinguish between effects of psychiatric symptoms or trauma exposure per se (lindley, carlson, & benoit, 2004; muhtz, wester, yassouridis, wiedemann, & kellner, 2008; simeon et al ., 2007). Second, although the issue of trauma exposure in control subjects is important, many studies did not address this issue . Some studies screened their eligible control subjects on trauma exposure (either during childhood, adulthood or both), and included only ne controls (lindley et al . . Occasionally, te controls with a comparable type of trauma - exposure, e.g., military veterans with deployment related trauma exposure, were included (de kloet et al ., 2007, 2008; golier, schmeidler, legge, & yehuda, 2007). Often, however, the presence or absence of trauma exposure in non - psychiatric controls was not taken into account (baker et al ., 2005; smith et al ., 1989; yehuda, golier, halligan, meaney, & bierer, 2004; yehuda et al ., 1993). These different ways of selecting and handling trauma exposure in control groups between studies makes comparison and interpreting the mixed results difficult . This was a major reason for conducting a meta - analysis on studies investigating trauma exposure and hpa - axis regulation in te and ne subjects without psychiatric disorders . To study the additional effect of ptsd, we also carried out a meta - analysis on studies investigating ptsd patients and te subjects . In the following sections, we will discuss the findings from this dissertation within a broader perspective of vulnerability and resilience . Furthermore, we will discuss our experiences in relation to the inclusion of the te participants . Consistent with our hypothesis and with findings from previous studies, we found a blunted cortisol and acth response to the dex / crh challenge test in mentally healthy adult women with a history of moderate to severe childhood trauma exposure . Preclinical studies have shown that in rodents, very early in life, a time window exists during which low basal levels of corticosterone (with the same role in rodents as cortisol in humans) and hypo - responsiveness of the hpa - axis are crucial for the normal development of the brain circuitry (de kloet et al ., 2005). This window of vulnerability is known as the stress hypo - responsive period (shrp), and prolonged activation of the hpa - axis is only resorted to under severe physiological and psychological stress (sapolsky & meaney, 1986). The shrp in rats is sustained by specific components of maternal care: licking and grooming, and delivery of milk to the pup (suchecki et al ., 1993). Manipulations that alter the licking and grooming (maternal separation) result in long - term alterations in hpa - axis regulation and in emotional behaviour (sanchez et al ., 2001). Similar findings on variations in maternal behaviour during infancy have been reported in non - human primate studies (coplan et al ., 1996). Mothers with low foraging demands (lfd) could obtain food without effort, whereas mothers with constantly high foraging demand (hfd) had to complete a daily task to obtain their food . In the variable foraging demand (vfd) condition, the mothers were exposed to unpredictable conditions with respect to food access, resulting in diminished perception of security and a reduction of maternal care of the infants . As adults, the vfd - reared macaques exhibited more trait anxiety than the lfd and hfd- reared macaques . In addition, dysregulation of the hpa - axis was reported, in particular, increased corticotropin releasing factor concentrations and decreased adrenal activity (coplan et al ., 1996). In humans, during the first year of life, the hpa - axis also seems to have a hypo - responsive period . Several studies have shown that hpa - axis activity in early human development is under strong psychosocial regulation and that a healthy attachment style is an important protective factor from developing poor hpa - axis regulation (gunnar & donzella, 2002). Children who received insensitive, unresponsive care were more susceptible to cortisol elevations after a stressor than children who were securely attached to a caregiver (tarullo & gunnar, 2006). The fact that the women in our study were exposed to sexual and physical abuse as well as physical and emotional neglect during a time in their lives when hpa - axis regulation was vulnerable to change, may, therefore, explain our findings of a blunted cortisol response to stress in this group . Our findings suggest that exposure to traumatic events during childhood alters the regulation of the hpa - axis . The results in our studies on military veterans differ from those on civilian subjects, suggesting that trauma exposure affects military personnel in other ways than it does civilian railway personnel, or, alternatively, that military personnel have a different level of hpa - axis - related resilience on average than civilian railway personnel . There may be several explanations for the findings of a blunted cortisol response in train drivers and conductors and no dysregulation in military veterans . An important purpose of military training is to let the soldiers get used to stressful circumstances and to keep their cool in times of danger . Another explanation may be that people who are drawn to choosing a profession in which they are confronted with higher levels of stress and, potentially, even trauma, may have different personality structures (as well as hpa - axis set points) than people who do not make this choice . As ptsd was first diagnosed in combat veterans, it seems indicated to investigate hpa - axis regulation after trauma exposure in military veterans . However, this rather obvious choice has some pitfalls: there is a good chance that other factors such as personality, trained adaptation to stress, and resilience play a significant role in regulation of the hpa - axis in military personnel . Another explanation for the blunted cortisol response to awakening we observed in te railway personnel may be the fact that 25% of these men were working irregular shifts as opposed to none of the ne civilian controls . As the effect of working irregular shifts on hpa - axis regulation griefahn & robens, 2008; kudielka, buchtal, uhde, & wust, 2007), we did not make this an exclusion criterion . It may however be possible that working irregular shifts has a dysregulating effect on the circadian rhythm, and therefore on the circadian cortisol release . A third explanation for the difference in findings in the two groups may be the fact that railway personnel are still functioning in the environment where the traumatic events took place (in the trains, on the platforms), perhaps resulting in an increased fight - or - flight readiness, a normal response after stress . The types of adult trauma and time since trauma may have different effects on hpa - axis regulation . These differences, however, may well be indissociable from the effects of personality traits and lifestyle factors . Therefore, we would recommend studying the effect of trauma exposure in large cohorts of individuals from the general population . The most important psychosocial risk factors for the development of psychiatric disorders such as ptsd after trauma exposure are a history of psychopathology, prior trauma, trauma severity, a family history of psychiatric disorders, a lack of social support, and additional life stress (brewin, andrews, & valentine, 2000; ozer, best, & lipsey, 2003). In addition, we know that trauma exposure during childhood increases the vulnerability to the development of ptsd and mdd in adulthood, especially in women (heim, newport, bonsall, miller, & nemeroff, 2001; kessler, davis, & kendler, 1997; kessler et al ., 1995). The presence of risk factors does not necessarily mean that a person will develop a psychiatric disorder after exposure to trauma . Also, the absence of risk factors does not automatically imply that a person is not susceptible to the development of psychiatric disorders after trauma . The currently known risk factors can only explain a portion of the risk for ptsd in te populations . Both risk and resilience factors are closely related and may reflect twin sides of adaptation to trauma (agaibi & wilson, 2005). In the studies described in this dissertation, we selected the participants who were likely to have high resilience against trauma - related psychopathology, as we only included individuals with multiple trauma exposure, without a history of psychiatric disorders as assessed with the mini international neuropsychiatric interview (m.i.n.i .) (van vliet & de beurs, 2007) and without current psychological complaints as assessed with the bsi (de beurs & zitman, 2006). All participants scored well below the cut - off for psychopathological caseness on the bsi . By selecting the te participants in this way, we were able to study the effect of trauma exposure per se on hpa - axis regulation . Most studies on resilience after trauma exposure have relied on assessments of people after the traumatic event has occurred . Determination of true resilience factors would require prospective studies in which these factors are assessed prior to the onset of a trauma (hoge, austin, & pollack, 2007). As far as we are aware, a large prospective study is currently carried out amongst dutch military men and women who have been deployed to afghanistan between 2005 and 2008 (prismo). All participants were assessed on psychological- and hpa - axis functioning prior to, during and directly after deployment, as well as during follow - up assessments after 6 months and 1, 2, 5, and 10 years . This study will provide more information on resilience to the development of ptsd and other stress - related psychiatric disorders . Schematic outline of trauma exposure, hpa - axis regulation, resilience, and vulnerability for developing psychiatric disorders, and the mediating role of the hpa - axis . Alternatively, study designs with twins could help unravel the genetic and environmental factors involved in vulnerability and resilience . Twin research to date suggests that (1) exposure to assaultive trauma is moderately heritable whereas exposure to non - assaultive trauma is not, (2) ptsd symptoms are moderately heritable, and (3) comorbidity of ptsd with other disorders may be partly due to shared genetic and environmental influences (afifi, asmundson, taylor, & jang, 2010). As in many studies, recruitment of the participants was a major task and more difficult than expected . We sought to include healthy individuals with a history of exposure to psychological trauma in childhood as well as in adulthood . First, we set out to recruit healthy individuals with a history of childhood trauma exposure . An advertisement was placed in a well read dutch women's magazine (libelle). We asked for te as well as ne individuals without current or lifetime psychiatric disorders . The response was somewhat disappointing: 42 women and six men responded to the advertisement . Almost half of them were not eligible because of nightshift work, current stress or medical issues such as thyroid problems . At the same time as recruiting individuals with a history of childhood trauma exposure, we placed an interview and an advertisement in the most read nationwide newspaper (de telegraaf) to recruit individuals with a history of trauma exposure during adulthood and without current or lifetime psychiatric disorders . The response was unsatisfactory as well; a mere 50 individuals (the majority of whom were women) responded . First, the most likely explanation may be that well - functioning te individuals are more inclined to forget the negative impact of the traumatic event (engelhard, hout, & mcnally, 2008), and as a result, do not remember the event as being traumatic (i.e., bias for not remembering traumatic events), and, therefore, do not recognise themselves in the description of the advertisement . . A final explanation may be that participation in the study required too much time and effort (as it included crh infusion and blood sampling), especially considering the fact that we only wanted to include healthy people, who generally have other obligations and limited time . Of the people who did respond to our advertisement, many were not eligible to participate because they were (again or still) going through a stressful period (e.g., divorce, illness of partner or child) or they were using medication that interfered with hpa - axis regulation . The next step in recruiting the necessary individuals for our study was to approach organizations through which large numbers of te healthy people could be reached . First, we contacted the dutch veterans institute, where we found veterans with and without deployment related trauma exposure . Second, we contacted the ns (nederlandse spoorwegen [dutch railways]), the main dutch railway company, to find te train drivers and conductors . During inclusion of the railway personnel, we discovered that some of the men (as not enough women were eligible) worked irregular shifts, either morning shifts, day shifts or evening shifts . None worked night shifts . To obtain a well - matched control group, we hoped to include train drivers and conductors without exposure to traumatic events . Unfortunately, due to reasons unknown to us, the dutch railway company did not give us permission to contact ne healthy train drivers and conductors and, therefore, we had to include a sample of ne men from the general community, who were not working in irregular shifts . Recruiting te individuals without psychiatric disorders also was a challenge . Those who chose to respond to the advertisements might have had more close relatives with ptsd or trauma exposure, specific personality traits, or other unmeasured characteristics that may have confounded associations . Recruitment of participants among specific groups with a high incidence of trauma exposure may have led to a confounding influence of personality traits or other lifestyle factors that are relevant to these specific groups . However, an advantage of contacting and selecting potentially eligible participants instead of having them respond to an advertisement is that self - selection bias plays a lesser role . The overall conclusion of this dissertation is that trauma - exposure during childhood is associated with an attenuated cortisol response after the dex / crh challenge test in women . In contrast, trauma exposure during adulthood was not associated with alterations in hpa - axis regulation after the dex / crh test . We did, however, find a blunted salivary cortisol response to awakening in male railway personnel compared to unexposed controls . Even though our results on childhood trauma exposure are based on just one study, pre - clinical studies using rodents and non - human primates have given us more understanding of the neuroendocrine consequences of early life stress and indicate that hpa - axis response to stress may be influenced by early adversity (shea, walsh, macmillan, & steiner, 2005). Also, recent studies support our findings (carpenter et al ., 2007; elzinga, roelofs, tollenaar, bakvis, van pelt et al ., 2008; meinlschmidt & heim, 2005) of lower cortisol levels in te subjects compared to ne controls with respect to childhood trauma . Similar to the results of our two empirical studies on trauma exposure during adulthood, the literature on adulthood trauma exposure and its association with hpa - axis regulation and ptsd is inconsistent . This inconsistency may, for one, be the result of some studies including te control subjects whereas other studies included ne control subjects, and still other studies included both te and ne controls . To explore the complex and subtle relationship between trauma exposure and hpa - axis regulation further we focussed exclusively on adult trauma exposure and not childhood trauma in these meta - analyses, because: (1) adulthood trauma exposure may differently impact the hpa - axis than childhood trauma exposure, (2) there are many groups of people who are at risk for trauma exposure during adulthood (e.g., military personnel, police officers, fire - fighters, rescue workers, health care workers), and 3) less is known about adulthood trauma . Based on the results from our meta - analyses of studies examining trauma exposure during adulthood and hpa - axis regulation in individuals with and without ptsd, we concluded that basal hpa - axis dysregulation in te adults is neither associated with trauma exposure nor with ptsd symptomatology . Cortisol suppression after dexamethasone, however, was higher in te healthy individuals compared to ne healthy controls . This suggests that trauma exposure during adulthood may be associated with more delicate neuroendocrine hpa - axis dysregulation involving the hypothalamic - pituitary feedback system . Because the findings on the dexamethasone suppression test (dst) were based on only 5 studies, they should be confirmed by future studies . Resilience to psychiatric disorders after trauma exposure during childhood is associated with hpa - axis alterations that are demonstrable in adult women . Resilience and vulnerability to ptsd after trauma exposure during adulthood, however, were not clearly associated with hpa - axis alterations . The association between childhood trauma exposure, resilience and vulnerability in which the hpa - axis plays a mediating role, and the association between adulthood trauma exposure and resilience and vulnerability without the mediating role of the hpa - axis . Even though our studies on trauma exposure during adulthood do not clearly show an association between trauma and hpa - axis regulation, studies assessing the effect of administering cortisol to patients post - trauma show that the development of subsequent ptsd can be influenced (aerni et al ., 2004; schelling et al ., 2001, schelling et al ., 2004 this forms another line of evidence that the (modulating) role of the hpa - axis is of importance . Since the association between adulthood trauma and resilience cannot be explained by hpa - axis regulation, other factors must play a role . As described in the introduction of this dissertation, the autonomic nervous system (ans) is another important part of the stress system, in addition to the hpa - axis . The most studied outcome measures of the ans are heart rate, blood pressure and skin conductivity . In addition a recent study in patients with generalized social anxiety disorder (gsad) showed that not cortisol but salivary alpha - amylase (saa), a relatively new marker of autonomic activity, was found to be higher in basal, non - stimulated conditions, as well as after the dst (van veen et al ., 2008). The findings in this study suggest that in gsad there is an increased activity of the ans but not of the hpa - axis . This hyperactivity of the ans is in line with the clinically observed somatic symptoms of hyperarousal in gsad such as trembling, blushing and perspiration . As symptoms of chronic hyperarousal of the ans often are present in ptsd, the ans may play a role in resilience and vulnerability after trauma exposure . When an individual experiences a traumatic event, the brain activates the ans to meet the threat of a traumatic event, which is a normal, healthy, adaptive survival response . Sometimes, however, the ans continues to be chronically aroused even though the threat has passed and has been survived a key symptom of ptsd . Also, in a study among depressed adult women with a history of childhood trauma, hyperreactivity of the ans was reported (heim et al ., 2000). The assessment of the ans in studies investigating the relation between trauma exposure and the resilience or vulnerability to psychopathology may provide new information on why some people develop psychiatric disorders and others do not . Beside the ans, the experience of a traumatic event may disrupt major beliefs regarding personal invulnerability, benevolence of the world, meaning, self - worth, and relations with others . An individual may feel vigilant, depressed, powerless, vulnerable or guilty about not being able to change the situation, and these feelings may colour the way the individual sees the world . Based on the findings and conclusions from the studies that were presented in this dissertation, we suggest that it should become good practice to include a te and/or a ne control group in studies examining psychiatric disorders and hpa - axis regulation . In studies examining the relationship between trauma exposure and stress regulation, adequate trauma assessment, not only of the te subjects but also of the controls should take place . This trauma assessment should not only cover trauma exposure in adulthood but more importantly, also trauma exposure during childhood . Even though this dissertation mainly refers to ptsd, trauma assessment during childhood as well as during adulthood should also be carried out in studies involving mdd and hpa - axis regulation . Also, a meta - analysis on the effects of childhood trauma exposure on hpa - axis regulation in healthy individuals is needed to confirm the findings we presented in chapter 2 . Furthermore, in future studies on trauma exposure, stress regulation, and psychopathology, it is important not to focus solely on the hpa - axis but to shift attention to other principal stress - axes (e.g., the ans) and their interaction with the hpa - axis to fully understand the neural effects of stress . As mentioned earlier, assessment of alpha - amylase may be studied as a marker of ans functioning . Another example for further study is the neuropeptide oxytocin that plays a role in mediating social affiliation, attachment, social support, and maternal behaviour (young & wang, 2004). Oxytocin also has a protective effect against stress and anxiety, and may therefore increase resilience to psychiatric disorders . A preliminary association has been found between decreased oxytocin concentrations and trauma exposure in childhood, suggesting that oxytocin may somehow be involved in the mechanism translating early adversity into adult vulnerability to stress and disease, including depression and anxiety disorders (heim et al ., 2009). As far as psychological factors are concerned, enhancing resilience in individuals at risk of trauma exposure as well as in te individuals by focusing on interpersonal factors such as attachment and interpersonal relationships may prove to be a valuable addition to the exposure techniques that are widely used as treatment for ptsd (markowitz, milrod, bleiberg, & marshall, 2009). It is important for future studies to assess hpa - axis functioning with uniform protocols (e.g. Cortisol sampling at specific time points and over more than one day) and also to use the same outcome measures and statistical analysis techniques (e.g. Area under the curve, glm repeated measurements). In addition, because of the issue of compliance to the salivary cortisol sampling procedure, cortisol sampling monitoring devices should be developed and used . In addition to all the cross - sectional studies that have been discussed in this dissertation, prospective (longitudinal) studies, for instance in groups of people with a high risk of trauma exposure, will lead to more understanding of the relationship between trauma exposure, psychiatric disorders and stress regulation . Finally, an increasing number of studies on the influence of genetics and epigenetics on the development of psychiatric disorders and resilience after trauma exposure are being published . On the one hand, genetic factors can influence the risk of exposure to some forms of trauma, perhaps through individual differences in personality that influence environmental choices (stein, jang, taylor, vernon, & livesley, 2002). On the other hand, animal studies show that neglect and exposure to stressors may alter gene expression in the brain, leading to increased stress and anxiety in rats (francis & meaney, 1999). In general, future research involving genetics may deepen our understanding of the complex links among genes, brain, cognition, emotion, and the environment.
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Hemopericardium is an effusion of blood into the pericardial sac and it is due to heart disease (heart rupture as a consequence of necrosis or traumas) or to an intrapericardial aortic rupture . In both cases, death may occur as a result of cardiac tamponade due to the accumulation of blood in the pericardium, which leads to an obstructive shock . As the external pressure on the heart increases, the distending or transmural pressure (external - intracavitary pressure) decreases and consequently the intracavitary pressure rises for compensation, leading to an impaired venous return and an elevation of the venous pressure . If the external pressure is so high to exceed the ventricular pressure during diastole, diastolic ventricular collapse and a cardiac arrest may occur . The obstructive shock results in a moderate / severe cyanosis of the face and neck. During the last few years, with the introduction of post - mortem computer tomography (pmct), a valuable contribute has been given to forensic investigations in making, contributing and supporting the diagnosis of several violent and natural causes of death, leading to the introduction of this technique in some offices of forensic sciences as a standard procedure before the traditional autopsy and in sporadic cases even to substitute it . In this paper, a case of death due to cardiac tamponade as a consequence of hemopericardium is reported, in which pmct was useful in order to understand the dynamic and cause of death, before the post - mortem examination . The body was still warm and no signs of trauma or other injuries were found . He had a medical history of hepatitis c and hypertension with a poor pharmacological response . Before the autopsy a pmct examination was performed about 3 h after the discovery of the corpse . The obtained dicom scans were rendered in 2d and 3d images using the open source software osirix on a maxosx computer . A board certified radiologist interpreted all radiological images . The pmct report was available before the autopsy examination and showed an intrapericardial aortic dissection together with a periaortic hematoma, a sickle - shaped intramural hematoma and a false lumen . A hemopericardium was also shown, consisting in fluid and clotted blood, which differentiated to each other because of their densities (figure 1). Even before the traditional postmortem examination, we were able to declare death due to a cardiac tamponade as a consequence of hemopericardium in a type - a aortic dissection according to stanford classification . At the external examination of the body, no signs of trauma were observed . At the autopsy examination, the features observed by the pmct scans were totally confirmed . On opening the pericardial sac, a cardiac tamponade was present, consisting in a large size hematoma of clotted blood (800 g) and fluid blood (500 g) (figure 2a). After a block dissection of the heart together with the ascendant aorta, and a careful dissection of the soft tissues of these anatomical structures, an intrapericardial aortic dissection was found (figure 2b). (a): a hypodense area compared with the lumen (blue arrow in the red circle); (b): a false lumen together with an intramural hematoma displayed as a hyperdense area (blue arrow in the red circle); and (c & d): a hemopericardium consisting in fluid blood (blue circle) and clotted blood (red asterisk). (a): the presence of the hemopericardium confirmed the pmct findings when the pericardial sac was opened; (b): site of aortic dissection (indicated by the probe) involving the ascending aorta in its intrapericardial tract . Aortic dissection is by far the most common and serious condition affecting the aorta . Among these pathologies, the ascendant aortic dissection is the most severe condition that may lead to death in a large number of cases, due to the rapidity of the pathological process . The dissection seems to be strictly related to the cystic medial necrosis, a disorder of large arteries, in particular the aorta, characterized by an accumulation of basophilic ground substance in the media with cyst - like lesions . It usually results from chronic hypertension or from rare conditions (such as marfan's syndrome, aortic coarctation and bicuspid aortic valve). Whatever the mechanism, it is often brought about by degenerative changes in the aortic wall . Since dissection can involve any aortic segment, the disease can manifest itself through a variety of clinical presentations . In fact, when aortic dissection occurs, aortic branches occlusion may happen . In case of dissections of the ascending aorta, the major aortic branches are occluded, resulting in rapidly fatal complications such as cardiac tamponade, major stroke, or massive myocardial infarction. If the fluid occurs rapidly (as may occur after trauma or myocardial rupture), as little as 100 ml can cause tamponade . However, the data reported in literature about the amount of blood able to cause death in acute cases are discordant . Frequently, a quantity of 300400 ml of blood is found in bodies, even if other reports show a greater volume. Aortic dissections can be classified by the location of the tear . According to the stanford classification, there are two types of aortic dissections: (1) type a aortic dissections involve the ascending aorta and frequently needs emergency surgery . These types of dissections are often found in subjects with a history of high blood pressure, an ascending aortic aneurysm, connective tissue disorder (e.g., marfan syndrome, bicuspid aortic valve, or a family history of aortic dissections). (2) type b aortic dissections do not involve the ascending aorta (involvement of aortic arch without involvement of the ascending aorta is considered type b or simply called arch dissection but is not a type a dissection). These types of dissections may be managed conservatively with blood pressure and heart rate control . Therefore, the case here reported can be classified as a type a aortic dissection . The evaluation in living subjects of cardiac tamponade due to a hemopericardium, using diagnostic imaging techniques with special regard to the ct, allows a good interpretation of the anatomy and the pathology of the pericardium ., in post - mortem investigations, few studies have used pmct imaging to investigate cardiac tamponade in the last decade, (table 1) and the results obtained show the feasibility of using the pmct technique to diagnose haemopericardium and cardiac tamponade in cadavers . It must also be considered that since in in - vivo imaging of aortic dissections the contrast - enhancement of the lumen is essential to detect critical radiological features, it is possible that these findings can be missed during a standard pmct without contrast . To overcome this problem, a pmct - angiography can be carried out, even if the use of this technique is currently limited . Regarding this topic, f: female; m: male; pmct: post - mortem computer tomography . Bello, et al . Have recently reported a case involving a 72 year - old man, in which multi - phase pmct angiography was helpful in defining the diagnosis, detecting a hemopericardium and the ruptured wall situated in the posterior part of the left ventricle . The autopsy was then performed, totally confirming the ct angiography findings . In the present case, this pathology was the trigger for the development of the ascendant aortic dissection resulting in a rapid cardiac tamponade, causing the man's death . According to the italian jurisdiction, when a subject (even a foreigner) dies in italy and questions concerning the cause and the manner of death arise, the prosecutor requires the forensic pathologist to perform a full postmortem examination, consisting in external and internal examination . The family of the deceased cannot refuse the autopsy . Because the deceased was jewish, his family objected to the autopsy in observance of their religion, however, the prosecutor ordered that the autopsy had to be carried out, notwithstanding the fact that the pmct was able to demonstrate the cause of death, because it was not considered a technique reliable and accurate enough to determine the cause of death . According to this conclusion, the autopsy was performed, confirming the ct findings . According to us, a pmct examination in always necessary in all cases of violent deaths, such as gunshot wounds, asphyxia, sharp force injuries, etc ., in such cases, the pmct must be considered as a support to the traditional postmortem examination that must always to be carried out . In case of natural deaths, a pcmt should always be performed whenever possible, because it sometimes can give a clue regarding the cause of death, making the autopsy virtually not mandatory (in the countries where it is allowed). For example, regarding to sudden natural deaths in the absence of traumas or in deaths due to infectious diseases, the real effectiveness of a full autopsy should be evaluated taking into account the potential risks for the pathologist during the autopsies . In these cases, a full pmct examination, together with an accurate clinical history of the deceased, exhaustive investigative reports concerning the circumstances surrounding the death and toxicological analyses from biological samples collected at the external examination, is a valuable tool that can alone led to determine the cause of death . The forensic pathologist, on the basis of the available information, to his own experience and also to the specific justice issues concerning the case, should be able to decide when and why the pmct alone is enough to clarify the cause of death or if a full postmortem examination is required . Furthermore, ethical and religious issues should be taken into consideration in such cases, when the traditional autopsy is not really necessary . A pmct approach in these cases can be useful to reduce the manipulation of the bodies, allowing families to observe their own religions . However, despite the rapid growth of forensic radiology, we must underline that a multidisciplinary approach in suspicious cases is always required . On the one hand, the pmct should become a standard in forensic practice in order to be performed whenever possible . On the other hand, if that happens, it must be avoided that the pmct led the pathologist to focus his attention only on the main radiological findings, giving less importance to other features, hence missing possible secondary diseases . Specifically, a pmct - guided postmortem examination must always to be carried out as fully as possible . In conclusion, pmct should be considered as an essential support to the traditional autopsy of violent deaths (and not its replacement), becoming as important as histopathological and toxicological analyses, in order to allow the forensic pathologist to have an overall view of all the features concerning the case . In natural deaths, conversely, when no specific issues arise, the pmct alone should be virtually considered as a valuable technique to clarify the cause of death, to reduce potential risks for the pathologist and also to overcome ethical and religious issues.
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The purpose of this ancillary study was to determine if the risk for diabetes complications in the action to control cardiovascular risk in diabetes (accord) glycemia trial differed among individuals with lower or higher hba1c levels than predicted by fasting plasma glucose (fpg). Accord participants were middle - aged and older people with type 2 diabetes and established cardiovascular disease (cvd) or known cardiovascular risk factors (1). The trial tested whether intensive treatment targeting hba1c levels of less than 6% (42 mmol / mol) would reduce the rate of cardiovascular events compared with a strategy targeting hba1c levels between 7.0% and 7.9% (53 and 63 mmol / mol). This hypothesis was not supported: intensive treatment failed to improve primary cardiovascular outcomes and was instead associated with 22% greater total mortality compared with standard treatment . The accord trial thus demonstrated that increased mortality was a previously unrecognized harm of intensive glucose - lowering therapy in high - risk patients with type 2 diabetes . Although symptomatic severe hypoglycemia was associated with an increased risk of death in the intensive and standard treatment groups, differences in hba1c or rates of hypoglycemia between the two groups did not explain the greater mortality observed in the intensive - treatment group (2). Treating patients with diabetes with drugs that lower blood glucose levels inherently increases the risk for hypoglycemia . Intensively treating patients with diabetes to a low hba1c target implicitly assumes that all patients will have roughly the same blood glucose level when they reach the target . Miller et al . (3) paradoxically reported that accord participants with higher hba1c levels had greater risk for hypoglycemia . If hba1c were an unbiased estimate of blood glucose, this observation would incongruously suggest that participants with higher blood glucose levels had greater risk for hypoglycemia . Numerous studies have shown, however, that some patients with diabetes have hba1c levels that are persistently lower or higher than predicted compared with other individuals with similar blood glucose levels (410). We reasoned that intensive treatment to a one - size - fits - all hba1c target of less than 6% (42 mmol / mol) may have inadvertently and disproportionately produced adverse outcomes in a subgroup of accord patients with diabetes with lower blood glucose levels than their hba1c would predict . To test this hypothesis, we used the hemoglobin glycation index (hgi) to identify accord participants with incongruous hba1c and fpg at baseline . Hgi is the calculated difference between an individual s observed hba1c and a predicted hba1c derived by inserting the individual s blood glucose concentration into a population regression equation describing the linear relationship between hba1c and blood glucose (hgi = observed hba1c predicted hba1c) (4,9). Assessment of hgi in the diabetes control and complications trial (dcct) showed that patients with type 1 diabetes with a high hgi had a threefold greater risk for retinopathy and a sixfold greater risk for nephropathy (6). Most prior hgi research calculated predicted hba1c based on mean blood glucose (self - monitored (4,9), timed profiles (6), or continuous glucose monitoring (11)). Hgis calculated using all glucose data downloaded from patient meters were highly correlated with hgis calculated using only prebreakfast glucose data (4). The feasibility of using fpg to calculate the hgi in patients with type 2 diabetes was previously proposed (12). (5) is calculated in exactly the same way as hgi except fructosamine replaces directly measured glucose for obtaining a predicted hba1c . Several studies have shown that patients with type 2 diabetes with a high glycation gap have greater risk for microvascular or macrovascular complications (5,10,13,14). Hgi and the glycation gap are strongly positively correlated, which suggests they reflect the same biological phenomenon (15). Patients with diabetes with low and high hgi have hba1c levels that are lower or higher than predicted, respectively, compared with other patients with similar blood glucose levels . We hypothesized that intensive treatment produced disproportionately lower blood glucose levels and increased hypoglycemia in the subgroup of accord participants with a high hgi . Also, higher baseline hba1c and higher average on - treatment hba1c were both strong predictors of mortality associated with intensive treatment in accord (16,17). Because hgi was associated with increased complications risk in patients with type 1 diabetes in the dcct, we also hypothesized that patients with type 2 diabetes with high hgi in accord might also have a greater risk for primary cardiovascular outcomes, total mortality, and microvascular disease . This research was approved by the institutional review boards at the louisiana state university health sciences center and children s hospital, new orleans, la . Accord was a multicenter, randomized clinical trial that used a double 2 2 factorial design to incorporate intervention trials for glycemia, hyperlipidemia, and hypertension . The lipid intervention trial tested in 5,518 participants the hypothesis that a hypolipidemic agent would improve outcomes in subjects with good glycemic control . The blood pressure intervention trial tested in the remaining 4,733 participants the hypothesis that a therapeutic strategy that targets a systolic blood pressure of <120 mmhg will reduce the rate of cvd events compared with a strategy that targets a systolic blood pressure of <140 mmhg . All participants enrolled in the accord trial had type 2 diabetes and had experienced a prior cardiovascular event or had other evidence of high risk for cvd . Median follow - up time was 5.0 years (mean 5.0; range 0.018.4). The rationale, study design, inclusion criteria, and other details of the accord trial are described elsewhere (1,1820). The glycemia intervention trial included all 10,251 accord participants, who were randomly assigned at baseline to a standard treatment group or an intensive treatment group . Any antihyperglycemic agent or combination of agents approved by regulatory authorities could be used as considered appropriate to achieve protocol - mandated target hba1c levels of 7.0% to 7.9% (53 to 63 mmol / mol) in the standard treatment group, or less than 6.0% (42 mmol / mol) in the intensive treatment group . During the trial, excess mortality was demonstrated at 3.5 years of average follow - up, at which time all participants in the intensive treatment group were converted to the standard treatment regimen (20). Data for this ancillary study were obtained from the accord coordinating center on all enrolled participants . Only data from participants with fpg and hba1c recorded at baseline (n = 10,125) were used in these analyses . The prespecified primary outcome for accord was a composite of the first occurrence of nonfatal myocardial infarction (mi), nonfatal stroke, or death from cardiovascular causes (18). Causes of cardiovascular death included fatal mi, congestive heart failure, documented arrhythmia, death after invasive cardiovascular interventions, death after noncardiovascular surgery, fatal stroke, unexpected death due to ischemic cvd occurring less than 24 h after the onset of symptoms, and death due to other vascular diseases such as pulmonary emboli or abdominal aortic aneurysm rupture . Primary and secondary outcomes were assessed from baseline through the last set of scheduled study visits (march june 2009) (20). Symptomatic, severe hypoglycemia requiring any assistance was defined as an episode of hypoglycemia with a documented blood glucose concentration of less than 50 mg / dl (2.8 mmol / l) in which the participant reported receiving medical care or assistance from another individual or recovery with carbohydrate treatment . Symptomatic, severe hypoglycemia requiring medical assistance was defined as an episode of hypoglycemia in which the participant received care at a hospital or emergency department or from medical personnel . Hypoglycemia assessment included hypoglycemia events from baseline up to the transition period when intensive glycemic intervention was terminated (february 2008). Baseline fpg and hba1c data from a random subsample of 1,000 accord participants were used to estimate the linear relationship between fpg and hba1c in the study population . A predicted hba1c was calculated for the remaining 9,125 participants by inserting the baseline fpg into the subsample linear regression equation (hba1c = 0.009 fpg [mg / dl] + 6.8). The 9,125 participants were then assigned to low, moderate, or high hgi subgroups based on baseline hgi and hgi cut points that divided the population into three equally sized subgroups (low hgi 0.520 [n = 3,041], 33.3%; moderate hgi 0.520 to 0.202 [n = 3,042], 33.3%; high hgi> 0.202 the use of a tertile classification system is by convention for consistency with previous hgi studies . We compared hgi classifications using simple linear regression with hgi classifications based on cubic spline regression and observed more than 90% identity . Baseline characteristics and other variables were compared among low, moderate, and high hgi subgroups . Group comparisons used anova or kruskal - wallis tests for normally distributed and nonnormally distributed continuous variables, respectively, and tests were used for categorical variables . Kaplan - meier curves and log - rank tests post hoc analyses were performed to compare risk of primary outcomes, total mortality, and hypoglycemia between the intensive and standard glycemia treatment groups and among hgi subgroups with adjustment for covariates . Tests of statistical significance were based on a two - tailed type 1 error at p <0.05 . The interaction term of hgi subgroup with glycemia treatment was added to each model, and a likelihood ratio test was applied . Whenever the interaction test did not meet the criteria for statistical significance, a bonferroni correction for multiple comparisons was applied when evaluating the effect of treatment by subgroup . Proportional hazards assumptions were assessed by cumulative sums martingale residuals over follow - up times using kolmogorov - type supremum tests and no violation was found . Covariates included baseline characteristics, study location among the seven clinical - center networks, and for other accord intervention assignments (the blood pressure trial, assignment to the intensive blood pressure intervention group, or the lipid trial, assignment to receive fibrate in the lipid trial). Sensitivity analyses were performed to determine hazard ratio stability after including intervention assignments in the cox model as stratifying factors rather than as covariates . Baseline characteristics were age, sex, ethnicity, education level, medical history (smoking history, duration of diabetes, retinopathy detected at baseline, history of cvd, high risk of congestive heart failure, evidence of significant atherosclerosis, albuminuria), and laboratory and clinical measures at baseline (fpg, diastolic blood pressure, estimated glomerular filtration rate, ldl - cholesterol, hdl - cholesterol, and triglycerides). We used cox regression in reporting our results to account for the fact that accord participants were monitored for different lengths of time, leading to a changing denominator over time that is not adequately modeled by the constant average denominator imposed by the logistic model . Accord was a multicenter, randomized clinical trial that used a double 2 2 factorial design to incorporate intervention trials for glycemia, hyperlipidemia, and hypertension . The lipid intervention trial tested in 5,518 participants the hypothesis that a hypolipidemic agent would improve outcomes in subjects with good glycemic control . The blood pressure intervention trial tested in the remaining 4,733 participants the hypothesis that a therapeutic strategy that targets a systolic blood pressure of <120 mmhg will reduce the rate of cvd events compared with a strategy that targets a systolic blood pressure of <140 mmhg . All participants enrolled in the accord trial had type 2 diabetes and had experienced a prior cardiovascular event or had other evidence of high risk for cvd . Median follow - up time was 5.0 years (mean 5.0; range 0.018.4). The rationale, study design, inclusion criteria, and other details of the accord trial are described elsewhere (1,1820). The glycemia intervention trial included all 10,251 accord participants, who were randomly assigned at baseline to a standard treatment group or an intensive treatment group . Any antihyperglycemic agent or combination of agents approved by regulatory authorities could be used as considered appropriate to achieve protocol - mandated target hba1c levels of 7.0% to 7.9% (53 to 63 mmol / mol) in the standard treatment group, or less than 6.0% (42 mmol / mol) in the intensive treatment group . During the trial, excess mortality was demonstrated at 3.5 years of average follow - up, at which time all participants in the intensive treatment group were converted to the standard treatment regimen (20). Data for this ancillary study were obtained from the accord coordinating center on all enrolled participants . Only data from participants with fpg and hba1c recorded at baseline (n = 10,125) were used in these analyses . The prespecified primary outcome for accord was a composite of the first occurrence of nonfatal myocardial infarction (mi), nonfatal stroke, or death from cardiovascular causes (18). Causes of cardiovascular death included fatal mi, congestive heart failure, documented arrhythmia, death after invasive cardiovascular interventions, death after noncardiovascular surgery, fatal stroke, unexpected death due to ischemic cvd occurring less than 24 h after the onset of symptoms, and death due to other vascular diseases such as pulmonary emboli or abdominal aortic aneurysm rupture . Primary and secondary outcomes were assessed from baseline through the last set of scheduled study visits (march june 2009) (20). Symptomatic, severe hypoglycemia requiring any assistance was defined as an episode of hypoglycemia with a documented blood glucose concentration of less than 50 mg / dl (2.8 mmol / l) in which the participant reported receiving medical care or assistance from another individual or recovery with carbohydrate treatment . Symptomatic, severe hypoglycemia requiring medical assistance was defined as an episode of hypoglycemia in which the participant received care at a hospital or emergency department or from medical personnel . Hypoglycemia assessment included hypoglycemia events from baseline up to the transition period when intensive glycemic intervention was terminated (february 2008). Baseline fpg and hba1c data from a random subsample of 1,000 accord participants were used to estimate the linear relationship between fpg and hba1c in the study population . A predicted hba1c was calculated for the remaining 9,125 participants by inserting the baseline fpg into the subsample linear regression equation (hba1c = 0.009 fpg [mg / dl] + 6.8). The 9,125 participants were then assigned to low, moderate, or high hgi subgroups based on baseline hgi and hgi cut points that divided the population into three equally sized subgroups (low hgi 0.520 [n = 3,041], 33.3%; moderate hgi 0.520 to 0.202 [n = 3,042], 33.3%; high hgi> 0.202 [n = 3,042], 33.3%). The use of a tertile classification system is by convention for consistency with previous hgi studies . We compared hgi classifications using simple linear regression with hgi classifications based on cubic spline regression and observed more than 90% identity . Baseline characteristics and other variables were compared among low, moderate, and high hgi subgroups . Group comparisons used anova or kruskal - wallis tests for normally distributed and nonnormally distributed continuous variables, respectively, and tests were used for categorical variables . Kaplan - meier curves and log - rank tests were used to compare the distribution of time to first event . Post hoc analyses were performed to compare risk of primary outcomes, total mortality, and hypoglycemia between the intensive and standard glycemia treatment groups and among hgi subgroups with adjustment for covariates . Tests of statistical significance were based on a two - tailed type 1 error at p <0.05 . The interaction term of hgi subgroup with glycemia treatment was added to each model, and a likelihood ratio test was applied . Whenever the interaction test did not meet the criteria for statistical significance, a bonferroni correction for multiple comparisons was applied when evaluating the effect of treatment by subgroup . Proportional hazards assumptions were assessed by cumulative sums martingale residuals over follow - up times using kolmogorov - type supremum tests and no violation was found . Covariates included baseline characteristics, study location among the seven clinical - center networks, and for other accord intervention assignments (the blood pressure trial, assignment to the intensive blood pressure intervention group, or the lipid trial, assignment to receive fibrate in the lipid trial). Sensitivity analyses were performed to determine hazard ratio stability after including intervention assignments in the cox model as stratifying factors rather than as covariates . Baseline characteristics were age, sex, ethnicity, education level, medical history (smoking history, duration of diabetes, retinopathy detected at baseline, history of cvd, high risk of congestive heart failure, evidence of significant atherosclerosis, albuminuria), and laboratory and clinical measures at baseline (fpg, diastolic blood pressure, estimated glomerular filtration rate, ldl - cholesterol, hdl - cholesterol, and triglycerides). We used cox regression in reporting our results to account for the fact that accord participants were monitored for different lengths of time, leading to a changing denominator over time that is not adequately modeled by the constant average denominator imposed by the logistic model . Figure 1c shows the linear relationship between hba1c and fpg in the accord population at baseline . Figure 1a and b shows that the frequency distribution of hba1c was markedly different in the low, moderate, and high hgi subgroups . Selected baseline demographic, biochemical, and clinical characteristics are compared among the hgi subgroups in table 1 . Of particular note, there were disproportionately more black and hispanic participants and fewer white participants in the high hgi subgroup . Furthermore, high hgi participants were younger, had longer duration of diabetes, were more likely to already have retinopathy and a history of albuminuria at baseline, and were more likely to have used insulin before the start of the study . The low, moderate, and high hgi subgroups have green, blue, or red lines, respectively . Baseline characteristics of accord participants by hgi subgroup gfr, glomerular filtration rate; iqr, interquartile range . Unless otherwise noted, values are means sd for continuous variables or number (%) for categorical variables . Overall differences between hgi groups using anova, kruskal - wallis tests, or tests . 2b) treatment, mean hba1c remained significantly different (p <0.001) between hgi subgroups and was highest in the high hgi subgroup . In contrast, the mean fpg was significantly lower (p <0.001) in the moderate and high hgi subgroups after 1 year of standard treatment (fig . 2a) and was not different (p> 0.05) between hgi subgroups after 1 year of intensive treatment (fig . 2b). The number of participants above or below the hba1c intensification thresholds varied after 1 year in the standard treatment arm: 43.0% of high hgi participants remained above the 8% (64 mmol / mol) intensification threshold compared with only 30.6% of moderate and 21.9% of low hgi participants . After 1 year in the intensive treatment arm, 88.7% of high hgi participants remained above the 6% (42 mmol / mol) intensification threshold compared with 83.9% of moderate and 72.7% of low hgi participants . Barring mitigating circumstances, such as a recent hypoglycemic event, having disproportionately more participants above the intensification threshold should have resulted in greater treatment intensification at the 1-year visit . Mean (95% ci) hba1c () and fpg levels () for low, moderate, and high hgi subgroups after 1 year of standard (a) or intensive (b) glycemia treatment . For each panel, hba1c or fpg values with different superscripts (a, b, c) are significantly different (p <0.05). 3) and cox regression analyses (table 2) show that the effect of intensive treatment differed markedly among the hgi subgroups . For example, if we ignore hgi, the event rate for primary outcomes was not significantly different (p = 0.09) between the intensive (9.8%) and standard (10.7%) treatment groups (table 2 and fig . A statistically significant interaction was detected between treatment and hgi (p = 0.0091). Subsequent hgi subgroup analysis showed that intensive treatment reduced primary outcomes by 25% (p = 0.02) in the low hgi subgroup and by 23% (p = 0.02) in the moderate hgi subgroup (table 2 and fig . 3b). In contrast, primary outcomes in the high hgi subgroup were not significantly different between the standard and intensive glycemia treatment groups (p = 0.20). Kaplan - meier curves for primary outcomes, total mortality, and hypoglycemia requiring any assistance . Proportions of participants free of the specified outcome over time are compared between standard and intensive treatment groups (panels a, c, d) and among the hgi subgroups (panels b, d, f). Standard treatment is depicted by orange dashed lines and intensive treatment by solid purple lines . Low, moderate, and high hgi subgroups have green, blue, or red lines, respectively . Risk and adjusted hazard ratios of composite primary outcomes, total mortality, and hypoglycemia by glycemia treatment group and hgi subgroup first occurrence of nonfatal mi, nonfatal stroke, or death from cardiovascular events . Covariates include age, sex, race, education, diabetes duration, history of smoking, previous cvd, high risk of heart failure, history of arterial stenosis, history of albuminuria, retinopathy at baseline, estimated glomerular filtration rate, hdl - cholesterol, ldl - cholesterol, and intervention trial (hypertension treatment, intensive vs. standard; hypertension trial vs. lipid trial; hyperlipidemia treatment, fibrate vs. no fibrate). Covariates include age, sex, race, education, history of smoking, statin uses, insulin uses, previous cvd, history of albuminuria, history of arterial stenosis, retinopathy at baseline, and intervention trial (hypertension treatment, intensive vs. standard; hypertension trial vs. lipid trial; hyperlipidemia treatment, fibrate vs. no fibrate). Covariates include age, sex, race, education, diabetes duration, living alone or not, insulin uses, previous cvd, retinopathy at baseline, neuropathy at baseline, and estimated glomerular filtration rate . Bonferroni correction for multiple comparisons . Ignoring hgi again, total mortality (table 2 and fig . 3c) was significantly greater (p = 0.02) in the intensive treatment group (7.7%) compared with the standard treatment group (6.3%). Hgi subgroup analysis showed that although intensive treatment significantly increased total mortality by 41% (p = 0.02) in the high hgi subgroup, it had no effect on mortality in the moderate or low hgi subgroups compared with standard treatment (table 2 and fig . 3d). Ignoring hgi once more, hypoglycemia requiring any assistance was more than threefold greater (p <0.001) in the intensive treatment group compared with the standard treatment group (table 2 and fig . Hgi subgroup analysis showed that the incidence of hypoglycemia was progressively higher in the low, moderate, and high hgi subgroups in the intensive (14.5, 16.8, and 18.8%, respectively) and standard (3.7, 4.5, and 7.5%, respectively) glycemia treatment groups (table 2 and fig . Sensitivity analyses that included other intervention assignments as stratifying factors in the cox models, rather than as covariates, showed that the estimated hrs for primary outcomes, total mortality, and hypoglycemia remained stable . Interindividual variation in hba1c caused by factors other than blood glucose concentration appears to be partly hereditary (21,22) and has been reported in patients with type 1 diabetes (49,23,24), with type 2 diabetes (1214,2527), and without diabetes (2832). Pediatric patients with type 1 diabetes with higher hgi had higher levels of skin advanced glycation end products (33). Levels that are persistently lower or higher than expected among individuals with similar blood glucose levels have been detected in studies where blood glucose was estimated based on fpg (12), self - monitored mean blood glucose (4,8,9,34), 7-point mean blood glucose profile sets (6,35), continuous glucose monitoring (7,11), or fructosamine (5,26,27). Unlike other methods for estimating blood glucose, continuous glucose monitoring and fructosamine are relatively free of sampling bias yet produce similar results when assessing interindividual variation in hba1c . The present studies indicate that subjects in accord with high hgis had more retinopathy and nephropathy at baseline, as previously reported in the dcct (6). The differences in drug use observed between hgi subgroups at baseline could be related to the fact that insulin is more likely to be prescribed for individuals with persistently higher hba1c (high hgi). Alternatively, different drugs have been shown to influence the quantitative relationship between hba1c and blood glucose concentration (36), which could in turn influence the hgi . Higher hgi among black accord participants supports our previous observation of racial variation in hgi in children with type 1 diabetes (8). Evidence of clinically significant interindividual variation in the quantitative relationship between blood glucose concentration and hba1c markedly complicates the use of hba1c for the diagnosis and management of diabetes, especially in mixed - race populations (3742). Our present analyses examine the accord glycemia trial through a new lens, namely, the hgi calculated using fpg and hba1c measured at baseline . As previously reported by the accord investigators, we observed no difference in primary outcomes between the standard and intensive treatment groups despite our use of a slightly different data set (we omitted patients without hba1c or fpg at baseline) and different statistical methods . Hgi subgroup analysis indicated, however, that intensive treatment actually improved primary outcomes in low and moderate hgi participants, a beneficial effect that was offset and masked by the apparently detrimental effects of intensive treatment in the high hgi subgroup . Although total mortality was significantly higher in the intensive treatment group, hgi subgroup analysis showed that the higher mortality associated with intensive treatment was restricted to the high hgi subgroup . Collectively, these observations show that hgi identifies two subpopulations in accord, one that experiences benefits and one that experiences harms from the same intensive glucose - lowering strategy . The risk for hypoglycemia was greatest in the high hgi subgroup in the standard and intensive treatment groups . More high hgi participants remained above the protocol - mandated hba1c treatment thresholds after 1 year of standard or intensive treatment . Despite higher mean hba1c, high hgi participants in both glycemia treatment groups had mean fpg values after 1 year that were as low as or lower than those observed in low hgi participants . Collectively, these observations are consistent with our hypothesis that intensive treatment may have inadvertently caused high hgi participants to receive more intensive treatment and could explain the otherwise paradoxical results reported by miller et al . The twin goals of diabetes management are to keep blood glucose levels low enough to limit the development of long - term diabetes complications but high enough to avoid hypoglycemia . After the accord trial reported that intensive treatment increased mortality and hypoglycemia, the american diabetes association (ada) recommended that hba1c treatment goals for individual patients should be personalized according to characteristics such as age and frequency of hypoglycemia, while also reiterating that lowering hba1c generally helps prevent or delay long - term complications (43). Our observations of markedly different outcomes in the different hgi subgroups in response to intensive treatment strongly supports the ada recommendation for more personalized diabetes management and suggests that hgi could be used to help individualize treatment goals . Exactly how the results of this study might be used to reinterpret other clinical trials or how the results might be applied in future trial designs or in clinical practice remains to be determined . One reason is that accord participants were older than the general public and selected for elevated risk for cvd . This could explain why the baseline regression equation in accord is markedly different from the linear regression equations reported by studies such as the a1c - derived average glucose (adag) study (44). As such, the results may not be generalizable to other clinical trials or to the general population with diabetes and we cannot recommend the use of the accord regression equation in other populations . Furthermore, only one other study has used fpg to assess the hgi in patients with type 2 diabetes, and these results were only reported in a meeting abstract (12). All other prior hgi and glycation gap studies in patients with diabetes used some estimate of mean blood glucose or fructosamine . Additional studies of hgi in other clinical trials could help determine how best to use the hgi in clinical practice . Intensive treatment to a low hba1c target of less than 6% (42 mmol / mol) cannot be recommended for all patients with type 2 diabetes because primary cardiovascular outcomes, total mortality, and hypoglycemia were all adversely affected in high hgi participants in accord . Further studies should determine if the observed beneficial effects of intensive treatment on primary outcomes in low and moderate hgi participants outweigh any detrimental effects that might be caused by the increase in hypoglycemia associated with intensive treatment . Our results confirm that hba1c is not a one - size - fits - all indicator of blood glucose concentration and suggest that failure to take this into account can result in suboptimal diabetes care . Our present analyses examine the accord glycemia trial through a new lens, namely, the hgi calculated using fpg and hba1c measured at baseline . As previously reported by the accord investigators, we observed no difference in primary outcomes between the standard and intensive treatment groups despite our use of a slightly different data set (we omitted patients without hba1c or fpg at baseline) and different statistical methods . Hgi subgroup analysis indicated, however, that intensive treatment actually improved primary outcomes in low and moderate hgi participants, a beneficial effect that was offset and masked by the apparently detrimental effects of intensive treatment in the high hgi subgroup . Although total mortality was significantly higher in the intensive treatment group, hgi subgroup analysis showed that the higher mortality associated with intensive treatment was restricted to the high hgi subgroup . Collectively, these observations show that hgi identifies two subpopulations in accord, one that experiences benefits and one that experiences harms from the same intensive glucose - lowering strategy . The risk for hypoglycemia was greatest in the high hgi subgroup in the standard and intensive treatment groups . More high hgi participants remained above the protocol - mandated hba1c treatment thresholds after 1 year of standard or intensive treatment . Despite higher mean hba1c, high hgi participants in both glycemia treatment groups had mean fpg values after 1 year that were as low as or lower than those observed in low hgi participants . Collectively, these observations are consistent with our hypothesis that intensive treatment may have inadvertently caused high hgi participants to receive more intensive treatment and could explain the otherwise paradoxical results reported by miller et al . The twin goals of diabetes management are to keep blood glucose levels low enough to limit the development of long - term diabetes complications but high enough to avoid hypoglycemia . After the accord trial reported that intensive treatment increased mortality and hypoglycemia, the american diabetes association (ada) recommended that hba1c treatment goals for individual patients should be personalized according to characteristics such as age and frequency of hypoglycemia, while also reiterating that lowering hba1c generally helps prevent or delay long - term complications (43). Our observations of markedly different outcomes in the different hgi subgroups in response to intensive treatment strongly supports the ada recommendation for more personalized diabetes management and suggests that hgi could be used to help individualize treatment goals . Exactly how the results of this study might be used to reinterpret other clinical trials or how the results might be applied in future trial designs or in clinical practice remains to be determined . One reason is that accord participants were older than the general public and selected for elevated risk for cvd . This could explain why the baseline regression equation in accord is markedly different from the linear regression equations reported by studies such as the a1c - derived average glucose (adag) study (44). As such, the results may not be generalizable to other clinical trials or to the general population with diabetes and we cannot recommend the use of the accord regression equation in other populations . Furthermore, only one other study has used fpg to assess the hgi in patients with type 2 diabetes, and these results were only reported in a meeting abstract (12). All other prior hgi and glycation gap studies in patients with diabetes used some estimate of mean blood glucose or fructosamine . Additional studies of hgi in other clinical trials could help determine how best to use the hgi in clinical practice . Intensive treatment to a low hba1c target of less than 6% (42 mmol / mol) cannot be recommended for all patients with type 2 diabetes because primary cardiovascular outcomes, total mortality, and hypoglycemia were all adversely affected in high hgi participants in accord . Further studies should determine if the observed beneficial effects of intensive treatment on primary outcomes in low and moderate hgi participants outweigh any detrimental effects that might be caused by the increase in hypoglycemia associated with intensive treatment . Our results confirm that hba1c is not a one - size - fits - all indicator of blood glucose concentration and suggest that failure to take this into account can result in suboptimal diabetes care.
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During root canal treatment, clinicians face various unwanted procedural mishaps that can occur at any stage of treatment . Of all, instrument fracture within the root canal system and more rarely fractured piece protruding beyond the apex are among the most troublesome and frustrating errors . Fractured instrument extending beyond rotary nickel titanium (niti) files are extensively used for cleaning and shaping of the root canals because of their higher flexibility compared to stainless steel (ss) files . Despite the superior qualities of niti rotary files, niti rotary files fracture because of excessive cyclic fatigue, torsional failure or a combination of both while most of ss instruments fracture due to excessive torque . Various factors that predispose the files to fracture are instrument design, dynamics of instrument use, the manufacturing process, canal configuration, cleaning and sterilization process and frequency of usage of instrument . A previous study documented no adverse effect of the broken fragment retained in the root canal system on healing of endodontically treated teeth while another study reported lower rate of healing when broken instrument remained in the canal . Presence of preoperative periapical radiolucency, inadequate size and apical extent of fractured instrument are some of the prognostic factors affecting the outcome of root canal treatment in such cases . Proper cleaning and shaping of the root canals are hindered or prevented by presence of fractured fragments inside the root canal . Retrieval of fractured instruments is usually very difficult and impossible at times, with a reported success rate of 55 to 79% . Several devices and techniques have been introduced for retrieval of separated instruments such as ruddle irs (dentsply, tulsa, ok, usa), masseran endodontic kit (micro - mega, lynnewood, washington, usa) and the cancellier instrument removal system (sybronendo, orange, ca, usa). Newly developed ultrasonic tips used with piezoelectric ultrasonic units are used for conservative removal of dentin surrounding the separated instrument; moreover, their vibrations facilitate the removal of fractured instrument . The endo success retreatment kit (satelec acteon, mrignac, france) was recently developed for use in satelec acteon piezoelectric ultrasonic device (satelec acteon, mrignac france) to assist in retrieval of fractured instruments, amongst many other uses . Endo success retreatment kit consists of six titanium - niobium mini - tips, designed for retreatment available in different lengths and tapers . Herein, we describe a clinical scenario of instrument retrieval broken beyond the apex of a mandibular molar tooth using endo success ultrasonic tips . A 38 year - old female patient was referred by her local dentist to the endodontics department of manubhai patel dental hospital, for retrieval of a fragment of a niti protaper file (f1, dentsply maillefer, ballaigues, switzerland) broken during root canal enlargement and embedded in the distal canal of the mandibular right first molar extending beyond the apex (tooth #46). During clinical examination, there was an access cavity filled with a temporary filling material and the tooth was sensitive to percussion . One of the two intra - oral periapical (iopa) radiographs brought by the patient revealed incomplete root canal treatment of the right mandibular second premolar and first molar (fig . 1b), showed attempted endodontic retreatment in both premolar and molar and also the separated instrument in the distal canal of the mandibular right first molar extending beyond the apex . (a) pre - operative radiograph (b) retreatment and the fractured file after rinsing the patient s mouth with 0.2% chlorhexidine solution, local anaesthesia was administered and isolation was done with rubber dam . Access cavity was modified using a safe - end fissure bur (dentsply, maillefer, ballaigues, switzerland) to obtain a straight line access to the canals . Then, using a modified gates glidden drill (size 3, dentsply maillefer, ballaigues, switzerland), a staging platform was created . This was done to expose the file and the surrounding dentin to allow thinner ultrasonic tips to trough deeper around the file . After staging, et25 tip of endo success retreatment kit was attached to the ultrasonic device and was activated first at the inner dentinal wall of the distal canal to create a tiny pocket approximately 1.0 mm deep from the severed surface of the file fragment . Once this narrow space was obtained, a shallow groove was cut along the outer dentinal wall such that there was no obstruction to keep the fragment from being pulled coronally . Then, two h files were inserted in an attempt to grab the fractured fragment and pull it out with an anti - clockwise motion . Edta solution was introduced inside the canal to enhance the cavitation and acoustic streaming effect of ultrasonics . Ultrasonic vibration was applied to the separated file in the space created between the fragment and the inner wall of the canal, and moved in push and pull motions until the separated instrument jumped out of the canal . (a) retrieved rotary niti file (b) the 7 mm rotary niti file fragment retrieved (c) retrieved h file (d) the 2 mm h file fragment retrieved (e) obturation (f) one year follow - up the retrieved file fragment was 7 mm long (fig . 2b). But still, the fractured h file was inside the canal, which was retrieved using the ultrasonic vibration . Again, a radiograph was taken and retrieval of both fractured instruments was ensured (fig . After instrument retrieval, working length was determined using an apex locator (propex, dentsply, maillefer, ballaigues, switzerland) and radiographs . The root canals were cleaned and shaped in a crown - down manner using rotary niti files (protaper, dentsply maillefer, ballaigues, switzerland). Next, 2.5% sodium hypochlorite and 2% chlorohexidine were used for irrigating the root canals and calcium hydroxide (calcicur, voco, cuxhaven, germany) as an intracanal medicament was placed . In the second visit, obturation was carried out by lateral compaction technique using gutta percha points (protaper, dentsply, maillefer, ballaigues, switzerland) and ah - plus sealer (dentsply, maillefer, ballaigues, switzerland). 2e) and the patient was referred to her general dental practitioner for the permanent coronal restoration of this tooth and endodontic retreatment of the second premolar . Fracture of an endodontic instrument during root canal treatment hinders further cleaning and shaping of the root canal system . Such inability to further clean and shape the root canal system can compromise the outcome of the treatment . In such cases, it is said that the prognosis depends on the condition of the root canal (vital or non - vital), canal anatomy, type of pulpal pathology, periapical status, degree of cleaning and shaping at the time of separation, the level of file separation in the canal and type of fractured instrument . The prognosis of these teeth is generally lower than that of a tooth with normal endodontic treatment . Orthograde and surgical approaches are the two methods recommended for managing cases with broken instruments . Bypassing the instrument, removing the instrument or preparation of the canal and obturation to the level of the fractured instrument are phases of an orthograde approach . In our case as the patient was referred by some other dentist, we did not know the actual extent of canal disinfection when the instrument broke, and thus, bypassing or retrieving the separated instrument deemed necessary . As the separated instrument extended beyond the apex, bypassing the instrument or obturation to the level of the fractured instrument would not serve the purpose . Considering the non - surgical endodontics being the more conservative approach, the retrieval of instrument was attempted . Diameter, length and position of the fragment within the root canal influence the nonsurgical removal of a broken instrument . Also, the thickness of root dentin, the depth of external concavities and the root canal anatomy influence the removal of the broken fragment . Instruments that lie in the straight portions of the canal can be typically removed . In this case report, instrument was fractured in the distal canal, which was a straight canal with the least curvature . Material type of the fractured instrument is also an important factor to be considered during its removal . The ss files do not fracture upon removal with ultrasonics, while niti instruments may undergo further fracture due to heat build - up when ultrasonic devices are used for their removal . The ss fragments will show early movement as they absorb the ultrasonic energy bodily, while in case of niti fragments, only the point of contact with the tip absorbs the energy . Tu et al . Reported a case in whom, a separated ni - ti instrument was retrieved using several ultrasonic tips under a dental operating microscope from the distolingual root canal of a mandibular first molar . The instrument was separated at the middle third of the canal whereas in our case the file broke in the apical third extending beyond the apex, which made its retrieval more difficult . Recently, a case was reported by shenoy et al, where a separated hand instrument extended beyond the apex was retrieved from the mesiobuccal canal of a second molar . They extracted the tooth atraumatically, retrieved the instrument and reimplanted the tooth again in the socket . In our case, several methods and instrument retrieval systems have been proposed for retrieval of broken instruments from the root canals . However, none of them can guarantee 100% success or can be considered the gold standard for instrument retrieval . Due to various advantages of ultrasonics in instrument retrieval such as minimal dentin damage and compatible tip designs, which can reach the apical third of the canal, however, one must consider that with the advent of rotary niti files, the occurrence of broken instruments has increased, especially in the hands of inexperienced clinicians . Proper training of new techniques and adherence to the established principles and guidelines of clinical usage can reduce the incidence of niti instrument fracture . The ultrasonic technique was successful in removing the rotary niti file fractured beyond the apex and the stainless steel h file from a mandibular molar tooth.
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The occurrence of foreign bodies such as metal screws, staple pins, darning needles, pencil leads, beads and tooth picks lodged in the exposed pulp chambers of carious or traumatically injured deciduous and permanent teeth has been reported . Most often, these cases are diagnosed accidentally on radiographic examination of the tooth which may be associated with infection, pain, swelling and recurrent abscesses as a sequelae to the pulpal exposure and lodgement of the foreign body . Clinical and radiographic examinations are necessary to confirm the presence, size, location and the type of the foreign object . Two cases of foreign objects found within the pulp chambers of the deciduous teeth with their management are presented here . A 10-year - old girl reported with a chief complaint of pain and pus discharge from the upper front tooth on the right side . Patient gave a history of pain since 10 days and pus discharge from the tooth since last 2 days . On clinical examination, there was discoloration and open pulp chamber in relation to right maxillary deciduous canine (53) [figure 1]. Radiographic examination of the tooth revealed a radio - opaque object resembling twisted pieces of metal wire lodged within the pulp chamber and root canal of 53 [figure 2]. The tooth was extracted under local anesthesia [figure 3]. On removal of the foreign objects in relation to the tooth, it was found that there were two staple pins and a small piece of aluminium foil [figure 4]. The child, who had previously denied placing inserting within the tooth, later confessed that she had placed the objects to remove food particles and to get relief from pain . Photograph of discolored deciduous maxillary canine on the right side radiograph showing the foreign object in the tooth photograph showing the metallic object in the extracted tooth two staple pins and pieces of aluminum foil recovered from the tooth a 5-year - old male child was brought to the department by their parents with a complaint of pain in relation to decay in the upper front teeth . Clinical examination revealed that the deciduous right maxillary central incisor (51) was grossly decayed with open pulp chamber [figure 5]. Intraoral periapical radiographs of the maxillary anterior region examination revealed the presence of a radio - opaque foreign object within the pulp chamber and root canal of 51 [figure 6]. The tooth was extracted under local anesthesia and the object was retrieved from the canal, which was found to be the broken head of a sewing needle [figure 7]. Photograph showing decayed maxillary incisors radiograph showing the foreign object in the tooth resembling the head of sewing needle head of sewing needle recovered from the tooth a 10-year - old girl reported with a chief complaint of pain and pus discharge from the upper front tooth on the right side . Patient gave a history of pain since 10 days and pus discharge from the tooth since last 2 days . On clinical examination, there was discoloration and open pulp chamber in relation to right maxillary deciduous canine (53) [figure 1]. Radiographic examination of the tooth revealed a radio - opaque object resembling twisted pieces of metal wire lodged within the pulp chamber and root canal of 53 [figure 2]. The tooth was extracted under local anesthesia [figure 3]. On removal of the foreign objects in relation to the tooth, it was found that there were two staple pins and a small piece of aluminium foil [figure 4]. The child, who had previously denied placing inserting within the tooth, later confessed that she had placed the objects to remove food particles and to get relief from pain . Photograph of discolored deciduous maxillary canine on the right side radiograph showing the foreign object in the tooth photograph showing the metallic object in the extracted tooth two staple pins and pieces of aluminum foil recovered from the tooth a 5-year - old male child was brought to the department by their parents with a complaint of pain in relation to decay in the upper front teeth . Clinical examination revealed that the deciduous right maxillary central incisor (51) was grossly decayed with open pulp chamber [figure 5]. Intraoral periapical radiographs of the maxillary anterior region examination revealed the presence of a radio - opaque foreign object within the pulp chamber and root canal of 51 [figure 6]. The tooth was extracted under local anesthesia and the object was retrieved from the canal, which was found to be the broken head of a sewing needle [figure 7]. Photograph showing decayed maxillary incisors radiograph showing the foreign object in the tooth resembling the head of sewing needle head of sewing needle recovered from the tooth as children often tend to have the habit of inserting foreign objects in the oral cavity, there are more chances of finding foreign objects in their teeth . Sometimes, children do not reveal to their parents due to fear . Grossman, gelfman, and harris reported retrieval of indelible ink pencil tips, brads, a tooth pick, adsorbent points, tomato seed, pins, wooden toothpick, a pencil tip, plastic objects, toothbrush bristles and crayons from the root canals of anterior teeth left open for drainage . Mcauliffe has suggested various radiographic methods to be followed to localize a radio - opaque foreign object, such as parallax views, vertex occlusal views, triangulation techniques, stereo radiography and tomography . The steglitz forceps have also been described for use of removal of silver points from the root canal . There is a description of an assembly of a disposable injection needle and thin steel wire loop, formed by passing the wire through the needle being used . This assembly was used along with a mosquito hemostat to tighten the loop around the object . In the cases in the first case, the patient first denied of inserting anything in the tooth but on further questioning she admitted of inserting staple pins, foils and papers in the tooth . As the prognosis was poor, the tooth was extracted . On removal of the foreign objects from the extracted tooth, we found two staple pins and a piece of aluminum foil . In the second case, the child came with a complaint of pain in the primary central incisor . On routine radiographic examination, broken head of sewing needle was found . Again here, the parents were not aware of the fact that child had inserted sewing needle in the tooth . But the parents were not willing to come again and were insisting for the removal of the tooth . Though the presence of foreign objects retrieved from the root canals and pulp chambers of the permanent teeth have been reported, the presence of foreign objects found in the deciduous teeth is an uncommon situation . Timely diagnosis and management of foreign object embedded in the tooth should be done to avoid further complication.
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Inflammatory bowel disease (ibd) is a chronic, relapsing, idiopathic inflammation of the gastrointestinal tract . Subtypes of ibd include crohn's disease (cd), ulcerative colitis (uc), and inflammatory bowel disease unclassified (ibd - u). The majority of epidemiological studies on the incidence and prevalence of ibd relate to the adult population . Historically, europe and north america have been considered high incidence areas while asia, africa, and the middle east have been considered low incidence areas [2, 3]. Emerging data has suggested that the incidence of ibd is increasing globally in both developed and developing countries . One study from central saudi arabia on the epidemiology of juvenile onset ibd estimated an incidence of 0.5 per 100 000 per year and a prevalence of 5/100 000 . While this is significantly lower than the incidence rates of 11.43/100 000 per year reported in north america, comparison with older data nevertheless suggests an increasing incidence . The emergence of chronic inflammatory diseases such as ibd has been closely linked to social and economic development and it has been postulated that the westernisation of society accounts for the increasing incidence of ibd in countries where it was once considered rare . The importance of ethnic, racial, and geographic factors in ibd is illustrated by the considerable literature citing varying risks of developing ibd in different ethnic populations . It has been well established that the ashkenazi jewish population have a higher risk of developing ibd than other ethnic groups [7, 8]. Several studies also show that ethnic groups with low rates of ibd in their home country have a much higher incidence of ibd following immigration to western countries . For instance, studies on migrant populations found a higher incidence of ibd in south asians than non - south asians in the pediatric population of british columbia and the adult population of leicester [911]. Recent studies have found the overall incidence rate of ibd in australia to be among the highest reported . In addition, phavichitr et al . Found that the incidence of pediatric cd in victorian children rose from 0.128 per 100 000 per year in 1971 to 2.0 per 100 000 per year in 2001 . However, there are no reports on the epidemiology of ibd in specific ethnic groups in australia . Given the fact that australia is a multicultural society with significant emigration of families from the middle east this may be relevant . Therefore the objectives of the current study were to examine the clinical characteristics and management of ibd in children of middle eastern descent diagnosed at the sydney children's hospital randwick (schr). A retrospective chart review was undertaken on all patients identified as being of middle eastern ethnicity on the schr ibd database . Ibd specific data collection began at schr in 1987 and data was complete up to the year 2011 at the time of review . At diagnosis, children were considered to be of middle eastern ethnicity if one or both parents self - identified as being of middle eastern ethnicity or from any of the following countries: egypt, iran, iraq, israel, jordan, kuwait, lebanon, oman, qatar, saudi arabia, syria, united arab emirates, west bank and gaza, and yemen . Children with israeli ancestry and jewish ethnicity were not included in this study due to the well - defined predisposition to ibd in this population . A control group of patients of non - middle eastern descent was also identified from the schr ibd database: these were matched to patients of middle eastern descent according to age at diagnosis, gender, and disease type . The project was approved by the south eastern sydney and illawarra area health service research ethics committee . Information collected at diagnosis for the study and control groups included family history of ibd in first degree relatives, smoking exposure history, residential postal code, age at diagnosis, symptoms at presentation, duration of symptoms at presentation, specific blood tests (erythrocyte sedimentation rate (esr), c - reactive protein (crp), platelets, albumin, haematocrit, alanine transaminase (alt), aspartate transaminase (ast)), disease location, extraintestinal manifestations, pediatric crohn's disease activity index (pcdai) or pediatric ulcerative colitis activity index (pucai) score, height, and weight . Disease management information was also collected including whether they received the following treatments: exclusive enteral nutrition (een), corticosteroids, aminosalicylates, thiopurines, methotrexate, biological, tacrolimus, or surgical intervention . Disease location was classified according to the montreal classification of l1 (terminal ileum), l2 (colon), l3 (ileocolonic), and l4 (upper gastrointestinal (gi)) for cd . Symptoms at presentation were grouped under the following categories: abdominal pain, diarrhoea, mucus and/or blood in stools, weight loss, per rectal bleeding, and loss of appetite . Height and weight measurements at diagnosis were converted to height for age z - scores and weight for age z - scores using the centre for disease control application epiinfo, based on the cdc-2000 charts . Point incidence and point prevalence were calculated for both the middle eastern study group and the control group for the schr catchment area . For this purpose the catchment of the schr was defined as the local government areas (lga) of the south eastern sydney and illawarra area health service, which includes botany, hurstville, kogarah, randwick, rockdale, sutherland, sydney, waverley, wollongong, and woollahra . Population information for the lgas was obtained from the australian bureau of statistics 2006 census data . Ancestry information in the 2006 census data was collected by similar means to data in the schr database, where ancestry was defined by self - reporting of familial ancestry and birthplace . The census data was sorted by ancestry, lga, and age (016 years). The schr ibd database was used to identify patients who resided in the defined catchment area and had active inflammatory bowel disease in 2006 to calculate point prevalence . The database was also used to identify those patients within the catchment area who were diagnosed in 2006 to calculate the point incidence . The remoteness area category was calculated for each patient from residential postal codes based on the australian standard geographical classification - remoteness area (asgc - ra). The asgc - ra is a hierarchical classification system of geographical areas developed by the australian bureau of statistics (abs) that provides a common framework of statistical geography . The categories used were ra1 (major cities of australia), ra2 (inner regional australia), ra3 (outer regional australia), ra4 (remote australia), and ra5 (very remote australia). A fisher's exact test was used to compare the two groups with regard to smoking exposure history, family history, symptoms at presentation, extraintestinal manifestations, and disease location . The management of ibd was analysed by fisher's exact tests, with uc and cd being analysed separately . An unpaired t - test was used to compare platelets, albumin, haematocrit, pcdai, height for age z - scores, and weight for age z - scores between the two groups . The mann - whitney test was used to compare the groups for esr, crp, alt, and ast . The relative risk (rr) was calculated for incidence and prevalence; a result was considered significant if the confidence intervals (ci) did not embrace a relative risk of one . Of the 441 patients on the schr ibd database, 35 (7.9%) were identified as being of middle eastern ethnicity . However, 11 of the 35 were excluded from this retrospective study as files for these patients were unavailable . Therefore a final cohort of 24 patients of middle eastern ethnicity (both parents of middle eastern ethnicity) and 24 non - middle eastern controls were included in this study . Of the 24 patients of middle eastern ethnicity, 14 (58.3%) had cd, 7 (29.2%) had uc, and the remainder had ibd - u (table 1). Fifteen (62.5%) of the group were male: 9 of these had cd and 4 had uc and 4 had ibd - u . Twenty (83.3%) patients were born in australia, 2 (8.3%) were born in lebanon, and 2 were born in the usa . The mean age at diagnosis overall was 9.8 years (range, 0.715.7). Four patients (16.6%) were diagnosed under the age of five years and 11 patients (45.8%) were diagnosed before the age of 10 years . There was mean of 92 days (range 23227 days) between date of diagnosis of the middle eastern ethnicity patients and their matched controls . Identified as middle eastern ethnicity, 16 had parents identified as lebanese, 3 egyptian, 2 turkish, and 1 algerian, and 2 parents did not provide a country of birth but self - identified as middle eastern ethnicity . Of the controls, 18 had parents identified as caucasian, 2 indian, and 1 caucasian - jewish, and 3 did not provide a country of birth but self - identified as non - middle eastern ethnicity . There was no difference in family history of ibd in first - degree relatives of middle eastern (5/22; 2 unknown; 22.7%) and control (2/22; 2 unknown; 9.1%) patients . There was no difference in smoking exposure history between the two groups, middle eastern (7/21; 33.3%) and control patients (5/19; 26.3%). All middle eastern patients (24/24; 100%) were living in ra1 (major city), while the controls had fewer patients (17/24; 70.8%) in ra1 and a greater distribution over ra2 (4/24; 16.7% inner regional australia) and ra3 (3/24; 12.5% outer regional australia) areas (p = 0.017). Symptom duration prior to diagnosis did not vary between the middle eastern (median 8, range 1208 weeks) and control groups (median 16, range 2104 weeks) (p = 0.37). Abdominal pain and diarrhoea were the most common symptoms at presentation for both groups (table 2). Erythrocyte sedimentation rate (esr) at diagnosis was more elevated in middle eastern children compared to controls (p = 0.02); however, all other standard blood results were similar in both groups (table 3). Alt and ast values were lower in the middle eastern group compared to the control group (p = 0.03 and p = 0.02, resp .) (table 3). Pcdai scores at diagnosis were significantly higher in the middle eastern group (mean 37, sd 13) compared to the control group (mean 27, sd 11; p = 0.033) (figure 1). Height for age z - score and weight for age z - scores at diagnosis were similar between the groups . Two (8.3%) of the middle eastern patients and one (4.2%) control had height for age z - score indicating stunted growth (<2 sd). There was a lower incidence of colonic disease (l2) (p = 0.01) in the middle eastern group with cd compared with the control group (table 4). Upper gi disease was present in 10 (71%) of the controls and 13 (93%) of the middle eastern cd patients . Terminal ileal location (l1), ileocolonic disease (l3), and upper gi tract involvement (l4) were similar in both patient groups (table 4). There was no difference between the groups for disease location in uc as most patients in both groups had pancolitis (e3). The incidence of ibd in the schr catchment area in 2006 for the middle eastern pediatrics population (aged 016 years) was higher (33.1 per 100 000 children per year) compared to the control group (4.3 per 100 000 children per year). The relative risk analysis, although indicating a high risk of ibd with middle eastern ethnicity, does not reach significance (rr 7.63, 95% ci 0.9565.01) (figure 2). However the prevalence of ibd in the middle eastern pediatric population was significantly higher at 165.4 per 100 000 children compared to the control prevalence rates of 28.7 per 100 000 children (rr 5.76, 95% ci 2.3014.43) (figure 2). Overall, there were no differences in the use of standard medical therapies between the groups (p> 0.05 for all). In addition, there was no difference in surgical management between the two groups (p> 0.05). However, considering the children with cd separately, the use of thiopurines was significantly higher in the middle eastern group for the management of cd (p = 0.002) (table 5). There was no difference in use of corticosteroids, aminosalicylates, biologicals, or tacrolimus for management of cd between the groups (table 5). There was also no difference for any of the therapies between the groups for the management of uc . This is the first study comparing the incidence, presentation, and management of ibd in a middle eastern pediatric population now residing in a the estimated incidence rate for ibd in this population in 2006 was among the highest reported in the pediatric literature and was almost 8 times higher than that observed for the non - middle eastern population in the same location . There is great variation in the literature on the difference in incidence between different populations . However our findings are consistent with those of other studies that have identified higher risk of ibd in specific groups such as south asians and ashkenazi jews [7, 9]. Accurate prevalence and incidence rates of pediatric ibd for many middle eastern countries have not been reported in the literature . It has been postulated that the low incidence of ibd in developing countries is attributed to poor sanitation and hygiene and greater exposure to microorganisms during childhood . The recent rise in incidence in both developed and developing countries has coincided with improvements in hygiene over the twentieth century and the move from a lifestyle of high microbial exposure to low microbial exposure [6, 16]. Previous studies have reported people who emigrate to western industrialised countries are at higher risk of developing ibd . Similar findings of increased incidence upon migration have been reported in patients of south asian origin upon emigration to canada and the uk . Interestingly, 83.3% of the middle eastern patients in the current study were born in australia, adding support to the theory that the 2nd generation of immigrants to industrialised countries is most at risk of developing ibd . We have presented point incidence and point prevalence; however, there are a number of limitations that must be considered when assessing this data . The cohort was limited to the geographical catchment area of one pediatric centre in sydney, australia . Taking into consideration the limitations of this dataset, the small sample size and potential confounders due to immigration, emigration, and referrals outside of the catchment area, the results presented here may have either overestimated or underestimated the incidence of ibd in this population . Ahuja and tandon suggested studies that relying on the reporting of pediatric hospitals, such as the current study, may lead to an underestimation of incidence and an overestimation of disease severity . The control cohort were matched based on age, gender, and type of disease; therefore, 24 controls were included from 406 non - middle eastern patients listed in the ibd database . Several patients listed in the ibd database were excluded from the incidence and prevalence calculations as they came from outside the schr catchment area . Although the numbers are likely to be small, it is also possible that several pediatric ibd patients, both middle eastern and non - middle eastern, from within the catchment area were attending other hospitals or were being treated as private patients and as such were not included in the schr database . Therefore, we propose that these initial findings indicate that a further population based cohort study is warranted . The gender preponderance (higher number of boys) observed among these middle eastern children contrasts with studies of the adult ibd population where there are a slightly greater proportion of females with cd, although a recent report by el mouzan et al . Of childhood - onset ibd in saudi arabia also reports a higher predominance of males with cd at 56% . Nevertheless this pattern of disease distribution (58.3% cd, 29.2% uc, and 12.5% ibd - u) is consistent with recent british, canadian, and american studies of pediatric cd populations [1921]. Middle eastern children with cd had significantly less disease restricted to the colon (l2) than the controls while there were similar levels of terminal ileal disease, ileocolonic disease, and upper gastrointestinal involvement between the groups . In contrast to this, studies in other ethnic populations have found more extensive colonic disease than the general ibd population . However, this is consistent with a low incidence of colonic disease and high incidence of ileocolonic disease that has been observed in a series of kuwaiti children with ibd . In the current study, excluding upper gastrointestinal involvement (l4), ileocolonic disease (l3) was the most common presentation site affected in middle eastern cd patients . Studies estimate that upper gastrointestinal involvement occurs in the range of 3080% in children and less than 10% of adults with cd [2326]. In concordance with this, a large proportion of both groups in the current study had upper gi (l4) involvement . The comparatively high use of thiopurines in the treatment of cd in the middle eastern patients is suggestive of a more severe disease requiring immunosuppressive treatment . The efficacy of thiopurines in maintaining clinical remission in cd is well established with trial data supporting the introduction of thiopurines in children with moderately severe disease at diagnosis . Interestingly, in the current study, the severity of disease was not reflected in the duration of symptoms, symptoms at presentation, or surgical management, as there were no differences between the two groups . However the middle eastern patients had higher cd activity scores and esr at diagnosis than the controls . This finding, along with the higher thiopurine use, is suggestive of a more active disease in middle eastern children . The mean age at diagnosis for this cohort of patients was 9.8 years, which was slightly lower than reported in the literature . Family history appears to be one of the most important factors that confer risk for the development of ibd . No difference was established in family history rates between the middle eastern (22.7%) and non - middle eastern patients (9.1%). Recent publications of pediatric ibd in saudi arabia report family history rates of 15.3% and 9.4% . Further, the incidence of a positive family history in the middle eastern patients was comparable to that observed in a series of kuwaiti children . Despite this, there is variability in the family history rates reported which likely reflects the small numbers of patients in these reports . Therefore further investigation is required to determine if middle eastern children are at the same or greater risk of ibd than non - middle eastern children . Consanguinity data was not available for either the middle eastern or non - middle eastern cohorts and could not be considered when assessing family history rates . Altered linear growth is commonly present in children at the time of presentation with ibd [29, 30]. A pediatric study in kuwait found that growth failure was a significant problem in their patients at presentation . In contrast to this, only 8.3% of the middle eastern children and 4.2% of controls had height for age z - scores indicative of stunted growth . This inconsistency may be due to the small sample size or may represent shorter symptom duration prior to diagnosis and therefore less impact upon linear growth . Although the pubertal status of the children at diagnosis in the current retrospective study was not available, many of the children were of a prepubertal age . In addition to the limitations provided by a retrospective study design, the sample size of the current study also limited more complete full data interpretation . The sample size also likely influenced the interpretation of the incidence rates between the two groups: this lack of significance may reflect a type 2 error . In conclusion, the present study indicated that middle eastern patients were less likely to have disease restricted to the colon than the control children . Further the middle eastern children had higher cd activity at diagnosis and also required a higher incidence of immunosuppressive treatment . This data is consistent with a more severe phenotype of cd in middle eastern children . Although there were limitations with the dataset used to calculate point incidence and prevalence, the calculated values indicated that there is a higher point incidence and prevalence of ibd in middle eastern children attending the schr . Likely these patterns of disease in an ethnic group now resident in australia reflect the interactions between environmental and genetic factors . Further epidemiological and genetic investigations of such populations with high incidence of disease are required to better understand the aetiology of pediatric ibd.
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As more and more protein sequences become available with their structures undetermined, recognizing functional signatures directly from sequences is particularly desirable in functional proteomics (13). Automatic discovery of patterns in unaligned biological sequences is an important problem in molecular biology (48). For a good review on the mining algorithms, the readers can refer to refs (911). When compared with the approaches based on multiple sequence alignment in identifying functional regions, pattern mining algorithms have the advantage of automatically determining the subset of sequences involved in the final mining results (12). The derived patterns are useful in many research issues in bioinformatics, including automatic functional annotation of sequences, database search of homologues, detection of functional sites and prediction of hot regions in protein protein interactions (2,1315). Frequently used models include regular expressions, profiles and hidden markov models (hmms) (10). This paper focuses on discovery of patterns expressed in regular expression and considers only exact components in a pattern . An exact component permits only one specific amino acid in one position, such as the capital letters in the pattern n - r - x(5,19)-y - x - g - x(3)-d . In this example, x(3) are called rigid gaps, a gap of fixed length, which are composed of one and three wildcards, respectively . On the other hand, x(5,19) is a flexible gap, a gap of flexible length, which admits at least five successive wildcards and at most 19 successive wildcards in between the exact components r and y. a flexible gap handles the don't - care regions where large insertions and deletions might happen during evolution, while a rigid gap deals with the conservative substitutions allowed in biological sequences . When only exact elements are considered in mining process, the derived patterns are usually very sparse, in which the pattern elements are interleaved with a large amount of gaps . Patterns of this type are hard to detect but are greatly appreciated because they concisely highlight the important residues associated with protein functional sites . In proteins, the conserved residues usually appear as clusters (it is called a block in this paper), and multiple clusters together constitute an important substructure . The conserved regions that strongly correlate with each other and conserved simultaneously are usually interleaved with large irregular gaps (15,16). In other words, the residues associated with a functional motif are not necessarily found in one region of the sequence (2,1519). This complicates the mining process and often confuses the approaches based on multiple sequence alignment . Regular expression is considered as a deterministic model contrary to the probabilistic models such as profiles and hmm (10). A deterministic pattern can be matched or not matched by a sequence . In the mining process, a pattern will be reported as long as it matches more than a user - specified percentage of the input sequence set . A pattern is said to be diagnostic for a family if it matches all the known sequences in the family, and no other known sequence (9). However, a diagnostic pattern does not always correspond to a functional signature . By setting the minimum support constraint as a lower value, magiic - pro can discover patterns that really present as functional signatures but are only conserved in a subset of input sequences . Discovering sparse and flexible patterns which are conserved in only a subset of input sequences is a time - consuming task due to the large search space of solutions . So many related studies employ other constraints in addition to the minimum support constraint to expedite the mining process . Mining algorithms that consider only short conserved words (4,5,17,18) or rigid gaps (68,12), such as web service teiresias (12), are efficient and effective in identifying short motifs . On the other hand, the pratt (21) algorithm introduced the concept of gap flexibility to enlarge the search space . Furthermore, it has been shown by experiments in our recent work that considering large flexibilities causes the failure of pratt to deliver satisfied results within an acceptable time (16). Different from the previous works, our approach considers two types of gaps to improve the mining efficiency, where the gaps within a conserved region are called an intra - block gap and the gaps in between two adjacent conserved regions are called inter - block gaps (16). Using two types of gap constraints for different purposes improves the efficiency of mining process while keeping high accuracy of mining results . The server magiic - pro further employs rigid intra - block gaps instead of the flexible ones proposed in (16) since it has been observed in protein sequences that insertions and deletions are seldom present in highly conserved regions (2,12). Our experimental results also reveal that considering only rigid gaps within a block is useful in eliminating noisy patterns . Magiic - pro provides many useful tools for examining and visualizing the derived patterns, which will be described in detail later . After that, we will show by experiments that magiic - pro is efficient and effective in identifying functional sites and predicting hot regions in protein protein interactions . The web service magiic - pro is in particular designed for mining protein sequences, where the kernel algorithm executing sequential pattern mining is based on our previously developed algorithm magiic (16) incorporated with several state of the art data mining techniques . Magiic - pro first quickly identifies rigid gapped blocks by bounded - prefix growth technique of magiic . After that, the candidate blocks are concatenated into patterns with large irregular gaps by exploiting the antimonotone characteristic of this problem (19,20). Finally, a newly proposed bounded - gap closure checking scheme developed based on (22) is executed to eliminate patterns that can be covered by other super patterns with the same occurrences . After the mining process terminates, magiic - pro generates a pattern snapshot that shows all the derived patterns in alignment with the query protein . The residues present in different patterns are combined together to create a conservation plot, where the conservation level of each residue is determined by the percentage of total number of supporting proteins merged from different patterns . The conservation plot provides a whole picture about the conserved residues of a query protein . We assume that every user of magiic - pro has a protein sequence of interest at hand . Magiic - pro takes a protein sequence as input, and helps the users to prepare the training data for pattern mining . The task of collecting relative sequences of the query protein can be achieved by using swiss - prot annotations or executing the psi - blast program . Once the query protein and the training data have been determined, the mining process is executed using the parameters described in the following subsection . A pattern will be reported as long as it matches at least a certain number of sequences . The support constraint is critical to the mining results, but it might not be possible to know in advance by what percentage level a satisfied pattern can be discovered . Since lower values bring more patterns, the users are suggested to start with a large support constraint, e.g. 90%, and magiic - pro will decrease it gradually until a desired number of patterns have been found . In addition to the minimum support constraint, magiic - pro has some other parameters for advanced users . Before going into the details assume that a pattern is consisted of pattern elements as a sequence, and each successive pair of elements is either interleaved with a gap or not . In this work, small and rigid gaps are considered as intra - block gaps, while large and flexible gaps are treated as inter - block gaps . The notation x(a, b), a <b, is used for a flexible gap with minimum length of a and maximum length of b, and x(a) stands for a rigid gap with a fixed length of a. the wildcard x(a) is omitted if a = 0, and is written as x if a=1, i.e. X = x(1). The maximum length of an intra - block gap (default value=3);the maximum relative flexibility of an inter - block gap with respect to the length of the inter - block gap present in the query protein (default value=30%); the maximum length of an intra - block gap (default value=3); the maximum relative flexibility of an inter - block gap with respect to the length of the inter - block gap present in the query protein (default value=30%); the second group of the advanced parameters specifies the size or length constraints . The minimum number of elements in a block (default value = 3);the minimum number of blocks in a pattern (default value = 2); the minimum number of elements in a block (default value = 3); the minimum number of blocks in a pattern (default value = 2); we argue that a pattern should have at least two blocks to be meaningful, because an important region is seldom to be conserved singly either from structural or functional aspects . In this way after the mining process finishes, the users can first take a look on the conservation plot and pattern snapshot . As shown in the figure 1a, the locations of the conserved regions are summarized in the complete conservation plot derived from all the patterns . It can be observed in figure 1a that there are nine conserved regions in the query protein . In the same web page, the users are provided with an interactive interface to collect patterns of interest in a pattern snapshot . Different from the conservation plot, a pattern snapshot in addition tells which pattern blocks are simultaneously conserved during evolution . The users are suggested to browse the lists of the top 10 high - support and top 10 large - size patterns . The size of a pattern is defined as the number of exact components it contains . A pattern with a high support usually highlight the most highly conserved residues that are related to a functional region, while a longer pattern with a lower support in general provides a complete signature with respect to a functional site . (b) top 10 high - support patterns with three or more blocks . (c) top 10 large - size patterns with three or more blocks examples of pattern snapshots and the conservation plot provided by magiic - pro . (b) top 10 high - support patterns with three or more blocks . (c) top 10 large - size patterns with three or more blocks here we use the same example from figure 1a to explain how the interactive snapshot can facilitate examining the mining results . In figure 1b, we first examine the top 10 high - support patterns with 3 or more blocks . Similar patterns can be considered as being associated with the same functional site, but each of them is distinct from the others because the sets of supporting sequences are different . The top one pattern in figure 1b identifies the most three important regions of this query protein, which are related to the binding sites of the ligands fad and nap, denoted as the blocks 1, 2 and 3 in both figures 1a and 2 . Next, we can request the top ten large - size patterns with three or more blocks . It is observed in figure 1c that blocks 4 and 5 are the next most conserved blocks that are simultaneously conserved with blocks 1, 2, and 3, and the further next is the block 6 . The top one large - size pattern in figure 1c is plotted with an available pdb structure in figure 2, showing that these six blocks together constitute a complete signature regarding the binding sites of ligands fad and nap . Figure 2.a pattern plotted with an available structure of the oxidoreducatase fad / nad(p)-binding protein . Structures are shown with the conserved pattern blocks plotted with sticks in different colors, block r - x - y - s - x(2)-s highlighted in green, block g - t - g - x - a - p in yellow, block g - x(3)-l - x(2)-g in pink, block a - x - s - r in orange, block k - x - y - x - q in deep pink, and block y - x - c - g in purple, the ligand fad plotted with ball - and - stick representation in blue, and the ligand nap with ball - and - stick representation in brown . Structures are shown with the conserved pattern blocks plotted with sticks in different colors, block r - x - y - s - x(2)-s highlighted in green, block g - t - g - x - a - p in yellow, block g - x(3)-l - x(2)-g in pink, block a - x - s - r in orange, block k - x - y - x - q in deep pink, and block y - x - c - g in purple, the ligand fad plotted with ball - and - stick representation in blue, and the ligand nap with ball - and - stick representation in brown . (pdb code:1qfy: a, query protein: p10933, fenr1_pea). To facilitate studying the patterns of interest, we provide five useful links for each pattern . Second, the derived pattern can be plotted with a protein structure if there are pdb entries available for any of the supporting sequences . Third, the derived pattern can be fed to the scanprosite web service to check its selectivity, the ability to reject false positive matches . Fourth, the users can perform a multiple sequence alignment on the segments of supporting sequences that are associated with the selected pattern . This helps the user to construct a more generalized pattern with amino acid substitutions considered . Fifth, magiic - pro aligns each excluded sequence with the segment of the query protein . We assume that every user of magiic - pro has a protein sequence of interest at hand . Magiic - pro takes a protein sequence as input, and helps the users to prepare the training data for pattern mining . The task of collecting relative sequences of the query protein can be achieved by using swiss - prot annotations or executing the psi - blast program . Once the query protein and the training data have been determined, the mining process is executed using the parameters described in the following subsection . A pattern will be reported as long as it matches at least a certain number of sequences . The support constraint is critical to the mining results, but it might not be possible to know in advance by what percentage level a satisfied pattern can be discovered . Since lower values bring more patterns, the users are suggested to start with a large support constraint, e.g. 90%, and magiic - pro will decrease it gradually until a desired number of patterns have been found . In addition to the minimum support constraint, magiic - pro has some other parameters for advanced users . Before going into the details assume that a pattern is consisted of pattern elements as a sequence, and each successive pair of elements is either interleaved with a gap or not . In this work, small and rigid gaps are considered as intra - block gaps, while large and flexible gaps are treated as inter - block gaps . The notation x(a, b), a <b, is used for a flexible gap with minimum length of a and maximum length of b, and x(a) stands for a rigid gap with a fixed length of a. the wildcard x(a) is omitted if a = 0, and is written as x if a=1, i.e. X = x(1). The maximum length of an intra - block gap (default value=3);the maximum relative flexibility of an inter - block gap with respect to the length of the inter - block gap present in the query protein (default value=30%); the maximum length of an intra - block gap (default value=3); the maximum relative flexibility of an inter - block gap with respect to the length of the inter - block gap present in the query protein (default value=30%); the second group of the advanced parameters specifies the size or length constraints . The minimum number of elements in a block (default value = 3);the minimum number of blocks in a pattern (default value = 2); the minimum number of elements in a block (default value = 3); the minimum number of blocks in a pattern (default value = 2); we argue that a pattern should have at least two blocks to be meaningful, because an important region is seldom to be conserved singly either from structural or functional aspects . In this way after the mining process finishes, the users can first take a look on the conservation plot and pattern snapshot . As shown in the figure 1a, the locations of the conserved regions are summarized in the complete conservation plot derived from all the patterns . It can be observed in figure 1a that there are nine conserved regions in the query protein . In the same web page, the users are provided with an interactive interface to collect patterns of interest in a pattern snapshot . Different from the conservation plot, a pattern snapshot in addition tells which pattern blocks are simultaneously conserved during evolution . The users are suggested to browse the lists of the top 10 high - support and top 10 large - size patterns . The size of a pattern is defined as the number of exact components it contains . A pattern with a high support usually highlight the most highly conserved residues that are related to a functional region, while a longer pattern with a lower support in general provides a complete signature with respect to a functional site . Figure 1.examples of pattern snapshots and the conservation plot provided by magiic - pro . ((b) top 10 high - support patterns with three or more blocks . (c) top 10 large - size patterns with three or more blocks examples of pattern snapshots and the conservation plot provided by magiic - pro . ((b) top 10 high - support patterns with three or more blocks . (c) top 10 large - size patterns with three or more blocks here we use the same example from figure 1a to explain how the interactive snapshot can facilitate examining the mining results . In figure 1b, we first examine the top 10 high - support patterns with 3 or more blocks . Similar patterns can be considered as being associated with the same functional site, but each of them is distinct from the others because the sets of supporting sequences are different . The top one pattern in figure 1b identifies the most three important regions of this query protein, which are related to the binding sites of the ligands fad and nap, denoted as the blocks 1, 2 and 3 in both figures 1a and 2 . Next, we can request the top ten large - size patterns with three or more blocks . It is observed in figure 1c that blocks 4 and 5 are the next most conserved blocks that are simultaneously conserved with blocks 1, 2, and 3, and the further next is the block 6 . The top one large - size pattern in figure 1c is plotted with an available pdb structure in figure 2, showing that these six blocks together constitute a complete signature regarding the binding sites of ligands fad and nap . Figure 2.a pattern plotted with an available structure of the oxidoreducatase fad / nad(p)-binding protein . Structures are shown with the conserved pattern blocks plotted with sticks in different colors, block r - x - y - s - x(2)-s highlighted in green, block g - t - g - x - a - p in yellow, block g - x(3)-l - x(2)-g in pink, block a - x - s - r in orange, block k - x - y - x - q in deep pink, and block y - x - c - g in purple, the ligand fad plotted with ball - and - stick representation in blue, and the ligand nap with ball - and - stick representation in brown . Structures are shown with the conserved pattern blocks plotted with sticks in different colors, block r - x - y - s - x(2)-s highlighted in green, block g - t - g - x - a - p in yellow, block g - x(3)-l - x(2)-g in pink, block a - x - s - r in orange, block k - x - y - x - q in deep pink, and block y - x - c - g in purple, the ligand fad plotted with ball - and - stick representation in blue, and the ligand nap with ball - and - stick representation in brown . (pdb code:1qfy: a, query protein: p10933, fenr1_pea). To facilitate studying the patterns of interest second, the derived pattern can be plotted with a protein structure if there are pdb entries available for any of the supporting sequences . Third, the derived pattern can be fed to the scanprosite web service to check its selectivity, the ability to reject false positive matches . Fourth, the users can perform a multiple sequence alignment on the segments of supporting sequences that are associated with the selected pattern . This helps the user to construct a more generalized pattern with amino acid substitutions considered . Fifth, magiic - pro aligns each excluded sequence with the segment of the query protein . In this section, we first demonstrate the efficiency of magiic - pro in identifying long patterns based on the 13 datasets with different levels of similarities listed in table 1 . With the default settings of the advanced parameters, magiic - pro starts the search by setting the minimum support constraint as 90%, and decreases this constraint step by step until at least one pattern have been found . At this stage, we observed that most of the patterns with the maximum support are related to a functional site of the query protein but do not serve as a complete signature of a functional site . In order to find patterns with more conserved blocks involved, we continued decreasing the minimum support constraint and stopped the process when the calculation time of a single mining task is longer than 60 s. table 1 reports the minimum support where we stopped for each dataset, as well as the searching time used in the latest search . Table 1 also shows the number of blocks generated in the first stage of the mining process and the number of derived patterns with at least two blocks . It is observed in table 1 that a large amount of single blocks do not collaborate with other blocks to form a longer pattern . The number of patterns converges rapidly when the number of blocks in a pattern increases . The patterns found in the top 10 high - support and the top 10 large - size lists of each dataset demonstrate the potential of magiic - pro in identifying functional sites and hot regions in protein protein interactions . Owing to the limited space, we only show one experimental result in the following paragraph, while the others are provided on the web page of magiic - pro . Table 1.analysis of the efficiency of magiic - proquery proteinsize of training datasetting of minimum support (%) no . Of blocks derived in the first stageno . Of the derived patterns with different number of blockstime used (in s)23456o14965 (stk6_human) serine / threonine - protein kinase 619106023427p51656 (dhb1_mouse) estradiol 17-beta - dehydrogenase 149420211462349p19120 (hsp7c_bovin) heat shock cognate 71 kda protein47390115312p00962 (syq_ecoli) glutaminyl - trna synthetase34670751244p10933 (fenr1_pea) ferredoxin nadp reductase2802014901112948618192723p08622 (dnaj_ecoli) chaperone protein dnaj275807986786655p25910 (blab_bacfr) beta - lactamase type ii precursor267527128601392624p27142 (kad_bacst) adenylate kinase24380146178332p22887 (ndkc_dicdi) nucleoside diphosphate kinase2337078187169371p09372 (grpe_ecoli) protein grpe1953047915331836599239p00730 (cbpa1_bovin) carboxypeptidase a1 precursor57501414972931p08692 (arsc1_ecoli) arsenate reductase5170187<1p35568 (irs1_human) insulin receptor substrate 12580202<1the symbol hyphen stands for no patterns found. Analysis of the efficiency of magiic - pro the symbol hyphen stands for no patterns found. Here we use the case of query protein p00730 to illustrate that the long patterns found by magiic - pro are biologically meaningful . The pattern in figure 3a constitutes the pocket for inf (n-(hydroxyaminocarbonyl) phenylalanine) and the zinc ions . A longer pattern with a lower support (28 sequences) is plotted in figure 3b . It is of interest that this pattern constitutes the substructure which presents its importance from another protein (p04852) in the complex with the lci protein . This small example shows the necessity of finding motifs with different conservation levels that match different subsets of sequences in the training data . On the other hand, the diagnostic patterns provided in the prosite database simply capture the signature regarding the zinc binding site . Patterns are shown in sticks with different blocks plotted by distinct colors, lci protein in ribbons, and zinc ions in crimson spheres . (a) the pattern with a high support is plotted with the structure of the bovine pancreatic carboxypeptidase a complexed with the ligand inf, which is plotted in ball - and - sticks representation and colored in cpk (1hdq.pdb, p00730) (b) a longer pattern with a lower support provides the contact regions when interacting with the protein lci, shown with the structure of another protein p48052 in complex with lci, where the ligand glu is plotted in ball - and - sticks representation and colored in cpk . Patterns are shown in sticks with different blocks plotted by distinct colors, lci protein in ribbons, and zinc ions in crimson spheres . (a) the pattern with a high support is plotted with the structure of the bovine pancreatic carboxypeptidase a complexed with the ligand inf, which is plotted in ball - and - sticks representation and colored in cpk (1hdq.pdb, p00730) (b) a longer pattern with a lower support provides the contact regions when interacting with the protein lci, shown with the structure of another protein p48052 in complex with lci, where the ligand glu is plotted in ball - and - sticks representation and colored in cpk . (1dtd.pdb, p48052) since the minimum number of the elements in a block is suggested to be set as 3, it might happen that some residue or two of the residues are conserved but cannot be found by magiic - pro in its primitive results . In this case, the users are suggested to perform a multiple sequence alignment for an interested pattern on the matched segments of supporting sequences through the link provided by magiic - pro . By this way, the derived patterns can be enhanced with multiple sequence alignment to have both singly conserved residues and conservative substitutions well considered . Since the minimum number of the elements in a block is suggested to be set as 3, it might happen that some residue or two of the residues are conserved but cannot be found by magiic - pro in its primitive results . In this case, the users are suggested to perform a multiple sequence alignment for an interested pattern on the matched segments of supporting sequences through the link provided by magiic - pro . By this way, the derived patterns can be enhanced with multiple sequence alignment to have both singly conserved residues and conservative substitutions well considered . Detecting functional signatures directly from primary information is a challenging task . The mining process is tedious especially when the users have no prior knowledge about the query protein that can be used to judge how the mining results are . Magiic - pro quickly guides the biologists directly to the most highly conserved regions, and after that the users can extend the derived patterns by using the advanced parameters to refine the mining results . The derived patterns are useful in prediction of protein functions and structures, protein ligand interactions, and protein - protein interactions.
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It has long been appreciated that the regulation of biological processes involves a complex orchestration of genes in networks . However, typical analytic frameworks for genome - wide association studies (gwas) have assumed a simplified genetic architecture, largely considering independent additive contributions to genetic risk . While recent studies have supported a large contribution of additive genetic variance to complex traits (yang et al ., 2010; lee et al ., 2011a), missing heritability remains, which has led to a range of potential explanations (eichler et al ., 2010). One such explanation is the existence of epistatic (gene by gene) effects, which are, in fact, not precluded even in the presence of sizeable additive genetic variance (hill et al ., 2008). In this opinion and review, we propose that there is room for additional mining of datasets generated in the gwas era using a paradigm of networks that aggregate gene gene interactions and main effects . We argue that network approaches may have utility not only for discovery, but also for further characterizing relationships with genetic effects that may have been identified through standard analytic means . The recognition that numerous genetic variants may act in concert to modulate disease susceptibility has led to the development of gene set enrichment and pathway approaches (torkamani et al ., 2008; shi et al ., 2009; liu et al ., 2010). The small but consistent effect of many snps in gene sets suggests evidence at the population level of the coordination of genes known to interact through particular biological pathways . Gene set enrichment approaches are able to identify these coordination effects despite typically relying on small effect single - marker association evidence . Gene interaction effects also show small effect sizes, but evidence for coordination from main effects suggests that the aggregation of gene gene interactions and main effects in epistasis networks may lead to even more consistent pathway enrichment . Epistasis networks may also be used to test the hypothesis of a network paradigm of evolution and disease susceptibility . A recent study comparing the connectivity changes of arabidopsis networks following gene duplication events suggests a possible model of evolution acting at the level of the interactome network (dreze et al ., 2011). These studies suggest improved understanding of disease susceptibility may be achieved by conceptualizing the genotype to phenotype mapping as a network of coupled gene gene interactions and main effects . We refer to these as genetic association interaction networks (gain) or epistasis networks; however, the networks we describe model main effects as well as epistasis . In a recent gain analysis of a study of the immune response to smallpox vaccine, we identified a new association in the rxra gene reflecting a large number of gene gene interactions (davis et al ., 2010). Variants in figure 1 after the ability to connect other actors to him in a few jumps in a network constructed by shared movie credit . Such variants may be important to a phenotype, not because of their individual effect, but because of their overall influence in modulating the effect of other variants . Another analogy from popular culture is lady gaga, who has over one million twitter followers . Even a small perturbation from such a node may have a large downstream effect due to a cascade through the subnetworks of followers . The brando node would be easily found by a standard single - locus statistic, but the kevin bacon node would only be revealed by an epistasis network approach due to its many small gene gene interactions . The epistasis network may also be useful for identifying new mechanisms for known effects, such as the connection of the brando node to the pathway represented by the subnetwork below it . Returning to acting as an analogy, marlon brando variants (the usual target of the gwas approach) may or may not have a large network centrality, but are deemed important because of their individual (main) effect . Variants could include the cyp2c9 and vkor1c polymorphisms involved in warfarin metabolism (limdi et al ., 2010), the wealth of human leukocyte antigen (hla) alleles associated with immunologic phenotypes (lechler and warrens, 2000; blackwell et al ., 2009), or apol1 variants associated with kidney disease in african - americans (genovese et al ., 2010). While the majority of variants identified to date do not exhibit such individually strong effects, most are only likely in linkage disequilibrium (ld) with causal alleles; given that the gwas approach relies upon tagsnps to efficiently span the entire genome . Thus, there is an expectation that additional brando snps (presumably functional variants tied to more penetrant phenotypic effects) will be found by fine - mapping experiments (such as the immunochip for autoimmune disorders (trynka et al ., 2011), trans - racial mapping (rosenberg et al ., 2010; teo et al ., recent studies incorporating next - generation sequencing in age - related macular degeneration (raychaudhuri et al ., 2011) and inflammatory bowel disease (rivas et al .,, when considering the issue of the missing heritability, it is important to remember that causal variants are expected to occur at lower frequencies compared to the tagsnps that have been identified thus far (visscher et al ., 2011). Therefore, the existence of stronger effects will be offset to an unknown extent by a lower prevalence of causal alleles, potentially restricting the total variability that will be explained at the population level . It seems unlikely that identifying less frequent, causal alleles will immediately fill the missing heritability void, suggesting that complementary approaches, such as understanding variation in a network context, could have important implications in biological mechanistic research and translational medicine . For example, even for well - known genetic factors such as hla, there is a growing recognition of epistatic considerations, such as the interplay between hla class i polymorphisms and the killer cell immunoglobulin receptor (kir) gene complex within the context of infectious disease, autoimmune disorders, cancer, and bone marrow transplantation outcomes (kulkarni et al ., 2008; thus, network analysis is not restricted to discovery . Even in cases where there are known genetic effects, network analysis provides an opportunity to more fully characterize the genetic etiology by identifying the full network of interactions with known susceptibility variants . The focus of this review is on empirical network approaches, where connections are based on gene gene interactions (epistasis) estimated from gwas data . However, we pause to mention prior knowledge networks (pkns), such as ingenuity pathway analysis (www.ingenuity.com), which represent the most popular network analysis approach applied to gwas to date (burgner et al ., 2009). Similar to gene set enrichment approaches, pkns typically utilize statistical measures of marginal association to select genes to include in a putative network, with connections then constructed from various knowledge bases including protein protein interactions (ppis), co - occurrence in literature mining, or co - expression in microarray experiments ., 2011b) uses evidence from pkns based on selected candidate pathways combined with odds ratios from gwas in order to boost the importance of neglected genes . Prior knowledge based strategies have also been used to focus searches for epistatic effects in multiple sclerosis (bush et al ., 2011) and bipolar disorder (moskvina et al ., 2011) found that a prioritization strategy did not lead to an enrichment of replicable statistical interactions, suggesting a large risk of false positive associations . These contrasting results could reflect the issue that connections are typically defined independently of the environmental / disease context; leading to differences in performance as a function of the particular trait under study . While the knowledge bases for pkns are constantly improving in quality and scope, there is likely a need for empirical network approaches that are able to account for the conditional dependence of gene connections on the specific disease under study . For example, in studies of infection or vaccination, it is reasonable to hypothesize that genetic effects in networks may be dependent on the particular antigenic perturbation . There has been a growth in the use of pathway - based approaches to gwas to shed light on biological processes and identify new candidate disease genes . These approaches identify enriched pathways from a broader set of significant genes rather than focus only on a few of the most significant snps . For example, pathway - based approaches and pathway clustering have identified important processes in breast cancer (torkamani et al ., 2008; menashe et al ., 2010) and in the diseases from the wtccc gwas (torkamani et al ., 2008) we propose that such pathway - based approaches to gwas may benefit from the prioritization of genes based on the aggregation of gene gene interactions as well as single - locus effects . A data - driven epistasis network approach may increase the discovery of enriched disease - specific pathways and new candidate gene targets in gwas over single - locus prioritization alone . Gene interaction networks in this way may require some modifications to pathway analysis because enrichment scores typically rely on permutation to determine the statistical significance of enrichment, and data - driven networks are much more computationally intensive than single - locus calculations . Previous data - driven network approaches for gwas have used shannon information theory for epistasis calculations and network construction (jakulin and bratko, 2004; moore et al ., 2006; mckinney et al ., 2009; davis et al ., 2010). However, casting the network in the widely used statistical framework of a general linear model (glm) has some advantages over information theory . For example, use of a glm framework provides the flexibility to handle environmental covariates, longitudinal data, missing data, censoring, and cluster structure (e.g., family studies) through the inclusion of appropriate random effects . As an example, we present a likelihood ratio test of association between disease and a genetic locus, allowing for the possibility that the genetic effect may be modified by another genetic factor . Control data, but it is straightforward to develop similar tests for non - dichotomous phenotypes and other designs (e.g., family studies). The coefficient bb gives the baseline risk of disease and coefficients b1 and b2 correct for main effects in the interaction regression model . Gene edge weights b12 in the gain matrix, we are interested in the b12 regression coefficients that are statistically different from zero . The statistical framework also allows false discovery rate (fdr) procedures to be applied to correct for multiple gene fdr would be a more computationally efficient procedure than the permutation approached used in mckinney et al . The diagonal element bii of the gain is simply the main effect regression coefficient without interactions . Additional terms may be added to the regression equation to define interactions between other factors, such as environment or gene expression, to create a heterogeneous network or to correct for these factors . Heterogeneous interaction networks represent an important frontier in genetics because they may improve our understanding of the interplay between genetic and environmental modifiers of susceptibility . Once an epistasis graph like figure 1 is calculated, one may identify the most influential or central snps or other factors in the network . Such snp hubs in the disease - specific network may be potential targets for therapy or diagnostics, and the rankings may be used for a more sensitive pathway enrichment analysis . Various measures of node centrality have been proposed, with one of the most powerful and computationally tractable being eigenvector centrality the basis of google s pagerank (page et al ., 1999). Eigenvector centrality calculates the steady state eigenvector of a markov transition matrix whose elements represent the probability to move from one node to another . We recently designed a transition matrix method based on the main effects and interactions in gain and an eigenvector centrality algorithm we call snprank (davis et al ., 2010). In a disease risk interaction network, the network is specific to the disease (context sensitive), and each node and edge contains information about disease risk . The diagonal of b captures main (brando) effects, while the off diagonals represent gene the factors dj (the node degree) and tr(b) (the trace of b) normalize t to a stochastic matrix . The elements of b could be calculated using approaches such as information theory or from coefficients of statistical models . Simple recursion is used to compute t s steady - state eigenvector, whose elements are the rankings of each node . 2) also includes self - interactions or main effects when i = j. in contrast, google s pagerank does not permit self - interactions because this would represent a website including links to itself and would artificially inflate the site s importance . 2) includes a parameter, typically chosen close to 1 so that the main effect is not overwhelmed by the potentially large number of gene gene interactions a snp may have . We find in the range [0.8, 0.9] gives the highest internal consistency of the snprank scores, which we estimate by splitting data randomly into halves and calculating the kendall tau (kendall, 1938) rank correlation of the snpranks . We typically set to 0.85; however, = 1 is also a reasonable simplification to (eq . The parameter can also be used to blend prior knowledge from canonical pathways or protein protein interactions . A helpful way to conceptualize the way the algorithm scores the importance of genes is to think of the snprank centrality algorithm as simulating ants that follow paths through the network that have the most disease risk information, either due to gene gene interactions or main effects encoded in the edges and nodes of the network . More pheromones are deposited at more frequently visited genetic or environmental nodes, and the amount of pheromone is proportional to the rank score of the node . The steady state eigenvector, v (eigenvalue = 1), of the snprank transition matrix is given by tv = v, where the elements of v are the snprank score of each snp . The elements of v represent a probability field, and, thus, it is possible to use quantile plots and similar approaches to identify snps with snpranks that deviate from a uniform probability distribution . One can also see from the eigenvalue equation that the snprank importance of the ith snp, vi, is influenced by the entire network: in other words, the snprank of the ith snp is a function of all interaction coefficients bij (j i), the main effect (self interaction), bii, of the ith snp, and the recursively estimated importance of each snp vj (j i). This approach does not include pure higher order interactions, like three - way etc . But when ranking an individual snp, this approach models the interaction with every other snp in the network and the snp s main effect . An important point for future research is the extent to which network approaches offer improved statistical power to detect complex genetic effects . In the case of pkns, power critically depends on the extent to which a particular dataset fits with the current state of biological knowledge . Similar to a correctly constructed informative prior distribution in bayesian modeling, pkns will have an advantage in situations where the data is congruent with prior expectations . Much of this is due to limitations in simulation methodology, as it is currently difficult to generate datasets with a complex and realistic latent structure . While some inroads have been made (himmelstein et al ., 2011), because approaches to characterize complex effects are in their infancy, it is also not clear what constitutes a realistic level of complexity . Therefore, it is imperative that advancements in network methodologies be pursued in tandem with research into appropriate simulation frameworks . We do note, however, that approaches like snprank operate on network representation typically defined in a pair - wise fashion . Therefore, they implicitly inherit some of the features of the approach used to construct this pair - wise representation, whether based on information theory, statistical modeling, or other approaches (cordell, 2009). The power of network approaches thus could be improved by finding the best approach or combination of approaches for detecting pair - wise interactions (ritchie et al ., 2001; fan et al ., 2011). In addition to this inheritance, snprank may also boost power to detect associations by aggregating numerous interactions and main effects (selinger - leneman et al . Strategies for replication of a single snp are well accepted (kraft et al ., 2009), but the replication strategy for a network is less obvious because of the complex entanglement of all snps in the network . One level of replication would be to test for replication of a gene set in an independent cohort based on gene set enrichment from the network prioritization . Ld is another area that requires further investigation because the common strategy of ld pruning could run the risk of excluding interactions . Finally, both of these issues simultaneously become pertinent within the context of comparisons across racial / ethnic populations (teo et al ., 2010) as differential patterns of ld could complicate the interpretation of edges connecting specific haplotype tagging variants . Previous data - driven network approaches for gwas have used shannon information theory for epistasis calculations and network construction (jakulin and bratko, 2004; moore et al ., 2006; however, casting the network in the widely used statistical framework of a general linear model (glm) has some advantages over information theory . For example, use of a glm framework provides the flexibility to handle environmental covariates, longitudinal data, missing data, censoring, and cluster structure (e.g., family studies) through the inclusion of appropriate random effects . As an example, we present a likelihood ratio test of association between disease and a genetic locus, allowing for the possibility that the genetic effect may be modified by another genetic factor . Control data, but it is straightforward to develop similar tests for non - dichotomous phenotypes and other designs (e.g., family studies). The coefficient bb gives the baseline risk of disease and coefficients b1 and b2 correct for main effects in the interaction regression model . Gene edge weights b12 in the gain matrix, we are interested in the b12 regression coefficients that are statistically different from zero . The statistical framework also allows false discovery rate (fdr) procedures to be applied to correct for multiple gene gene hypotheses . Fdr would be a more computationally efficient procedure than the permutation approached used in mckinney et al . The diagonal element bii of the gain is simply the main effect regression coefficient without interactions . Additional terms may be added to the regression equation to define interactions between other factors, such as environment or gene expression, to create a heterogeneous network or to correct for these factors . Heterogeneous interaction networks represent an important frontier in genetics because they may improve our understanding of the interplay between genetic and environmental modifiers of susceptibility . Once an epistasis graph like figure 1 is calculated, one may identify the most influential or central snps or other factors in the network . Such snp hubs in the disease - specific network may be potential targets for therapy or diagnostics, and the rankings may be used for a more sensitive pathway enrichment analysis . Various measures of node centrality have been proposed, with one of the most powerful and computationally tractable being eigenvector centrality the basis of google s pagerank (page et al ., 1999). Eigenvector centrality calculates the steady state eigenvector of a markov transition matrix whose elements represent the probability to move from one node to another . We recently designed a transition matrix method based on the main effects and interactions in gain and an eigenvector centrality algorithm we call snprank (davis et al ., 2010). In a disease risk interaction network, the network is specific to the disease (context sensitive), and each node and edge contains information about disease risk . The diagonal of b captures main (brando) effects, while the off diagonals represent gene the factors dj (the node degree) and tr(b) (the trace of b) normalize t to a stochastic matrix . The elements of b could be calculated using approaches such as information theory or from coefficients of statistical models . Simple recursion is used to compute t s steady - state eigenvector, whose elements are the rankings of each node . 2) also includes self - interactions or main effects when i = j. in contrast, google s pagerank does not permit self - interactions because this would represent a website including links to itself and would artificially inflate the site s importance . 2) includes a parameter, typically chosen close to 1 so that the main effect is not overwhelmed by the potentially large number of gene gene interactions a snp may have . We find in the range [0.8, 0.9] gives the highest internal consistency of the snprank scores, which we estimate by splitting data randomly into halves and calculating the kendall tau (kendall, 1938) rank correlation of the snpranks . We typically set to 0.85; however, = 1 is also a reasonable simplification to (eq . The parameter can also be used to blend prior knowledge from canonical pathways or protein protein interactions . A helpful way to conceptualize the way the algorithm scores the importance of genes is to think of the snprank centrality algorithm as simulating ants that follow paths through the network that have the most disease risk information, either due to gene gene interactions or main effects encoded in the edges and nodes of the network . More pheromones are deposited at more frequently visited genetic or environmental nodes, and the amount of pheromone is proportional to the rank score of the node . The steady state eigenvector, v (eigenvalue = 1), of the snprank transition matrix is given by tv = v, where the elements of v are the snprank score of each snp . The elements of v represent a probability field, and, thus, it is possible to use quantile plots and similar approaches to identify snps with snpranks that deviate from a uniform probability distribution . One can also see from the eigenvalue equation that the snprank importance of the ith snp, vi, is influenced by the entire network: in other words, the snprank of the ith snp is a function of all interaction coefficients bij (j i), the main effect (self interaction), bii, of the ith snp, and the recursively estimated importance of each snp vj (j i). This approach does not include pure higher order interactions, like three - way etc . But when ranking an individual snp, this approach models the interaction with every other snp in the network and the snp s main effect . An important point for future research is the extent to which network approaches offer improved statistical power to detect complex genetic effects . In the case of pkns, power critically depends on the extent to which a particular dataset fits with the current state of biological knowledge . Similar to a correctly constructed informative prior distribution in bayesian modeling, pkns will have an advantage in situations where the data is congruent with prior expectations . Much of this is due to limitations in simulation methodology, as it is currently difficult to generate datasets with a complex and realistic latent structure . While some inroads have been made (himmelstein et al ., 2011), because approaches to characterize complex effects are in their infancy, it is also not clear what constitutes a realistic level of complexity . Therefore, it is imperative that advancements in network methodologies be pursued in tandem with research into appropriate simulation frameworks . We do note, however, that approaches like snprank operate on network representation typically defined in a pair - wise fashion . Therefore, they implicitly inherit some of the features of the approach used to construct this pair - wise representation, whether based on information theory, statistical modeling, or other approaches (cordell, 2009). The power of network approaches thus could be improved by finding the best approach or combination of approaches for detecting pair - wise interactions (ritchie et al ., 2001; fan et al ., in addition to this inheritance, snprank may also boost power to detect associations by aggregating numerous interactions and main effects (selinger - leneman et al ., 2003). Strategies for replication of a single snp are well accepted (kraft et al ., 2009), but the replication strategy for a network is less obvious because of the complex entanglement of all snps in the network . One level of replication would be to test for replication of a gene set in an independent cohort based on gene set enrichment from the network prioritization . Ld is another area that requires further investigation because the common strategy of ld pruning could run the risk of excluding interactions . Finally, both of these issues simultaneously become pertinent within the context of comparisons across racial / ethnic populations (teo et al ., 2010) as differential patterns of ld could complicate the interpretation of edges connecting specific haplotype tagging variants . Pathway and gene set enrichment approaches have demonstrated the utility of aggregating information from many moderate - sized single - locus effects . While such approaches assume implicitly that the targeted genetic architecture reflects a complex interacting system, the prioritization of genes for enrichment typically relies on single - locus effects . We propose network models of gwas data, built up from many single - locus and gene gene interactions . We anticipate systems level network approaches to gwas will reveal new mechanisms and improve our understanding of the complex relationship between genotypes and phenotypes . However, there are numerous statistical and bioinformatics challenges that remain to be addressed to realize this systems level understanding . The two network approaches for gwas discussed in this brief review prior knowledge and gene pkns are able to leverage information from different scales, such as ppi or gene co - expression, though these data sources may lack specificity to the disease under study . Specificity could be achieved by calculating the interaction between genes conditional on the phenotype at hand . Thus, the best approach may be to integrate prior knowledge with epistasis networks, perhaps through a bayesian formalism . As the biological connections of pkns and canonical pathways continue to improve in specificity, the integration of these networks with new interactions discovered in empirical networks will likely be a powerful combination . Previous data - driven epistasis networks have modeled interactions with shannon information theory measures because of the computational efficiency and power to detect interactions . An advantage of regression - based empirical networks is the ability to incorporate variation from other data types (gene expression, methylation, copy number), covariates, and environmental factors . Another open question is how much of the missing heritability is explained by network / epistatic effects . Answering such questions will require the development of statistical models that provide a coherent predictive mechanism based on a highly interacting network . There is also a methodological need for tools to construct and understand features of empirical networks, such as subnetwork motifs, hubs, and node degree distributions . One well - studied degree distribution is the power law, which corresponds to the notion of a small world network (bassett and bullmore, 2006). It remains to be seen whether the degree distribution of epistasis networks exhibits a power law and how ld would affect the degree distribution . A power law edge distribution of small world networks implies that most nodes have a small number of edges, but there are a few nodes with a large number of connections (hubs). The small world property allows one to traverse from one node to any other in relatively few steps and the network is robust to random attack or mutation . The need for the application and development of algorithms for characterizing networks is not unique to genomics . The importance of network concepts and algorithms is recognized throughout biology, notably in neuroscience where there is current interest in resting - state functional connectivity networks from fmri data (braun et al ., 2011). Thus, there is an opportunity to adapt methods and integrate data from other domains for genomic data . Both data from brain connectivity and gene network analysis for other genomic data seem to exhibit characteristics of small world networks (barabasi and oltvai, 2004), and it will be important to determine whether this is also reflected in the structure of gwas data . We are in the early stages of understanding how the network of epistatic and main effects synthesizes with biological networks and pathways . Knowledge of hubs, centralities and other properties of disease risk epistasis networks provide a new path toward identifying critical nodes in the network that may act as therapeutic targets or disease risk predictors . And a data - driven interaction network paradigm of gwas and deeper sequencing may lead to new insights into the mechanisms of evolution and complexity . 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.
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Malaria has been one of the major challenges to global health during the past century . In 1900, 58% of the world's land area more than a million deaths annually have been attributed to malaria throughout the latter half of the 20th century, most in children younger than 5 years, with countries of sub - saharan africa bearing the largest toll . However, mounting evidence suggests a decline in the global burden of malaria, a decrease that began in the mid 20th century in some regions but was most notable in parts of africa over the past decade . This reduction has led to renewed focus among the malaria community on a goal of malaria elimination in many countries . While malaria has been in decline, the geographical range and disease burden of another tropical infectious disease has been on the rise . Dengue has emerged as an increasing public health problem over the past 50 years, particularly in southeast asia and central and south america, with an unknown but possibly substantial level of transmission in africa . Like malaria, dengue is a vector - borne disease of the tropics and is a major cause of morbidity in endemic areas, particularly in children and young adults; however, the scale of dengue morbidity and mortality is uncertain and thought to be less than that of malaria . Dengue is caused by four distinct but related viruses (serotypes denv 14) that are transmitted among people by aedes mosquitoes . The disease burden and geographical range of dengue have expanded, from about 15 000 cases reported annually from fewer than ten countries during the 1960s to almost one million cases a year across more than 60 countries in 20002005 . As a result, dengue has been identified as an important threat to global public health . In view of this rising challenge, could lessons learned from global efforts to control malaria help inform strategies to prevent and perhaps reverse the spread of dengue? In this personal view, we compare and contrast malaria and dengue with respect to epidemiology, current and future interventions available for prevention and control, and their prioritisation as global health issues, in terms of funding, capacity, and international collaborations . We also argue that improved data on the range and endemicity of dengue are a vital component of global prevention and control efforts . The geographical ranges of both malaria and dengue are limited by the spatial extent of the competent vectors particular species of anopheles and aedes mosquitoes, respectively . The bionomics of the vectors shapes the epidemiology of each disease . With a few exceptions, anopheles mosquitoes favour rural environments, mainly because of their larval habitat requirements . By contrast, the primary vector of dengue, aedes aegypti, thrives in urban environments where abundant container breeding sites in and around human habitations allow immature vectors to develop and adults to feed and rest close to high densities of humans, their preferred host for blood meals . Although aedes mosquitoes have a restricted flight range of about 100 m in the field, passive transport of ae aegypti by land and, in immature stages, by sea led to their re - establishment in countries of south and central america, from which they had previously been eliminated in the mid 20th century, and dispersal throughout southeast asia during and after world war 2 . The geographical range of a secondary dengue vector, aedes albopictus, has also expanded substantially over the past 30 years, but it is a less efficient vector and is not currently seen as a major contributor to or risk factor for increased dengue transmission . The growing mobility of viraemic people, both within endemic settings and into new regions by increased domestic and international travel and migration, has been key in driving the global expansion of dengue in recent decades . This movement has created conditions in which multiple virus serotypes cocirculate, leading to an increase in the risk of sequential infections and severe disease . By contrast, the ecological requirements of anopheles mosquitoes have not facilitated their dispersal, and the unprecedented urbanisation that has characterised the past century is associated with reduced risk of malaria transmission, at least in the african setting . The global burden of a disease is a function of both its geographical range and the intensity of transmission in affected areas . By both these measures, the global burden of malaria has unequivocally decreased over the past century, although this decline has not been consistent across all malaria - endemic countries . Serious efforts to define the geographical limits and intensity of malaria transmission go back to the mid 20th century, when global control and eradication efforts were gathering momentum . A renewed effort to quantify the magnitude and distribution of the burden of malaria has seen new epidemiological and cartographic techniques applied to multiple collated data sources to model the spatial extent of malaria transmission and so to estimate populations at risk of exposure . These calculations place 24 billion people living in 87 countries at risk of plasmodium falciparum infection in 2007, resulting in around 450 million clinical cases of p falciparum malaria annually . The inclusion of uncertainty intervals around estimates has been a major step forward with these cartographic methods . For dengue, assessments of the spatial extent of transmission have been based largely on empirical data of reported dengue cases from endemic and epidemic settings, with models then fitted to correlate the observed distribution with environmental and climatic characteristics . Who estimates that 50 million dengue virus infections occur every year across about 100 countries, representing a population at risk of 25 billion people, although this number could be an underestimate of the true burden . The most recent assessment of the global distribution of dengue identifies 128 countries with good evidence of transmission and puts almost four billion people at risk . The most commonly used measure of malaria endemicity is the parasite prevalence rate, which represents the proportion of a population with malaria parasites detectable in their blood . This measure has been used widely in malaria surveys throughout the past century and has been used to generate the first evidence - based global map of malaria endemicity in 2007, recently updated for 2010 (figure). Another key metric of malaria transmission risk is the entomological inoculation rate, which represents the rate at which people are bitten by infectious mosquitoes . The relationship between the entomological inoculation rate and the parasite prevalence rate is non - linear . Empirical measurements for the entomological inoculation rate have been gathered less routinely and consistently than for the parasite prevalence rate, making the former a less useful measure for global endemicity mapping . Consequently, the entomological inoculation rate and other important metrics for malaria have been inferred with modelled relationships between them and extensive maps of parasite rates . Epidemiological data on the global burden of dengue rely almost entirely on reports of clinically apparent disease, derived from national surveillance systems and, in a few cases, from prospective longitudinal studies . The figure shows a map of dengue risk that combines disease notification and outbreak data from international organisations, case reports on returning travellers, published scientific literature on dengue occurrence, and a biological model of environmental suitability . Serological data from longitudinal studies permit estimation of infection incidence in a population, including the ratio of symptomatic to inapparent infections . This type of study depends, however, on follow - up of cohorts, which needs far greater investment of time and money compared with cross - sectional surveys that are used to obtain estimates of the malaria parasite prevalence rate . Traditional indicators of the abundance of aedes mosquitoes, based on immature vector stages (house index, container index, breteau index), are collected routinely in many dengue - endemic countries, but their correlation with human infection and disease is poor . Counts of ae aegypti pupae per person might correlate more closely with adult vector density and, therefore, potential for dengue transmission . Direct measurement of the density of adult ae aegypti with pcr to ascertain the proportion infected with dengue virus would be most informative, but this approach is logistically and financially demanding to do on a sufficiently large scale in view of the difficulty in sampling adult vectors and the expected large variance in both adult numbers and prevalence of infection . For both malaria and dengue, the relationship between the risk of infection and the risk of disease is non - linear and depends on host immune status and age at infection . Clinical case numbers are relevant to the prediction of demand for diagnostic tests, health - care services, and treatments . Who also defines laboratory - confirmed clinical dengue cases of any severity as the most appropriate endpoint for dengue vaccine trials . However, for describing transmission extent and intensity, especially when making comparisons between countries and over time, reliance on case - burden data is fraught with issues of inconsistent reporting patterns (both spatially and temporally), differences in clinical case definitions, over - reporting when laboratory testing is not routine, and under - reporting of patients who do not present to health services or who are managed as outpatients only . A measure of the incidence of infection, rather than disease, might also be an appropriate endpoint for trials of dengue vector - control interventions in the community; not only is active surveillance of clinical outcomes more resource - intensive than cross - sectional blood sampling but also a large (and variable) proportion of prevented infections are likely to be asymptomatic . Therefore, a smaller sample size will be needed to show an effect on infection rates, compared with a clinical effect of the same size, because of the higher overall event rate for infections versus clinical cases . What alternative metric could be used to measure dengue endemicity, equivalent to the parasite rate for malaria? Virological markers of dengue such as viraemia and presence of the ns1 antigen in blood are short - lived compared with untreated malaria parasitaemia, disappearing about 1 week after onset of clinical symptoms . Furthermore, the magnitude and duration of viraemia varies with severity of disease, virus serotype, and host immune status . Cross - sectional age - stratified serological surveys of dengue - specific igg can indicate the prevalence of past exposure to dengue virus but are confounded by antibodies directed against other flaviviruses, where these cocirculate . Cross - sectional seroprevalence surveys of dengue - specific igm might indicate recent infection with dengue virus or other flaviviruses . Aside from potential low specificity, interpretation is complicated by the variable kinetics of the igm response, most importantly, the difference between first and subsequent infections, making comparison of population - based igm surveys between epidemiological settings difficult . Population - based surveys of dengue neutralising antibody, measured by the plaque reduction neutralisation test, would provide the most sensitive and specific information on virus transmission patterns, including serotype - specific data and multiple heterotypic exposures . However the plaque reduction neutralisation test is substantially more resource - intensive than standard igm and igg immunoassays . Finding the appropriate metric to measure the endemic level of dengue is a clear research priority . Success in controlling malaria over the past century has been attributed predominantly to widespread implementation of insecticide - treated bednets, household spraying of residual insecticides, and effective drugs to reduce mortality and interrupt transmission . The countries in which little progress has been made with malaria control are commonly those where political instability, war, or economic underdevelopment have hindered widespread implementation of these interventions . The situation with dengue is different; vector control is the only currently available approach for prevention and control and is pursued mainly through reduction of larval development sites, via environmental clean - up campaigns to dispose of discarded or unnecessary water containers, and prevention of mosquito access to breeding sites . Other methods include treatment of water - storage vessels with larvicide or predacious copepods to kill larval stages . The effectiveness of these interventions has been demonstrated at a local community level but rarely on a large scale or across diverse epidemiological settings (not since the ae aegypti eradication campaign of the 1950s), with singapore and cuba perhaps the only exceptions . Success of such efforts depends on sustained community support and participation . However, even when mosquito populations have been reduced drastically, as in singapore, cases of dengue still occur, with evidence of increasing risk of clinical disease associated with older age at first infection . Killing adult mosquitoes has a theoretically greater effect on transmission than does targeting larvae . Space - spraying of insecticide to kill adult vectors in and around households is popular because it represents a highly visible response to localised outbreaks of dengue, but a sustained effect on virus transmission has not been demonstrated . Indoor residual spraying of insecticide has a long history of use in malaria control, and its importance as a key intervention for interruption of malaria transmission has been reaffirmed by who . Many behavioural characteristics of anopheles vectors that make indoor residual spraying an effective malaria intervention, such as their anthropophagic biting preferences and tendency to rest and feed indoors, are common also to aedes dengue vectors . There is some evidence that high household coverage of indoor residual spraying in an outbreak setting could reduce dengue transmission . Use of this method to control yellow fever in the americas in the mid 20th century had a concomitant and striking effect on dengue transmission, but there are very few reports of the application of indoor residual spraying specifically to control dengue . The preference of aedes vectors for daytime activity and feeding means that insecticide - treated bednets are ineffective for dengue control . Findings of several small trials of other insecticide - treated materials, such as curtains and water - jar covers, indicate a reduction in indices of household vectors, and larger trials are warranted to investigate effectiveness in a range of epidemiological settings . International guidelines recommend parasitological confirmation, when possible, of all suspected cases of malaria and prompt initiation of treatment to prevent progression to severe disease . Timeliness is also very important for effective clinical management of dengue; progression from an acute febrile phase to non - complicated recovery, or through a critical phase characterised by thrombocytopenia and capillary permeability with potential for haemorrhage and shock, takes place over 37 days . Unlike malaria, no specific treatment for dengue is available, and clinical management entails close haematological monitoring, fluid - replacement therapy as required, and recognition of warning signs of severe disease . Although serological, molecular, and rapid diagnostic tests for dengue are widely available, the expense, waiting time, and large case numbers mean that clinical management and case reporting in most endemic settings is based on clinical diagnosis alone . Increasing availability of rapid diagnostic tests could theoretically improve timeliness and accuracy of dengue diagnoses . However, studies of the effect of test results on clinical management and outcome of dengue cases, including cost - effectiveness studies, are needed to inform recommendations for widespread use . Demand for routine diagnostic testing for dengue could increase substantially if an antiviral drug were available . Research efforts towards vaccines against malaria and dengue are similarly complicated by (among other challenges) the antigenic or serotypic variability of the organisms . A longlasting highly effective dengue vaccine should be much easier to develop than an equivalent malaria vaccine because of the relative antigenic complexity of the two pathogens and the longevity of immune responses to viral infections, compared with those to malaria parasites . However, developers of a dengue vaccine must contend with the theoretical risk of severe disease associated with sequential infection with a heterologous serotype and, thus, aim to develop a tetravalent vaccine . Candidate vaccines for malaria and dengue are in phase 3 field trials, but despite publication of promising clinical trial data for the leading malaria vaccine candidate, a substantial vaccine - mediated reduction in the global burden of either disease is not imminent . Thus, vector control, effective diagnosis, and clinical management remain the cornerstones of control for both diseases, for the foreseeable future . The challenge now in malaria control is equitable and effective implementation of interventions that have proven efficacy . However, to tackle the increasing burden of dengue, well designed and controlled field trials are needed of both existing and novel vector - control interventions, linked to detailed epidemiological data, to improve the evidence base and inform local and national dengue - control strategies . Further challenges for evaluation of dengue interventions might include the effect of human movement on patterns of transmission, and the pronounced temporal and spatial heterogeneity in transmission, which will necessitate very large cluster - randomised study designs . These issues are also likely to be challenges for malaria control though the elimination or eradication phases . Malaria control throughout the past century has been a combined effort of national, regional, and international programmes . The global malaria eradication programme launched in 1955 by who was the largest coordinated international public health campaign in history . With an intensive strategy of vector control using residual insecticides, combined with detection and treatment of cases, 22 countries in the americas and 27 in europe achieved malaria elimination between 1950 and 1978 . Despite these successes, the goal of elimination was not met universally and was never proposed for sub - saharan africa; in 1969, who's strategy was revised to one of control . Efforts to control dengue also benefited from an elimination campaign in the mid 20th century; in 1947, the pan - american health organization adopted a proposal by brazil for a so - called hemispheric (pan - american) strategy to remove the ae aegypti vector . Although the aim of this campaign was eradication of urban yellow fever, which shares the same vector as dengue, the successful elimination by 1967 of ae aegypti from all countries of the americas (except for the usa, venezuela, and the caribbean region) saw a substantial reduction in dengue morbidity across this region . Unfortunately, this campaign had the same outcome as the global malaria eradication programme, with a reversion to a strategy of control because of a combination of reduced political will, insufficient financing to sustain intensive control efforts, and increasing decentralisation of national public health authorities, among other factors . The ae aegypti vector re - established itself in areas from which it had been eliminated, with a resultant rise in dengue epidemics in the americas throughout the 1970s and 1980s . In southeast asia, an elimination goal for dengue or its vector has never been proposed formally . Efforts to control malaria during the past 15 years have intensified after the development of international initiatives to coordinate and finance the scale - up of interventions, beginning with the roll back malaria partnership, launched in 1998, and the global fund for aids, tuberculosis, and malaria, founded in 2002 . More recently, the bill and melinda gates foundation has allocated substantial funds to malaria control and eradication efforts . These initiatives recognise the need for external funding and support to malaria - endemic countries to achieve coverage of interventions at a level that will affect transmission and morbidity . An estimated us$99 billion was committed by international donor agencies for malaria control in endemic countries between 2002 and 2010 . By contrast, vector - control interventions for dengue remain the financial and logistical responsibility of national control programmes in endemic countries, which are funded from national budgets with no substantial or sustained external sources of financing . Dengue is a high public health priority in endemic countries, but the main target of spending is responsive vector - control activities around reported cases, combined with passive case surveillance and some routine virus and vector surveillance . Budgets are usually insufficient to implement these actions fully, let alone to sustain breeding source reduction activities, larval control, and environmental management, which might be more effective but are highly resource - intensive . It is difficult to see how the continuing geographical spread and increasing intensity of dengue transmission can begin to be reversed without support in coordination and financing from outside endemic countries . Support should include applied research to improve the evidence base for existing vector - control techniques, for novel interventions such as transfection of wolbachia spp into ae aegypti to suppress dengue transmission, and for strategic planning to implement and finance a future vaccine . Even when a dengue vaccine becomes a reality, external assistance for financing and implementation will be needed by some endemic countries, as will continued concerted efforts in vector control . Unlike malaria, which receded from southern europe in the mid 20th century, aedes mosquitoes, and possibly dengue, could continue to expand into warmer areas of high - income countries, including australia, the usa, and southern europe . This possibility should provide any additional impetus needed for dengue to be viewed as more than a neglected tropical disease . The burden of morbidity, mortality, and economic loss attributable to dengue is not comparable with that caused by malaria . However, the coordinated initiatives for funding of regional and global collaborative research and control activities, which have proven effective to address the global burden of malaria, could also drive similar gains in dengue control . Based on lessons learned from malaria control, we propose that development of better methods to quantify dengue endemicity and disease burden, permitting comparisons across countries and regions, is an essential step towards halting the current rise in disease range and intensity . We must be able to quantify these increases accurately so we can establish baselines against which future trends can be compared . Analysis of the clinical and demographic profile of acute cases can tell us much about local dengue transmission dynamics, but improved indices of transmission including means of accounting for asymptomatic infections that can be measured at a population level and within specific subgroups would provide a much more complete picture of local transmission patterns . Use of serological markers of dengue infection in epidemiological studies has some limitations, but age - stratified serosurveys of neutralising antibodies against the virus probably represent the best equivalent to the malaria parasite prevalence survey for population - based estimates of the incidence of infection . Improved entomological measures of risk for dengue transmission, based on density of the adult vector and infection prevalence, could complement these estimates but might also be inferred, similar to malaria, from measurement of incidence in human populations . Improved estimates of the dengue disease burden would inform economic analyses of vector - control activities and future vaccination strategies . Effective implementation of these interventions could be achievable within the national budgets of a few dengue endemic countries, but many national dengue control programmes would benefit from coordinated international funding to achieve adequate coverage, as has proven effective in malaria control . This fact reinforces the importance of developing improved indicators of local, regional, and global dengue endemicity and disease burden, to advocate for funding directed to areas of greatest need, to identify locations where interventions are most likely to succeed, and to monitor future progress of disease prevention efforts, including vaccines . Sih is funded by a senior research fellowship from the wellcome trust and is supported by the li ka shing foundation, the rapidd program of the science and technology directorate, department of homeland security, and the fogarty international center, national institutes of health . This research was also supported partly by the idams project (grant no 281803) within the 7th framework programme of the european commission . The sponsors had no role in preparation of the manuscript or the decision to publish . We thank cameron simmons, oliver brady, peter gething, thomas scott, philip mccall, and jeremy farrar for valuable comments and suggestions during the preparation of this manuscript; and katherine battle for proofreading.
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Toxoplasmosis occurs in advanced stages of human immunodeficiency virus (hiv) infection and is the most common opportunistic infection of the central nervous system in patients with the acquired immunodeficiency syndrome (aids), especially in those patients with a cd4 + count <200 cells / mm . The most common clinical presentation of cerebral toxoplasmosis in patients with hiv infection is headache, focal neurological deficits and seizures . We present two cases with stroke - like presentation of cerebral toxoplasmosis from our centre . A 30-year - old right - handed nigerian woman was admitted to the university of calabar teaching hospital, nigeria, with a 1-week history of inability to move the left upper and lower limbs of acute onset . There was a history of headache, but no seizures, vomiting or loss of consciousness . She was not known to be hypertensive or diabetic, and she does not drink alcohol nor does she smoke . The patient was diagnosed to be hiv positive about 2 years prior to this admission and commenced on antiretroviral drugs (arv) but defaulted on therapy for about 1 year prior to the current presentation . Her latest cd4 + count was not known . On neurologic examination, she showed left upper motor neuron facial nerve palsy and increased tone and brisk reflexes in both left upper and lower limbs . Power was grade 0/5 and grade 1/5 in the left upper and lower limbs, respectively, on the medical research council scale, with a positive babinski sign on the left . A brain ct scan revealed a rounded ring - enhancing hypodense lesion in the parafalcine region of the right parietal cortex (fig . A complete blood count revealed low haemoglobin (8 g / dl), esr 80 mm in the first hour, and cd4 + count 18 cells / mm . Iu / l and toxoplasma igg was 202.0 iu / l, both markedly raised . A diagnosis of stroke - like syndrome due to cerebral toxoplasmosis in the background of hiv infection was made and the patient was commenced on pyrimethamine (200 mg at start, then 50 mg daily), clindamycin (600 mg every 6 h), folic acid (10 mg daily), septrin (960 mg b.i.d . ), amoxicillin / clavulanic acid (625 mg t.d.s . ), and haematinics . Two weeks after treatment, the patient resumed arv therapy with truvada and lopinavir / ritonavir . Power gradually improved to 4/5 in both limbs, and she started ambulating and was discharged to follow - up as an outpatient . A 35-year - old female was referred to the university of calabar teaching hospital, nigeria, with a 2-day history of left - sided weakness affecting her leg and arm and focal seizures involving her left upper limb . She had a history of hiv infection, diagnosed 4 years prior to the current presentation and treated with arv drugs . Her last cd4 + count (6 months before) was 28 cells / mm . On neurologic examination, there was left hemiparesis (grade 3:5 on the medical research council scale for both left upper and lower limbs) with increased tone and brisk reflexes and upgoing plantar reflexes (babinski sign). An urgent brain ct showed a rounded marginally enhancing hyperdense lesion with perilesional oedema in the right thalamus (fig . There was obliteration of the posterior horn of the ipsilateral lateral ventricle by the aforementioned mass . A similar but ill - defined lesion with more extensive perilesional oedema blood tests showed haemoglobin 10 g / dl, esr 106 mm in the first hour, cd4 + count 24 cells / mm and toxoplasma igg antibody 300 iu / l . Based on these findings, a diagnosis of cerebral toxoplasmosis presenting with a stroke - like syndrome, on the background of hiv treatment failure, was made . The patient was discharged home to continue follow - up at the medical outpatient department.
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Microtubules are polymers of /-tubulin dimers that stochastically switch between phases of growth and shortening . These dynamics are generally reported by four parameters: the rates of growth and shortening and the frequencies of switching between these phases, referred to as catastrophes and rescues1 . Conventional analysis of intracellular microtubule dynamics involves microinjection2 or expression of fluorescently labeled tubulin3 to homogenously label the microtubule network, and time - consuming, computer - assisted hand - tracking of individual microtubule ends over time2,4 . Although computational modeling has improved the efficiency and reliability of this method5, the analysis remains limited to microtubules near the cell periphery, where ends of labeled microtubules can be observed over sufficient periods of time . Sub - stoichiometric labeling6 allows microtubule observation deeper in the cell and could permit end tracking relative to the microtubule lattice, but the identification of microtubule ends and filaments in this context is an unresolved challenge; to date, only a few papers describe hand - measured data from limited numbers of microtubules4,7 . An alternative strategy to visualize microtubule polymerization utilizes proteins that specifically recognize a structural property of growing microtubule plus ends, such as fluorescently - tagged end binding proteins 1 and 3 (eb1- or eb3-egfp) 810 . The exponential decay of available binding sites results in the characteristic comet - like fluorescence profiles of egfp - tagged ebs . Fast turnover11 causes rapid loss of eb fluorescence from non - polymerizing microtubule ends and rapid appearance of eb comets when microtubules start growing . Here, we describe a computational strategy to extract microtubule growth rates from eb1-egfp time - lapse image sequences and introduce an approach to infer parameters of microtubule dynamics in pause and shortening phases without visible comets . We detect eb1 comets, track the comets to measure microtubule growth rates, and spatiotemporally cluster eb1 growth tracks into more complete trajectories, which include inferred shortening and pause phases (supplementary fig . 1). This method allows analysis of a large population of intracellular microtubule ends in a spatially unbiased manner . To identify eb1-egfp comets in each frame of a time - lapse sequence (fig . 1a), we calculated the difference of two gaussian (dog) transformations with standard deviations 1 and 2 adjusted to enhance the band of image frequencies associated with eb1-egfp comets (supplementary note 1 and fig 1c) and individual objects likely corresponding to eb1 comets were then generated by connected component labeling of the thresholded dog image (fig . 1c). To discriminate against selected objects that are not true comets, we selected by template matching objects that conform to the average shape of eb1-egfp comets (supplementary note 3 and fig k2 were introduced to adjust the automatically detected threshold if required for specific imaging conditions . All adjustable parameters are summarized in supplementary table 1 . Because the average eb1 comet shape is determined for each image, the detection is robust against variations throughout the time - lapse sequence, for example during acute application of microtubule polymerization inhibitors that reduce the polymerization rate (fig . 1d). A list of eb1 comets was thus obtained for each frame (fig . 1e), including the position of the object centroid, the magnitude of eb1-egfp comet eccentricity and the angular direction of its main axis . To validate the detection algorithm, the positions of ~120 hand - detected eb1-egfp comets were compared to automatically detected objects for different pairs of 2 and k1 because these parameters are most sensitive to imaging conditions (fig . Because the detection algorithm is sensitive to image intensity variations at the size scale of eb1-egfp comets, in confocal images in which a low eb1-egfp signal is evident along the length of microtubules, k1 was increased to k1> 2 to avoid a large number of false positive detections . In widefield images, however, in which eb1-egfp along the microtubule lattice is obfuscated by out - of - focus light, the default pixel intensity threshold of k1 = 1 worked robustly (supplementary fig . The detection error was quantified by the percentage of false positives (100(1-m / d)) and the percentage of false negatives (100(1-m / g), where d is the number of computer - detected comets, g is the number of hand - selected comets, and m is the number of matches between those two sets). There was low correlation between 2 and false positive detections over most of the range of 2 values tested . Similarly, false negative detections did not follow a clear trend, but a shallow minimum was evident around 2 = 4 indicating that detection was most successful when 2 matched the expected length scale of eb1-egfp comets (fig 1f). The detection performance was similar in widefield images, and was largely independent of the precise shape of the comet as the algorithm was successfully applied to other plus - end tracking proteins (supplementary fig . 2). Detection was also largely insensitive to simulated high frequency pixel noise (supplementary fig . 3). A small number of undetectable eb1-egfp comets is expected in every image due to proximity or overlap, which results in objects that do not pass the template matching of the detection algorithm . However, these events should not introduce a systematic error in the detected eb1-egfp comet population . The elongated shape of eb1-egfp comets implies that the comet centroid is systematically located behind the very tip of the microtubule . For growth rate measurements this offset is irrelevant as long as it remains constant over time . To assess the magnitude of potential temporal variation in the offset we determined the difference between computationally - defined eb1-egfp comet positions and manually detected intensity maxima, which co - localize with the microtubule tip . As expected, the difference increased with comet eccentricity (fig . 1 g). Perpendicular to the microtubule direction it was small (0.49 0.94 pixel) and below the accuracy of manual detection . For highly elongated comets, the difference was 2.95 1.26 pixel along the direction of the microtubule (fig . 1h); we estimated that the standard deviation of this difference was sufficiently small to have minor impact on the analysis of microtubule growth (supplementary note 4). Because the elongation of the eb1-egfp comet depends on the rate of microtubule growth10 indeed, we found that the fastest comets had a high eccentricity, although the distribution also included many slower comets with equally high eccentricity (fig . In contrast, no obvious correlation existed between eccentricity and eb1-egfp peak intensity (fig . Overall, this indicates complex kinetics of eb1 interactions with microtubule ends or fluctuations of microtubule end structures in cells . Detected eb1-egfp comets were tracked using a kalman filter - based multi - object tracking algorithm13 (supplementary note 5 and fig . As expected for random switching from growth to shortening, the track lifetime follows an exponential decay14 with a mean lifetime = 3.6 frames (fig . Because the number of tracks with a lifetime of 2 frames was higher than expected, we suspected that a significant subpopulation of very short tracks resulted from detection or tracking errors . Analysis of the variance of the growth rate histogram as a function of minimal lifetime further indicated a very high standard deviation for short tracks (fig . Thus, subsequent analysis only included growth tracks with a lifetime of 4 frames and greater . To evaluate the combined performance of the detection and tracking algorithms we compared computer - generated tracks with simulated differences in spatial (supplementary fig . We found that the algorithm performed robustly at magnifications that satisfy the nyquist sampling criterion (effective pixel size of <100 nm), and that a temporal sampling of 12 frames s was sufficient to achieve good track reconstruction of microtubule growth . Microtubules are stiff15 and over the short time intervals used to observe microtubule polymerization dynamics they translocate little . As a result, microtubule shortening, rescues and thus, time - shifted, nearly parallel eb1-egfp tracks with significant spatial overlap have a high probability of belonging to the same microtubule . To detect events of microtubule shortening and pausing in addition to the microtubule growth directly visible in eb1-egfp sequences and to infer parameters of their associated dynamics, we developed a computational scheme to cluster tracks that fulfill defined geometrical and temporal constraints based on a priori assumptions about intracellular microtubule behavior (fig . 3a). Given an eb1-egfp growth track terminating at time t, we considered all track initiations in the interval [t+1, t+tmax] in cones with a forward opening angle = 45 and a backward opening angle = 10 as candidate links that could potentially represent a continuation of the terminated growth (forward link) or shortening (backward link), respectively . The narrow backward cone reflects the tendency of microtubules to shorten along the preceding growth trajectory4 . For each candidate link we then calculated the maximal allowable distance for forward (cfwd) and backward gaps (cbwd) as: (1)cfwd = vmaxmin(tgap, tmax) (2)cbwd = min(vmaxtgap, vmedtmax) vmax represents the 95 percentile of the speed distribution from all participating growth tracks, and tgap is the time between termination and initiation of the two candidate tracks . Equation (1) assumes that short time gaps mainly result from temporary occlusion of growing microtubule ends due to noise, overlap, or out - of - focus motion, where the comet reappears at a distance proportional to the growth rate and tgap . Such pauses a microtubule end undergoes a random walk of short growth and shortening events, and the comet reappears at a distance proportional to the square root of time points without detectable eb1-egfp comet . In equation (2), takes into account that microtubule shortening is generally faster than growth3,4 . Vmedtmax, where vmed is the median of the growth track speed distribution, safeguards against the combinatorial explosion of candidate links in dense eb1 comet fields . Furthermore, candidate links were considered only when the directional change i, j between the two associated tracks was less than max = 60 (fig . 3b), because microtubules rarely bend more than 60 over short distances16 . From the set of candidate links that fulfilled the distance and angular criteria we then selected the largest possible subset of links with overall minimal cost using linear assignment17,18 . The cost for an individual candidate link ci, j was defined as (3)ci, j=i, j(||cos(i, j)||||cos(i, j)||), which gives preference to pairs of tracks in close proximity (i, j), with a low bending angle (i, j), and with small transversal shifts (i, j). Although the methods currently used may generate some false positive links, the selection is fairly robust because all tracks compete for links simultaneously . In a typical eb1-egfp time - lapse sequence, each growth track termination associated with ~200 candidate links that fulfilled the temporal (tmax) and angular criteria (, and max). Applying the distance cutoffs (cfwd and cbwd) reduced this to an average of 3 competing track initiations . As expected, the majority of links were positioned in the cell periphery where microtubules more frequently switch between growth and shortening (fig . Forward gaps during which eb1-egfp comets are invisible could be due to periods of pause or very slow growth during which the eb1-egfp signal becomes too dim to be detected, or due to very fast - growing eb1-egfp comets that are temporarily out - of - focus . We therefore calculated the forward gap speed vfwd as the distance between the last point in the terminating track and the first point in the linked growth track divided by the gap duration (fig . Typically, less than 15% of the forward gaps had a speed larger than the slowest 30% of the growth tracks . A small forward translocation is expected even for true pause events because of the latency of detectable eb1-egfp comet formation at the end of a newly growing microtubule (supplementary note 6). Similar to forward gaps, backward gaps contain information about microtubule shortening events, and as above we calculated a backward gap speed vbwd (fig . The histograms of both vfwd and vbwd sharply decayed towards the last bin below the maximally allowable velocity . (1) and (2) were set sufficiently large to capture the majority of clusters of aligned growth tracks (fig ., we recorded dual - wavelength time - lapse sequences of cells expressing both eb1-egfp and mcherry - tubulin (fig . 3e). Because mcherry - labeled microtubules were too dense in the cell interior to directly observe dynamic ends (supplementary fig . 6), we hand - tracked 19 microtubules in a peripheral cell region in which microtubule ends were clearly visible (fig . Detection and tracking of eb1-egfp comets was accurate as indicated by the overlap of hand- and computer - tracked growth (fig . Although clustered growth tracks and hand - tracked microtubule trajectories were similar, qualitative comparison showed that many shortening events were missed (fig . This was expected because the clustering algorithm cannot detect shortening that is not preceded or followed by a growth track within the distance and angle criteria . However, a high percentage of false negatives is acceptable as long as the population of microtubules analyzed is sufficiently large and unbiased to provide a representative readout of overall microtubule dynamics . To quantitatively test this, we compared hand - tracked rates of growth, pause, and shortening, and pause durations to those obtained by computational analysis . Importantly, the definition of growth, pause, and shrinkage events by hand - tracking relies on user - defined judgment as to when a microtubule undergoes a change in velocity . The definition of these events by growth track clustering relies on the detection of eb1-egfp comets as molecular markers of microtubule growth . Therefore, prior to a comparison of hand- and computer - tracking, the hand - tracked measurements had to be corrected to account for the differences in assumptions (supplementary note 7). Even after these corrections, hand - tracked growth and shortening rates were significantly higher than the computer - inferred rates (table 1). This deviation originated in the fundamentally different definitions of growth, pause, and shortening events in hand- and computer - tracking . Specifically, computer - tracked growth events contain a subset of events where the microtubule end moves very slowly without loss of the eb1-egfp comet . Similarly, computer - inferred backward gaps contain very slow events that would be classified as pauses in hand - tracked trajectories, in addition to phases of true microtubule shortening . We determined a threshold for a computer - generated growth track or backward gap to be accepted as a significant growth or shortening event by analysis of the velocity distribution of forward gaps, which includes both pauses and gaps due to unobserved growth events (supplementary note 8). This filtering resulted in excellent agreement between computer - inferred and hand - tracked growth and shortening rates (table 1). Thus, although comparisons between computational analysis and hand - tracking cannot be made directly, reasonable assumptions about the limitations of both methods confirmed that the growth and shortening rates we inferred are representative of microtubule polymerization dynamics . A discrepancy between computer - inferred and hand - tracked pause durations remained (table 1), likely due to the latency of eb1-egfp comet formation and disappearance (supplementary note 6). Thus, pause durations inferred by the clustering algorithm are not directly comparable to hand - tracking of homogenously labeled microtubules . Similarly, because shortening events interrupted by pauses will be combined into a single phase, the computer - inferred vbwd is not a direct measure of the microtubule depolymerization rate . Nevertheless, we expect that molecular conditions that change microtubule polymerization dynamics will result in relative changes of these computer - inferred parameters . The clustering algorithm also generated erroneous connections between growth tracks not belonging to the same microtubule . To quantify the frequency of such events, we overlaid clustered trajectories generated with two different stringencies onto to mcherry - tubulin and eb1-egfp channels (videos 46) and verified computer - assigned growth, pause, and shortening phases, as well as catastrophes and rescues . Within the region analyzed (fig . 3e), the software accumulated 475 microtubule growth tracks lasting at least 4 frames . At the less stringent parameter set (=45, =60) 329 tracks clustered into 118 trajectories with an average lifetime of 17 seconds . For the more stringent parameters (=15, =15) 226 tracks clustered into 91 trajectories with a 14 second average lifetime . Only 8% of the trajectories were identical between the two analyses, indicating the sensitivity of the clustering process in selecting viable track connections (fig . Because of the high microtubule density, about half of the computer - assigned events could not be observed clearly . However, if we only considered events that we could visually confirm as true or false, our algorithm showed good performance (supplementary table 2). As expected, the high stringency parameter set was more accurate, and with both parameter sets, the software more robustly connected microtubule growth tracks interrupted by pause rather than shortening events . However, the better performance at high stringency comes at the expense of less data, which bears the risk that some of the heterogeneity of microtubule behavior is lost . We next tested whether the computational analysis detected well - characterized responses to pharmacological microtubule dynamics inhibitors . At low concentrations, nocodazole inhibits both growth and shortening rates and increases the time microtubules spend in pause19, and we detected statistically significant effects on these parameters at concentrations as low as 10 nm (fig . This demonstrates that although our computational analysis is sufficiently sensitive to detect expected differences in microtubule polymerization dynamics . In addition, the results obtained with two different clustering parameter sets were similar, indicating that false positives are sufficiently low so as to not significantly affect correctly clustered microtubule growth tracks . Armed with our new software tool, we examined two long - standing questions in the field: first, we tested whether tubulin acetylation21 alters microtubule polymerization dynamics . We treated cells with trichostatin a (tsa), a broad specificity histone deacetylase inhibitor that inhibits the tubulin - specific deacetylase hdac622 (fig . 5a). We could not detect statistically significant differences in microtubule dynamics when cells were treated with tsa alone . However, in cells simultaneously incubated with 50 nm nocodazole the inferred shortening rate in tsa - treated cells decreased significantly (fig . We thus determined the distance from the cell edge at which the difference in the median microtubule growth rate between the edge and interior microtubule populations was largest . In three cells analyzed, this distance varied between 3.7 m and 6.4 m (fig 5c, d). The median growth rate in the cell body was 19.722.7 m / min compared to 16.617.1 m / min closer to the cell edge . 5e) but at the cell edge a larger fraction of the links between growth tracks were backward links (4460% in comparison to 3543% in the cell interior), consistent with the observation that peripheral microtubules more frequently switch between growth and shortening23,25 . We recently reported that gsk3 inactivation may be required for spatial microtubule dynamics regulation in migrating epithelial cells24, but we did not measure significant shifts in the spatial gradient of growth rates between control cells and cells expressing constitutively active gsk3(s9a). Here, we first confirmed this result using our new method for identifying spatial gradients in microtubule dynamics (fig 5f). Our clustering algorithm then revealed a large decrease in catastrophe probability at the cell edge and a decrease in the percentage of time microtubules spent in the shortening phase (fig . This demonstrates that gsk3 regulates specific aspects of microtubule polymerization dynamics and is consistent with the hypothesis that gsk3 inactivation increases cell edge microtubule interactions with the cortical cytoskeleton24 . Importantly, the clustering algorithm was able to detect these differences even though growth and shortening rates in the cell edge of control and gsk3(s9a)-expressing cells were not significantly different . To identify eb1-egfp comets in each frame of a time - lapse sequence (fig . 1a), we calculated the difference of two gaussian (dog) transformations with standard deviations 1 and 2 adjusted to enhance the band of image frequencies associated with eb1-egfp comets (supplementary note 1 and fig 1c) and individual objects likely corresponding to eb1 comets were then generated by connected component labeling of the thresholded dog image (fig . 1c). To discriminate against selected objects that are not true comets, we selected by template matching objects that conform to the average shape of eb1-egfp comets (supplementary note 3 and fig k2 were introduced to adjust the automatically detected threshold if required for specific imaging conditions . All adjustable parameters are summarized in supplementary table 1 . Because the average eb1 comet shape is determined for each image, the detection is robust against variations throughout the time - lapse sequence, for example during acute application of microtubule polymerization inhibitors that reduce the polymerization rate (fig . 1d). A list of eb1 comets was thus obtained for each frame (fig . 1e), including the position of the object centroid, the magnitude of eb1-egfp comet eccentricity and the angular direction of its main axis . To validate the detection algorithm, the positions of ~120 hand - detected eb1-egfp comets were compared to automatically detected objects for different pairs of 2 and k1 because these parameters are most sensitive to imaging conditions (fig . Because the detection algorithm is sensitive to image intensity variations at the size scale of eb1-egfp comets, in confocal images in which a low eb1-egfp signal is evident along the length of microtubules, k1 was increased to k1> 2 to avoid a large number of false positive detections . In widefield images, however, in which eb1-egfp along the microtubule lattice is obfuscated by out - of - focus light, the default pixel intensity threshold of k1 = 1 worked robustly (supplementary fig . The detection error was quantified by the percentage of false positives (100(1-m / d)) and the percentage of false negatives (100(1-m / g), where d is the number of computer - detected comets, g is the number of hand - selected comets, and m is the number of matches between those two sets). There was low correlation between 2 and false positive detections over most of the range of 2 values tested . Similarly, false negative detections did not follow a clear trend, but a shallow minimum was evident around 2 = 4 indicating that detection was most successful when 2 matched the expected length scale of eb1-egfp comets (fig 1f). The detection performance was similar in widefield images, and was largely independent of the precise shape of the comet as the algorithm was successfully applied to other plus - end tracking proteins (supplementary fig . 2). Detection was also largely insensitive to simulated high frequency pixel noise (supplementary fig . 3). A small number of undetectable eb1-egfp comets is expected in every image due to proximity or overlap, which results in objects that do not pass the template matching of the detection algorithm . However, these events should not introduce a systematic error in the detected eb1-egfp comet population . The elongated shape of eb1-egfp comets implies that the comet centroid is systematically located behind the very tip of the microtubule . For growth rate measurements this offset is irrelevant as long as it remains constant over time . To assess the magnitude of potential temporal variation in the offset we determined the difference between computationally - defined eb1-egfp comet positions and manually detected intensity maxima, which co - localize with the microtubule tip . As expected, the difference increased with comet eccentricity (fig . 1 g). Perpendicular to the microtubule direction it was small (0.49 0.94 pixel) and below the accuracy of manual detection . For highly elongated comets, the difference was 2.95 1.26 pixel along the direction of the microtubule (fig . 1h); we estimated that the standard deviation of this difference was sufficiently small to have minor impact on the analysis of microtubule growth (supplementary note 4). Because the elongation of the eb1-egfp comet depends on the rate of microtubule growth10 indeed, we found that the fastest comets had a high eccentricity, although the distribution also included many slower comets with equally high eccentricity (fig . In contrast, no obvious correlation existed between eccentricity and eb1-egfp peak intensity (fig . Overall, this indicates complex kinetics of eb1 interactions with microtubule ends or fluctuations of microtubule end structures in cells . Detected eb1-egfp comets were tracked using a kalman filter - based multi - object tracking algorithm13 (supplementary note 5 and fig . As expected for random switching from growth to shortening, the track lifetime follows an exponential decay14 with a mean lifetime = 3.6 frames (fig . Because the number of tracks with a lifetime of 2 frames was higher than expected, we suspected that a significant subpopulation of very short tracks resulted from detection or tracking errors . Analysis of the variance of the growth rate histogram as a function of minimal lifetime further indicated a very high standard deviation for short tracks (fig . Thus, subsequent analysis only included growth tracks with a lifetime of 4 frames and greater . To evaluate the combined performance of the detection and tracking algorithms we compared computer - generated tracks with simulated differences in spatial (supplementary fig . We found that the algorithm performed robustly at magnifications that satisfy the nyquist sampling criterion (effective pixel size of <100 nm), and that a temporal sampling of 12 frames s was sufficient to achieve good track reconstruction of microtubule growth . Microtubules are stiff15 and over the short time intervals used to observe microtubule polymerization dynamics they translocate little . As a result, microtubule shortening, rescues and thus, time - shifted, nearly parallel eb1-egfp tracks with significant spatial overlap have a high probability of belonging to the same microtubule . To detect events of microtubule shortening and pausing in addition to the microtubule growth directly visible in eb1-egfp sequences and to infer parameters of their associated dynamics, we developed a computational scheme to cluster tracks that fulfill defined geometrical and temporal constraints based on a priori assumptions about intracellular microtubule behavior (fig . We considered all track initiations in the interval [t+1, t+tmax] in cones with a forward opening angle = 45 and a backward opening angle = 10 as candidate links that could potentially represent a continuation of the terminated growth (forward link) or shortening (backward link), respectively . The narrow backward cone reflects the tendency of microtubules to shorten along the preceding growth trajectory4 . For each candidate link we then calculated the maximal allowable distance for forward (cfwd) and backward gaps (cbwd) as: (1)cfwd = vmaxmin(tgap, tmax) (2)cbwd = min(vmaxtgap, vmedtmax) vmax represents the 95 percentile of the speed distribution from all participating growth tracks, and tgap is the time between termination and initiation of the two candidate tracks . Equation (1) assumes that short time gaps mainly result from temporary occlusion of growing microtubule ends due to noise, overlap, or out - of - focus motion, where the comet reappears at a distance proportional to the growth rate and tgap . Such pauses a microtubule end undergoes a random walk of short growth and shortening events, and the comet reappears at a distance proportional to the square root of time points without detectable eb1-egfp comet . In equation (2), takes into account that microtubule shortening is generally faster than growth3,4 . Vmedtmax, where vmed is the median of the growth track speed distribution, safeguards against the combinatorial explosion of candidate links in dense eb1 comet fields . Furthermore, candidate links were considered only when the directional change i, j between the two associated tracks was less than max = 60 (fig . 3b), because microtubules rarely bend more than 60 over short distances16 . From the set of candidate links that fulfilled the distance and angular criteria we then selected the largest possible subset of links with overall minimal cost using linear assignment17,18 . The cost for an individual candidate link ci, j was defined as (3)ci, j=i, j(||cos(i, j)||||cos(i, j)||), which gives preference to pairs of tracks in close proximity (i, j), with a low bending angle (i, j), and with small transversal shifts (i, j). Although the methods currently used may generate some false positive links, the selection is fairly robust because all tracks compete for links simultaneously . In a typical eb1-egfp time - lapse sequence, each growth track termination associated with ~200 candidate links that fulfilled the temporal (tmax) and angular criteria (, and max). Applying the distance cutoffs (cfwd and cbwd) reduced this to an average of 3 competing track initiations . As expected, the majority of links were positioned in the cell periphery where microtubules more frequently switch between growth and shortening (fig . Forward gaps during which eb1-egfp comets are invisible could be due to periods of pause or very slow growth during which the eb1-egfp signal becomes too dim to be detected, or due to very fast - growing eb1-egfp comets that are temporarily out - of - focus . We therefore calculated the forward gap speed vfwd as the distance between the last point in the terminating track and the first point in the linked growth track divided by the gap duration (fig . 3a, c). Typically, less than 15% of the forward gaps had a speed larger than the slowest 30% of the growth tracks . A small forward translocation is expected even for true pause events because of the latency of detectable eb1-egfp comet formation at the end of a newly growing microtubule (supplementary note 6). Similar to forward gaps, backward gaps contain information about microtubule shortening events, and as above we calculated a backward gap speed vbwd (fig . The histograms of both vfwd and vbwd sharply decayed towards the last bin below the maximally allowable velocity . (1) and (2) were set sufficiently large to capture the majority of clusters of aligned growth tracks (fig . To evaluate the performance of growth track clustering, we recorded dual - wavelength time - lapse sequences of cells expressing both eb1-egfp and mcherry - tubulin (fig . 3e). Because mcherry - labeled microtubules were too dense in the cell interior to directly observe dynamic ends (supplementary fig . 6), we hand - tracked 19 microtubules in a peripheral cell region in which microtubule ends were clearly visible (fig . 3f and video 2). Detection and tracking of eb1-egfp comets was accurate as indicated by the overlap of hand- and computer - tracked growth (fig . Although clustered growth tracks and hand - tracked microtubule trajectories were similar, qualitative comparison showed that many shortening events were missed (fig . This was expected because the clustering algorithm cannot detect shortening that is not preceded or followed by a growth track within the distance and angle criteria . However, a high percentage of false negatives is acceptable as long as the population of microtubules analyzed is sufficiently large and unbiased to provide a representative readout of overall microtubule dynamics . To quantitatively test this, we compared hand - tracked rates of growth, pause, and shortening, and pause durations to those obtained by computational analysis . Importantly, the definition of growth, pause, and shrinkage events by hand - tracking relies on user - defined judgment as to when a microtubule undergoes a change in velocity . The definition of these events by growth track clustering relies on the detection of eb1-egfp comets as molecular markers of microtubule growth . Therefore, prior to a comparison of hand- and computer - tracking, the hand - tracked measurements had to be corrected to account for the differences in assumptions (supplementary note 7). Even after these corrections, hand - tracked growth and shortening rates were significantly higher than the computer - inferred rates (table 1). This deviation originated in the fundamentally different definitions of growth, pause, and shortening events in hand- and computer - tracking . Specifically, computer - tracked growth events contain a subset of events where the microtubule end moves very slowly without loss of the eb1-egfp comet . Similarly, computer - inferred backward gaps contain very slow events that would be classified as pauses in hand - tracked trajectories, in addition to phases of true microtubule shortening . We determined a threshold for a computer - generated growth track or backward gap to be accepted as a significant growth or shortening event by analysis of the velocity distribution of forward gaps, which includes both pauses and gaps due to unobserved growth events (supplementary note 8). This filtering resulted in excellent agreement between computer - inferred and hand - tracked growth and shortening rates (table 1). Thus, although comparisons between computational analysis and hand - tracking cannot be made directly, reasonable assumptions about the limitations of both methods confirmed that the growth and shortening rates we inferred are representative of microtubule polymerization dynamics . A discrepancy between computer - inferred and hand - tracked pause durations remained (table 1), likely due to the latency of eb1-egfp comet formation and disappearance (supplementary note 6). Thus, pause durations inferred by the clustering algorithm are not directly comparable to hand - tracking of homogenously labeled microtubules . Similarly, because shortening events interrupted by pauses will be combined into a single phase, the computer - inferred vbwd is not a direct measure of the microtubule depolymerization rate . Nevertheless, we expect that molecular conditions that change microtubule polymerization dynamics will result in relative changes of these computer - inferred parameters . The clustering algorithm also generated erroneous connections between growth tracks not belonging to the same microtubule . To quantify the frequency of such events, we overlaid clustered trajectories generated with two different stringencies onto to mcherry - tubulin and eb1-egfp channels (videos 46) and verified computer - assigned growth, pause, and shortening phases, as well as catastrophes and rescues . Within the region analyzed (fig . 3e), the software accumulated 475 microtubule growth tracks lasting at least 4 frames . At the less stringent parameter set (=45, =60) 329 tracks clustered into 118 trajectories with an average lifetime of 17 seconds . For the more stringent parameters (=15, =15) 226 tracks clustered into 91 trajectories with a 14 second average lifetime . Only 8% of the trajectories were identical between the two analyses, indicating the sensitivity of the clustering process in selecting viable track connections (fig . Because of the high microtubule density, about half of the computer - assigned events could not be observed clearly . However, if we only considered events that we could visually confirm as true or false, our algorithm showed good performance (supplementary table 2). As expected, the high stringency parameter set was more accurate, and with both parameter sets, the software more robustly connected microtubule growth tracks interrupted by pause rather than shortening events . However, the better performance at high stringency comes at the expense of less data, which bears the risk that some of the heterogeneity of microtubule behavior is lost . We next tested whether the computational analysis detected well - characterized responses to pharmacological microtubule dynamics inhibitors . At low concentrations, nocodazole inhibits both growth and shortening rates and increases the time microtubules spend in pause19, and we detected statistically significant effects on these parameters at concentrations as low as 10 nm (fig . This demonstrates that although our computational analysis is sufficiently sensitive to detect expected differences in microtubule polymerization dynamics . In addition, the results obtained with two different clustering parameter sets were similar, indicating that false positives are sufficiently low so as to not significantly affect correctly clustered microtubule growth tracks . Armed with our new software tool, we examined two long - standing questions in the field: first, we tested whether tubulin acetylation21 alters microtubule polymerization dynamics . We treated cells with trichostatin a (tsa), a broad specificity histone deacetylase inhibitor that inhibits the tubulin - specific deacetylase hdac622 (fig . 5a). We could not detect statistically significant differences in microtubule dynamics when cells were treated with tsa alone . However, in cells simultaneously incubated with 50 nm nocodazole the inferred shortening rate in tsa - treated cells decreased significantly (fig . We thus determined the distance from the cell edge at which the difference in the median microtubule growth rate between the edge and interior microtubule populations was largest . In three cells analyzed, this distance varied between 3.7 m and 6.4 m (fig 5c, d). The median growth rate in the cell body was 19.722.7 m / min compared to 16.617.1 m / min closer to the cell edge . 5e) but at the cell edge a larger fraction of the links between growth tracks were backward links (4460% in comparison to 3543% in the cell interior), consistent with the observation that peripheral microtubules more frequently switch between growth and shortening23,25 . We recently reported that gsk3 inactivation may be required for spatial microtubule dynamics regulation in migrating epithelial cells24, but we did not measure significant shifts in the spatial gradient of growth rates between control cells and cells expressing constitutively active gsk3(s9a). Here, we first confirmed this result using our new method for identifying spatial gradients in microtubule dynamics (fig 5f). Our clustering algorithm then revealed a large decrease in catastrophe probability at the cell edge and a decrease in the percentage of time microtubules spent in the shortening phase (fig . This demonstrates that gsk3 regulates specific aspects of microtubule polymerization dynamics and is consistent with the hypothesis that gsk3 inactivation increases cell edge microtubule interactions with the cortical cytoskeleton24 . Importantly, the clustering algorithm was able to detect these differences even though growth and shortening rates in the cell edge of control and gsk3(s9a)-expressing cells were not significantly different . We present a framework for the analysis of intracellular microtubule polymerization dynamics based on automatic tracking of a microtubule plus end marker such as eb1-egfp, and geometric clustering of growth tracks . Because we never observed growing microtubules without associated eb1, and no condition has been reported that selectively removes eb1 from a subset of growing microtubule ends, eb1-egfp is a faithful reporter of the entire intracellular microtubule population . Since eb1-egfp overexpression may influence microtubule dynamics by enhancing growth rates26, stabilizing microtubules against catastrophes27, or by disrupting the localization of other plus end - tracking proteins28, it is important that our algorithm allows comet detection at low expression levels . The algorithm may be used with other plus end markers such as egfp - clip-170, allowing independent experimental validation . In addition, because a universal eb1-binding motif has been identified29, artificial plus end trackers will likely be available soon that only minimally affect microtubule dynamics . Because eb1-egfp only labels growing microtubules, microtubule behavior has to be interpolated when microtubules stop growing . We introduced several assumptions about the average behavior of microtubules, including maximum rates, allowed angles for direction changes, and the observation that microtubules shorten along the track previously defined by the growing end, to link growth tracks probably belonging to the same microtubule . Thus, our algorithm produces a relatively low number of false positives, but a high number of false negatives . Although we have performed an extensive validation of our clustering algorithm by visual comparison with time - lapse sequences that included homogeneously labeled microtubules, we currently have no formal way to quantify the frequency of clustering errors . This is an important caveat because different imaging conditions or different intracellular densities of growing microtubule ends may affect clustering efficiency . Thus, when using this method, thorough experimental controls must be performed to avoid the propagation of systematic errors (supplementary note 9). It is also important to note that measurements inferred by cluster analysis are not directly comparable to traditionally reported parameters of microtubule dynamics because the assumptions underlying both analysis methods are fundamentally different . For example, terminal shortening cannot be detected by our approach, and backward gaps may include pauses and short growth phases that do not produce a detectable eb1-egfp comet . Nevertheless, our data show that large numbers of growing microtubule ends measured throughout the cell reveal robust and spatially differentiated variations in microtubule polymerization behavior between experimental conditions . Our approach will thus complement and enhance conventional analysis, which typically reports the dynamics of very few microtubules tracked at the cell edge . There is no mathematical reason precluding the adaptation of our approach to the analysis of microtubule dynamics in more physiological three dimensional cell culture systems . In fact, the generalization of tracking and clustering has already been demonstrated in a study of kinetochore dynamics in mitotic spindles30 . Detection of highly eccentric eb1-egfp comets in 3d will be more complex because of the unequal optical resolution in lateral and axial directions . Thus, the comet shape in 3d will depend on microtubule orientation, and the detection bandpass filter used here will have to be replaced by more sophisticated, orientation invariant methods . However, full 3d analysis will also require faster acquisition at sufficient temporal and axial resolution and further development of brighter, more photostable fluorescent probes . However, the ease of eb1-egfp imaging makes our method attractive for high - content screening applications, in which microtubule dynamics may be relevant indicators of cell state.
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Keratomycosis caused by filamentous fungi rapidly progress deep into the stroma with edematous infiltrations, often accompanied by a severe anterior chamber inflammation . In contrast, some species of filamentous fungi, such as alternaria, can cause mild superficial corneal infections . Unfortunately, these are frequently misdiagnosed as a bacterial corneal infection (suzuki et al 2004). Here, we report a superficial keratomycosis caused by the filamentous fungus, beauveria bassiana (b. bassiana), and its successful treatment with voriconazole (vcz). An 80-year - old woman struck her left eye with the frame of her glasses . Due to continuing ocular pain and hyperemia, she was referred to our hospital 9 days after the original injury . The patient was also suffering from recurrent diabetic iritis and continuously used topical antibiotics and corticosteroids . At the time of her first visit to our hospital, there was ulceration of the corneal epithelium at the 5 oclock region along with slight superficial infiltration, slight edema, and ciliary injection . No endothelial plaques or any immune rings although a part of the descemet s membrane was folded, there was only slight anterior chamber inflammation (figure 1). A corneal scraping smear indicated the presence of gram - positive fungal septate hyphae with budding (figure 2a), which led to a diagnosis of keratomycosis by filamentous fungus . Positive staining by fungiflora y also supported the keratomycosis diagnosis . From the culture of the corneal scraping, we were able to isolate a whitish yellow colony, with the fungus exhibiting zigzag rachis and oval conidia, which are characteristics of b. bassiana in slide cultures (figure 2b). Two different microbiology laboratories, the department of the laboratory for clinical investigation at osaka university hospital and the chiba university research center for pathogenic fungi and microbial toxicoses, independently confirmed the presence of the organism . The patient was managed with topical 0.1% miconazole (mcz), 1% vcz once an hour, and 1% pimaricin (pmr) ointment administered once a day along with oral itraconazole (itcz: 100 mg / day). Although there was gradual amelioration of the lesion, the presence of a band - shaped calcium deposit interrupted the re - epithelization, requiring the deposit to be scraped off on two different occasions during the healing process . The lesion healed with only mild scarring remaining after two months . In order to determine the minimum inhibitory concentration (mic) required for each of the antimycotic agents, asty and e - test in vitro susceptibility tests the results for the mic tests were: amphotericin - b: 8.0, 5-fc: 64, fluconazole (fcz): 256, itcz: 0.25, mcz: 0.5, mica - fangin (mcfg): 0.5, and vcz: 0.5 (g / ml). In addition, the organism was also found to be susceptible to topically applied antimycotic agents that included, 0.5% vcz, 5% pmr, and 0.5% mcz, which were administered by the disk method (figure 3). Vcz is a powerful antimycotic agent that is used against filamentous fungi (ozbek et al 2006; sponsel et al 2006; bunya et al 2007; lee et al 2007; thiel et al 2007). Although treatments of five cases of beauveria corneal infection, four cases of b. bassiana, and one case of b. alba have been previously reported, for most of these patients, they also required an additional surgical procedure, such as penetrating keratoplasty or keratectomy, to completely manage the keratomycosis (ishibashi et al 1984;mcdonnell et al 1984; sachs et al 1985; low et al 1997; kisla et al 2000). In order for a successful treatment to be performed without any additional surgical procedures, there needs to be an effective therapeutic regimen for antimycotic agents that can be applied in cases involving filamentous fungi . For example, in the present case, we administered vcz and our data indicated that vcz might have been the crucial factor that was responsible for the success of the treatment . We decided to confirm the sensitivity of the antimycotic agents at their topically applied concentrations, as the sensitivity judged by the clsi standard is based upon serum concentrations, which might not always be identical to the sensitivity of the topically applied antimycotic agents . Although mcz, pmr, itcz were also effective in the treatment of our patient, the result of sensitivity test using disk method indicates that vcz was the most effective agent for b. bassiana infections . It is also important to emphasize two characteristics of b. bassiana keratomycosis that were observed in our patient . First, we clinically noted that the keratomycosis caused by b. bassiana was restricted to the superficial cornea . This differs from other filamentous fungi, as the pathogenesis for b. bassiana is relatively weak and does not cause severe stromal infiltration or anterior chamber inflammation, as has been reported for the rabbit model (ishibashi et al 1987). Since the lesion caused by b. bassiana extended horizontally and not vertically within the stroma, endothelial plaque and anterior inflammation were not evoked . On the contrary, aspergillus and fusarium, which are representative filamentous fungi of keratomycosis, will in general quickly invade deeply into the corneal stroma and induce endothelial plaque and hypopyon . The second notable characteristic of b. bassiana in slide cultures is the presence of septate hyphae with budding and zigzag filaments at the apex . This characteristic appearance can be used to distinguish it from the features of aspergillus and fusarium . Thus, an early smear diagnosis based on these characteristics might help to ensure a successful treatment with antimycotic agents . However, b. bassiana is found in plants and in the soil, and because this organism is entomopathogenic, it is used as a biologic for pesticides (posada et al 2007; safavi et al 2007) thus, the possibility exists that while the patient was working outdoors, the frame of her eyeglasses was contaminated and upon the ocular injury that was caused by the frame of her eyeglasses, the fungi were able to invade through the damaged tissue . Our patient had a long history of recurrent diabetic iritis and continuously used topical corticosteroid . This immunocompromized situation also triggered the colonization of the fungi . In conclusion, a weak pathogenic filamentous fungus with zig - zag rachis and budding, as determined by smear, may account for b. bassiana . Vcz proved to be a powerful tool that not only was successfully used in the current case, but also in the future might be an agent that can be used therapeutically to treat similarly infected types of patients.
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Therefore, prevalence of depression, anxiety, phobia, and obsessive disorders are increased during pregnancy . About 18% of pregnant women are depressed during pregnancy while 14% of them experience depression for the first time during pregnancy . Anxiety is also other common disorder during pregnancy with a prevalence of around 18% . The results of an interview - based study revealed that more than one third of pregnant women had comorbid symptoms of depression and anxiety . Untreated gestational depression and anxiety can affect mothers relationships with their infants and other family members and may lead to different negative consequences for both mother and fetus . For instance, they are associated with intrauterine growth retardation, infant s depressive behaviors, depression during adolescence, low birth weight, premature delivery, mothers postnatal depression, and lower accountability to infants . Anxiety can also predispose children to behavioral, emotional, and cognitive disorders later in their life . Given their high prevalence and negative consequences, effective and timely management of depression and anxiety during pregnancy is of paramount importance . However, studies have shown that neither pregnant women nor psychiatrists pay attention to the management of depression and anxiety . Chan et al ., reported that pregnant women consider inattentiveness as a right option for management of depression . Currently, psychological problems are treated mainly by using psychotherapy and drug therapy . Given their unique conditions and their concerns over the negative effects of medications on fetus, most pregnant women prefer psychotherapy over drug therapy . Two effective psychotherapeutic interventions which have been used for pregnant women are cognitive behavior therapy (cbt) and interpersonal therapy . However, these interventions have not been developed for managing comorbid psychological problems such as depression and anxiety . In recent years, mindfulness - based interventions were developed and used for managing psychological problems among pregnant women . One of these interventions is mindfulness - integrated cognitive behavior therapy (micbt) which integrates mindfulness - based techniques with cognitive behavior therapy . It is a structured treatment strategy which trains clients to internalize their attention in order to regulate their emotions and attention and then externalize and use their regulated emotions and attention for managing their problems . Previous studies have shown the effectiveness of micbt in alleviating depression and hyperglycemia among patients with type ii diabetes mellitus, reducing students procrastination, perfectionism, and worry . However, to our knowledge, the effects of micbt have not yet been evaluated among pregnant women . The aim of present study was to examine the effect of mindfulness - integrated cognitive behavior therapy on depression and anxiety among pregnant women . We used the cluster random sampling method to select three healthcare centers from all healthcare centers which had the necessary facilities for conducting the study and were located in kashan, iran . Study population included of all pregnant women referring to akramian, taleghani, and ketabchi health centers . We referred to the study setting and compiled a list of all pregnant women with a gestational age of one to six months . As all pregnant women had already referred to the health centers since the fifth week of their pregnancy to receive prenatal care services, the lowest gestational age was six weeks . On the other side, the length of our intervention was eight weeks with a one - month follow - up and thus, the highest gestational age was considered to be six months . Pregnant women with higher gestational ages were excluded due to the likelihood of going into labor before the end of the follow - up period and the probable confounding effects of labor on their mental status . Accordingly, we only selected the pregnant women who had a gestational age of one to six months . Then, midwives working in the study setting were asked to inform pregnant women about the study . The inclusion criteria consisted of pregnant women in the one to six months of gestational age, had at least a high school degree, acquired a score of greater than 13 in the edinburgh depression scale and a score of greater than 16 in the beck anxiety inventory, and had no history of psychological disorders or chronic physical problems, have not received psychotherapy or drug therapy during the last six months preceding the study, and women whose depression and anxiety were not secondary to certain known causes such as grief, marital conflict, divorce, or unwanted pregnancy . The exclusion criteria were having no desire for continuing participation in the study, having two or more absences from the study intervention sessions, and having a premature delivery . With an alpha of 0.05, a beta of 0.1, and an effect size of 0.8 (25), the cohen s formula for sample size calculation revealed that 33 participants were necessary for each study group . However, by considering an attrition rate of 10%, we recruited 40 participants to each group . Participants were randomly allocated into experimental and control groups using block randomization method, by using units of 4 blocks . The study primary outcomes were depression and anxiety which were measured three times: before the intervention (t1), immediately after (t2), and one month after intervention (t3). The measurement tools were a demographic questionnaire, the edinburgh postnatal depression scale, and the beck anxiety inventory . The edinburgh postnatal depression scale (epds) had been previously used for measuring pre- and postnatal depression among women . Items 1, 2, and 4 are scored from 0 to 3 while the other items are scored reversely . The cronbach s alpha of the persian epds has been reported to be 0.92 . We also assessed the reliability of the epds which yielded a cronbach s alpha of 0.82 (n= 100). The beck anxiety inventory (bai) is a 21-item questionnaire for measuring anxiety severity . Each bai item represents one of the common manifestations of anxiety . On each item, a respondent can choose one of the four points of not at all, the cutoff scores of the bai are as follows: 07: no or minimal anxiety; 815: mild anxiety; 1625: moderate anxiety; and 2663: severe anxiety . The validity and the reliability of the persian bai we also found that the cronbach s alpha of the inventory was 0.78 (n= 1513). The study intervention was a micbt program which was implemented in eight 90-minute sessions . An overview of micbt, the flow of the program, and the contents of the next sessions; session 2.the basic principles of mindfulness, the components of cbt, and mindful breathing; session 3.mindful breathing (continued), step - by - step body scanning exercises, and awareness of visceral sensations; session 4.body scanning exercises (continued), behavior therapy techniques (such as problem solving), and the relationship of mindfulness with cbt; session 5.body scanning exercises (continued); session 6.interpersonal skills, assertiveness, and role play; session 7.acceptance and management of suffering in daily life; session 8.review and evaluation . The intervention was performed by a msc in clinical psychology (first author) who had received specialized training in this area under the supervision of a phd in clinical psychology . Participants in the experimental group received micbt while participants in the control group received only routine prenatal care services such as weight control, blood pressure monitoring, and delivery - related educations . At the end of the study participants in control group received training manual of intervention sessions . Descriptive statistics measures such as frequency, mean, and standard deviation as well as statistical tests such as repeated - measures analysis of variance (anova) were used for data analysis by using spss (version 11.5) software . This study was approved by the ethics committee of kashan university of medical sciences, kashan, iran, with an approval code of p/13/0/3/4005, december 17, 2014 and is registered in the iranian registry of clinical trials with the irct2015012920869n1 code . Before implementing the intervention, participants were asked to fill out and sign the informed consent form of the study . Eighty pregnant women participated in this study (40 person for each group). Ten participants from the experimental were excluded due to their poor attendance at micbt sessions . Moreover, seven participants from the control were also excluded because of developing pregnancy - related physical problems or their failure to complete the study questionnaires at either t2 or t3 . Finally, data analysis was performed on the data retrieved from 63 participants (30 women in the experimental and 33 women in the control group) (figure 1). Chi - square and independent - samples t - test were used for demographic variables (age, body mass index and gestational age were analyzedusing the t - test, and educational level, previous childbirth and job were analyzed using the chi - square test).the of participants' mean age in the experimental and control groups were 26.0 (5.8) and 26.7 (4.5), respectively (p <0.05). The mean of participants gestational age in the experimental and control groups were respectively 15.0 (1.1) and 15 (1.2) weeks (p <0.05; table 1). Study groups did not differ significantly from each other concerning variables such as age, education, number of deliveries, gestational age, body mass index, and employment status (p <0.05; table 1). Clinical trial flowchart data are presented as n (%), bindependent - samples t - test was used . Table 2 shows the mean scores of participants depression and anxiety at the three measurement time - points of t1, t2, and t3 . At t1, the mean scores of depression and anxiety in the experimental and the control groups were 16.83 (2.7) vs. 16.33 (2.64) and 19.76 (6.33) and 20.24 (6.11), respectively . The differences between the study groups regarding the pretest mean scores of depression and anxiety were not statistically significant (p<0.05). However, the results of the repeated - measures anova test for comparing the variations of depression and anxiety scores showed that at t2 and t3, the mean scores of anxiety and depression in the experimental group were significantly lower than the control group (p <0.001; table 2). Repeated measures anova variations of depression scores in the two study groups variations of anxiety scores in the two study groups the results of this study showed that mindfulness - based interventions produced positive effects during pregnancy . For instance, vieten and astin implemented an eight - session mindfulness - based educational program for women who were in the second or third trimester of their pregnancy ., also conducted a pilot study to investigate the effects of mindfulness - based training on psychological distress among ten multiparous pregnant women . 3 the control groups were respectively in the 1228 and 1729 weeks of their pregnancy . They used epds and the depression anxiety stress scale (dass) for data collection and reported that mind fulness - based intervention significantly reduced the epds and the dass scores . In another study, duncan and bardacke employed a one - group non - controlled design and implemented a mindfulness - based educational program for 27 pregnant women . The women were in the third trimester of pregnancy and most of them (92.6%) were experiencing their first pregnancy . The findings revealed that mindfulness was effective in alleviating depression, anxiety, and negative affect and in enhancing positive affect and well - being . Given the facts that their study was non - controlled and some of their participants had previously participated in yoga training courses, the findings reported by duncan and bardacke might have been affected by participants previous experiences . Recently, a study was conducted on 39 women with major depression disorder (27 women) and bipolar - spectrum disorder (12 women) in order to assess the effects of an eight - week mindfulness - based cognitive therapy . The participating women either were pregnant (12 individuals), had planned for pregnancy (11 individuals), or were in their postnatal period (26 ones). The findings showed that mindfulness - based cognitive therapy significantly alleviated major depression disorder and had no significant effect on bipolar - spectrum disorder . As mentioned earlier, depression and anxiety are comorbid conditions.therefore, the effectiveness of treatments can be enhanced through adopting treatment strategies which can alleviate both of them . This is of paramount importance to pregnant women because simultaneous and rapid alleviation of mother s depression and anxiety can reduce the negative effects of these two conditions on fetus . Mindfulness is rather a new concept in psychotherapy and has attracted therapists and researchers attention in recent years . It teaches people to focus on the present moment, identify their own experiences, cultivate a non judgment attitude, accept their experiences, reduce reactivity towards them, and effectively manage them . Mindful attention requires preventing habitual reactions and creating non judgment attitude towards, acceptance of, and detach from internal experiences . People who receive cbt need executive functions such as keeping attention, deliberate actions, affect regulation, behavior control, problem solving, and motivation in order to get the most from cbt . Executive functions are mainly performed in the prefrontal area of the brain . When people access their executive functions and use them for self - regulation, their distress is reduced . On the other hand, people suffering from depression experience distress due to having limited access to their executive functions . Accordingly, during sensitive courses of emotional excitements such as pregnancy, executive functions can be used and promoted for alleviating depression and anxiety . The effectiveness of cognitive behavior therapies can be enhanced through integrating mindfulness - based executive functions . Moreover, emotional dysregulation are related to the dysfunction of the front limbic region, i.e. Where prefrontal activity is reduced and amygdale is stimulated . Evidence shows that integrating cbt with mindfulness - based interventions produces more significant results compared with single therapy . Great effectiveness of mindfulness - based interventions has required clinicians to use these interventions to enhance the effectiveness of their treatments . Micbt is one of these interventions and further investigations are still needed for assessing and confirming their effectiveness . One of the study limitations was related to lack of background knowledge about the study subject matter due to its novelty . Mindfulness interventions are among the most appropriate and effective strategies for managing psychological problems . Nonetheless, given their novelty, few studies have been conducted so far on these interventions . Moreover, we could not retrieve a random sample of pregnant women from all healthcare centers located in kashan, iran . We also were not able to assess the long - term effects of micbt in the postnatal period . Future studies are recommended to perform long - term follow - up assessments in order to evaluate the effects of micbt on different pregnancy - related outcomes such as emotional well - being, postnatal depression, and mother - infant relationships . The results of this study indicated that micbt is effective in alleviating pregnant women s depression and anxiety even for one month after implementing it . We wish to thank the research deputy of kashan university of medical sciences, kashan, iran, for financial supporting of this project . Moreover, authors are grateful to staffs of akramian, taleghani, and ketabchi health centers, and dr.
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Giant cell pancreatic cancer was first described by sommers and meissner in 1954 . In the literature, these rare tumours have been divided into two subtypes: osteoclast - like giant cell and pleomorphic giant cell carcinoma of the pancreas . Although a number of reviews have shown possible prognostic differences between these two subtypes, the most recent world health organisation (who) classification places the neoplams in the same category, undifferentiated carcinoma with osteoclast like giant cells . In any case, unless detected early, the majority of cases have a very poor prognosis often worse than pancreatic adenocarcinoma . In this report, we review the literature in the area and present a unique case of a patient with known alcohol abuse who developed metastatic pleomorphic giant cell cancer of the pancreas within months of a diagnosis of a pancreatic serous cystadenoma . The case also highlights the challenges in managing pancreatic cystic lesions and emphasizes the importance in considering rare forms of pancreatic cystic masses when the findings are atypical for the presentation . A 44-year - old man, who was a known alcoholic, presented to the first department of surgery, university of athens with symptoms of epigastric abdominal pain, vomiting, and weight loss . Biochemical analysis revealed alanine aminotransferase (alt) 172 u / l, gamma - glutamyl transpeptidase (-gt) 163 u / l, alkaline phosphates (alp) 464 u / l, and c - reactive protein (crp) 84.90 mg / l . The serum levels of various tumour markers were not increased: alfa feto protein (afp) 1.6, carbohydrate antigen 19 - 9 (ca19 - 9) 12.3, carbohydrate antigen 72 - 4 (ca72 - 4) 1.8, and carcinoembryonic antigen (cea) 3.6 . A subsequent computed tomography (ct) scan revealed the presence of a 4 cm 4.2 cm cystic lesion in the body and tail of the pancreas . There was also a minor increase in diameter of the peripheral segment of the pancreatic duct and disseminated damage of the pancreatic parenchyma, suggestive of multiple episodes of pancreatitis in the past . Fine - needle aspiration biopsies with endoscopic ultrasound guidance (eus) of the pancreatic lesion were performed . U / l), and cytology was consistent with the diagnosis of a serous cystadenoma . Tumour markers (cea, afp, and ca 19 - 9) were negative . The patient's symptoms subsequently improved with conservative management only: fluid resuscitation, analgesia, and antiemetics . Six days after admission, the patient was discharged with outpatient followup with repeat scanning . Four months followed without symptoms, and a second ct scan indicated only a small increase (4 cm 4.5 cm) in the pancreatic cystic lesion . A repeat ct in addition to magnetic resonance imaging (mri) and magnetic resonance cholangiopancreatography (mrcp) revealed only marginal enlargement in the pancreatic cystic lesion, but now there were also multiple lesions in the liver ranging from a few milimetres to 2.5 cm in diameter (figure 1). The patient underwent laparoscopy and biopsy which revealed infiltration of the liver tissue by a giant cell carcinoma . The growth pattern was diffuse with pseudospaces, and the stroma was loose and abundant with inflammatory infiltrates (figures 2(a) and 2(b)). The neoplastic cells showed cytokeratin 7 (figure 3(a)) and cytokeratin 19 (figure 3(b)) immunopositivity . The morphological and immunohistochemical features from the hepatic, pancreatic, and lymph node biopsies revealed the diagnosis of pleomorphic giant cell carcinoma, with the organ of origin the pancreas . At this advanced stage, surgical resection was not possible and the patient died four months later . Cystic lesions of the pancreas are an increasingly common finding with modern radiological investigations, although pancreatic cystic neoplasms remain rare and account for only 10%15% of these cysts [4, 5]. Once identified, the initial step in managing cystic lesions is differentiating a pancreatic pseudocyst from a cystic neoplasm . A careful review of the clinical background of the patient is paramount, with previous documented pancreatitis or identifiable risk factors for pancreatitis (chronic alcohol consumption, history of gall stones, or a strong family history of pancreatitis) an essential starting point . If these factors are present the cystic lesion is more likely a pseudocyst, but it may also be the first presentation of a neoplastic lesion . The patient demographics, and the cyst size, site, and quantity provide valuable information in predicting the nature of the lesion . Ultimately a combination of ct, mri, mrcp, endoscopic retrograde cholangiopancreatography (ercp), or eus with biopsy provides the diagnosis in most cases, with eus the most fashionable approach at present [68]. The patient in this report was a 44-year - old male with known excess alcohol consumption, and ct findings are consistent with previous pancreatitis . Although the clinical features were suggestive of a pseudocyst, an eus with biopsy was performed and while the amylase was elevated, the cytology suggested a serous cystadenoma . Serous cystadenomas are largely benign lesions which present more frequently in middle aged / elderly females without a history of pancreatitis are evenly distributed throughout the pancreatic gland and have a low amylase level and low tumour markers (specifically cea) [9, 10]. It is worthwhile noting however that the diagnostic accuracy of ct for pancreatic cysts has been reported to range from 2090% and the sensitivity for analyzing pancreatic cystic fluid shows a range from 5093% [1113]. In this instance, the patient clinically improved and with cytology demonstrating a serous cystadenoma; close observation was deemed the most appropriate management . Interestingly, tseng et al . Recommend excision of large (> 4 cm) serous cystadenomas irrespective of symptoms, which goes against the management of this 4.4 cm cyst . Although serous cystadenomas are considered effectively benign, there have been a number of single - case reports highlighting the presence of malignant features [15, 16]. However, there are no reported cases in the english literature of a coexistent serous cystadenoma and giant cell pancreatic cancer, as was the case here . Giant cell pancreatic cancer is a rare neoplasm, characterised by the presence of giant cells, hypervascularity, and an inflammatory response . It accounts for 2%12.8% of all cases of pancreatic malignancies, and despite active intervention, patients usually die within months of diagnosis . The neoplasm has been subdivided into two groups, osteoclast - like and pleomorphic giant cell pancreatic cancer . Indeed, a third grouping, known as mixed type, has highlighted the possibility that these tumours may indeed represent a morphological spectrum with osteoclast - like giant cell tumours at one end and pleomorphic giant cell tumours at the other . Classic osteoclast - like giant - cell tumours have a predominant population of osteoclast - like giant cells and abundant hemosiderin granules whereas pleomorphic giant cell pancreatic neoplasms have more pleomorphic multinucleated giant - cells and mononuclear cells . The clinical features of pleomorphic giant cell carcinoma are comparable to those of pancreatic adenocarcinoma with abdominal pain and weight loss the most prevalent [17, 20]. Cancer site has a role to play here, with head of pancreas cancers presenting more frequently with jaundice . Although there does not appear to be a preferred pancreatic site, even though some studies report higher prevalence in the body and tail . The mean age of onset is 65 years, and there appears to be a male predominance . Elevated inflammatory markers are present in the majority of cases, and ct findings often show large irregular hypodense masses (majority> 6 cm) [17, 19, 20]. The survival range for pleomorphic giant cell pancreatic cancer ranges from several weeks in advanced unresectable disease to 25 months [21, 22]. The osteoclast - like giant cell variant may have a better prognosis (due to reduced prevalence of metastasis), but the evidence for this is inconclusive [20, 22, 23]. There have been at least two reported cases of osteoclast - like giant cell pancreatic tumours presenting as pseudocyst lesions and a similar number as mucinous cystadenomas [2426]. There has been one case of a mixed (osteoclast - like and pleomorphic) giant cell pancreatic cancer presenting as a pseudocyst . There have been no reported cases of an association between serous cystadenoma and any form of giant cell pancreatic cancer, which we report here . The diagnostic accuracy of ct, eus with biopsy and cytology is quite high, depending on the papers cited [69]. However, it is more than possible for a neoplastic cyst to be missed on a single biopsy, which is plausible in the case here . However, considering the clinical improvement in the patient's condition, background history of alcohol excess + / episodes of pancreatitis and stable disease on repeat scanning, the role of conservative management could be justified . It may also be suggested that in the eight months from initial diagnosis an aggressive pleomorphic giant cell cancer may have developed at or near the site of the presumed cystadenoma rather than a direct association between the two . The management of this case may have been different on reflection of the radiological and cytological findings considering the clinical background of the patient . Surgical resection at the initial presentation may have identified the neoplasm and altered the outcome for the patient . Overall the case emphasizes the challenge in managing pancreatic cystic lesions and suggests lowering the threshold for surgical resection in atypical cases . The report discusses an unusual case of pleomorphic giant cell cancer of the pancreas which presented initially as a pancreatic cystic lesion and was diagnosed as a serous cystadenoma . The case highlights the challenges in managing pancreatic cystic lesions and emphasizes the importance of considering less common forms of pancreatic cystic masses when the findings are atypical with the presentation . Surgical excision in these cases over conservative steps may be the most appropriate management.
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Local anesthetic agents are chemicals that reversibly block the transmission1 of action potential of nerve membrane . An essential pre - requisite to success in dentistry is to achieve good quality local anesthesia (la). Local anesthetic agents are normally associated with absence of pain during surgical intervention in bone and soft tissue . There are many local anesthetic agents, lignocaine being the gold standard2 available with the wide selection of vaso - constrictive agents that improve the clinical efficacy and the duration la . Lignocaine diffuses readily through interstitial tissues and lipid rich nerves, giving rapid onset of action . Its vasodilating effect is more than that of prilocaine and mepivacaine.3 adrenaline prolongs the duration as well as the depth of anesthesia . It is effective in preventing or minimizing blood loss during surgical procedures . Due to vaso - constrictive effects of adrenaline, absorption of la and systemic toxicity are reduced . If adrenaline is not added to lignocaine, vasodilating effect of lignocaine limits pulpal anesthesia to only 5 - 10 min . 0.2 mg adrenaline is a safe maximum dose in healthy patients and it is best to limit the total dose to 0.04 mg in cardiac patients . It should be kept to a minimum amount capable of producing adequate results . Systemically adrenaline like drugs can cause a number of cardiovascular disturbances while most are short lived, permanent injury or even death may follow drug induced ventricular fibrillation, myocardial infarction or cerebro - vascular accidents.4 to compare the efficacy of 2% lignocaine with two different concentrations of adrenaline - 1:80000 and 1:200000.to study the cardiovascular effects of la with adrenaline in two different concentrations.to recommend the ideal concentration of adrenaline to the elderly and asa iii and asa iv risk patients with the history of cardiovascular problems . To compare the efficacy of 2% lignocaine with two different concentrations of adrenaline - 1:80000 and 1:200000 . To study the cardiovascular effects of la with adrenaline in two different concentrations . To recommend the ideal concentration of adrenaline to the elderly and asa iii and asa iv risk patients with the history of cardiovascular problems . To compare the efficacy of 2% lignocaine with two different concentrations of adrenaline - 1:80000 and 1:200000.to study the cardiovascular effects of la with adrenaline in two different concentrations.to recommend the ideal concentration of adrenaline to the elderly and asa iii and asa iv risk patients with the history of cardiovascular problems . To compare the efficacy of 2% lignocaine with two different concentrations of adrenaline - 1:80000 and 1:200000 . To study the cardiovascular effects of la with adrenaline in two different concentrations . To recommend the ideal concentration of adrenaline to the elderly and asa iii and asa iv risk patients with the history of cardiovascular problems . Irrespective of the number of teeth extracted, inferior alveolar and lingual nerve blocks were administered (long buccal whenever necessary). Unilateral extractions were carried out in a single sitting and the other side was done on next visit . The patients who had any systemic illness like hypertension, cardiac problems and diabetes etc . Were excluded from the study . The following data were collected during the procedure: time of administration of la, onset of anesthesia noted as subjective and objective symptoms (subject: tingling and numbness in the lower lip and tongue, objective: absence of pain on instrumentation).amount of la used.pulse rate and blood pressure (bp)were recorded using automated multi - nodular monitor - before the administration and immediately, 10 mins, 30 mins and 60 mins.evaluation of analgesia was done by the operator as successful, partial success and failure.intra operatively pain was scored on visual analogue scale (vas)5 as reported by the patient . The vas was presented in the form of a printed ruler numbered 0-10.any sign of systemic toxicity was recorded . Time of administration of la, onset of anesthesia noted as subjective and objective symptoms (subject: tingling and numbness in the lower lip and tongue, objective: absence of pain on instrumentation). Pulse rate and blood pressure (bp)were recorded using automated multi - nodular monitor - before the administration and immediately, 10 mins, 30 mins and 60 mins . Evaluation of analgesia was done by the operator as successful, partial success and failure . Intra operatively pain was scored on visual analogue scale (vas)5 as reported by the patient . The vas was presented in the form of a printed ruler numbered 0 - 10 . Safety and efficacy of the two solutions with two different concentrations of adrenaline were studied in 40 patients . No systemic toxicity was observed . As observed in the table 1 and graph 1, there was no significant change in both the groups in the point of view of time of onset . With regard to the duration of action of la, 1:80000 adrenaline concentrations showed more than that of 1:200000 . This is due to the faster absorption of la when used with less concentration of adrenaline . The amount of la used for both the groups does not show any significant change . As shown in the table 1, there was no change in the vas scale as both the solutions had same efficacy . Time of onset, duration, amount of la used and pain (vas) comparison in two groups . Comparing the onset of anesthesia in two groups subjectively and objectively . There was significant rise in the pulse rate immediately when la with 1:80000 adrenaline concentrations was used and it came to the normal gradually after 60 min as seen in the graph 2 . Variations in pulse rate . But when la with 1:200000 adrenaline concentrations was used, there was no significant rise in the pulse rate . While assessing the systolic bp, there was rise significantly when la with 1:80000 adrenaline concentrations was used whereas there was no major change observed when la with 1:200000 was used as shown in graph 3 . There was slight rise in diastolic bp when la with 1:80000 adrenaline was used but there was decrease when la with 1:200000 adrenaline concentration as seen in the graph 4 . Though there are many la agents available in the market, lignocaine is the most widely used in dentistry . Most of the time, la agents are used with vasoconstrictors, though the concentrations may vary . The presence of a vasoconstrictor in the anesthetic cartridge has a major influence on the duration of anesthesia.6 the ability of vasoconstrictors to retard the systemic absorption of injected la agents is the basis for their widespread use.4 vasoconstrictors employed in local anesthetic solutions have the potential for interacting with the wide variety of drugs.7 physiological responses associated with local anesthetic solutions containing a vasoconstrictor have included changes in heart rate and bp, dysarrythmias, ischemic changes (st segment and t wave), the release of endogenous catecholamines, endocrine response to surgery and hypokalemia.8 local aesthetic agents with adrenaline as the vasoconstrictor used for the surgical soft tissue and bone interventions in the oral region tend to cause more post - operative pain than la without adrenaline as the vasoconstrictor.9 in our study, comparison of two different concentrations of adrenaline is evaluated (efficacy as well as cardiovascular effects). So the epinephrine concentration does not affect the clinical efficacy of local anesthetic agent as reported by the study conducted by santos et al . And dagher et al.10,11 the time of onset of anesthesia as well as the amount of la used in our study is similar to the study conducted by malamed et al.12 significant cardiovascular effects were observed in the study as seen in the statistical analysis, there was significant rise in the mean pulse rate when 1:80000 adrenaline used whereas no significant change observed in 1:200000 used . There was significant rise in the systolic and diastolic bp when la with 1:80000 adrenaline used while 1:200000 adrenaline did not bring any significant change . (2001)demonstrated significant cardiovascular changes 10 mins after the injection of lidocaine with the higher adrenaline concentration of 1:80000.8 gregorio et al . Has reported after his study that it is important to stress that with articaine and other local anesthetic solutions in general, 1:100000 and 1:50000 epinephrine concentrations are associated with greater cardiovascular stimulation than 1:200000 epinephrine formulations.13 for an adult healthy patient, la with any of the concentration of adrenaline can be used as the efficacy is not altered in both the solutions . Since the duration of anesthesia is significantly different for both the solutions, la with 1:80000 is preferred in case of long procedure . For hypertensive patients, some physicians advise la without adrenaline but la with 1:200000 could be used taking the advantage of adrenaline and avoiding complications as the adrenaline concentration is insignificant . The present study of two types of 2% lignocaine with two different concentrations showed that both of them have the same efficacy . Coming to the cardiovascular effects, 1:80000 adrenaline concentrations showed significant rise in pulse rate as well as bp as compared with the other drug . For the cardiac and elderly patients, when treating any patient taking medication, one should be aware of the potential medical complications and always use the least concentrated solution of vasoconstrictor that allows for deep anesthesia during a period of time . La with a lower concentration of adrenaline will not compromise the profundity and success of anesthesia and would be safe for this group of patients.
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A pleasant and pleasurable first dental visit of the child is important in establishing a bond of trust between him and the dentist, thus ensuring a successful outcome of the ensuing treatment . Achieving a child's co - operation to deliver children avoid dental treatment mainly due to fear and anxiety resulting from anticipated pain, fear of unfamiliar surroundings, bright lights, loud noises, sharp instruments etc ., and it is more so at the time of the first visit . Since young children are curious by nature, simple methods like tender love and care (tlc), modeling and tell show and do (tsd), in which a pediatric dentist is well trained, can be successfully used on a majority of children for an introduction to the operatory and familiarization . These methods, however, may not prove to be as successful in the very young potentially un - cooperative children <4 years of age, leaving a large number of procedures, many a times, unaccomplished or compromised . This group makes up a sizeable number of pediatric dental patients visiting the out - patient department for treatment . A handful of other cases, which also remain outside this domain, are children with inherent behavioral problems and young, anxious children reporting with dental emergencies requiring immediate attention . General anesthesia, apart from being an expensive procedure, requires a hospital set - up and is sometimes a poor compromise between the extent of treatment and difficulties associated with it . It therefore, remains a good choice for children requiring extensive treatment or children not found fit for sedation usually due to an underlying medical problem or sometimes, parental demand . The various agents currently being used, the world over, for sedation / anesthesia in pediatric dentistry, are propofol, ketamine, midazolam, n2o - o2 analgesia and sevofluorane . These drugs / agents can be administered via, intravenous, oral, rectal, intranasal, and inhalation routes . Out of various routes, it is the oral route, which is considered the most acceptable and convenient . Midazolam, via the oral / oral - transmucosal route is currently a popular agent among pediatric dentists for sedating young children as it is a short acting benzodiazepine, which is efficacious, has a rapid onset of action, an excellent safety profile, a reliable dose dependent anxiolysis and a low - grade anterograde amnestic effect . The limitations of oral midazolam include a poor depth of sedation, poor analgesia, respiratory depression and a short duration of action . There is no study in the literature, which has studied the effect of midazolam sedation on common anxiety provoking stimuli in dentistry . The purpose of this study was to evaluate if 0.5 mg / kg midazolam via the oral - transmucosal route is efficacious in significantly reducing the child's anxiety at different procedural steps of a class ii restorative procedure compared with placebo, as measured by the venham's clinical anxiety scale . A sample of 40 healthy, american society of anesthesiologists i, children aged 3 - 4 years having at least one carious primary mandibular molar requiring a class ii amalgam restoration and no previous dental history were selected from the out - patient department of oral health sciences centre, postgraduate institute of medical education and research . The children were randomly divided into experimental and control groups comprising of 20 children each, using block randomization technique . Written informed consent was obtained from the parents of children involved in the study . On the day of the procedure, the children in the experimental group (group i) received 0.5 mg / kg body weight of midazolam (ranbaxy, 1 mg / ml vial) mixed in strawberry syrup and those in the control group (group ii) received the same syrup mixed in saline, 15 min prior to having been taken inside the operatory by the principal investigator (pi). The solutions were administered by the pi in increments, using a bowl and spoon . The study was double - blind in nature with both the investigator and the parents / child not aware of the group to which they belonged . To maintain the blind nature of the study the test and control solutions were prepared by a co - investigator and were of similar consistency . The clinical procedure for both groups comprised of the following steps: entry into operatory (oe); 15 min after administration of the test solution the child was brought into the operatory accompanied by the anesthetist and the chief investigator to be seated in the dental chair, administration of local anesthesia (la); local anesthetic gel (xylocaine gelly) was applied on the site of injection on the side of the tooth being restored, followed by a classical inferior alveolar nerve block in that region with 2% lignocaine hydrochloride having 1: 80,000 dilution of adrenaline, using a 26 gauge sterile needle, rubber dam application (rda); amalgam being a technique sensitive material all cases were treated under rubber dam . In most cases clamp no . 8a was used for primary second molars and premolar clamps no . 1 and 2 for primary first molars, operative procedure (op); in the selected carious mandibular molars, a class ii mesio - occlusal or disto - occlusal cavity was prepared depending on the location of carious lesion followed by restoration with amalgam, by a single investigator (pi). The anxiety levels were evaluated by the pi using the venham's clinical anxiety scale (1977) [table 1], first at baseline as the pretreatment anxiety scores and then at the beginning and termination of each one of the treatment steps as during treatment anxiety scores . Behavior management techniques (bmt) such as tlc, tsd, distraction were used in both groups, voice control and physical restraint were used only when the child showed extremely un - coperative behavior corresponding to venham's score of 4, leading to interference in treatment . The parameters such as total treatment time, depth of sedation and acceptability of the drug were also recorded and have been shared in a previously published part of the same study . A trained anesthetist, at baseline and subsequently at every 15 min interval monitored all children for blood pressure, respiratory rate, and oxygen saturation during the entire procedure . Student's t - test at 5% significance level was used for analysis . For intragroup analysis, paired t - test the two groups were found to be relatively well matched according to the baseline anxiety levels assessed using the venham's scale . Though the baseline anxiety levels were slightly greater in the midazolam group, the difference was statistically not significant [table 2]. There was a highly significant (p <0.001) reduction in anxiety levels in the midazolam group from the baseline levels till the time the child was brought into operatory 15 min after administration of the test solution . In the behavior management group comparison between baseline anxiety and anxiety level on entry into operatory 15 minutes after administration of the test solution the baseline and final values corresponded to the anxiety levels at the start and end of each procedural step . It can be appreciated that the majority of the times the mean final values were always lower than the baseline values in both groups [table 3]. This trend was seen for all steps in group i. the group ii showed a similar pattern for step oe step of group ii where the final anxiety levels were higher than baseline, and a difference was also seen in the step though a somewhat similar trend was seen in the two groups in terms of reduction from the baseline anxiety levels to the final anxiety levels, it was, however, noted that both baseline and final anxiety levels remained lesser in group i throughout the clinical procedure as compared to group ii [table 3 and 3a]. This intergroup difference was significant for oe, and la administration (p <0.01) and statistically not significant for rda and op . Mean anxiety scores during treatment comparison of baseline and final value it was interesting to note that in both the groups the baseline anxiety levels of the succeeding stage always exceeded the final values of the previous stage until the step rda [table 3 and 3b]. This trend was, however, reversed in case of the op step in both groups where the baseline anxiety levels were lower than the final levels after rda (p> 0.1). Comparison of the final value of one step with baseline of next step further, difference in anxiety levels was derived by subtracting the final values from baseline values for each clinical step in the two groups . The mean values for oe were found to be 0.10 for group i and - 0.20 for group ii respectively . The negative value for group ii indicated that instead of a decrease in anxiety score from baseline to final there was an increase in anxiety [table 3 and 3c]. In the steps that followed, that is, la and rda, the decrease in anxiety was found to be of a greater degree in group ii than in group i (p> 0.1). The children in group ii did not show any change in the anxiety level during the start and end of op compared with a 0.26 decrease in group i (p> 0.1). Intergroup comparison of difference of final and baseline anxiety levels, for each step in group i; bringing the child into operatory and administration of la, could be accomplished in all the cases compared with group ii where it was not possible to bring two children inside the operatory, and another five children refused la administration, making a total of seven children in whom this step could not be accomplished . One child in group i refused rda (never reached the next clinical step) and one more did not allow the op to be completed, thus having unaccomplished procedures in two cases . In group ii, the total number of children in whom rda could not be accomplished were 12, and there was one child who refused the op making a total of 13 children in whom cavity cutting and filling remained unaccomplished [table 4]. Number of unaccomplished procedural steps in children with incompatible treatment it was observed that almost all children in group i could be managed using tsd only, except for two children who required use of voice control and physical restraint during rda . On the contrary, in group ii, two children required restraint even during oe, three during administration of la and two during rda . None of the children in this group, however, required restraint for the op [table 5]. The differences in the use of type of bmt between the two groups were, however, not found to be statistically significant (p> 0.1). The two groups were found to be relatively well matched according to the baseline anxiety levels assessed using the venham's scale . Though the baseline anxiety levels were slightly greater in the midazolam group, the difference was statistically not significant [table 2]. There was a highly significant (p <0.001) reduction in anxiety levels in the midazolam group from the baseline levels till the time the child was brought into operatory 15 min after administration of the test solution . In the behavior management group comparison between baseline anxiety and anxiety level on entry into operatory 15 minutes after administration of the test solution the baseline and final values corresponded to the anxiety levels at the start and end of each procedural step . It can be appreciated that the majority of the times the mean final values were always lower than the baseline values in both groups [table 3]. This trend was seen for all steps in group i. the group ii showed a similar pattern for step oe step of group ii where the final anxiety levels were higher than baseline, and a difference was also seen in the step though a somewhat similar trend was seen in the two groups in terms of reduction from the baseline anxiety levels to the final anxiety levels, it was, however, noted that both baseline and final anxiety levels remained lesser in group i throughout the clinical procedure as compared to group ii [table 3 and 3a]. This intergroup difference was significant for oe, and la administration (p <0.01) and statistically not significant for rda and op . Mean anxiety scores during treatment comparison of baseline and final value it was interesting to note that in both the groups the baseline anxiety levels of the succeeding stage always exceeded the final values of the previous stage until the step rda [table 3 and 3b]. This trend was, however, reversed in case of the op step in both groups where the baseline anxiety levels were lower than the final levels after rda (p> 0.1). Comparison of the final value of one step with baseline of next step further, difference in anxiety levels was derived by subtracting the final values from baseline values for each clinical step in the two groups . The mean values for oe were found to be 0.10 for group i and - 0.20 for group ii respectively . The negative value for group ii indicated that instead of a decrease in anxiety score from baseline to final there was an increase in anxiety [table 3 and 3c]. In the steps that followed, that is, la and rda, the decrease in anxiety was found to be of a greater degree in group ii than in group i (p> 0.1). The children in group ii did not show any change in the anxiety level during the start and end of op compared with a 0.26 decrease in group i (p> 0.1). In group i; bringing the child into operatory and administration of la, could be accomplished in all the cases compared with group ii where it was not possible to bring two children inside the operatory, and another five children refused la administration, making a total of seven children in whom this step could not be accomplished . One child in group i refused rda (never reached the next clinical step) and one more did not allow the op to be completed, thus having unaccomplished procedures in two cases . In group ii, the total number of children in whom rda could not be accomplished were 12, and there was one child who refused the op making a total of 13 children in whom cavity cutting and filling remained unaccomplished [table 4]. It was observed that almost all children in group i could be managed using tsd only, except for two children who required use of voice control and physical restraint during rda . On the contrary, in group ii, two children required restraint even during oe, three during administration of la and two during rda . None of the children in this group, however, required restraint for the op [table 5]. The differences in the use of type of bmt between the two groups were, however, not found to be statistically significant (p> 0.1). The class ii amalgam restoration was a moderate time duration procedure and therefore performed in a single sitting . The categorization of clinical procedures into different stages enabled evaluation of the changes in anxiety levels due to the introduction of different stimuli during a particular stage, e.g. Oe until patient sits on chair, represented a situation where child was exposed to a new environment, but not yet exposed to any procedure, administration of la, which is known to be the most feared dental procedure, application of rubber dam, which represented a noninvasive but new and anxiety - provoking procedure by virtue of its appearance, and restorative procedure, which involved a moderately lengthy procedure with some painful moments . The final anxiety levels at the end of each step in the experimental group were always found to be lower or same in comparison with the baseline levels recorded at the start of that step . The trend was also seen in all procedural steps in the control group except on entry, showing that children in the two groups were in a more relaxed state after completion of a clinical step . This effect could not be attributed to midazolam sedation alone, as the trend was similar in the two groups for the two most fear evoking stimuli, that is, la and rda . The decrease in final anxiety could therefore, be a result of bmt employed during the steps, which was similar in the two groups . The effect of midazolam sedation, however, becomes apparent when we compare the increase in final anxiety levels at on entry in the control group to a decreased value in the experimental group [table 3]. It shows that midazolam in conjunction with behavior management is more helpful in relaxing the child initially than behavior management alone, thus increasing the chances of successful and easy accomplishment of further treatment steps . The effect of midazolam sedation in decreasing the anxiety levels in children becomes even more apparent when the baseline and final anxiety levels were compared for each step between the two groups . It was observed that the anxiety levels remained lower in group i as compared to group ii, throughout the treatment . This difference was statistically significant for steps on entry and la and not significant for the steps rda and op . The effect could be totally attributed to the sedative effect of midazolam, which significantly reduced the child's anxiety right at the first step, that is, oe and lasted until the last step thereby maintaining a lower anxiety state throughout the procedure . Though the anxiety levels in group i increased with an introduction of each new stimulus, they still remained much lower than that in group ii . Therefore, the anxiety levels in the two groups showed a somewhat similar trend, but at different levels [graph 1]. When the anxiety levels at the end of one step (final anxiety level) were compared with the anxiety level at the start of the succeeding step (baseline anxiety level), it was observed that after the child was brought inside the operatory, introduction of the la needle and syringe and rubber dam caused a significant increase in anxiety levels in both groups . Therefore, even though midazolam produced a lesser anxiety state in group i, introduction of a new fear evoking stimuli produced a significant increase in anxiety levels even in this group, similar to that of group ii, where no sedation was present . However, it is appreciable that even after an increase in anxiety levels, children remained in a much more manageable state in the experimental group, leading to significantly greater number of completed procedures . A total number of case that could not be completed even under the effect of midazolam (group i) were 2 out of 20 (90% successful) and 13 out of 20 could not be completed using only routine bmts (group ii), bringing their success rate to 35% only . Moreover, the successfully accomplished procedures in group i required physical restraint as a means of behavior management only in 10% of cases compared with 59% of times in group ii . Mean anxiety scores during treatment midazolam, via the oral route in a dose of 0.5 mg / kg body weight has been shown to have very few side effects in the literature . Paradoxical reactions of midazolam have also been recorded in children, which include hallucinations, agitation, inconsolable crying, restlessness and disorientation . In our study, the vital parameters of the sedated children remained stable and within normal limits . For any sedation procedure, however, individual variations are always present and the change in depth of sedation can never be predicted . From the above discussion of this study, it can be concluded that midazolam via the oral - transmucosal route in a dose of 0.5 mg / kg is an effective anxiolytic drug or sedative agent for successful accomplishment of a moderate time duration procedure like a class ii restoration (approximately 30 min). It significantly reduces a child's anxiety right at the beginning of the procedure, which causes the children to remain within normal limits of behavior management, even in cases of heightened anxiety during invasive procedures like la and fear evoking ones like rda . Midazolam was seen to relax the child and improve his disposition, showing a synergistic effect with bmts, as it becomes easier to manipulate and coax children under sedative effect into accepting more difficult procedural steps and thus increasing the chances of a successful treatment.
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Peyronie disease (pd) is a connective tissue disorder that is characterized by localized fibrotic plaques in the tunica albuginea, most commonly on the dorsal surface of the penis, that result in penile bending and often pain . As an initial trigger, an inflammatory process and subsequent aberrant wound healing by repetitive trauma to the penis during intercourse has gained widespread acceptance . This inflammatory process itself is self - limited and the pain typically resolves with time . Unfortunately, by progression, the penile deformity remains in 90% to 95% of patients, with surgery remaining as the sole conclusive treatment for this sequel . In addition, pd is frequently associated with erectile dysfunction (ed); in a recent retrospective study of 1,001 patients with pd, 58.1% of patients reported having ed . Diagnosis of pd is based on sexual history and careful physical examination of the penis, which are sufficient for establishment of the diagnosis . In contrast, owing mainly to an incomplete understanding of the exact pathogenesis, the optimal management of pd remains a clinical dilemma, particularly the nonsurgical approach . Despite a wide spectrum of currently available treatment options, including oral agents, intralesional injection, extracorporeal shock wave therapy, and external traction therapy [8 - 11], none of these has demonstrated conclusive effects and most studies of these treatments did not have a placebo - controlled design . To date, none of these treatment options carries a grade a recommendation according to contemporary western guidelines; thus, the clinical strategy for management of pd is primarily dependent on the preferences of physicians and patients . However, incorrect and outdated information on this condition from the first - encountered physician may cause misdiagnosis or mislead the patient into unrealistic beliefs, as demonstrated in a recent survey on primary care physicians and urologists . With this background, we conducted a survey to elucidate the actual diagnosis and treatment patterns, including nonsurgical and surgical approaches, by korean urologists . We also intended to assess each treatment modality, as preliminary data for creation of further local guidelines on pd, by evaluation not only of the urologists' perceptions from the point of view of the suitability of the treatment for pd, but also of patient satisfaction with each management modality investigated by the urologist . A probability sample was taken from the korean urological association registry of physicians, and a specially designed questionnaire was e - mailed to 2,421 randomly selected urologists . The purpose of the survey was to explore the practice characteristics and attitudes of each urologist . In this study, the researchers observed human subjects set forth in the helsinki declaration of the compliance with ethical principles of medical research . The survey contained 56 questions on pd - related symptoms and diagnosis of pd (n=13), methods for management of pd (n=37), and general questions about demographics (n=6). In questions on the available method used in the diagnosis and treatment of pd, multiple - choice was permitted, given the current uncertainty in the management of pd . As an attempt to identify proper methods for treatment of pd, we asked the urologists about their perceptions from the point of view of the suitability of each treatment and modality, which was divided into a scale with five grades (1, below 20%; 2, 20%-39%; 3, 40%-59%; 4, 60%-79%; and 5, over 80%), and patient satisfaction, which was estimated by use of a scale from 0 to 10 (0, no satisfaction; 10, full satisfaction). Responses were received from 385 practicing urologists (15.9%). Among them, 263 were from nontraining hospitals, including 68.3% of responses (231/385) from primary care urologists and 122 responses from university - training hospitals (21.7%). The median duration after certification as an urologist was 12 years (range, 0.41 years), and 59% (227/385) had clinical experience of more than 10 years . Of the respondents, 66% (255/385) had treated fewer than five patients with pd per year, whereas 16.6% of urologists saw more than 10 pd patients (64/385). The most bothersome symptom causing patients to visit the urology clinic was penile curvature (75.1%, 289/385), followed by painful erection (13.5%, 52/385), difficulty in penetration (4.2%, 16/385), and ed (2.1%, 8/385). Plaques were palpable in approximately half of cases (193/383). Regarding the interval from development of symptoms to seeking a specialist, 47.5% of urologists (182/383) answered that their patients visited between 6 and 12 months from the development of symptoms, followed by 3 to 6 months (32.6%, 125/383). Regarding the natural course, most urologists (87.8%, 338/383) believed that pd is a progressive disease . Similarly, most urologists (82.2%, 315/383) responded that spontaneous healing in pd occurred in fewer than 20% of patients . For these two questions, the working year after certification of the urologist or the type of institution they worked for showed no statistical difference (table 1). On a multiple - choice question regarding diagnostic method for pd, the responses were, in order, history taking with physical examination (97.9%, 375/385), international index of erectile function (iief) questionnaires (39.7%, 152/385), combined intracavernous injection and stimulation (34.7%, 133/385), and duplex sonography (28.2%, 108/385). As for the proper timing to initiate management of pd regardless of medical or surgical approaches, 44.1% of urologists (169/383) responded " when penile curvature or pain occurred, " followed by " when the patient wants " (38.1%, 146/383) and " when the penile nodule was identified " vitamin e was the most preferred initial medical management for 80.2% of respondents (307/385), followed by phosphodiesterase-5 (pde-5) inhibitors (27.4%, 105/383), potaba (aminobenzoate potassium; 20.1%, 77/383), carnitine (16.7%, 64/383), colchicine (11.7%, 45/383), tamoxifen (10.4%, 40/383), and pentoxifylline (7.0%, 27/383). However, among the three most common of these, the urologists' perception from the point of view of the suitability of treatment and patient satisfaction with the treatment were significantly different (p<0.001 and p<0.001, respectively, by chi - square test) (fig . Seventy - two urologists (277/385) replied positively on the use of intralesional injection . Regarding combination with an oral agent, 41.8% of urologists (160/383) initiated injection when oral medication had failed; however, 35.3% started injection from the beginning of use of an oral agent (136/383). The most preferred injection protocol was that administered on a weekly basis (89.6%, 240/268), with duration of less than 12 weeks (94.9%, 263/277). The preferred injection agent was, in order, corticosteroid (72.2%, 200/277), verapamil (45.1%, 125/277), and interferon (3.2%, 9/277); however, the type of agent did not have an effect on either urologists' perception regarding the suitability of treatment or patient satisfaction (p=0.485 and p=0.498) (fig . Urologists who responded considered surgical treatment, particularly when initial oral and injection therapy had failed (67.6%, 259/383). Thirty - eight percent of respondents (148/383) performed surgery for pd in their own clinic, whereas the others (61%) did not . The most frequently performed procedure was plication (84.1%, 190/226), followed by excision and graft (42.9%, 97/226), penile prosthesis implantation (14.2%, 32/226), and others (1.3%, 3/226). Among these procedures, urologists' perception and patient satisfaction were significantly different (p=0.001 and p=0.002) (fig . 3a, b), favoring penile prosthesis implantation . Among the most popular treatments in each modality among oral agents, intralesional injection, and surgery, although pd has been recognized for over 200 years, no consensus exists, particularly with regard to the standard treatment of this condition . On the basis of contemporary western guidelines, surgery is the only recommended treatment option, and plication techniques have been used almost exclusively in cases of isolated penile curvature with high curvature correction rates . In contrast, the nonsurgical modalities have shown little progress and have not kept pace with the surgical options in the treatment of pd . To date, there are no u.s . Food and drug administration - approved, nonsurgical options . Nevertheless, it is also true that there is interest in pd, mainly as a result of improved recognition, widespread use of pde-5 inhibitors, and increasing sexual activity of older men and resultant injury of the penis . Indeed, the prevalence of pd appears to be 3 to 10 fold higher than previously estimated . By definition, patients being seen in our clinic complaining of pd are highly motivated; hence, the actual occurrence of this disease within the population may be higher owing to patients' reluctance to come to their physician for treatment and diagnosis of this embarrassing condition . Besides its efficacy in the treatment of deformity, surgery is indicated when pd is stable for at least 3 months, which is usually the case after 12 months from the onset of symptoms, and intercourse is compromised as a result of deformity . In addition, surgery may be associated with complications and the possibility of penile shortening . Therefore, based on limited placebo - controlled clinical trial support, physicians usually have no choice but to recommend nonsurgical options, reserving surgery for patients in the chronic phase of pd with deformity and interference in sexual function . To the best of our knowledge, this is the first nationwide survey to address the practice patterns and general perceptions about pd of urologists in korea, as well as patient satisfaction with the management of pd . First, the korean urologists' perceptions regarding the natural course of pd and diagnostic approaches were analogous with currently available western guidelines . The majority of urologists (87.8%) believed that pd is a progressive condition, and more than 81% of respondents believed that spontaneous healing in pd occurs in fewer than 20% of patients . These observations are quite the opposite of the outcomes reported by larochelle and levine, whose group conducted a survey on pd in the united states . In their study, 17% of primary care physicians and 38% of urologists believed that the disease resolves spontaneously in more than 50% of cases . It is also notable that in this survey, these notions were not affected by the duration in practice as a urological specialist or the type of medical institution in which the urologist worked (table 1). This is in contrast with the depictions by larochelle and levine, in which urologists who had been in practice for more than 10 years were more likely to have incorrect assumptions about pd than were urologists in practice for less than 10 years . Regarding diagnostic approaches, most of the respondents in this survey performed physical examination, and approximately 40% evaluated potentially concomitant ed by use of the iief questionnaire . This also contrasts with the findings of the survey conducted by larochelle and levine, where nearly one in two primary care physicians and one in six urologists did not perform routine examination of the patient's penis . Second, regarding a nonsurgical approach, whereas vitamin e was used primarily for initial oral management by more than 80% of respondents, its reported efficacy was relatively lower both according to the physicians' perception of treatment suitability and patient satisfaction in comparison with other oral agents . In contrast, for intralesional injection, physicians' perception of treatment suitability and patient satisfaction were not affected by the agents injected . Among the most common choices in oral and intralesional injection, corticosteroid injection was significantly better perceived by both patients and physicians in comparison with vitamin e. however, this outcome should be interpreted with caution, considering that there is currently no conservative treatment that eventually results in complete relief of all symptoms, including pain, plaque formation, and penile curvature . Indeed, several double - blind, placebo - controlled trials on the use of vitamin e, potaba, propoleum, tamoxifen, colchicine, acetyl - l - carnitine, propionyl - l - carnitine, and omega-3 fatty acids for the treatment of pd have been conducted, usually with minor or little proven effect [17 - 23]. With regard to intralesional injection therapy, whereas 90% of the studies reported positive outcomes, most of those studies did not offer convincing evidence - based data, hampered by their small patient populations . Despite initial promising results, in a recent placebo - controlled, single - blind trial of intralesional verapamil injection, no significant improvements were observed in penile curvature, plaque size, or penile pain . The most peculiar finding in this survey was that the highest grade in both patient satisfaction and physicians' perception of the suitability of treatment was achieved by the surgical approach, as shown in fig . 4 . Currently, owing to a lack of any nonsurgical management that can definitively alter the progression of the disorder, surgical intervention is the only efficacious treatment for pd . If there is ed that is not responding to pharmacologic treatment, the best option from contemporary guidelines is the implantation of an inflatable penile prosthesis, with or without an associated procedure over the penis (modeling, plication, or even grafting plus the prosthesis). Indeed, for the surgical techniques, the urologists' perception and patient satisfaction were significantly different, favoring penile prosthesis implantation with a relatively higher grade in both aspects (fig . 3). Finally, in this survey, the duration of time to a doctor visit was relatively shorter than that reported in western countries, where approximately one - third of pd patients did not see a doctor until 4 years after the emergence of penile symptoms . This is also in contrast with a widely accepted notion that members of the asian population are highly sexually conservative and less sexually active . While distinctions including medical insurance systems, accessibility to medical suppliers, and cultural differences should be considered, this aspect reflects the urgent need for active treatment for patients suffering from this embarrassing condition . A strength of the current study is the recruitment of a reply from 385 acting urologists; to the best of our knowledge, this is the largest number of urologists recruited by use of a detailed survey, particularly on pd . A possible criticism of this study is the response rate of 15.9%, which is far from that required to attain representativeness among practicing korean urologists . Because the categories on the questionnaire used in the survey were not designed to permit direct comparison, limited information can be obtained, particularly on the diagnostic approach and efficacy of treatment . In addition, many other factors in terms of patient or physician demographics that were not investigated in this survey may have an effect on the outcomes . Indeed, because these data were obtained only from urologists from the physician's point of view, the actual characteristics and responses of the patients remain obscure . In the future, conduct of local population - based studies and randomized controlled trials will be needed; until then, approaches based on currently available guidelines are still recommended . Eventually, the development of integrated curricular and specialized guidelines for korean males supported by local data on treatment of pd may be required . The results of our current survey provided insights into the clinical practice of korean urologists in the treatment of pd . The urologists' recognition of pd is in line with current understanding of this disease, and the diagnosis was based mainly on history taking and physical examination . Among various treatment approaches, surgery was the most effective modality from the perspective of both the urologists' perceptions regarding the suitability of treatment and patient satisfaction . These observations indicate the need for development of practical local guidelines based on solid clinical data and to ensure that these guidelines are widely promoted and accepted by the urological community.
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We report a case of a 67-year - old male who presented with a cecocentral scotoma caused by a septic embolus from subacute bacterial endocarditis (sbe). A 67-year - old man presented with sudden, painless decreased vision in the left eye . A dilated fundoscopic exam, humphrey visual field test, transthoracic echocardiogram, abdominal computed tomography (ct), and blood cultures were all performed . A dilated fundoscopic exam revealed temporal segmental optic disc pallor on the left, and humphrey visual field testing demonstrated a dense left cecocentral scotoma . 9f) and palpitations, transthoracic echocardiogram revealed valvular vegetations, and contrast ct of the abdomen revealed an abscess in the dome of the liver likely due to an infectious thrombus . This case illustrates that a sudden cecocentral scotoma may be the initial manifestation of sbe . Subacute bacterial endocarditis (sbe) is an indolent microbial infection of the endocardium with the potential for systemic dissemination by way of septic emboli . We report the case of a 67-year - old male who presented with a cecocentral scotoma caused by an infectious embolus from sbe . A 67-year - old man presented to our emergency department (ed) complaining of sudden, painless decreased vision in the left eye for 3 days . He reported no other systemic or ocular symptoms, including no fever, weight loss, pain on chewing, joint pain, or skin rashes . Other than treatment for dental caries 1 week prior to presentation, he had not sought medical attention for over 10 years . On examination, the patient correctly identified nine of nine ishihara color plates on the right but only four of nine color plates on the left . Dilated fundoscopic exam revealed sharp, nonedematous, nonglaucomatous - appearing optic disks bilaterally but with temporal segmental pallor on the left . The remainder of the peripheral posterior exam was within normal limits, including no roth or cotton wool spots, no hollenhorst plaques, no retinal edema or hemorrhage, and no neovascularization . Other than a mildly elevated temperature to 100.1f, all other vital signs were within normal limits, and there were no focal neurologic findings . Laboratory testing revealed a sedimentation rate of 80 and a white blood cell count of 11,000/l with 78% neutrophils . Platelet count was 225,000/l, and the hematocrit and basic metabolic panel were within normal limits . As computed tomography (ct) and/or magnetic resonance imaging (mri) were not acutely available, we performed humphrey visual field testing, which demonstrated a dense left cecocentral scotoma (figure 1). The differential diagnosis of a pale nerve with a cecocentral scotoma includes an embolic event, optic neuritis, toxic optic neuropathy, and nutritional deficiency . The patient was admitted to the intensive care unit for further medical workup, including further imaging and blood studies . Seventy - two hours later, the patient returned to the ed with increased temperature (103.9f), palpitations, and stable decreased visual acuity of the left eye and was therefore readmitted . Transthoracic echocardiogram revealed valvular vegetations (not shown), and contrast ct of the abdomen revealed an abscess in the dome of the liver that was likely due to an infectious thrombus (figure 2). Following intravenous antibiotic treatment, his systemic condition steadily improved over 1 week, warranting discharge from the hospital . The patient continued oral antibiotic therapy for 3 additional weeks until repeat blood cultures showed no evidence of residual infection . On serial yearly follow - up examinations almost a decade after the original insult, the patient s low vision status on the left persists with decreased snellen vision, reproducible red desaturation, and decreased color plates on that side . Follow - up intravenous fluoroscein angiography has remained unremarkable, with recent fundus photos of the left eye (figure 3) showing inferotemporal disk pallor . Follow - up mri was not acquired, as the patient s examination and visual defect remained stable over many years . We postulate that an infectious embolus from endocarditis impeded circulation, causing ischemia and segmental infarction of the left optic nerve and affecting the maculopapillary bundle . We report the first case of a cecocentral scotoma presenting as the initial manifestation of sbe . It is important for both the ophthalmologist and general practitioner to recognize that infective endocarditis has varied presentations, ranging from general malaise to loss of appetite . If left untreated, it can be fatal.1 it is commonly caused by relatively avirulent viridans streptococci organisms that have a propensity to adhere to damaged heart valves and endothelium.2 infective endocarditis is typically handled on an inpatient basis to employ the use of intravenous bacteriocidal antibiotics tailored to the specific organism found in cultures and also to closely monitor the patient.3 another option is to administer antibiotics on an outpatient basis but only after treatment has already been started in the hospital and a full initial assessment performed.1 an embolic event due to sbe is a frequent and potentially life - threatening complication of the disease . It has been reported that neurological complications due to embolic events may be as high as 20%40%.3 mri may show infarctions scattered throughout the brain secondary to emboli . Ocular manifestations of embolic disease can include roth spots, retinal hemorrhages, and infectious vitritis.3 because sbe often presents as a fever of unknown origin, ophthalmic signs of the disease may often present prior to a confirmatory echocardiogram or blood culture but are often visually insignificant . Some studies have demonstrated premacular hemorrhage as the first sign of sbe, whereas others have reported patients with seizures, headaches, and roth spots prior to being diagnosed with sbe.4,5,6 systemic embolization of infective thrombi reportedly occurs in 45%65% cases of sbe.7 we postulate that visual symptoms and signs occur from turbulent flow within the small vascular channels supplying the optic nerve head, causing local ischemia and thereby leading to infarction of the retinal nerve fiber layer . The standard of practice is to rely on echocardiograms as a diagnostic and prognostic tool to guide our management of sbe; however, our ability to predict the risk of embolic events is limited.8 in our case, the sudden appearance of a cecocentral scotoma in the absence of systemic symptoms and grossly abnormal laboratory values was the harbinger of future embolic events in this patient and the first manifestation of a serious generalized infectious process . Our case confirms that ophthalmic events may indeed herald sbe and that ophthalmologists should consider systemic workup (in concert with primary care physicians) for patients who present with sudden cecocentral scotomas or other visual field defects.
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Paclobutrazol (chemical structure as in figure 1) was a plant growth regulator registered for the reduction of terminal growth and pruning volume, the inhibition of gibberellins and sterol biosynthesis, and hence the rate of cell division [1, 2]. Due to its toxicity, the agreed adi (accepted daily intake) and arfd (acute reference dose) were all 0.1 mg / kg bw / day . According to reg . Number 396/2005 (ec) annex i, the mrl for paclobutrazol in fruit was 0.5 mg / kg, and the national food safety quality standard (gb 2763 - 2014, china) was 0.5 mg / kg in wheat and rice . However, no information was available concerning the residual status and pattern of paclobutrazol in potato . Growth inhibition characteristics had been reported for paclobutrazol with soil drenches, soil sprays, and foliar sprays . Cross - comparisons among these studies were difficult since they were conducted under different growth conditions (field, greenhouse, and growth chamber). Two other factors that influenced the paclobutrazol residues were absorption rate and longevity or persistence of the compound in treated plant tissue and soil . Traditionally, gas chromatography (gc) and gas chromatography - mass spectrometry (gc / ms) was once popular for analyses of paclobutrazol, but gc / ms required prederivatization and target compounds may be decomposed by injection at high temperatures . More recently, hplc - based methods for analyses of paclobutrazol had been published in plant [69] and pear pulps using uv detection and different cleanup steps in order to get recoveries of 70% at the 0.01 mg / kg level . Thus, using these conventional techniques, it was necessary to perform one or more cleanup steps to decrease interferences and preconcentration steps in order to obtain adequate detection levels . Nowadays, lc coupled to tandem ms (ms - ms) [1113] had also been applied to plant growth regulator analysis in fruits as a powerful confirmation tool, improving the sensitivity and reducing the sample pretreatment steps, such as solid phase extraction (spe) techniques . Recently, the liquid - liquid extraction with low temperature partitioning (lle - ltp) was promising for multiresidues analysis since this technique presented some advantages in relation to other extraction techniques, such as practicability, reduced number of steps, and low consumption of organic solvents, as well as being reliable and selective [11, 14]. In this study, it was to develop and validate a uhplc - ms / ms method for determination of paclobutrazol in potato and soil, taking advantage of liquid - liquid extraction at low temperature . In addition, the paclobutrazol residue in potato and soil and the dynamics dissipation in soil were investigated through field trials . The purpose of this study was to evaluate the influence of paclobutrazol residue in potato, and a mrl of paclobutrazol in potato was recommended . A high - performance liquid chromatography- (hplc-) grade acetonitrile and methanol were obtained from merck co. ltd . Sodium chloride and sodium acetate of analytical grade were obtained from the chemical reagent company (shanghai, china). Highly purified water (milli - q, millipore, bedford, ma) was used throughout the preparation of the mobile phase . Stock solution was prepared at 1000 mg / l in methanol and stored in dark vials at 20c . For the calibration curve, matrix - matched calibration was used with five series concentrations of extraction solution (0.001, 0.01, 0.05, 0.1, and 0.5 mg ultimate residue experiments were conducted in experimental fields located in songming agriculture demonstration garden, yunnan province, china, from april 18 to june 29, 2013 . The soil was sandy clay loam, with content of sand 46%, clay 28%, organic matter 2.02%, and ph 6.94 . The experiments were designed according to ny / t 7882004 (guideline on pesticide residue trials) issued by the ministry of agriculture, china . According to the usage guide, paclobutrazol formulation spray application was recommended one time during the early flowering stage of potato (in 45 days). Plants were treated with 3 dosage levels, 40 g (available ingredient)/ha (the low dosage, recommended dosage), 60 g (available ingredient)/ha (the middle dosage, 1.5 times the recommended dosage), and 100 g (available ingredient)/ha (the high dosage, 2.5 times the recommended dosage). Treatments were consisted of foliar sprayed (with or without thick polypropylene soil cover), and the tuber on the paclobutrazol residual soil with 60 g (available ingredient)/ha, and the control plants were treated with distilled water . Foliar sprays were applied with and without a ground cover to evaluate the impact of foliar adsorption and indirect soil absorption from foliar runoff on soil residue . To check the possibility of paclobutrazol contamination from surface and peel of tuber, twenty tubers (4.8 kg) were sprayed with paclobutrazol (1.0 mg / l), five of which were rinsed with 500 ml distilled water and 100 ml acetonitrile; then the rinsing acetonitrile was determined by uhplc - ms / ms after 30 min . Another five samples were peeled; then the peel and pulp were analyzed for comparison with potato harvest . As reported paper, potato was harvested about one month after flowering stage, so the tuber and soil were collected to determine final residue at ten days before harvest (22 days), harvest time (32 days), and eight days after harvest (40 days) after foliar spraying . To determine dissipation rate of paclobutrazol in soil, soil samples of approximately 500 g from depth of 010 cm were collected randomly from five points in each plot at 1 h, 1, 3, 7, 14, 21, 32, 40, and 50 days after soil spray . The soil samples were air - dried, clod broken, mixed, and sieved through a 2 mm sieve as described by sharma . Soil: 10 g soil sample was weighed into a 100 ml centrifuge tube and then 50 ml acetonitrile was added . The centrifuge tube was extracted in an ultrasonic bath for 10 min and centrifuged at 3000 g for 5 min . Afterwards, the tube was stored at low temperature (20c) in refrigerator (haier, qingdao, china) for 10 min to easily separate organic layers . Two - milliliter portions of the organic layer were filtered through 0.2 m membrane filters prior to analysis by uhplc - ms / ms . Potato: 25 g sample was weighed into a 100 ml centrifuge tube and extracted with 50 ml acetonitrile . The same procedure was followed as that described for soil up to uhplc - ms / ms . Sample analyses were performed on tandem mass spectrometry ab 4000 (ab sciex, ontario, canada) which consisted of a 1290 ultrahigh performance liquid chromatography (agilent technology, usa). A zorbax eclipse plus c18 column (50 mm 2.1 mm i.d ., 1.8 m particle size) was employed for the separation of the analyte and was maintained at 30c . The mobile phase was comprised of acetonitrile / water containing 0.1 percent formic acid (78/22, v / v) and was delivered at a constant flow of 0.3 ml / min . The injection volume was 1 l . The spectral acquisition was operated in positive electron spray ionization mode, and multiple reactions monitoring (mrm) was utilized . The gas temperature was set at 350c with a flow rate of 8.0 l / min . The precursor ion was m / z 294.2, and production ion was m / z 129.0, 70.0 for paclobutrazol, with relative intensity of 4.5%/68%, in which the most intense production ion m / z 70.0 was the quantitative ion, and collision energy and fragmentor were 110 and 50 ev, respectively . Solid - phase extraction (spe) was a preferred way to prevent matrix interference, but it was time - consuming with high cost . The advantage of liquid - liquid extraction with low temperature was that the sample components remained in the ice phase, whereas paclobutrazol was extracted by the completely transparent liquid, which was easily obtained and directly analyzed by uhplc - ms / ms . As it had been previously indicated, cho et al . The result showed that the sample would be frozen and separated easily under 20c to 10 min . So it can be operated under facile condition by home refrigerator . In order to gain a favorable extraction yield, the extraction yield of the various solvents for paclobutrazol was listed in the following sequence: acetonitrile (recovery 95%)> acetone (recovery 86%)> dichloromethane (recovery 65%)> n - hexane (recovery 10%). Furthermore, the ratio of acetonitrile to the sample showed that the best results were with 2: 1 (potato) and 5: 1 (soil), resulting in 85% recovery greater than other ratios with 1: 1 or less . The tuning solution was introduced into the electrospray ionization (esi) source by direct infusion . The main ions produced in ms and ms / ms were identified in positive ionization modes . The obtained precursor ions indicated a clear relation between the structure of paclobutrazol and its ability for positive ionization . The precursor ion was m / z 294.2, and production ion was m / z 294.2129.0 and 294.270.0 (quantitative ion). The composition of mobile phase can influence the performance of the ionization process in esi mode . So, different mobile phases (acid, base, and neutral) were tested . The result showed that the response of paclobutrazol can be increased in acid mobile phase (0.1% formic acid). The optimized mobile phase was acetonitrile / water containing 0.1% formic acid (78/22, v / v), as shown in figure 2 . For this reason, residues of paclobutrazol in samples were quantified with matrix - matched five - series standard calibration (0.001 mg / l, 0.5 mg the calibration curves showed good linearity with the following equations and relative coefficients: y = 1.95693 10 + 964964x, correlation coefficient r = 0.9976 . The instrument detection limit (lod) and limit of quantification (loq) were estimated through ten repetitive injections of standard solution, which can detect at a signal - to - noise ratio (s / n) of three multiples and ten multiples, respectively . The lod value was 0.5 g / kg, and the loq values were always 2 g / kg in the potato and 5 g / kg in soil . Repeatability and reproducibility were expressed as relative standard deviations (rsd) of retention time (rt) and peak area (ar). Reproducibility of rt and peak area was good for 1 month; all values were 3.4% (rt) and 5.2% (peak area) or less . Furthermore, repeatability of rt and peak area, which was evaluated on 1 day, proved more satisfactory, with values of 1.5% (rt and peak area) or less . The accuracy of the whole method was evaluated by the development of a recovery study carried out at three concentration levels (5, 50, and 100 g / kg). All experiments were carried out in quintuplicate at each level (results are shown in table 1). As it can be seen in table 1, recovery values were satisfactory, ranging between 83 and 106% with rsd lower than 10% . As it can be seen from the rsd values, the method was reproducible and applicable to the analysis of paclobutrazol in potato and soil . Foliar spray without cover treatment showed that amounts of paclobutrazol were absorbed by foliage and foliar runoff was absorbed by the soil . . Showed result that paclobutrazol residues in the soil increase due to falling leaves from nearby sprayed trees . Apparently, the amount of paclobutrazol absorbed by stolon, then transported to tuber, was larger than by foliar absorptive capacity . To check the possible contamination of paclobutrazol from surface and peel of tuber the content in organic solvent was very lower than the expected concentrations in whole potato . So the paclobutrazol residue in potato harvest was mainly from absorption and transport from soil, which was not removed by peeling . The result showed that the initial deposits of paclobutrazol in soil were 1.14 mg / kg . Except for a slight increase on 1st day, the residue value showed a steady decrease and was below 0.22 mg / kg on 50th day . The dissipation equation of paclobutrazol was y = 1.0784e, r = 0.9403 . To establish a recommended maximum residue limit (mrl) of paclobutrazol in potato, two guideline values, with need maximum daily intake (nedi) and acceptable daily intake (adi), the value of adi with paclobutrazol was 0.1 mg / kg bw / day . Neda was derived on the supervised trials median residue (stmr) or processing factor (stmr - p) and food intake rate (f). Safe level of exposure (mrl) does not exceed the estimated level of actual exposure (adi). The equation was (1)nedi=stmristmr - pifi . According to the investigation of nutrition and health by chinese health ministry in 2002, the f of potato was 0.0496 kg, stmr in potato harvest (recommended dosage: high dosage level) was 00.521 mg / kg, and then the high value nedi (0.0258 mg) was not beyond 5% the value adi 63 (adult weight) of 6.3 mg . Liquid - liquid extraction with low temperature partitioning was developed and validated for analysis of paclobutrazol in potato and soil using uhplc - ms / ms . The optimal chromatographic separation and sensitivity were successfully applied to the analysis of paclobutrazol residue in potato and soil . According to risk assessment with nedi and adi,
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Currently, endoscopic submucosal dissection (esd) is widely used as an alternative to surgical resection in patients with early - stage gastric cancer or adenoma.1 since esd enables en bloc resection of large lesions, esd results in the creation of larger artificial ulcers . Kakushima et al2,3 have reported that gastric ulcers induced by esd will heal within 8 weeks, regardless of size, location, helicobacter pylori (h. pylori) infection, or the extent of gastric atrophy . Size reduction in the ulcers occurs by contraction in the early phase, and then regenerative mucosa covers the remaining mucosal defect within 8 weeks.4 however, at 4 weeks, the ulcers of all cases remain in a healing stage.2 oh et al5 reported that the initial ulcer size affects ulcer healing using proton pump inhibitors (ppi) at 4 weeks post - esd . If the size of the post - esd gastric ulcer is larger than predicted, ppi administration alone might not be sufficient for the ulcer to heal within 4 weeks . Esomeprazole6 is the s - isomer of omeprazole and was developed with the aim of improving the pharmacokinetic and pharmacodynamic profiles of racemic omeprazole . There is no report about the efficacy of post - esd gastric ulcers by esomeprazole . On the other hand, rebamipide, which is a mucosal - protective antiulcer drug, promotes healing rates at 8 weeks for patients with esd derived artificial ulcer.7 kato et al8 showed that the combination of ppi plus rebamipide was more effective than the ppi alone for treating ulcers larger than 20 mm within 4 weeks of esd . Woon geon shin et al9 showed that ppi and rebamipide combination therapy had a superior 4-week esd - induced ulcer healing rate and quality of ulcer healing compared with ppi monotherapy . Thus, ppi plus rebamipide combination therapy was generally effective for a 4-week esd - induced ulcer healing rate, but in larger esd - induced ulcers there were some issues that need to be addressed . Therefore, in the current study we assessed the efficacy of esomeprazole plus rebamipide combination therapy for esd - induced ulcer healing compared with omeprazole plus rebamipide combination therapy . A total of 153 patients who underwent esd for adenoma or early - stage gastric cancer at saiseikai wakayama hospital from september 2007 to august 2012 were included in this study . Of the 153 patients, 75 were excluded from analysis because they had been treated with other ppis or had major organ failure . Patients receiving antiplatelet or anticoagulant agents were asked to stop these medications at least 4 days before study procedures took place . Esd was indicated in patients with adenoma accompanied by any degree of dysplasia and in patients with early superficial gastric cancer . We conducted a case - control study to compare healing rates within 4 weeks effected by esomeprazole plus rebamipide (group e) and omeprazole plus rebamipide (group o). The esd technique has been precisely described elsewhere.1,10 and all esd was performed by a single endoscopist . The knives used in esd included flusknife, (dk2618jb, 1.5 mm type; fujinon, tokyo, japan) and insulation - tipped (it) diathermic knife (kd-10 l; olympus, tokyo, japan)1 in this study . The ulcer created after resection was carefully examined, and any visible vessels were coagulated by hemostatic forceps (radial jaw3 hot biopsy forseps; boston scientific, tokyo, japan). The resected specimen was immediately pinned flat to a rubber plate to measure the size . The ulcer area was approximated from multiplication of the long diameter and the diameter perpendicular to the long diameter of the resected specimen . The sizes of the artificial ulcers were divided into normal size (area <1,200 mm) or large size (area 1,200 mm).11 multifragment resection was considered incomplete, even when the lesion was completely removed endoscopically . Post procedure - related bleeding was defined as that when hematemesis, melena, or hemoglobin concentration decreased by more than 2 g / dl were observed . All bleeding was controlled by endoscopic treatments such as hemoclipping, epinephrine injection, electrocoagulation, argon plasma coagulation (apc), and hemostatic forceps . Perforation was diagnosed endoscopically by direct observation of mesenteric fat just after resection or by the presence of free air on radiographs or the ct image.12 after esd, all patients received intravenous administration of 20 mg omeprazole (omepral injection; astra zeneca co, osaka, japan) daily for the first 2 days, followed by 4 weeks of drug treatment . The group e was administered 20 mg oral esomeprazole (nexium; astra zeneca co, osaka, japan) and 300 mg oral rebamipide (mucosta; otsuka pharmaceutical, tokyo, japan) daily, whereas the group o was treated daily with 20 mg oral omeprazole (omepral; astra zeneca co, osaka, japan) and 300 mg oral rebamipide . Esophagogastroduodenoscopy was performed on the operative day, postoperative day (pod) 1, pod 7 and pod 28 in order to record the healing rates of each artificial ulcer, as well as any immediate complications . Ulcer stages were assessed using a six - stage system as proposed by sakita and fukutomi (table 1) at 28 days after the esd.13 statistical comparisons of the patients were performed using the test for categorical data and student s t - test for numerical data . Data are expressed as mean sd . Differences in the categorical variables between two groups were examined with the test . Data regarding the clinical and endoscopic features of the patients are outlined in table 2 . There were no significant differences between the two groups with respect to ulcer size, location of ulcer, tissue size, histopathology (included histopathology of subgroup) and positive h. pylori, except for age, gender and procedure time . Complications included post - procedure related bleeding in one patient from group e on the second day after esd . 39 percent and 27 percent of the patients had s1 stage disease after 4 weeks of group o and e and there were no significant differences between the two groups with respect to healing rate of s1 stage . To evaluate the effect of rebamipide plus ppi in large - sized or normal - sized ulcers, we performed a subgroup analysis of healing rates between the two groups . In group o, the healing rate of s1 stage in the large - sized ulcer was significantly lower than that of the normal - sized ulcer . By contrast, there were no significant healing rate differences between large - sized ulcer and normal - sized ulcer for the s1 stage in group e. in large - sized ulcers, a significantly higher healing rate of s1 stage were observed in the group e compared to group o, although there were no significant differences in normal - sized ulcers (table 3). During follow - up, endoscopic mucosal resection (emr) is widely applied for curative treatment of gastric neoplasms such as early gastric cancer or adenoma . Recently, emr has been replaced by esd, because it is difficult to achieve en bloc resection of specimens larger than 20 mm with emr; piecemeal resection leads to local recurrence with reported rates of about 15%.14,15 the development of esd has enabled performance of en bloc resections of lesions, irrespective of their size or location . Additionally, better pathological evaluation is achieved using en bloc specimens.16 however, there have been concerns regarding the technical difficulties of the procedure, the cost, the long operation time and the higher incidence of complications such as bleeding or perforation compared with conventional emr . Because ulcer dimensions are larger and the resection depths are greater than those associated with emr, the risk of bleeding is believed to be higher . Bleeding from the ulcer is the most serious complication during and after esd.17 green et al18 and berstad19 have shown that the intragastric ph should be 6.0 in order to allow platelet aggregation and prevent platelet disaggregation . Therefore, inhibitors of gastric acid secretion such as ppis and histamine-2-receptor antagonists (h2-ras) have been administered after endoscopic therapy for gastric neoplasms to keep the ph of gastric juice high and to induce rapid ulcer healing.17,2023 kakushima et al22 have shown that after esd, artificial ulcers treated by normal - dose ppi therapy healed within 8 weeks regardless of size and location . There is no consensus, however, regarding optimal treatment durations and drug regimens for relatively large esd - induced ulcers . Kakushima et al3 have also shown that 4 weeks of ppi treatment was not enough for a large post - esd ulcer to heal, and that 8 weeks was required . Oh et al5 had reported that the degree of healing of esd - induced ulcers was dependent on the initial ulcer size, indicating that the duration of treatment with ppi should be considered . Taking these data into consideration, it seems that the administration of a ppi alone may not be sufficient for a large esd - induced ulcer to heal within 4 weeks . Bleeding, which always occurs within 2 weeks of esd, is the most common complication with surgical - based artificial ulcers . The most effective strategy to prevent bleeding from an artificial ulcer after esd is to promote quick recovery from mechanical and artificial gastric mucosal wounds . In most patients, short - term administration of a ppi or h2-ras may be sufficient to heal artificial ulcers; however, in some patients the ulcer does not heal at an early stage, even after 8 weeks of ppi administration . This inability to heal may be due to severe atrophic gastritis, which commonly requires dissection of larger areas of mucosa . In addition, the more extensive dissection of the gastric submucosa, just above the muscularis propria, which is required for the assessment of cancer spread of lymphovascular invasion may also delay ulcer healing.7 although there have been several reports comparing ppis and h2ras for the prevention of delayed bleeding and the promotion of the healing of esd - induced ulcers, the reported results conflict.21,24,25 some authors report that ppi therapy is superior to h2-ras therapy21,24 while others find no differences between the 2 therapies.25,26 furthermore, several authors have reported that ppi and rebamipide combination therapy had a 48 week esd - induced ulcer healing rate compared with ppi monotherapy.7,27 other authors have reported that rebamipide promotes ulcer healing in large - sized esd - induced ulcers within 46 weeks after esd.8,11 these results could be explained by the regulatory action of rebamipide in the inflammatory processes . Rebamipide promotes the ulcer healing process by increasing the level of cytoprotective prostaglandin, epidermal growth factor, or nitric oxide, and decreasing the level of oxygen free radical . These actions of rebamipide could promote gastric mucosal blood flow at the ulcer margin, an important factor in ulcer healing, and accelerate mucosal or submucosal reconstruction of damaged structure.27 taking these data into consideration, it seems that the administration of a ppi alone may not be sufficient for a post esd - induced ulcer to heal within 4 weeks . In the present study, the number of ulcers that reached the scar stage was larger, but not significantly larger, in group e (27%) than in group o (39%) at 4 weeks after esd . Kato et al8 have reported that the combination of ppi plus rebamipide was significantly more effective than ppi alone for treating ulcers larger than 20 mm at 4 weeks after esd . The endpoint ulcers reached the scar stage in the ppi group (36%) and in the combination group (68%). There may have been a difference in the ulcer scarring rate because of possible differences in the baseline data such as the use of anti - platelet or inflammatory drugs . However, several authors have reported that artificial ulcers reached the scar stage at 4 weeks after emr / esd in 17% of the ppi group,28 15% of the ppi group,17 and approximately 10% of the ppi group,5,29 which were similar to our scarring rate results in both groups at 4 weeks . On the other hand, there is no report about the efficacy of post esd induced ulcers by esomeprazole . Esomeprazole was developed as a single optical isomer of racemic omeprazole and, accordingly, has demonstrated some pharmacological advantages . In particular, a higher oral bioavailability is thought to contribute to the greater degree of acid suppression with esomeprazole than omeprazole, and differences in metabolism pathways are thought to contribute to less interpatient variability with esomeprazole.6 findings from studies in healthy volunteers, patients with gastro - esophageal reflux disease (gord) or those with continuous nsaid therapy have shown that, by day 5, once - daily oral esomeprazole at doses of 20 or 40 mg is more effective at increasing intragastric ph to> 4 than once - daily lansoprazole, omeprazole, pantoprazole or rabeprazole . During day 5, the mean percentage of time that intragastric ph was> 4 with daily esomeprazole 40 mg was significantly greater than that with comparator ppis.6 therefore, we assessed the efficacy of esomeprazole plus rebamipide combination therapy for esd - induced ulcer healing compared with omeprazole plus rebamipide combination therapy . In our results, there were no significant differences between the two groups with respect to healing rate of s1 stage at 4 weeks after esd, but in subgroup analysis, regarding large - sized ulcers, a significantly higher healing rate of the s1 stage was observed in group e compared to group o. this result suggests that esomeprazole plus rebamipide combination therapy was found to be more effective than omeprazole plus rebamipide combination therapy for large esd - induced artificial ulcers (1200 cm). Because rapid ulcer healing through clot stabilization at an elevated intragastric ph is required, a strong acid suppressant such as esomeprazole is more effective with respect to healing rate of s1 stage at 4 weeks after esd . Although a ppi is certain to be the most useful drug with healing effects for post esd - induced ulcer, recent reports have confirmed a number of patients who are ppi - refractory (resulting from ppi metabolization, such as that occurring via cyp2c19).30 therefore, clinical research on therapeutic options other than acid - suppressing agents has been needed . There appears to be less variability in the pharmacokinetics of esomeprazole in the overall population compared with other ppis, because esomeprazole appears to be less dependent on cyp2c19 genetics.6 thus, at this point, although cyp2c19 genotyping of the patients could not be performed in this study, esomeprazole may show a high degree of stability in the treatment of post esd - induced ulcer . Large - scale, controlled studies are needed to verify the effectiveness of 4 weeks of esomeprazole plus rebamipide combination therapy in the prevention of late bleeding, as well as to investigate its effects on quality of ulcer healing . In conclusion, to the best of our knowledge, the present report is the first retrospective study to demonstrate that the safety and efficacy profiles of esomeprazole plus rebamipide and omeprazole plus rebamipide are similar for the treatment of esd - induced ulcers . Especially in large - sized ulcers, esomeprazole plus rebamipide promotes ulcer healing.
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The schedule shown in table 1 is for persons aged 0 through 18 years without delayed vaccination . For children and adolescents whose vaccination has been delayed for more than one month, the schedule should be referred to the catch - up immunization schedule (table 2). The schedule comprises two parts; for infants and children aged 0 to 6 years and for children and adolescents aged 7 to 18 years.
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According to china's ministry of health, china has approximately 4 million cataract victims, with 500,000 new cases being diagnosed each year . As a developing country, especially in rural china, poverty and limited access to health care, due to the uneven distribution of health care sources, can make it very difficult for these people to obtain proper treatment . Cataract surgical rate (csr) is still very low in rural china . Lifeline express hospital eye - train (lehet), the first charge - free cataract surgery project founded in 1997, is a quite important way to restore vision for the low - income rural people in china . Independent of cost or other factors, the first expectation from surgeons and patients is good postoperative visual outcomes . To meet these expectations, attention to accurate biometry measurements the biometry is indispensable to the surgeons and patients as it might indicate the prognosis and safety of the coming operation . In the biometric parameters, axial length (al) and corneal curvature are the most important however, the distribution and determinants of al have been assessed in only a few population - based studies of older persons [410], of which there is still no study of rural chinese population, especially in middle china, having cities with extremely long history . In 2011 and 2012, our hospital (peking university people's hospital, puph) had three missions of lehet in middle china . In this study, we explored the biometric parameters of adult cataract patients who had cataract surgeries on lehet in these missions and all were rural people . Our hospital, puph, had four missions of lehet, zhoukou in henan province and songyuan in jilin province in 2011, yuncheng in shanxi province and sanmenxia in henan province in 2012 . The sites were selected by the office of lehet, and they were blind to our hospital before the mission start . Yuncheng (n 35.03; e 111.01; altitude: 369.53 m) in shanxi province and zhoukou (n 33.62; e 114.66; altitude: 50.50 m) in henan province have thousands of years of history . Sanmenxia (n 34.77; e 111.20; altitude: 376.08 m) in henan province was built in the 1950s and also is a rural city . Residents in this new city partly immigrated from the whole of china, such as northeast china and west china . In 2011, the pure annual income of rural people was 5601.40 cny (about 889.11 usd) in shanxi province and 6604.03 cny (1048.26 usd) in henan province, much lower than beijing 14735.68 cny (about 2339.00 usd) cited from china statistical yearbook 2012 . Based on the sixth national census of china 2010 (http://www.stats.gov.cn/) and the 2010 annual survey data of china disabled persons' federation (http://www.cdpf.org.cn), cataract surgical rate (csr) was calculated as in table 1 . Any patients who wanted to have the charge - free cataract operations on lehet registered at the base hospital (a local hospital selected by the office of lehet). After the systemic and ocular examinations and signing the informed consent at the base hospital, the patients were sent to lehet . Preoperatively on lehet, all patients underwent a complete ophthalmological examination, that is, measurement of presenting visual acuity (va) by means of snellen charts (performed by the nurses from the base hospital), intraocular pressure evaluation (iop) by noncontact tonometer (canon tx-10/tx - f, tokyo, japan) by the trained nurses from puph, slit lamp examination (topcon sl-1e, tokyo, japan), and fundus examination (90 dioptre, volk optical, mentor, oh) with dilated pupil by the ophthalmologists from puph . Corneal curvature by auto - keratometer (nikon speedy - k, tokyo, japan), axial length (al) and b - scan by ultrasonic system (odm-2100, meda, tianjing, china), and corneal endothelial counting (cec) by specular microscope (topcon sp-3000p, tokyo, japan) were performed by the trained technicians from puph on the patients suitable for operation . The flatter (k1) and steeper corneal curvature (k2) were read directly from the auto - keratometer, and k was calculated as the average of k1 and k2 . Corneal radius (cr) was calculated from the formula cr (millimeter, mm) = 1000 0.3375/k (diopter, d). The srk / t formula for normal or long axial length (al more than 25.00 mm) and hoffer q formula for short axial length (al less than 22.00 mm) were used to calculate the power of intraocular lens (iol) and the estimated postoperative refractive errors were less than 0.25 d except patients with high myopia . Lehet was equipped with specular microscope, sp-3000p, in the first half of year 2012; the patients of zhoukou and part of yuncheng had no cec measurement . Exclusion criteria for this study are as follows: age less than 20 years, al equal to or more than 27.00 mm, and history of intraocular surgery . The study was in accordance with the tenets of the declaration of helsinki and has been approved by the institutional review board of puph . The student t - test was used to compare age and chi - square test was used to compare the female ratio between the groups . A p value less than 0.05 statistical analysis was performed using statistical product and service solutions software (spss version 20.0, armonk, new york, usa). 3828 cataract patients (3828 eyes) were enrolled in this study, including 1419 males and 2409 females (male: female = 1: 1.70) and 1984 right eyes and 1844 left eyes . There were no statistically significant differences between missions preoperatively in age, gender, and eye operated on . As in table 2, average age of these cataract patients was 69.50 8.05, which was 69.10 8.41 for males and 69.74 7.82 for females (p = 0.019), respectively . In detail, the average age was 68.55 8.12 for males and 69.57 8.07 for females in zhoukou (p = 0.056), 69.02 8.33 for males and 70.16 7.50 for females in yuncheng (p = 0.010), and 69.52 8.64 for males and 69.48 7.92 for females in sanmenxia (p = 0.933). Although the average age of females is older than males totally, that of males and females was of no difference for zhoukou and sanmenxia, except that of females which was older than that of males in yuncheng . As shown in tables 3, 4, and 5, not only for males or females, but also for total patients, the preoperative va (logmar) of these three groups is as follows: zhoukou> yuncheng> sanmenxia . The patients in sanmenxia had the best preoperative va, even in each gender, significantly . As shown in tables 3, 4, and 5, there was a statistically significant difference in preoperative iop between the patients of yuncheng and zhoukou, yuncheng and sanmenxia . The males, females, and total patients of yuncheng had lower preoperative iop compared with those in zhoukou or sanmenxia . As shown in figure 1 and tables 3, 4, and 5, the patients of zhoukou had lower k1 and k2, significantly . There was no statistically significant difference in k1 between those of yuncheng and sanmenxia, but k2 of yuncheng was higher than sanmenxia significantly . Respectively, both the males and females in zhoukou had lower k1 and k2 . However, for either the males or the females, there was no difference of k1 and k2 between those in yuncheng and sanmenxia . Average corneal power (k) is an important parameter to calculate the power of iol . In figure 1 and tables 3, 4, and 5, the patients in zhoukou had lower average corneal power (k) significantly compared with the other two groups, the same for male and female patients in zhoukou . But there was no significant difference in average corneal power (k) between those in yuncheng and sanmenxia, for either the males or the females . The difference between k1 and k2 could be used to indicate the corneal astigmatism, which has the effect on the postoperative visual acuity . In figure 1 and tables 3, 4, and 5, the difference of k1 and k2 for the patients was as follows: zhoukou> yuncheng> sanmenxia . But for the males except that |k1 k2| of zhoukou was higher than sanmenxia significantly, there was no significant difference between zhoukou and yuncheng or between yuncheng and sanmenxia . As seen in figure 2 and tables 3, 4, and 5, al for the patients was as follows: zhoukou <sanmenxia <yuncheng . For the males, al of zhoukou was shorter than the other two cities . For the females, al of yuncheng was longer than the other two sites . There was no significant difference in al between yuncheng and sanmenxia for males or between zhoukou and sanmenxia for females . The al / cr ratio is highly correlated with the spherical equivalent as a previous study . As seen in figure 2 and tables 3, 4, and 5, the patients in zhoukou had the smallest al / cr ratio closer to 3.0, and yuncheng and sanmenxia had similar ratio . Cec is a very important factor to decide the operation scheme and to predict prognosis . As there was no machine in zhoukou at that time, we only could compare cec between those in yuncheng and sanmenxia . As shown in figure 2 and tables 3, 4, and 5, cec of yuncheng was higher than sanmenxia, which was same result for the males . But for the females, there was no significant difference in cec between yuncheng and sanmenxia . This study explored the data of cataract patient, who had the free surgeries on lehet, on ocular biometry of chinese population in rural china . And our study provided the normative data on k1, k2, |k1 k2|, average corneal power (k), al, al / cr, and cec of this population; those were 43.74 1.64 d, 44.75 1.68 d, 1.02 0.86 d, 44.24 1.60 d, 23.04 1.49 mm, 3.03 0.12, and 2462.36 423.65/mm, respectively . Our study showed al in rural chinese population was normally distributed with a positive skew and a big kurtosis (1.417). Skew and kurtosis have been reported in the distribution of al in the reykjavik eye study, the singapore malay eye study, the singapore indian eye study, and fotedar et al . Hence, this is the first report of the appearance of big kurtosis in the distribution of al in rural chinese population . It is worthwhile comparing our findings with those of the tanjong pagar study on adult chinese population in singapore, which also used a - scan . The mean al in that study (23.23 1.17 mm) was a little longer than in our study (23.04 1.49 mm). Moreover, al in our study is shorter than latinos (23.38 mm) in los angeles with a - scan, malay people (23.55 mm) in singapore, indian people (23.45 mm) in singapore, and caucasian people (23.44) in the blue mountains area in australia with iolmaster, longer than another asian population (22.76 mm) in myanmar with ocuscan . The similarity of al in those studies with a - scan and ours is likely to be explained by the same method of al measurement . The difference in al of these studies might be explained by a greater degree of urbanization in singapore and subsequently a higher rate of axial myopia . Those three studies with iolmaster indicated that the race might have significant effect on al compared with region as the similarity of indian and caucasian people . The corneal power k1, corneal power k2, and k (average corneal power) in our study were not normally distributed with different skews and kurtosis . K2| in our study was normally distributed with a positive skew (2.704) and a significant kurtosis (13.317). Moreover, the preoperative visual acuities in the three missions of our study had the same trend as |k1 k2|, both of that of males and females are the same . There is evidence that the al / cr ratio of an emmetropic eye is usually very close to 3.0, and a higher al / cr ratio was reported to be a risk factor in myopia [15, 16]. However, few studies have reported the al / cr ratio . Compared with zhoukou, the patients in yuncheng and sanmenxia had similar al / cr ratio, also in males and in females . The singapore indian eye study showed that the al / cr ratio correlated more highly with the spherical equivalent than al alone . This correlation indicated that longer eyes are not necessarily myopic and worse presenting visual acuity, including those that are long because of overall body stature . The patients in zhoukou, who had shorter al and al / cr closer to 3.0, had the worst preoperative visual acuities . This indicated that in rural chinese population at least in the cataract patients the al / cr ratio, in other words, the spherical equivalent, had less effect on the visual acuity than |k1 k2|, the corneal astigmatism . In conclusion, this study provides normative ocular biometry in a large, representative rural chinese population . The corneal power k1, corneal power k2, and k (average corneal power) are not with normal distribution
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Body and pancreas weight were recorded, and blood glucose (glucometer elite, bayer, terrytown, nj), plasma insulin (enzyme - linked immunosorbent assay), and glucagon (radioimmunoassay) levels (joslin derc specialized assay core, boston, ma) were measured before experiments . Glucose tolerance tests, acute glucose - stimulated insulin secretion (gsis) tests, and insulin tolerance tests were performed as previously reported (27). Pancreata were harvested for immunohistochemical analyses and islet morphometry, and islets were isolated for rna extraction and in vitro gsis on batches of 15 size - matched islets as reported previously (28). Pancreas sections were used to assess -cell mass and size as described previously (27). -cell proliferation was assessed in pancreas sections as described previously (28), using anti - insulin (guinea - pig antibody, dako, carpinteria, ca), anti - brdu (rabbit polyclonal antibody, dako), or antiphosphohistone h3 antibodies (rabbit polyclonal antibody, upstate biotechnology, lake placid, ny). Other antibodies used include -catenin (rabbit polyclonal antibody, bd biosciences, oxford, u.k . ), glucagon (mouse monoclonal antibody, sigma - aldrich, st . Louis, mo), somatostatin (rabbit polyclonal antibody, abcam, cambridge, u.k . ), p21 (mouse monoclonal antibody, dako), insulin (mouse monoclonal antibody, biogenex, san ramon, ca), and dapi (sigma - aldrich) for nuclear staining . Quantitative real - time rt - pcr was performed on total rna samples extracted from islets and processed as described previously (28). Antibodies used include cyclin d2 (rabbit polyclonal antibody, santa cruz biotechnology inc ., santa cruz, ca), foxo1 (rabbit polyclonal antibody, cell signaling, danvers, ma), pancreatic duodenal homeobox-1 (pdx-1) (gift from c.v . Wright, vanderbilt university), p27 (mouse monoclonal antibody, bd biosciences), p21 (mouse monoclonal antibody, dako), and tubulin (mouse monoclonal antibody, abcam). All data are presented as mean sem and were analyzed using an anova or unpaired, two - tailed student t test as appropriate . Body and pancreas weight were recorded, and blood glucose (glucometer elite, bayer, terrytown, nj), plasma insulin (enzyme - linked immunosorbent assay), and glucagon (radioimmunoassay) levels (joslin derc specialized assay core, boston, ma) were measured before experiments . Glucose tolerance tests, acute glucose - stimulated insulin secretion (gsis) tests, and insulin tolerance tests were performed as previously reported (27). Pancreata were harvested for immunohistochemical analyses and islet morphometry, and islets were isolated for rna extraction and in vitro gsis on batches of 15 size - matched islets as reported previously (28). Pancreas sections were used to assess -cell mass and size as described previously (27). -cell proliferation was assessed in pancreas sections as described previously (28), using anti - insulin (guinea - pig antibody, dako, carpinteria, ca), anti - brdu (rabbit polyclonal antibody, dako), or antiphosphohistone h3 antibodies (rabbit polyclonal antibody, upstate biotechnology, lake placid, ny). Other antibodies used include -catenin (rabbit polyclonal antibody, bd biosciences, oxford, u.k . ), glucagon (mouse monoclonal antibody, sigma - aldrich, st . Louis, mo), somatostatin (rabbit polyclonal antibody, abcam, cambridge, u.k . ), p21 (mouse monoclonal antibody, dako), insulin (mouse monoclonal antibody, biogenex, san ramon, ca), and dapi (sigma - aldrich) for nuclear staining . Quantitative real - time rt - pcr was performed on total rna samples extracted from islets and processed as described previously (28). Antibodies used include cyclin d2 (rabbit polyclonal antibody, santa cruz biotechnology inc ., santa cruz, ca), foxo1 (rabbit polyclonal antibody, cell signaling, danvers, ma), pancreatic duodenal homeobox-1 (pdx-1) (gift from c.v . Wright, vanderbilt university), p27 (mouse monoclonal antibody, bd biosciences), p21 (mouse monoclonal antibody, dako), and tubulin (mouse monoclonal antibody, abcam). All data are presented as mean sem and were analyzed using an anova or unpaired, two - tailed student t test as appropriate . We compared the phenotype of mice at 3, 10, and 1214 months of age . The phenotype of p44tg mice becomes evident at 3 months, and the animals begin to succumb at 14 months of age (22). P44tg mice were lighter than their nontransgenic counterparts at all ages, as shown for 3 months of age in fig . The animals had already developed hypoinsulinemia at this age, which persisted throughout life (fig . 1b and 2c), suggesting overt diabetes that could contribute, in part, to death at this age . In contrast, all controls exhibited fed blood glucose levels <150 mg / dl at 12 months of age (fig . These data indicate that increased dosage of 40p53 leads to hypoinsulinemia and glucose intolerance as early as 3 months and worsens with age . Body weight (a), blood glucose (b), plasma insulin (c), and plasma glucagon (d) levels were measured at 3, 10, and 1214 months of age from random - fed control () and p44tg () (n = 59) mice . * p <0.05 for p44tg vs. control mice . #p <0.05 for control mice vs. different ages . Glucose tolerance tests performed at 2 months (a), 35 months (b), and 1014 months (c) of age (n = 68). Blood glucose was measured at 0, 10, 20, 30, 60, and 120 min after intraperitoneal injection of glucose . Insulin tolerance tests were performed in each group at 2 months (d), 34 months (e), and 1516 months (f) of age (n = 68). Glucose was measured at 0, 15, 30, 45, and 60 min after intraperitoneal injection of insulin . Ip, intraperitoneal; ipgtt, intraperitoneal glucose tolerance test; ipitt, intraperitoneal insulin tolerance test . Although both pancreas weight and number of islets exhibited an age - dependent increase in control mice, the total number of islets was less in p44tg mice even when normalized to total pancreas weight (fig . 1). Compared with 3 months of age, the differences became more marked at 1214 months . The reduced pancreatic weight and islet number in p44tg mice were associated with a dramatic decrease in -cell mass (fig . -cell mass was significantly lower in the p44tg mice and ranged between 11% (young) and 14% (old) in the age - matched control animals . Quantification of -cell size revealed a 30% decrease in the p44tg mice compared with controls at 3 months of age but showed the opposite in the older p44tg mice (fig . This represented a twofold increase in -cell size as the p44tg mice aged from 3 to 12 months . To explore the pathways that regulate -cell size, we considered previous reports that proteins in the insulin / igf-1 signaling pathways regulate -cell size via akt / mtor / s6k1 (1416). Quantitative real - time rt - pcr for these molecules did not reveal significant differences between groups at 3 months of age except for a decrease in insulin receptor (ir) expression in p44tg islets (fig . 3e). However, in islets from older p44tg mice, we observed increased gene expression of ir, irs1 (ir substrate-1), and especially akt1, mtor, and s6k1, suggesting an attempt at compensation by increasing the -cell size in the older mutant mice . This was not observed in the liver or white adipose tissue of p44tg mice (supplementary fig . A: pancreatic weight expressed as a percentage of the total body weight (n = 3). C and d: -cell size was assessed by coimmunostaining for -catenin (green) and insulin (red) with dapi (blue) in pancreas sections from control (n = 5) and p44tg (n = 5) mice at 3 and 1214 months of age as described in research design and methods . A representative islet for each group at magnification 40 is presented with the quantification of relative -cell size (mean sem from n 200 cells counted per mouse). E: real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . Results are normalized to tata - binding protein (tbp) and expressed relative to control mice . * p <0.05 for p44tg vs. control mice . #p <0.05 for 3- vs. 12- to 14-month - old control mice . (a high - quality digital representation of this figure is available in the online issue .) Examination of islet morphology by immunohistochemistry (fig . 4a) revealed the typical pattern of a core of insulin - containing cells surrounded by non-cells in islets from controls and in a majority of islets from p44tg mice at 3 months of age (fig . However, 35% of islets in the normoglycemic and hypoinsulinemic p44tg groups exhibited infiltration of - and -cells within the islet core and a relative decrease in the number of -cells (quantified in fig . These abnormal features, which were not observed at 2 months of age (supplementary fig . 2), were evident in 85% of the islets by 12 months of age in the p44tg group (fig . 4a, bottom). In parallel, 4c) revealed a significant increase in somatostatin gene expression that was consistent with a relative increase in -cells in p44tg mice (fig . A significant increase in the expression of insulin and glut 2 in 3-month - old transgenic mice suggested a compensatory effect at this younger age (fig . 4c, top) that was not evident in the older hyperglycemic group (fig . 4c, bottom). Because p44tg mice exhibit hypoinsulinemia and reduced -cell numbers, we evaluated -cell secretory function in vivo (fig . 2b), the gsis in isolated islets in vitro was not different between groups (fig . Reduction in insulin - positive cells and increase in somatostatin - positive cells in islets from p44tg mice . A: triple coimmunostaining for insulin (blue), somatostatin (green), and glucagon (red) on pancreas sections from control or p44tg mice at 3 and 1214 months of age as described in research design and methods (n = 5). B: the number of insulin - positive cells was counted in at least 10 islets randomly selected from pancreas sections from each mouse (n = 3 mice at each age per group). C: real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . Insulin was measured in plasma samples extracted from blood collected at 0, 2, and 5 min after intraperitoneal injection of glucose (3 g / kg body wt) from 45-month - old control and p44tg mice . Islets isolated from 45-month - old control and p44tg mice were incubated for 1 h with 3 or 16.7 mmol / l glucose as described in research design and methods . Bar graph depicts data obtained from three to five individual batches of size - matched islets from at least three independent mice per group . * p <0.05 for p44tg vs. control mice . #p <0.05 for 3- vs. 12- to 14-month - old control mice . (a high - quality digital representation of this figure is available in the online issue .) Next, we determined whether the decrease in -cell mass in p44tg mice (fig . Coimmunostaining of pancreas sections with antibodies against insulin and either one of two proliferation markers showed a decrease in the proliferation of -cells with age in control islets (fig . Brdu labeling demonstrated a virtual absence of replicating -cells in young and old p44tg mice compared with controls (fig . 5a). A decrease in phh3 immunoreactive -cells at 3 and 1214 months of age in p44tg mice confirmed reduced number of cells in the m or late g2 phases of the cell cycle (fig ., no significant difference was evident in -cell replication between the two groups (supplementary fig . -cell proliferation was assessed by coimmunostaining for brdu (a, green) and phh3 (b, green) with insulin (red) and dapi (blue) in pancreas sections from control and p44tg mice at 3 and 1214 months of age as described in research design and methods . A representative islet for each group at magnification 40 is presented (n = 5). P <0.05 for p44tg vs. control mice . #p <0.05 for 3- vs. 12- to 14-month - old control mice . (a high - quality digital representation of this figure is available in the online issue .) We next evaluated the effects of neogenesis (30) and apoptosis (31) in the maintenance of -cell mass . Single and clusters (8) of insulin+ cells (markers of neogenesis) were virtually absent in pancreatic ducts in 3-month - old control and p44tg mice . Although some insulin+ cells / clusters were observed in 12- to 14-month - old mice, no significant difference was evident between groups (number / mm pancreas: 0.3 0, control vs. 0.3 0.2, p44tg; p = ns, n = 3). Similarly, no significant differences were observed in insulin+ cells in extraductal pancreatic tissue (3 month; 14 0.3, control vs. 12.8 3.4, p44tg; 1214 month; 8.3 0.2, control vs. 10.2 2.4, p44tg; p = ns; n = 3), suggesting that neogenesis is not altered significantly when 40p53 is misexpressed . Although p53 has been studied in the context of apoptosis in multiple tissues, it has not been causally involved in -cell death during diabetes (32). Nevertheless, we performed the transferase - mediated dutp nick - end labeling (tunel) assay and found no significant differences in tunel+ -cells between groups (supplementary fig . These data provide strong evidence that the misexpression of 40p53 led to a marked reduction of -cell proliferation, consistent with reports for other cell types (22,24). P53 regulates the g1 to s phase progression through cell cycle inhibitors such as p21, but recent reports indicate that p53 also targets integral cell cycle proteins, such as cyclin e and cdk4 (18). We observed a significant decrease in the gene expression levels of cyclin d2, cdk2, and cyclin e1, and a significant increase in the level of cyclin e2 in 3-month - old p44tg mice (fig . Only cyclin e1 gene expression remained low; at the same time, however, cdk4 levels became significantly increased . The decrease in cyclin d2 gene expression at 3 months of age correlated with lower protein expression in isolated islets (fig . In old mice, despite normal levels of gene expression, cyclin d2 protein level was significantly decreased (fig . A: real time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . B: western blotting for cyclin d2 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . * p <0.05 for p44tg vs. control mice . We next examined the expression of foxo1, which regulates -cell proliferation (33), and two of its target proteins, pdx-1 (34) and p27 (35). Examination of gene and protein expression in whole islets revealed that foxo1 expression was significantly lower at 3 months but unchanged at 10 months in p44tg mice (fig . This decrease was less pronounced in old mice and correlated with fewer -cells in islets from the transgenic mice (fig . 4b). P27 gene and protein expression remained unchanged (fig . 7a and b). These data indicate that decreased -cell mass and proliferation observed in p44tg mice (figs . A: real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . B: western blotting for foxo1, pdx-1, and p27 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . * . Finally, we evaluated the gene expression of p53 and its protein targets (19). To measure the level of transcription of both p53 (endogenous) and 40p53 (endogenous and transgene) genes, we designed two sets of primers for quantitative rt - pcr because the use of specific primers to distinguish both genes was not possible . Np53 primers targeted the 5 end of the p53 transcript and amplified transcripts derived from the endogenous p53 gene only, whereas cp53 primers targeted sequences at the 3 end of rna and amplified transcripts derived from both the endogenous p53 gene and the transgene . We also measured the expression of several well - known p53 target genes (19), such as p21, 143 - 3, gadd45, bax, mdm2, pten, and the igf-1 receptor (igf-1r). We observed a significant decrease in endogenous p53 gene expression (np53 primers) that correlated with reduced gadd45 gene in islets from 3-month - old p44tg mice (fig . 8a, top). In islets from 12-month - old p44tg mice, we observed an increase in 40p53 expression (cp53 primers) that was associated with an enhanced expression of p21 and mdm2 (fig . Although bax gene expression was significantly elevated in p44tg islets at both ages, we did not observe significance differences between groups in the tunel assay (supplementary fig . The increase in gene expression of igf-1r that was evident in young p44tg islets did not persist in older mice, and we did not observe alterations in pten gene expression at either age (fig . However, we did find an increase in p21 protein by western blot analysis of p44tg islets (fig . Thus, the strong inhibition of -cell proliferation observed in p44tg islet cells correlated with a significant increase in p21, the main target of p53 in cell cycle regulation . Increased p21 expression in p44tg islets . A: real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . B: western blotting for p21 and quantification normalized to actin in islets from control and p44tg at 3 and 10 months of age . * c: quantification of p21-positive -cell nuclei in pancreas sections from control and p44tg mice at 3 and 1214 months of age (n = 5). * p <0.05 for p44tg vs. control mice (at least 810 islets from five mice per group). We compared the phenotype of mice at 3, 10, and 1214 months of age . The phenotype of p44tg mice becomes evident at 3 months, and the animals begin to succumb at 14 months of age (22). P44tg mice were lighter than their nontransgenic counterparts at all ages, as shown for 3 months of age in fig . The animals had already developed hypoinsulinemia at this age, which persisted throughout life (fig . 1b and 2c), suggesting overt diabetes that could contribute, in part, to death at this age . In contrast, all controls exhibited fed blood glucose levels <150 mg / dl at 12 months of age (fig . These data indicate that increased dosage of 40p53 leads to hypoinsulinemia and glucose intolerance as early as 3 months and worsens with age . Body weight (a), blood glucose (b), plasma insulin (c), and plasma glucagon (d) levels were measured at 3, 10, and 1214 months of age from random - fed control () and p44tg () (n = 59) mice . * p <0.05 for p44tg vs. control mice . #p <0.05 for control mice vs. different ages . Glucose tolerance tests performed at 2 months (a), 35 months (b), and 1014 months (c) of age (n = 68). Blood glucose was measured at 0, 10, 20, 30, 60, and 120 min after intraperitoneal injection of glucose . Insulin tolerance tests were performed in each group at 2 months (d), 34 months (e), and 1516 months (f) of age (n = 68). Glucose was measured at 0, 15, 30, 45, and 60 min after intraperitoneal injection of insulin . Ip, intraperitoneal; ipgtt, intraperitoneal glucose tolerance test; ipitt, intraperitoneal insulin tolerance test . Although both pancreas weight and number of islets exhibited an age - dependent increase in control mice, the total number of islets was less in p44tg mice even when normalized to total pancreas weight (fig . 1). Compared with 3 months of age, the differences became more marked at 1214 months . The reduced pancreatic weight and islet number in p44tg mice were associated with a dramatic decrease in -cell mass (fig . -cell mass was significantly lower in the p44tg mice and ranged between 11% (young) and 14% (old) in the age - matched control animals . Quantification of -cell size revealed a 30% decrease in the p44tg mice compared with controls at 3 months of age but showed the opposite in the older p44tg mice (fig . This represented a twofold increase in -cell size as the p44tg mice aged from 3 to 12 months . To explore the pathways that regulate -cell size, we considered previous reports that proteins in the insulin / igf-1 signaling pathways regulate -cell size via akt / mtor / s6k1 (1416). Quantitative real - time rt - pcr for these molecules did not reveal significant differences between groups at 3 months of age except for a decrease in insulin receptor (ir) expression in p44tg islets (fig . 3e). However, in islets from older p44tg mice, we observed increased gene expression of ir, irs1 (ir substrate-1), and especially akt1, mtor, and s6k1, suggesting an attempt at compensation by increasing the -cell size in the older mutant mice . This was not observed in the liver or white adipose tissue of p44tg mice (supplementary fig . A: pancreatic weight expressed as a percentage of the total body weight (n = 3). C and d: -cell size was assessed by coimmunostaining for -catenin (green) and insulin (red) with dapi (blue) in pancreas sections from control (n = 5) and p44tg (n = 5) mice at 3 and 1214 months of age as described in research design and methods . A representative islet for each group at magnification 40 is presented with the quantification of relative -cell size (mean sem from n 200 cells counted per mouse). E: real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . Results are normalized to tata - binding protein (tbp) and expressed relative to control mice . * p <0.05 for p44tg vs. control mice . #p <0.05 for 3- vs. 12- to 14-month - old control mice . (a high - quality digital representation of this figure is available in the online issue .) 4a) revealed the typical pattern of a core of insulin - containing cells surrounded by non-cells in islets from controls and in a majority of islets from p44tg mice at 3 months of age (fig . 4a, top). However, 35% of islets in the normoglycemic and hypoinsulinemic p44tg groups exhibited infiltration of - and -cells within the islet core and a relative decrease in the number of -cells (quantified in fig . These abnormal features, which were not observed at 2 months of age (supplementary fig . 2), were evident in 85% of the islets by 12 months of age in the p44tg group (fig . 4c) revealed a significant increase in somatostatin gene expression that was consistent with a relative increase in -cells in p44tg mice (fig . 4a), whereas glucagon gene expression (fig . 4c) and circulating glucagon levels (fig . A significant increase in the expression of insulin and glut 2 in 3-month - old transgenic mice suggested a compensatory effect at this younger age (fig . 4c, top) that was not evident in the older hyperglycemic group (fig . 4c, bottom). Because p44tg mice exhibit hypoinsulinemia and reduced -cell numbers, we evaluated -cell secretory function in vivo (fig . 2b), the gsis in isolated islets in vitro was not different between groups (fig . Reduction in insulin - positive cells and increase in somatostatin - positive cells in islets from p44tg mice . A: triple coimmunostaining for insulin (blue), somatostatin (green), and glucagon (red) on pancreas sections from control or p44tg mice at 3 and 1214 months of age as described in research design and methods (n = 5). B: the number of insulin - positive cells was counted in at least 10 islets randomly selected from pancreas sections from each mouse (n = 3 mice at each age per group). C: real - time rt - pcr on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . Results are normalized to tbp and expressed relative to controls . Insulin was measured in plasma samples extracted from blood collected at 0, 2, and 5 min after intraperitoneal injection of glucose (3 g / kg body wt) from 45-month - old control and p44tg mice . Islets isolated from 45-month - old control and p44tg mice were incubated for 1 h with 3 or 16.7 mmol bar graph depicts data obtained from three to five individual batches of size - matched islets from at least three independent mice per group . * . #p <0.05 for 3- vs. 12- to 14-month - old control mice . (a high - quality digital representation of this figure is available in the online issue .) Next, we determined whether the decrease in -cell mass in p44tg mice (fig . Coimmunostaining of pancreas sections with antibodies against insulin and either one of two proliferation markers showed a decrease in the proliferation of -cells with age in control islets (fig . Brdu labeling demonstrated a virtual absence of replicating -cells in young and old p44tg mice compared with controls (fig . 5a). A decrease in phh3 immunoreactive -cells at 3 and 1214 months of age in p44tg mice confirmed reduced number of cells in the m or late g2 phases of the cell cycle (fig ., no significant difference was evident in -cell replication between the two groups (supplementary fig . -cell proliferation was assessed by coimmunostaining for brdu (a, green) and phh3 (b, green) with insulin (red) and dapi (blue) in pancreas sections from control and p44tg mice at 3 and 1214 months of age as described in research design and methods . A representative islet for each group at magnification 40 is presented (n = 5). Quantification is shown on the right . * p <0.05 for p44tg vs. control mice . #p <0.05 for 3- vs. 12- to 14-month - old control mice . (a high - quality digital representation of this figure is available in the online issue .) We next evaluated the effects of neogenesis (30) and apoptosis (31) in the maintenance of -cell mass . Single and clusters (8) of insulin+ cells (markers of neogenesis) were virtually absent in pancreatic ducts in 3-month - old control and p44tg mice . Although some insulin+ cells / clusters were observed in 12- to 14-month - old mice, no significant difference was evident between groups (number / mm pancreas: 0.3 0, control vs. 0.3 0.2, p44tg; p = ns, n = 3). Similarly, no significant differences were observed in insulin+ cells in extraductal pancreatic tissue (3 month; 14 0.3, control vs. 12.8 3.4, p44tg; 1214 month; 8.3 0.2, control vs. 10.2 2.4, p44tg; p = ns; n = 3), suggesting that neogenesis is not altered significantly when 40p53 is misexpressed . Although p53 has been studied in the context of apoptosis in multiple tissues, it has not been causally involved in -cell death during diabetes (32). Nevertheless, we performed the transferase - mediated dutp nick - end labeling (tunel) assay and found no significant differences in tunel+ -cells between groups (supplementary fig . These data provide strong evidence that the misexpression of 40p53 led to a marked reduction of -cell proliferation, consistent with reports for other cell types (22,24). P53 regulates the g1 to s phase progression through cell cycle inhibitors such as p21, but recent reports indicate that p53 also targets integral cell cycle proteins, such as cyclin e and cdk4 (18). We observed a significant decrease in the gene expression levels of cyclin d2, cdk2, and cyclin e1, and a significant increase in the level of cyclin e2 in 3-month - old p44tg mice (fig . Only cyclin e1 gene expression remained low; at the same time, however, cdk4 levels became significantly increased . The decrease in cyclin d2 gene expression at 3 months of age correlated with lower protein expression in isolated islets (fig . In old mice, despite normal levels of gene expression, cyclin d2 protein level was significantly decreased (fig . A: real time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . B: western blotting for cyclin d2 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . * we next examined the expression of foxo1, which regulates -cell proliferation (33), and two of its target proteins, pdx-1 (34) and p27 (35). Examination of gene and protein expression in whole islets revealed that foxo1 expression was significantly lower at 3 months but unchanged at 10 months in p44tg mice (fig . This decrease was less pronounced in old mice and correlated with fewer -cells in islets from the transgenic mice (fig . 4b). P27 gene and protein expression remained unchanged (fig . 7a and b). These data indicate that decreased -cell mass and proliferation observed in p44tg mice (figs . A: real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . B: western blotting for foxo1, pdx-1, and p27 and quantification normalized to tubulin in islets from control and p44tg at 3 and 10 months of age . * finally, we evaluated the gene expression of p53 and its protein targets (19). To measure the level of transcription of both p53 (endogenous) and 40p53 (endogenous and transgene) genes, we designed two sets of primers for quantitative rt - pcr because the use of specific primers to distinguish both genes was not possible . Np53 primers targeted the 5 end of the p53 transcript and amplified transcripts derived from the endogenous p53 gene only, whereas cp53 primers targeted sequences at the 3 end of rna and amplified transcripts derived from both the endogenous p53 gene and the transgene . We also measured the expression of several well - known p53 target genes (19), such as p21, 143 - 3, gadd45, bax, mdm2, pten, and the igf-1 receptor (igf-1r). We observed a significant decrease in endogenous p53 gene expression (np53 primers) that correlated with reduced gadd45 gene in islets from 3-month - old p44tg mice (fig . 8a, top). In islets from 12-month - old p44tg mice, we observed an increase in 40p53 expression (cp53 primers) that was associated with an enhanced expression of p21 and mdm2 (fig . Although bax gene expression was significantly elevated in p44tg islets at both ages, we did not observe significance differences between groups in the tunel assay (supplementary fig . The increase in gene expression of igf-1r that was evident in young p44tg islets did not persist in older mice, and we did not observe alterations in pten gene expression at either age (fig . However, we did find an increase in p21 protein by western blot analysis of p44tg islets (fig . Thus, the strong inhibition of -cell proliferation observed in p44tg islet cells correlated with a significant increase in p21, the main target of p53 in cell cycle regulation . Increased p21 expression in p44tg islets . A: real - time rt - pcr was performed on rna extracted from islets of 3- and 10- to 12-month - old control (n = 38) and p44tg (n = 4) mice . B: western blotting for p21 and quantification normalized to actin in islets from control and p44tg at 3 and 10 months of age . * c: quantification of p21-positive -cell nuclei in pancreas sections from control and p44tg mice at 3 and 1214 months of age (n = 5). * p <0.05 for p44tg vs. control mice (at least 810 islets from five mice per group). By using p44tg mice with an ectopic p53 gene that encodes 40p53 (36), we have identified a role for the tumor suppressor p53 in the maintenance of normal -cell mass and glucose homeostasis . Although 40p53 has virtually no autonomous transcriptional activity, it can interact with full - length p53 to form tetramers that bind dna and activate or suppress target gene expression (36,37). Expression of ectopic 40p53 in the mouse alters the balance between the full - length and short isoforms that normally exists and hyperactivates p53 (22,36,38). This increased activity of p53 can account for the higher levels of p21 transcripts, which we observed in -cells from 1012-month - old p44tg mice . The expression of 40p53 in p44tg mice can also account for the higher level of mrna encoding the igf-1r, whose transcription is differentially regulated by wild - type and mutant p53 (39). An increased gene expression of igf-1r in liver and white adipose tissue from p44tg mice (supplementary fig . Similar to embryonic fibroblasts derived from p44tg mice (22), islets from young p44tg mice expressed significantly high levels of igf-1r, indicating impaired trans - suppression activity . Both the increased trans - activation of p21 in old islets and the decreased trans - suppression of the igf-1r in the young can be explained by differences in expression of the transgene encoding 40p53 with age, as detected by primers that amplify sequences encoding the cooh - terminal domain of the p53 protein . Younger p44tg mice expressed control levels of 40p53 and slightly lower levels of p53, whereas older mice expressed normal levels of p53 and higher levels of 40p53 . Biochemical experiments have demonstrated that the stability and activity of the p53 tetramer are exquisitely sensitive to the dose of 40p53, with low doses of 40p53 activating and high doses inactivating p53 function (40,41). The differential effects of 40p53 on p53-dependent trans - activation and trans - repression with age could also explain how p21 levels increase in both age groups, but by different mechanisms . In old mice, the increase would be due to a direct effect of p53 on the p21 promoter, leading to increased transcription of the p21 gene . In young mice, on the other hand, increased p21 could be an indirect effect of higher igf-1r expression and activation of igf signaling due to impaired igf-1r trans - repression . Stimulation of the igf-1 signal transduction pathway can increase p21 (42), and further work is necessary to delineate the link among 40p53/p53, p21, and igf-1r levels in -cell proliferation . -cell proliferation and glucose tolerance were impaired in 3-month - old p44tg animals and worsened to overt diabetes as the animals aged . Although random - fed blood glucose levels were normal in the transgenic mice at 3 and 10 months, the mutants displayed clear intolerance in response to a glucose challenge indicating deficiency in functional -cells . For example, sreenan et al . (43) report a reduced -cell mass before the onset of diabetes in the nonobese diabetic mouse, and tavana et al . (44) describe the phenotypes of 1-month - old mice doubly mutant for p53 and nonhomologous end - joining deficiency that exhibit an 50% decrease in -cell mass and yet manifest blood glucose levels that are not significantly different from controls . Further, obese and nondiabetic humans have been reported to express a wide range of -cell mass that is adequate to maintain euglycemia up to a specific threshold, and crossing the threshold correlates with fasting hyperglycemia and overt diabetes (45). In our study, it is possible that p44tg mice that are significantly leaner than controls at all ages only cross this threshold at 12 months of age when they begin to exhibit overt hyperglycemia . Thus, our data suggest that the balance between full - length and 40p53 isoforms plays a critical role in the maintenance of -cell proliferation and glucose homeostasis, and may be important in regulating the cell cycle during aging . Further support for a role for p53 in glucose homeostasis is evident from recent studies . (46) reported a role for adipose p53 in the regulation of insulin resistance . In our study, circulating proinflammatory cytokines, which are potential effectors in insulin resistance associated with diabetes, showed no significant differences between groups (supplementary fig . The cytokine expression profile in liver and white adipose tissue was also similar between groups . These data, along with the normal insulin sensitivity in the global p44tg mice, indicate that the glucose intolerance is due to intrinsic effects of p53 on -cell mass rather than to secondary effects of transgene expression in other insulin sensitive tissues . Indeed, (44) reported that dna double - strand breaks combined with an absence of p53-dependent apoptosis in mice leads to reduced -cell replication and severe age - dependent diabetes, supporting our hypothesis that p53 plays a role in the regulation of -cell proliferation . Although the mechanisms that regulate the dynamics of -cell turnover during aging are still being unraveled, several studies including our own support a role for replication as the major mechanism that underlies the compensatory growth response to insulin resistance (15,47). Two proteins that are important for this compensatory growth response are cyclin d2 (5,11,12) and pdx-1, the pancreatic duodenal homeobox domain protein (3). The lower expression of cyclin d2 in islets would potentially exacerbate the loss of -cells and lead to glucose intolerance and overt diabetes . In this study, the expression of pdx-1 was also altered in p44tg -cells . Although the gene expression of pdx-1 was increased in both young and old p44tg mice, the protein expression in islets was low, suggesting compensatory effects in gene expression or more dominant regulation of the transcription factor at the protein level . In old p44tg mice, however, there were fewer -cell nuclei in which pdx-1 could be detected . Immunohistochemistry (data not shown) also revealed that the localization of pdx-1 correlated inversely with that of foxo1, a transcription factor that regulates expression of this homeodomain protein (34). Because foxo1 transactivates cell cycle inhibitors (p21 and p27) (35) and represses cell cycle activators (cyclin d1 and d2), nuclear localization of foxo1 could have a major impact on cell cycle progression in aging -cells . It is also possible that foxo1 and p53 interact with deacetylase sirtuin 1 and function in a cooperative manner in an aging environment (48). Thus, the alterations in both pdx-1 and cyclin d2, in our model of accelerated aging, underscore the concept that these two proteins are involved in -cell replication during aging . Two cdkis, namely, p16 and p19, have been reported to be involved in -cell regenerative failure in diabetes (49). Our observations of enhanced expression of p16 and p19 genes confirm the premature aging phenotype of p44tg mice (supplementary fig . However, the regulatory link between p53 and the two aging markers in the maintenance or amplification of the phenotype requires further investigation . (22) previously described that young p44tg mice displayed signs of aging in bone . Further, the osteoblast - secreted molecule, osteocalcin, has recently been reported to modulate insulin secretion and insulin gene expression that correlated with expression of creb and neurod genes (50). In our study, we observed a mild but significant decrease in osteocalcin levels only in old p44tg mice; however, gene expression of insulin, creb, or neurod was unaltered (supplementary fig . 7). Finally, the significance of the presence of p53 in non-cell fractions obtained from dispersed islet cells (29) remains unclear . Although the relative increase in -cells in p44tg islets was not associated with an increase in glucagon gene expression or circulating glucagon levels, we observed a significant increase in the number of somatostatin - secreting -cells and gene expression in the mutant islets . The presence of a p53 response element in the somatostatin promoter (genomatix) warrants further studies to evaluate whether the increase in somatostatin actually decreases insulin secretion or limits -cell growth in a paracrine manner in the p44tg mice . In summary, increased dosage of 40p53 in mice promotes hypoinsulinemia and glucose intolerance, ultimately leading to overt diabetes with age and early death . The suppression of -cell proliferation secondary to changes in the expression of p21, cyclin d2, and pdx-1 in this mouse model of accelerated aging implicates impaired p53 function in the development of type 2 diabetes in the elderly.
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Hereditary gingival enlargement (hgf) is a rare condition with incidence of 1 in 750, 000 people . It may occur isolated or as a part of multisystem syndrome . It may present itself as an autosomal dominant which has been linked to chromosome 2p21-p22 and 5q13-q22 or recessive mode of inheritance . Although involvement of the sos-1 gene has been suggested recently . Families are affected across generations and a positive family history is always present in hgf . Hereditary gingival fibromatosis is a gradually progressive benign enlargement that affects the marginal, attached, and interdental gingiva . We are here presenting a rare case of a 13-year - old girl suffering from hereditary gingival fibromatosis along with surgical management to contribute the literature regarding this rare entity and moreover to emphasize the clinical significance of early management of gingival enlargement as if it is untreated timely could lead to pathological migration, abnormal jaw development, and various functional and esthetic problems . A 13-year - girl reported to department of periodontics, dr . Z.a.d.c . And hospital, amu, with a chief complaint of enlarged gums . She could not remember the duration of swelling but she said it was always present . Her medical history was non - contributory and she was not taking any medications regularly . Her family history revealed that her elder sister had also similar kind of gingival enlargement but that history ca nt be confirmed . Intraoral examination revealed fibrotic gingival overgrowth both on buccal and lingual / palatal aspects with firm consistency, increased stippling, and normal pinkish color . Gingival enlargement covers most part of teeth [figures 15]. Clinical examination also revealed retained deciduous canine and overlapping of maxillary central incisors and maxillary right canine placed highly in an arch . Radiographic examination was done to rule out any bony enlargement and general medical examination was advised . Panoramic radiographs shows retained right primary canine, no bone loss, or any kind of bony enlargement [figure 6]. Preoperative facial view showing gingival enlargement and overlapping of central incisor palatal view showing right retained deciduous canine right lateral view showing the migrated right permanent canine orthopantogram showing no bone loss and the right retained deciduous canine oral hygiene instructions were given, but the plaque removal was restricted by enlarged gingiva . Therefore, internal bevel gingivectomies were performed for all quadrants in the maxillary and mandibular arches . The surgical intervention was carried out using internal bevel and crevicular incisions to remove hyperplastic tissue and retained primary canine was extracted . The patient was given a prescription for an antibiotic for 24 h before the surgery and 5 days postoperatively (amoxicillin 500 mg, three times a day) to prevent postoperative bacteremia and an analgesic for three days postoperatively (aceclofenac 500 mg, twice a day) to relieve postoperative pain and a periodontal dressing (coe - pak) were placed and removed after 1 week . The interval between surgical procedures was 2 weeks . Excised tissue was sent for histopathological examination which revealed hyperkeratosis, elongated rete pegs, numerous bundles of collagen fibers, and mild inflammatory infiltrate [figure 7]. In the light of above features and family history, diagnosis of hgf postoperative record of 12 months shows improvement in position of both central incisors and right permanent canine [figures 812]. Histological picture showing collagen fibers and hyperdense connective tissue, deep rete pegs, acanthosis of epithelium postoperative facial view showing improvement in position of upper incisor and right maxillary canine postoperative lingual view postoperative palatal view postoperative left lateral view postoperative right lateral view oral hygiene instructions were given, but the plaque removal was restricted by enlarged gingiva . Therefore, internal bevel gingivectomies were performed for all quadrants in the maxillary and mandibular arches . The surgical intervention was carried out using internal bevel and crevicular incisions to remove hyperplastic tissue and retained primary canine was extracted . The patient was given a prescription for an antibiotic for 24 h before the surgery and 5 days postoperatively (amoxicillin 500 mg, three times a day) to prevent postoperative bacteremia and an analgesic for three days postoperatively (aceclofenac 500 mg, twice a day) to relieve postoperative pain and a periodontal dressing (coe - pak) were placed and removed after 1 week . Excised tissue was sent for histopathological examination which revealed hyperkeratosis, elongated rete pegs, numerous bundles of collagen fibers, and mild inflammatory infiltrate [figure 7]. In the light of above features and family history, diagnosis of hgf was made . Postoperative record of 12 months shows improvement in position of both central incisors and right permanent canine [figures 812]. Histological picture showing collagen fibers and hyperdense connective tissue, deep rete pegs, acanthosis of epithelium postoperative facial view showing improvement in position of upper incisor and right maxillary canine postoperative lingual view postoperative palatal view postoperative left lateral view postoperative right lateral view it is synonyms with the conditions like elephantiasis gingivae, fibromatosis gingivae, gigantism of the gingiva, congenital macrogingivae, hereditary gingival hyperplasia, and hypertrophic gingiva . Enlargement of gingiva can be due to use of medication like phenytoin, cyclosporine, and calcium channel blockers . Gingival fibromatosis can occur as an isolated nonsyndromic condition or be associated with other syndromes . The syndromes associated with gf include jones hartsfield, murray- puretic - drescher syndrome, zimmermann - laband, rutherfurd, cross, ramon, prune - belly syndrome associated with hearing deficiencies, hypertelorism, and supernumerary teeth . The laboratory investigations revealed no evidence of any systemic disorders such as leukemia, diabetes mellitus, or hormonal disorders . The histologic features observed in the present case had the typical appearance of gingival fibromatosis; hyperplasic dense fibrous connective tissue with acanthotic gingival epithelium . As the family history contributes to this case with no systemic and drug history and with no clinical features fulfilling these possible syndromes, diagnosis of isolated generalized hereditary gingival fibromatosis the gingival enlargement usually begins at the time of eruption of the permanent dentition or, less frequently, with the eruption of the primary dentition . This is consistent with this case . The most common effect due to enlargement was pathologic migration, diastema, and retained deciduous teeth which was present in reporting case . Due to severity of enlargement, gingival tissue covers almost entire crown causing esthetic and masticatory difficulties . After treating hyperplastic tissue, position of central incisor and upper right canine the most preferred method for excision is external bevel gingivectomy especially when there is no bone loss and only false pockets . However, in several reported cases there was no recurrence in a period of 2 years, 3 years, or even a 14-year follow - up . In the present case the clinical case report of a rare lesion of hereditary gingival fibromatosis presented here was managed successfully with surgical interventions with no recurrence . Gingival enlargement at this young age should be managed early to avoid pathological migration, malocclusion, esthetic, and functional complications.
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Lynch syndrome (ls) is an autosomal - dominant genetic predisposition to cancer, accounting for about 1%-5% of colorectal cancer (crc). Ls is caused by an inactivating germline mutation in a mismatch repair (mmr) gene, including mlh1, msh2, msh6, pms2, and epcam . Ls entails a lifetime crc risk of 60%-80% and displays distinct clinical phenotypes, such as early age of cancer onset, predominance of proximal crc, excessive synchronous and metachronous tumors, and various extra - colonic tumors of the endometrium, ovary, stomach, small bowel, pancreas, biliary tree, brain, and urothelium . The amsterdam criteria for a diagnosis of ls were developed by the international collaborative group on hereditary non - polyposis colorectal cancer (icg - hnpcc) in 1991, and were modified in 1999 because they included limited extra - colonic tumors and were relatively insensitive to germline mmr mutations . The bethesda criteria, which are more sensitive, are used together with the patient s microsatellite instability (msi) status to identify those who qualify for mmr mutation analysis . Both clinical diagnosis based on the amsterdam criteria and genetic diagnosis based on an mmr mutation analysis identify ls effectively . Since the establishment of the first hereditary colorectal tumor registry in 1925 at st mark s hospital, london, uk, the registration and systematic care of individuals with hereditary colorectal tumors has increased globally and been extended to families with familial adenomatous polyposis, ls, peutz - jeghers syndrome, and various rare hereditary gastrointestinal cancer syndromes . Well - organized registries have improved the effective management of patients with hereditary crc and their relatives . Furthermore, registry - based screening reduces the incidence and mortality of crc considerably in western countries [7 - 11]. However, such large - scale population - based screening and the early control of hereditary cancer can be maintained only by the activity of a national registry rather than single institutional or regional registries . The importance of central organization and ongoing funding in the management of hereditary cancer syndromes is well recognized . In contrast, there is no report on the performance of hereditary cancer registry in asian populations . The korean hereditary tumor registry (khtr) the khtr performs many important functions, including the registration of new patients with hereditary tumors, mutational screening of the genes responsible for specific hereditary tumors, and surveillance of at - risk relatives with presymptomatic genetic diagnoses . The khtr screens the genes responsible for hereditary tumors in various organs, including the colon, rectum, breast, ovary, stomach, eye, brain, bone, adrenal gland, and kidney . The khtr contains information on 186 different families affected by ls or suspected of ls, 98 different families affected by familial adenomatous polyposis, and many families with various other hereditary tumors, and it is currently one of the largest hereditary tumor registries in korea . This study evaluated the changes in various clinical features of ls patients in the registry in terms of the early detection of crc and the appropriate management of these patients over the past two decades, since the establishment of the khtr . Patients fulfilling the amsterdam ii criteria proposed by the icg - hnpcc were selected by pedigree review from 567 individuals in 186 different families affected with ls or suspected ls patients registered in the khtr . Patients not fulfilling the criteria, but whose genetic analysis confirmed a germline mutation in an mmr gene were also included . In addition to pedigree analyses, msi testing is routinely performed in clinical practice for all crc patients in major referring hospitals associated with our registry . Five microsatellite markers (bat-25, bat-26, d2s123, d5s346, and d17s250) are used to analyze paired normal and tumor dnas for msi . Tumors were classified as msi - high when at least two of the five markers displayed novel bands, msi - low when additional alleles were found with one of the five markers, and stable microsatellite when all microsatellite markers examined displayed identical patterns in both tumor and normal tissues . Genetic counseling is offered to patients with crc showing msi, and genetic testing is provided with the consent of the patient . Patients with crc showing msi are also tested for ls based on the results of genetic testing . The clinicopathologic data for all patients in the present study were available, including their demographic data, informative pedigree, details of treatment, and pathological stage of crc . The study patients are divided into two period groups: the period 1 group included patients diagnosed with ls in 1990 - 2004 and the period 2 group included patients diagnosed with ls in the past decade, 2005 - 2014 . The clinical characteristics of the two groups were compared to identify any changes that have occurred since the foundation of the khtr . The type of surgery used was classified into extended resection (subtotal colectomy, total colectomy, or restorative proctocolectomy with ileal pouch anal anastomosis) and segmental resection (right hemicolectomy, transverse colectomy, left hemicolectomy, anterior resection, low anterior resection, ultralow anterior resection, hartmann s operation, or abdominoperineal resection). The extent of the disease at diagnosis was classified by tnm stage (the seventh edition of the american joint committee on cancer staging manual). A chi - squared test or fisher exact test (where appropriate) was used for the inference of proportions . Null hypotheses of no difference were rejected if p - values were less than 0.05 . Patients fulfilling the amsterdam ii criteria proposed by the icg - hnpcc were selected by pedigree review from 567 individuals in 186 different families affected with ls or suspected ls patients registered in the khtr . Patients not fulfilling the criteria, but whose genetic analysis confirmed a germline mutation in an mmr gene were also included . In addition to pedigree analyses, msi testing is routinely performed in clinical practice for all crc patients in major referring hospitals associated with our registry . Five microsatellite markers (bat-25, bat-26, d2s123, d5s346, and d17s250) are used to analyze paired normal and tumor dnas for msi . Tumors were classified as msi - high when at least two of the five markers displayed novel bands, msi - low when additional alleles were found with one of the five markers, and stable microsatellite when all microsatellite markers examined displayed identical patterns in both tumor and normal tissues . Genetic counseling is offered to patients with crc showing msi, and genetic testing is provided with the consent of the patient . Patients with crc showing msi are also tested for ls based on the results of genetic testing . The clinicopathologic data for all patients in the present study were available, including their demographic data, informative pedigree, details of treatment, and pathological stage of crc . The study patients are divided into two period groups: the period 1 group included patients diagnosed with ls in 1990 - 2004 and the period 2 group included patients diagnosed with ls in the past decade, 2005 - 2014 . The clinical characteristics of the two groups were compared to identify any changes that have occurred since the foundation of the khtr . The type of surgery used was classified into extended resection (subtotal colectomy, total colectomy, or restorative proctocolectomy with ileal pouch anal anastomosis) and segmental resection (right hemicolectomy, transverse colectomy, left hemicolectomy, anterior resection, low anterior resection, ultralow anterior resection, hartmann s operation, or abdominoperineal resection). The extent of the disease at diagnosis was classified by tnm stage (the seventh edition of the american joint committee on cancer staging manual). A chi - squared test or fisher exact test (where appropriate) was used for the inference of proportions . Null hypotheses of no difference were rejected if p - values were less than 0.05 . One hundred and seventy - one ls patients fulfilling the amsterdam ii criteria for genetic testing during the study period were identified . Genetic testing for mmr mutations (in mlh1, msh2, pms2, or msh6) was performed in 141 of these 171 patients and germline mutations were identified in 60 (42%). Genetic testing confirmed mmr germline mutations in 11 of the 396 patients with no known family history, but who were close to the diagnostic clinical criteria, or had crc with msi - high . The distribution of germline mutations among mmr genes and mutation type (frameshift, missense) were not different between period groups . The period 1 group included 76 patients and the period 2 group included 106 patients . The mean age at diagnosis was 45.1 years (range, 13 to 85 years) for period 1 and 49.7 years (range, 20 to 84 years) for period 2 (p=0.015). The most common tumor site was the rectum (n=21, 27.6%) in the period 1 group and the sigmoid colon (n=33, 31.1%) in the period 2 group . However, the predominance of a proximal location was the same in the two groups . The extent of disease at diagnosis in the ls patients did not differ significantly over time (p=0.186) (table 1). Extended resection was performed in 55 patients (72.4%) and segmental resection in 21 patients (27.6%) of the period 1 group, and extended resection in 49 patients (46.2%) and segmental resection in 57 (53.8%) of the period 2 group (p=0.003) (table 2). This study shows that laparoscopic surgery is widely used for ls crc; the penetrance of laparoscopy in the period 2 group was 31.3% . Segmental resection was more frequently performed in patients registered in the khtr in the past decade, in patients with rectal cancer, or in older patients whose age at diagnosis was over 60 (table 3). Crc is one of the most common causes of cancer - related death in korea and its incidence has increased steeply over the past 25 years . Because crc displays familial clustering in up to 20%-30% of all cases, this increase makes it even more important to recognize hereditary crc syndrome in korea . Detecting hereditary crc not only allows the provision of the appropriate management to patients with hereditary crc, but also the identification of high - risk individuals among their family members . Offering them standard cancer surveillance can prevent advanced hereditary - crc - syndrome associated malignancies in affected familial members . Only a well - organized registry can accomplish these activities for the families affected by hereditary crc syndrome . Registry - based screening for ls is crucial, and over the past two decades nationwide or regional registries have been established in finland, germany, canada, denmark, and the netherlands . The danish hnpcc registry supervises the central registration of data on all families with hereditary crc, and reports that because of screening, ls patients with stage iii crc have a better overall survival rate than those with sporadic crc . The mortality of crc has been reduced] by a large - scale surveillance program for ls in the netherlands . A recent systematic review reported that registry - based screening is essential for reducing the incidence and mortality of crc in patients with ls, and the authors highlighted the importance of funding and managerial support for hereditary crc registries . The accumulation of extensive data on patients with hereditary cancer makes it possible to conduct large - scale research, providing medical evidence that can greatly improve clinical practice . In several countries, including finland (finnish hereditary colorectal cancer registry), germany (german hnpcc consortium), canada (hereditary cancer registry), and the netherlands (dutch cancer registry), the number of registered ls families with a known mutation exceeds 2,000 together with about 10,000 individuals who are confirmed mutation carriers . However, no reduction in the extent of the disease at diagnosis has been observed in the crc patients registered at the khtr since its foundation . The proportion of patients with advanced (regional or distant) disease at diagnosis in the period 2 group was still substantial (38.7% in the period 2 group vs. 27.6% in period 1, respectively), although the difference was not significant . The registry has been used in the management of hereditary colon cancer in collaboration with many cancer clinics over the past two decades . However, this study shows that the khtr, an institution - based registry, has not improved the overall mortality or early detection of ls patients . As an institution - based registry, it is impossible for the khtr to perform nationwide registration and systematic management for ls patients . To reduce the incidence and mortality of crc in patients with hereditary crc, including familial adenomatous polyposis and ls, registration and screening must extend beyond the regional registry level or a single institutional registry . Patients with ls should be notified of a regional genetics registry and managed in the context of that registry . As regional registries are extended, not only can patients with newly detected ls - associated cancer be provided with genetic counseling and testing at their regional or institutional registry, their at - risk relatives can be invited to genetic counseling and risk assessment, predictive testing, and appropriate screening at their own regional registry . It may also be easier to raise public awareness of hereditary crc throughout the country . It is highly significant that only about one third of ls patients develop the disease de novo, rather than inheriting the pathogenic mmr mutation from their parents . Despite this, most patients with hereditary crc are still managed on an individual basis in korea . Extended resection, such as subtotal colectomy, is generally favored over segmental resection in the curative surgery for ls patients with crc because segmental resection entails a greater risk of synchronous and metachronous crc . A retrospective study of ls patients with rectal cancer who underwent segmental resection found that the cumulative risk of metachronous colon cancer was 19% at 10 years, 47% at 20 years, and 69% at 30 years after surgical resection . Recent guidelines limit the use of segmental resection only to those patients for whom total colectomy is unsuitable, and only if regular postoperative surveillance is conducted . Rodriguez - bigas and moeslein suggested that treatment must be individualized for each patient because there has been no prospective or randomized control study suggesting that extended resection confers a survival benefit compared with segmental resection . Another study suggested that less extensive surgery should be considered for elderly patients, because the increase in life expectancy achieved with total colectomy rather than segmental resection in ls patients aged 67 years was only 0.3 years . For rectal cancer in ls the risk of metachronous colon cancer was reported as 15%-54% after segmental resection of rectal cancer, although quality of life concerns and defecation problems are substantial . The present study indicates that segmental resection is more frequently performed in patients with rectal cancer, or in older patients whose age at diagnosis was over 60 . Although not statistically significant, it is interesting that seven of 11 patients (63.6%) diagnosed with ls by genetic testing underwent segmental resection (table 3). The difficulties inherent in preoperative genetic testing in daily clinical practice may affect its contribution to surgical decision making . This study shows that increasing numbers of surgical procedures are currently performed with laparoscopy . As evidence of the safety and feasibility of the laparoscopic approach in crc surgery increases, expert opinion on the surgical management of crc in ls patients in conclusion, crc in patients with ls and registered at the khtr is still diagnosed at an advanced stage, more than two decades after the registry s establishment . This indicates that the impact of a single institutional registry on the screening and surveillance of hereditary tumors is limited . Segmental resection rather than extended resection has been performed more frequently in the past decade . A prompt nationwide effort to raise public awareness of hereditary crc and to increase the support for registries is required in korea.
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Wszyscy pacjenci mieli wykonane badania rtg i usg stawu skokowego do 7 dni od urazu . W badaniu rtg nie stwierdzono patologii u 129 pacjentw (63%); u 24 pacjentw (12%) stwierdzono zamanie awulsyjne kostki bocznej; u 36 pacjentw (17%) stwierdzono wysik w stawie skokowym grnym . W badaniu usg nie stwierdzono patologii u 19 pacjentw (9%); u 60 pacjentw (29%) stwierdzono zamanie awulsyjne kostki bocznej obejmujce przyczep wizada skokowo - strzakowego przedniego (atfl); u 34 pacjentw (17%) stwierdzono cakowite zerwanie atfl; u 51 pacjentw (25%) stwierdzono czciowe uszkodzenia atfl; inne uszkodzenia stanowiy 19% . Wykonane zabiegi operacyjne rekonstrukcji wizada skokowo - strzakowego przedniego (19) potwierdziy rozpoznania usg / rtg w 100% . Zerwania awulsyjne atfl, czyli obejmujce przyczep wizada, wystpuj u dzieci modszych (mediana 8 lat). Zerwania cakowite atfl (na przebiegu wizada, nieobejmujce przyczepu) wystpuj u dzieci starszych (mediana 14 lat). Ze wzgldu na istotnie ograniczon warto badania rtg w rozpoznaniu patologii stawu skokowego w wieych urazach skrtnych dzieci badanie wykorzystujce obrazowanie tkanek mikkich ultrasonograa zamania awulsyjne obejmujce przyczep atfl, przewaajce u dzieci modszych, wynikaj z niezakoczonego procesu kostnienia i wymagaj pilnej diagnostyki oraz konsultacji ortopedycznej . Sprain injuries are diagnosed in 6% of young people who practice sports, and the highest risk is associated with the following sports: soccer, rugby, hockey, handball, volleyball, basketball and squash . Most ankle sprain injuries involve the region of the lateral malleolus and its ligament complex (the lateral ligament complex of the ankle joint) which consists of three ligaments: anterior talofibular ligament (atfl), calcaneofibular ligament (cfl) and posterior talofibular ligament (ptfl). Sprained ankle is a result of the supination movement, i.e. Plantar flexion and inversion of the foot . This structure is a strong ligament and it is an element of the lateral ligament complex of the ankle joint . It attaches to the distal end of the fibula; the center of the enthesis is localized approximately 10 mm above the apex of the lateral malleolus . The talar enthesis of the atfl is localized on the lateral surface of the talus bone (fig . When the foot is in the anatomic position, the ligament runs nearly horizontally, but in the plantar flexion, the axis of the ligament is nearly parallel to the long axis of the shin bone . An injury to the ligament can concern its fibers (mid - substance complete or partial ligament tear) or the enthesis . In the latter situation, the bony attachment site of the ligament is detached (avulsion fracture of the fibula). Injuries in sprained ankle are assessed by x - ray and ultrasound (us) examinations, both of which are commonly available . The assessment of the degree to which the anterior talofibular ligament is damaged and the evaluation of coexisting injuries are important when planning the treatment . Surgery is indicated, for instance, when the anterior talofibular ligament is torn completely and the patient manifests clinical sings of ankle joint instability or when the ligaments of the ankle joint are damaged with a coexisting osteochondral fracture (e.g. Avulsion fracture of the lateral malleolus with the dislocation of an osteochondral fragment in children). When untreated, sprain injury can lead to joint instability, early degenerative changes and chronic joint pain . The aim of this paper is to draw attention to the usefulness of the ultrasound examination in the diagnosis of recent sprain injuries of the ankle joint . The retrospective analysis involved 206 patients (113 girls and 93 boys) who reported to the trauma outpatient clinic with recent ankle joint sprain . The average age of the patients was 10.6 . All of them were instantly examined with x - ray and us (within 7 days of sustaining injury). X - ray examinations were conducted with the use of a philips digital diagnost v.2 system with eleva workspot v.2 software . Ultrasound examinations were conducted with the use of a ge voluson e8 system using linear probes sp10 - 16d with the frequency of 718 mhz and an 11 l - d probe with the frequency of 410 mhz . X - ray examinations of the ankle joint were conducted in three views: a - p, lateral and mortise view (used for the assessment of the width of the tibiofibular syndesmosis and for the optimization of the talocrural joint space imaging). X - ray pictures were assessed by four radiologists experienced in diagnosing conditions within the muscoskeletal system . Ultrasound images were assessed by five radiologists experienced in diagnosing conditions within the muscoskeletal system . The following signs were assessed in each us examination: the presence of fluid / hematoma in the ankle joint, the image of the synovial membrane and the presence of injuries to the ligaments of the ankle joint: anterior and posterior tibiofibular ligament, anterior talofibular ligament, calcaneofibular ligament, medial ligament, chopart's joint ligaments (talonavicular and bifurcate ligament), as well as tendons of the peroneus muscles, tibialis muscles, extensor and flexor digitorum longus as well as the region of the epiphyseal cartilage of the fibula and the tuberosity of the fifth metatarsal bone . Moreover, bone fragments that could be detached in the mechanism of avulsion were actively searched for . Grade 1 referred to ligament edema / thickening, which results from elastic deformation of its fibers without the loss of their continuity (commonly known as stretched grade 2 was defined as injury in which some fibers are torn (edema, blurred fibrillar echotexture, areas of decreased echogenicity). Finally, grade 3 referred to complete rupture of the ligament (edema and ligament thickening, no evidence of fiber continuity, failure of function in a dynamic examination: ligament stumps or torn fibers move away from each other). A hematoma at the site of the ligament can be present for several days after sustaining injury . Avulsion fracture, i.e. Injury involving the attachment site, is a specific type of grade 3 damage . The structure of the ligament itself is usually assessed as normal or as in grade i injuries . Based on the clinical assessment and imaging findings, 19 patients were scheduled for a surgery to repair the anterior talofibular ligament . X - ray failed to visualize a pathology in 129 children (63%) (fig . 2). In 24 patients (12%), avulsion fracture of the lateral malleolus 3), and in 36 cases (17%), the examinations showed evidence of effusion in the ankle joint or thickening of the soft tissues adjacent to the lateral malleolus (fig . 4 and 5). A slight detached bone fragment, separated from the fibular outline, can be seen x - ray picture of the right ankle joint in the ap view . Thickening of the soft tissues adjacent to the lateral malleolus (arrow) x - ray picture of the right ankle joint in the lateral view . Radiological evidence of effusion in the talocrural joint (arrow) a us examination failed to visualize a pathology in 19 children (9%) (fig . 6; fig . 7). In 60 patients (29%), avulsion fracture of the lateral malleolus involving the atfl enthesis was found (fig . In 34 patients (17%), the atfl was found completely torn (fig . 9), i.e. The ligament itself was torn (not involving the enthesis). Partial atfl injury was diagnosed in 51 patients (25%), and other injuries in 19% of cases (fig . The course of the ligament (arrows) and ligament attachment sites (arrowheads). Marked fibrillar structure; stretched in a resting position avulsion injury of the anterior talofibular ligament at the fibular enthesis . Bone entheses are marked with arrowheads ultrasound image of complete tear of the anterior talofibular ligament . Swollen ligament stumps (arrows) bridged by thin scars the surgeries (19) conducted confirmed the us / x - ray diagnoses in 100% of cases (fig . Ligament stumps (arrowheads) based on the data, it can be concluded that avulsion atfl injuries are usually found in younger children (median: 8 years of age) in whom the ligament entheses are partially chondral attachments . In most cases (over 60%), the result of an x - ray examination was normal no visible structural changes within the joint, which considerably contrasts with the number of normal ultrasound results (9%) (fig . 2 and 6). Effusion in the ankle joint or thickened shadow of the soft tissues at the ankle level (fig . Atfl injuries, both those of the ligament only and those involving the enthesis (avulsion fractures), accounted for 70% of cases . Some patients with these injuries were scheduled for a surgery . It must be observed that in 36 patients, avulsion fracture was occult in x - ray, which accounts for 60% of patients with this injury . It merely enables one to make conclusions concerning soft tissue injuries based on the presence of joint effusion or soft tissue thickening . The sensitivity and specificity of both modalities (x - ray and us) can be compared only with respect to avulsion injuries when a separated bone fragment is visible in an x - ray picture . X - ray us sensitivity 40%100% x - ray examinations helped establish the correct diagnosis in 40% of cases with avulsion injury whereas this value reached 100% for a us examination . The specificity of both methods in detecting avulsion fractures is as follows: x - ray us specificity 100%100% all patients without avulsion fracture were diagnosed correctly, i.e. They obtained a negative result both in an x - ray and us examination . Ultrasonography is a valuable method to assess avulsion fractures, tiny fractures due to compression injury as well as coexistent soft tissue injuries in the ankle joint . Ultrasonography, as an inexpensive, easily available and noninvasive method, can be broadly used particularly in pediatric radiology, and its findings can significantly influence the therapeutic process . The final decision concerning the treatment is made by an orthopedists on the basis of the whole clinical picture, current patient condition and own experience . However, it is not used routinely to assess recent sprain injuries of the ankle joint in children because of its limited availability and long duration of scanning . Ultrasonography can be useful in the initial assessment of whether patients need an mri examination . The literature reports demonstrate that us and mri are equally sensitive in detecting ligament injuries in the ankle joint . X - ray and us examinations are used for the assessment of sprain injuries of the ankle joint in children and should be used as complementary tests in the emergency department . A us examination of the ankle joint is characterized by greater sensitivity in assessing avulsion injuries . Ultrasonography should be therefore considered the primary modality for the assessment of ligament injuries in the ankle joint since its findings can change the therapeutic process . Authors do not report any financial or personal links with other persons and organizations, which might affect negatively the content of this publication and/or claim authorship rights to this publication.
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Facioscapulohumeral muscular dystrophy (fshd) is a common inherited muscular dystrophy presented clinically with slowly progressive weakness and wasting of facial and limb muscles and rare bulbar muscle involvement . We present herein a known case of fshd presented with recent onset of severe bulbar symptoms and was found to have myasthenia gravis (mg) based on electrodiagnostic study, elevated level of acetylcholine receptor antibody and dramatic improvement with choline esterase inhibitor agents . Clinical presentation, electrodiagnostic and pathologic findings of this patient are described . A 70-year - old man presented to our department with complaint of 15-day history of progressive difficulty in chewing and dysartheria . He had a 50-year history of slowly progressive asymmetrical weakness of proximal upper limb muscles . Examinations revealed reduction in forces of bilateral orbicularis oculi muscles, weakness and wasting of bilateral triceps muscles especially on right side and bilateral winging of scapula more prominent on right side . The legs and pelvic girdle muscles had normal forces . Edrophonium test was performed and dysartheria and chewing difficulty showed dramatic improvement but had no effect on limb weakness . Muscle biopsy showed myopathic changes with invariability in muscle fiber size, intramuscular infiltration of chronic inflammatory cells, mostly lymphocytes, few hyaline fibers and prominent fat infiltration . Our patient's bulbar symptoms showed dramatic improvement following administration of choline - esterase inhibitor agents . A 70-year - old man presented to our department with complaint of 15-day history of progressive difficulty in chewing and dysartheria . He had a 50-year history of slowly progressive asymmetrical weakness of proximal upper limb muscles . Examinations revealed reduction in forces of bilateral orbicularis oculi muscles, weakness and wasting of bilateral triceps muscles especially on right side and bilateral winging of scapula more prominent on right side . The legs and pelvic girdle muscles had normal forces . Edrophonium test was performed and dysartheria and chewing difficulty showed dramatic improvement but had no effect on limb weakness . Muscle biopsy showed myopathic changes with invariability in muscle fiber size, intramuscular infiltration of chronic inflammatory cells, mostly lymphocytes, few hyaline fibers and prominent fat infiltration . Our patient's bulbar symptoms showed dramatic improvement following administration of choline - esterase inhibitor agents . It presents clinically with slowly progressive weakness and wasting of facial and shoulder girdle muscles and sometimes involvement of lower extremities . The clinical severity is wide ranging from asymptomatic individuals to wheel - chair dependent patients . Any unusual changes in course of disease or development of unusual symptoms should raise the possibility of concomitant disease . Sansone et al . Reported a 69-year - old known case of fshd who presented with sudden deterioration of limb weakness and development of bulbar symptoms and was found to have mg based on repetitive nerve stimulation, elevated level of acetylcholine receptor - binding antibody and dramatic improvement following immunomodulator administration . Reported a 50-year - old man with a 35 year history of fshd who presented with acute progressive weakness of lower extremities three weeks prior to admission . The patient was found to have mg based on decrement response on repetitive nerve stimulation, elevated level of acetylcholine receptor - binding antibody and improvement after thymectomy and administration of corticosteroid . In another report, mcgonigal et al . Presented a 56-year old newly diagnosed myasthenic patient who found to have 40-year history of progressive foot drop and although our case is a rare coexistence of fshd and mg, low prevalence of both diseases, may raise the possibility of the presence of other etiologies . Theoretically, it is related to breaking of immune tolerance to achrs as a result of muscle fiber degeneration . Patients with genetic myopathies may occasionally develop antibodies to achr . While these antibodies may not have pathogenic effects, their production is likely to be a consequence of sensitization to achr secondary to muscle fiber damage, rather than through an immune process in thymus . Muscle histopathological examination in some cases of fshd shows inflammatory changes, disproportionate to muscle fiber necrosis but the presence of mononuclear infiltration does not affect disease progression and the patients do not benefit from prednisone treatment . To the best of our knowledge, there are limited reports of concomitant occurrence of mg and fshd in literatures . Although the association of mg with fshd in our patient could be an incidental finding, it may raise the possibility of innate immune response, i.e. Autoinflammation in development of achr antibodies in genetic myopathies or it may suggest immune mechanisms in pathogenesis of fshd . Moreover, our experience may warrant concomitant neuromuscular disorder in patients with unusual symptoms of fshd.
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Technetium-99 m (tc-99 m) in complex with methylene - diphosphonate (mdp) is a commonly used substrate in radionuclide bone imaging . Bone scans utilizing this radiotracer are very sensitive and enable nuclear medicine physicians to screen for various osseous abnormalities and pathologies . Localization of the radiotracer is accomplished through the exchange of tc-99 m mdp for phosphate compounds during active bone remodeling, and aggregation is subsequently detected using a gamma camera . Even though the utilization of tc-99 m mdp bone scintigraphy is useful for identifying various physiologic abnormalities, its overall specificity is limited primarily by the lack of anatomic detail . It is therefore important to utilize the patient's medical history, in conjunction with other forms of imaging, to aid in differentiating potential pathologies . Here, we present a case in which tc-99 m mdp bone scintigraphy was utilized to raise concern for a serious abnormality despite relatively occult findings by conventional imaging modalities . A 70-year - old male presented with mild left hip pain and was found to have an elevated serum alkaline phosphatase (alk) level of 770 units / l . Unfortunately, due to a language barrier, a complete medical history could not be obtained . In light of the patient's elevated alk level, the planar whole body images [figure 1a] of the tc-99 m mdp bone scan demonstrated intense diffuse uptake extending from the left femoral head to the proximal shaft of the femur . Initially, we felt this type of appearance could be compatible with early paget's disease given the markedly elevated alk, despite the absence of apparent structural deformities . 70-year - old male with mild left hip pain and elevated alkaline phosphatase diagnosed with radiogenic osteosarcoma . (a) the planar whole body images of the technetium-99 m methylene diphosphonate bone scan demonstrates intense diffuse uptake extending from the left femoral head to the proximal shaft of the femur (double ended arrow). (b) the plain film of the left hip depicts three previously unreported fixation screws surrounded by osteopenic lesions (arrow) in the femoral neck and head . To confirm the initial diagnosis, the patient was sent directly for an x - ray [figure 1b]. However, the pattern of multifocal sclerosis (cotton wool appearance) that would be expected of paget's disease was not present . Instead, the radiograph demonstrated three previously unreported fixation screws, used to repair a femoral neck fracture (later discovered to have occurred 1 year ago). Surrounding the screws were poorly defined osteopenic, potentially lytic lesions in the femoral neck and femoral head [figure 1b]. Correlation of the tc-99 m mdp bone scan with the x - ray changed the initial diagnosis from suspected paget's disease to a differential of malignancy / metastasis, reparative phase of osteonecrosis, osteomyelitis, or reactive osteolysis to the fixation screws . Though simple loosening of the fixation screws could have explained the patient's mild hip pain, the activity demonstrated by the bone scan was much too diffuse and intense to support this diagnosis . To evaluate for metastatic disease, a computed tomography scan of the chest, abdomen, and pelvis was ordered the following day . Evaluation of the scan of the left hip was limited due to streak artifact from the screws; however, it was still possible to appreciate some mixed lytic / sclerotic changes in the native bone [figure 2a and b]. Even though these changes raised the suspicion of osseous malignancy or metastasis, they were not pathognomonic, as hardware loosening, osteopenia, and degenerative changes could have a similar appearance . However these findings in combination with the bone scan findings, provided enough suspicion of a pathology sinister enough to refer the patient for surgical hardware revision with potential biopsy . 70-year - old male with mild left hip pain and elevated alkaline phosphatase diagnosed with radiogenic osteosarcoma . (a and b) computed tomography of the pelvis reveal mixed lytic / sclerotic lesions (arrows) within the native bone, although evaluation of the left hip was limited due to streak artifact as seen on the two axial slices in figure 1 . Surgical exploration of the left hip revealed a grossly abnormal appearance of the left proximal femur with areas of hemorrhagic bone surrounding lytic lesions . Histologic study of the biopsy [figure 3] demonstrated sarcomatous, spindle shaped cells with adjacent tumor osteoid production; represented by eosinophilic, amorphous, fibrillary deposits between individual or small aggregates of tumor cells . 70-year - old male with mild left hip pain and elevated alkaline phosphatase diagnosed with radiogenic osteosarcoma . Histologic examination of the curettage (using hematoxylin and eosin stain, 10) reveals sarcomatous, spindle shaped cells (white arrows) with adjacent tumor osteoid production (black arrows); represented by eosinophilic, amorphous, fibrillary deposits (white arrows) between individual or small aggregates of tumor cells (black arrows). In context, the presence of osteosarcoma was highly unlikely as there have been no case reports of primary osteosarcomas occurring in a site of a fracture that was surgically repaired 1 year prior . Additionally, the distal femur would be a far more likely site for a primary osteogenic sarcoma to occur . However, upon further interrogation of the patient's past medical history, it was discovered that the patient had prostate cancer 11 years ago . Given the standard of care at that time, one could surmise that he was treated with 60 - 70 gy of targeted external beam radiation to the prostate . The dose that he received and the time frame since receiving his radiation would be compatible with radiogenic osteosarcoma as there is a latency period of about 10 years . The repaired stress fracture 1 year prior was potentially pathologic from the malignancy, but was most likely missed due to the slightly atypical radiographic appearance on the plain film x - ray performed in the surgeon's office . Tc-99 m mdp bone scans are very sensitive tests for detecting a wide array of abnormalities within the bone . Despite its comprehensive applicability, it is limited in identifying specific diseases and pathologies; often relying on the addition of other investigative modalities to reach a final diagnosis . Here, a tc-99 m mdp bone scan was used to identify the presence of abnormal bone remodeling, which led to further imaging work - up and confirmatory biopsy . Osteosarcomas are particularly sensitive to tc-99 m mdp bone scans as their pathology involves active bone remodeling . However, its presence here was unexpected due to the atypical location on the bone scan and the limited available medical history (due to a language barrier). When paired with the patient's medical history of radiation treatment and recent fracture, the femur would have been exposed to scatter and bremsstrahlung radiation from its proximity to the treatment field . Additionally, the 10-year latency period to developing malignancy is typical for post - external beam radiation therapy induced malignancies . The tc-99 m mdp bone scan demonstrated its value by raising the suspicion for a more sinister process in the left hip than was seen in radiographic appearance on plain film . This case demonstrated the value of tc-99 m mdp bone scintigraphy as an initial detector of bone abnormalities . Its sensitivity prevents it from identifying specific pathologies without the aid other investigative methods as depicted in this case . This case also underscores the importance of considering the bone scan findings in conjunction with the patient's clinical history and anatomic imaging . By using all this information in combination, we were able to sift through the differential and refer the patient for appropriate management.
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The success of endodontic treatment is mainly dependent on thorough cleaning, shaping and disinfection of root canal system . The irrigation protocol thereby plays a key role in the disinfection of the root canal space . A clean root canal system along with a three dimensional seal is the clinician's road to success . Subsequent to biomechanical preparation, an amorphous irregular layer known as the smear layer is formed on the root canal walls . It contains inorganic and organic substances that include fragments of odontoblastic process, microorganisms and necrotic debris . Its presence increases microflora and the inorganic toxins, decreases the sealing ability and increases the potential for bacterial survival and reproduction . Until date, no single irrigant has been capable of demonstrating both tissue dissolving as well as demineralizing properties . Current methods to remove the smear layer might involve the use of a chelating agent during irrigation or as a final rinse in combination with other irrigants having tissue dissolving properties . Sodium hypochlorite (naocl) is the main endodontic irrigant used to dissolve the organic portion of the smear layer . To remove the inorganic portion of the smear layer, a decalcifying agent is used, which can be either a chelator or an acid currently, all the products in the dental market sold to dissolve smear layer are based on ethylenediaminetetraacetic acid (edta) or citric acid . Therefore, it has been proposed to use peracetic acid (paa) instead of these classical decalcifying agents to dissolve the smear layer and also to disinfect the root canal system . Apart from edta and paa, a newly introduced qmix solution is also recommended chelator which is used in conjunction with naocl . Recent research indicates that qmix (dentsply tulsa dental, tulsa, ok), an experimental irrigant containing a mixture of a bisbiguanide antimicrobial agent, a polyaminocarboxylic acid calcium - chelating agent and a surfactant, might be as effective as edta at removing smear layers when used after an initial rinse with naocl . It is well - known that some chemicals used for endodontic irrigation are capable of causing alterations in the chemical composition of dentin . Any change in the ca: p ratio may in turn change the microhardness, permeability and solubility characteristics of dentin and may also adversely affect the sealing ability and adhesion of dental materials . No study until date has been done to compare the effect of paa, qmix and edta on calcium loss and microhardness of root dentin . Therefore, the present study has been undertaken to evaluate the effect of different irrigation regimens on calcium loss and its effect on the micro - hardness of the root dentin . Ten intact, single rooted human premolars, which were extracted for orthodontic reasons were used for this study . Teeth were stored for 1 week in formalin and then in normal saline until use . The teeth were decoronated at the cementoenamel junction using a high speed carbide bur under copious water irrigation . Thick transverse sections of 2 mm with a maximum and minimum width of 3 mm and 2 mm respectively were obtained from the coronal third of each root using a low - speed safe sided diamond disc . Each section was further divided into 4 quarters, each part constituting a sample specimen from the same tooth for each group . Specimens were horizontally embedded in autopolymerizing resin so as to expose the canal part of the dentin . The specimens were ground flat on a circular wet grinding machine with ascending grades of sic abrasive papers (320, 600, 1000, 1200 and 1500 grit) under constant water irrigation using leco grinder polisher . 17% edta was prepared by adding 17 g of disodium salt of edta powder into 100 ml of distilled water and favoring its dissolution by the addition of 5 n sodium hydroxide . 2.25% paa was prepared by dissolving equal volume of hydrogen peroxide (26%), acetic acid and acetylhydroxyperoxide in equilibrium . Nearly 5% naocl solution and qmix (dentsply, tulsa dental, ok, usa) were obtained as commercial preparations . The treatment groups were as follows: group 1 (control): 5% naocl for 5 min - distilled water for 5 mingroup 2: 5% naocl for 5 min-17% edta for 5 mingroup 3: 5% naocl for 5 min-2.25% paa for 5 mingroup 4: 5% naocl for 5 min - qmix for 5 min . Group 1 (control): 5% naocl for 5 min - distilled water for 5 min group 2: 5% naocl for 5 min-17% edta for 5 min group 3: 5% naocl for 5 min-2.25% paa for 5 min group 4: 5% naocl for 5 min - qmix for 5 min . All the specimens were immersed in a magnetic stirrer bath containing 10 ml of the first test solution for 5 min . They were then immersed into 10 ml of the second test solution of the respective group for another 5 min . Each time after irrigation of one specimen per group, the eluates were centrifuged at 4000 g for 10 min . Subsequently, 10 ml of the supernatant was transferred to a polypropylene tube with a lid and stored at 20c until further analysis . Once all the eluates had been collected, they were thawed and then analyzed for their calcium content using an atomic absorption spectrophotometer (lab india, india) with an air acetylene flame . For each specimen after the combined treatment, surface hardness of the root dentin was measured with a vickers hardness tester (hmv, shimadzu, japan). Hardness was measured under the load of 300 g with duration of 15 s. in each sample, three indentations were made . The representative hardness value for each sample was obtained as the average of the three indentation values . Mean values were compared among different study groups by using one - way anova followed by post hoc tukey test . Pearson's correlation was done to compare the relation between calcium loss and subsequent microhardness of root dentin . 17% edta was prepared by adding 17 g of disodium salt of edta powder into 100 ml of distilled water and favoring its dissolution by the addition of 5 n sodium hydroxide . 2.25% paa was prepared by dissolving equal volume of hydrogen peroxide (26%), acetic acid and acetylhydroxyperoxide in equilibrium . Nearly 5% naocl solution and qmix (dentsply, tulsa dental, ok, usa) were obtained as commercial preparations . The treatment groups were as follows: group 1 (control): 5% naocl for 5 min - distilled water for 5 mingroup 2: 5% naocl for 5 min-17% edta for 5 mingroup 3: 5% naocl for 5 min-2.25% paa for 5 mingroup 4: 5% naocl for 5 min - qmix for 5 min . Group 1 (control): 5% naocl for 5 min - distilled water for 5 min group 2: 5% naocl for 5 min-17% edta for 5 min group 3: 5% naocl for 5 min-2.25% paa for 5 min group 4: 5% naocl for 5 min - qmix for 5 min . All the specimens were immersed in a magnetic stirrer bath containing 10 ml of the first test solution for 5 min . They were then immersed into 10 ml of the second test solution of the respective group for another 5 min . Each time after irrigation of one specimen per group, the eluates were centrifuged at 4000 g for 10 min . Subsequently, 10 ml of the supernatant was transferred to a polypropylene tube with a lid and stored at 20c until further analysis . Once all the eluates had been collected, they were thawed and then analyzed for their calcium content using an atomic absorption spectrophotometer (lab india, india) with an air acetylene flame . For each specimen after the combined treatment, surface hardness of the root dentin was measured with a vickers hardness tester (hmv, shimadzu, japan). Hardness was measured under the load of 300 g with duration of 15 s. in each sample, three indentations were made . The representative hardness value for each sample was obtained as the average of the three indentation values . Mean values were compared among different study groups by using one - way anova followed by post hoc tukey test . Pearson's correlation was done to compare the relation between calcium loss and subsequent microhardness of root dentin . The mean calcium loss and dentin microhardness and its standard deviation along with intergroup comparison are given in table 1 . Intergroup comparison for calcium loss and microhardness mean calcium loss in naocl + distilled water group (2.79 0.97 ppm) and naocl + qmix group (5.72 0.91 ppm) are least followed by naocl + edta group (6.38 1.76) and maximum in naocl + paa group (9.00 0.55 ppm). There was a statistically significant difference between all groups except between naocl + qmix group and naocl + edta group (f = 50.215, df-3, 36; p <0.001). The mean dentin microhardness in naocl + distilled water group (77.39 2.16 vickers hardness number [vhn]) and naocl + qmix group (70.68 4.97 vhn) were maximum followed by naocl + edta group (69.70 4.14) and least in naocl + paa group (62.98 8.17 vkn). There was statistical significant difference between all the groups except between naocl + qmix group and naocl + edta group (f = 12.26, df-3, 36; p <0.001). A negative correlation existed between the calcium loss and reduction in the microhardness of root dentin . Dentin is composed of inorganic components of hard dental tissues, in which calcium and phosphorus are distributed in the form of hydroxyapatite crystals . The ca / p ratio in hydroxyapatite is approximately 1.67 and it depends on many factors such as level of mineralization, type of crystals, age of tissue and anatomical site . During biomechanical preparation, removal of the smear layer requires the use of irrigants that can dissolve both the organic and inorganic components . Since chelating agents act only on the inorganic component of the smear layer, therefore they were used in combination with naocl which acts on its organic component altering the mechanical and chemical properties of root dentin . However, reports have indicated that the use of edta and naocl may lead to dentinal erosion of the root canal walls . Further, it has been reported that surface treatment of dentin with different agents may cause alterations in the chemical and structural composition of dentin, which in turn may change its permeability and solubility characteristics and subsequently a loss of ca / p ratio of root dentin resulting in an impact on the microhardness . In the design of this study, the issue of biological variability among different teeth was addressed by comparing the effects of different solutions on the dentin sections from the same patient . This allowed the comparison of the demineralizing capacity of different irrigating solutions on identical sections having similar degree of calcification geometry and surface area . Also, coronal parts of the roots were used to prepare the dentin specimens because of the higher possibility of sclerotic dentin in apical root . The exposure time of all the irrigants was kept 5 min and was standardized for all the groups . Atomic absorption spectroscopy is the technique used in this study to evaluate the demineralization effect of the chelators used and to determine the concentration of calcium in each sample . It is a single element technique which is less cost - effective than newer multi - element, techniques such as inductively coupled plasma atomic emission spectrometry . In the present study, the vickers microhardness test was done to evaluate the surface changes of dental hard tissue specimens, treated with the chemical agents . Further microhardness determination can provide indirect evidence of mineral loss or gain in the dental hard tissues . A significant negative correlation between loss of calcium from root dentin and microhardness panighi and gsell found a simple linear correlation between hardness of dentin and calcium ion concentration and between hardness of dentin and wettability . In this study, irrigation with 5% naocl followed by distilled water as a final rinse eluted minimum amount of calcium from the dentin (2.7 ppm mean) and when compared with other groups, the difference was statistically significant . (2009) where naocl and distilled water hardly eluted any ca . In the control group, no chelating agent was used but still some calcium loss was seen as a result of its mechanical flushing action on smear layer formed on root dentin . Since chelating agents cause demineralization of dentin, resulting in its softening, we find that more ca loss was seen in groups having chelating agents . This might be the reason why all experimental groups showed significantly lower microhardness when compared with the control group . When the mean ca loss of group 3 (5% naocl - paa) was compared with group 2 (5% naocl + edta) and group 4 (5% naocl - qmix), the result showed that group 3 extracted significantly more ca ions . More ca loss shown by paa can be explained by the fact that because of its acidity, the calcium stays in solution and does not reprecipitate . Despite the weak chelating power of this agent, more amounts of ca ions were eluted from the root canal as compared to edta, which is a much stronger chelator but can only be in solution at a slightly alkaline ph . However, in our study the ph used was neutral and thus edta could not remain precipitated in the solution . (2009) also found that edta removed slightly more smear layer than paa (p <0.05). More ca loss shown by paa could have resulted in a significant reduction in microhardness when compared with edta and qmix group . An insignificant difference in ca loss and microhardness was seen between 5% naocl - edta and 5% naocl - qmix groups . As qmix contains edta in its composition along with chlorhexidine and a detergent, the effect of qmix on root dentin could have been almost similar to edta . A study carried out by dai et al . (2011) showed that the smear layer removing ability of qmix was comparable to that of 17% edta . From the present study, it can be concluded that: irrigation with 5% naocl + 2.25% paa caused the maximum calcium loss from root dentin and minimum microhardness.irrigation with 5% naocl + distilled water caused minimum calcium loss from root dentin and maximum microhardness.a reduction in the microhardness of root dentin was observed with increase in calcium loss from root dentin . Irrigation with 5% naocl + 2.25% paa caused the maximum calcium loss from root dentin and minimum microhardness . Irrigation with 5% naocl + distilled water caused minimum calcium loss from root dentin and maximum microhardness . A reduction in the microhardness of root dentin was observed with increase in calcium loss from root dentin.
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It is estimated to increase the prevalence rate of cancer about 45% by 2025 in developed countries . Iran is one of developing countries, in which recently occurred the significant population growth and economic and social status changes . Nearly, 38% of these cancer cases in both sexes were due to gastrointestinal tract, of which 65,000 cases suffered from esophagus cancer . Of yearly 35,000 death from cancer in iran, 5800 cases were related to this cancer . Esophagus cancer rarely occurs in western countries, but it is considered one of the eight most common cancers in the world . The highest incidence rate, 100180/100,000, was observed in the north of iran and north of china, while in the usa the rate was <5/100,000 . The lack of early symptoms and the strong bilateral lymph flow of esophagus make the diagnosis to take place in advanced stages . Both histological types of esophagus cancer (squamous cell carcinoma [scc] and adenocarcinoma [adc]) are very fatal, with 5 years survival <10% . In the past,, adc has increased, and recently constitutes 60% of the cancer cases . In developing countries, such as iran and china, recent studies in iran showed that the prevalence of adenocarcinoma (ads) is increasing . This may be the improvement of public health, and epidemic of obesity and gastro - intestinal reflex diseases . Environmental factors, including western lifestyles, are effective in the incidence and pathology of esophagus cancer, especially in developing countries such as iran . The lack of a comprehensive and population - based study on the incidence, pathological, and clinical aspects of esophagus cancer, this study investigated the epidemiological trend and pathological changes in esophagus cancer in iran . This secondary data analysis study was carried out based on longitudinal program in iran that have national registry of cancer (ncr) which is trying to identify all cases of cancer occurring in iran . Data used in this study were obtained from ncr, and disease control and prevention of ministry of health and medical education in iran for 20032008 . More details about cancer registry in iran were previously published in the international journal of preventive medicine . In this study, data on the incidence of esophageal cancer were selected according to the international classification of diseases - oncology with the code c15 for age groups and sex . Histological data, like squamous cell carcinoma nos, scc keratinizing, scc large cell nonkeratinizing, adenocarcinoma nos, carcinoma nos, also was extracted by sex and years of the study for the cancer . We calculated crude incidence rate and the age - standardized incidence rate (asir)/100,000 persons . To describe incidence time trends for 6 years studied, we carried out joinpoint regression analysis using the software joinpoint regression program, version 4.1.1.1 october 2014 . As well to evaluate the histological changes, were obtained the percentage allocated for kind of histological types . So to analysis histology change percentage trends for 6 years, we used the software joinpoint regression program and carried out joinpoint regression analysis for data analysis . The test of significance uses a monte carlo permutation method (i.e., it finds the best fit line). Joinpoint regression analysis involves fitting a series of joined straight lines on a log scale to the trends . The final model selected was the most parsimonious of these, with the estimated annual percent change (apc) based on the trend within each segment . The terms significant increase or significant decrease signify that the slope of the trend was statistically significant (p <0.05). This secondary data analysis study was carried out based on longitudinal program in iran that have national registry of cancer (ncr) which is trying to identify all cases of cancer occurring in iran . Data used in this study were obtained from ncr, and disease control and prevention of ministry of health and medical education in iran for 20032008 . More details about cancer registry in iran were previously published in the international journal of preventive medicine . In this study, data on the incidence of esophageal cancer were selected according to the international classification of diseases - oncology with the code c15 for age groups and sex . Histological data, like squamous cell carcinoma nos, scc keratinizing, scc large cell nonkeratinizing, adenocarcinoma nos, carcinoma nos, also was extracted by sex and years of the study for the cancer . We calculated crude incidence rate and the age - standardized incidence rate (asir)/100,000 persons . To describe incidence time trends for 6 years studied, we carried out joinpoint regression analysis using the software joinpoint regression program, version 4.1.1.1 october 2014 . As well to evaluate the histological changes, were obtained the percentage allocated for kind of histological types . So to analysis histology change percentage trends for 6 years, we used the software joinpoint regression program and carried out joinpoint regression analysis for data analysis . The test of significance uses a monte carlo permutation method (i.e., it finds the best fit line). Joinpoint regression analysis involves fitting a series of joined straight lines on a log scale to the trends . The final model selected was the most parsimonious of these, with the estimated annual percent change (apc) based on the trend within each segment . The terms significant increase or significant decrease signify that the slope of the trend was statistically significant (p <0.05). Of all cases, 45.72% (8311 cases) were females and 54.28% (9866 cases) males . The most common histological types related to squamous cell carcinoma nos and adenocarcinoma nos were 64.53% and 10.37%, respectively . Frequency, crude, and standardized incidence of esophagus cancer by sex, during the years 2003 - 2008 to evaluate the changes in the incidence of esophageal cancer, comparison of asir indicated an increasing trend . In other words, the standardized incidence rate increased from 4.93 to 7.77 for women and from 4.64 to 7.66 for men . Results of joinpoint analysis revealed that the trend of annual changes of incidence rate significantly increased in both sexes . The annual percentage changes (apc), the incidence rate was 7.9 (ci: 3.3 - 12.6) for women and 9.6 (ci: 6.0 - 13.2) for men [figure 1]. Joinpoint analysis for incidence of esophagus cancer (male and female) in iran, 20032008; age - standardized rate/100,000 (using world standard population) two type of histology (squamous cell carcinoma nos, and adenocarcinoma nos) included 87.3%and 86.56% of the histology of esophageal cancer in women and men, respectively . The frequency of five common histological types of esophageal cancer is shown in figure 2 . Distribution of histology esophageal cancer in iran the percentage allocated to histology types of esophageal cancer was not constant during years studied . The percentage allocated for histology of squamous cell carcinoma nos (scc), and scc large cell, and nonkeratinizing had a significant decreasing trend in both sexes (p <0.05). The type of adenocarcinoma nos increased in both sexes (p <0.05) [table 2]. Joinpoint analyses of cancer percentage allocated trend to the histology data for esophageal cancer in iran (2003 - 2008) to evaluate the changes in the incidence of esophageal cancer, comparison of asir indicated an increasing trend . In other words, the standardized incidence rate increased from 4.93 to 7.77 for women and from 4.64 to 7.66 for men . Results of joinpoint analysis revealed that the trend of annual changes of incidence rate significantly increased in both sexes . The annual percentage changes (apc), the incidence rate was 7.9 (ci: 3.3 - 12.6) for women and 9.6 (ci: 6.0 - 13.2) for men [figure 1]. Joinpoint analysis for incidence of esophagus cancer (male and female) in iran, 20032008; age - standardized rate/100,000 (using world standard population) two type of histology (squamous cell carcinoma nos, and adenocarcinoma nos) included 87.3%and 86.56% of the histology of esophageal cancer in women and men, respectively . The frequency of five common histological types of esophageal cancer is shown in figure 2 . Distribution of histology esophageal cancer in iran the percentage allocated to histology types of esophageal cancer was not constant during years studied . The percentage allocated for histology of squamous cell carcinoma nos (scc), and scc large cell, and nonkeratinizing had a significant decreasing trend in both sexes (p <0.05). The type of adenocarcinoma nos increased in both sexes (p <0.05) [table 2]. Joinpoint analyses of cancer percentage allocated trend to the histology data for esophageal cancer in iran (2003 - 2008) our study investigated the trend of incidence and pathological changes of esophagus cancer during 20032008 in iran . Our findings showed that there was an increasing trend of esophageal cancer over 6 years . The decreasing trend was quicker for scc, large cell, and nonkeratinizing than scc . Adc, nos had an increasing trend in both sexes . In this study, most cases were scc (64.53%) followed by the ad (10.37%). There was a significant decreasing trend for scc, while ad is significantly increased in both sexes . Ad incidence is enhancing in china . In developing countries, including iran and china, the prevalence of this type of cancer is decreasing in the world . Also scc type of esophagus cancer is declining, but adc is increasing in iran . Changes in the epidemiology of esophageal cancer in the world and iran occurred two to three decades after health promotion and cultural, economic and social development . Obesity and acid reflux from the stomach into the esophagus are possible causes of this cancer . In iran and other developing countries, lifestyle changes and westernization lead to become the incidence of cancer similar to the developed countries . The reported incidence rate of the cancer was 13.8/100,000 in men and 6.5/100,000 in women in developing countries . In developed countries, the rates were 6.5 and 1.3/100,000 in men and women, respectively . Our results indicated that the rates were 7.56 and 7.77/100,000 in men and women, respectively . . Age - standardized rate (asr) for women and men was 43.3 and -36.3/100,000, respectively, in the province of golestan . Asr was 15.4 and 14.4/100,000 in women and men, respectively, in the province of ardebil . In the province of semnan, the lowest in kerman province rate was 3 and 2.1/100,000 in women and men, respectively . The mentioned regions are the belt for esophageal cancer, especially the north east of iran . Unlike scc, with equal prevalence in men and women, male factor is considered an important risk factor for adc . According to globacan, our study revealed that the prevalence of ad was 2.64 times more in men than women . A number of case control studies in iran and india showed that drinking hot tea and coffee increases the risk of esophageal cancer . The results of case control studies conducted in golestan showed that drinking too hot has increased the risk of esophageal cancer about 10-fold . However, scientific evidences have not declared that regular consumption of vegetables and fresh fruits may reduce the risk of scc . A study performed in high - risk areas (such as golestan) in iran revealed that nitrosamine levels in the saliva of population were 4 times more than german population . There is a relationship between oral disease and tooth loss, and higher rates of mouth and stomach cancers . In china, latin america, eastern europe, and japan, similar to iran, inadequate oral hygiene was considered as a precursor of esophageal squamous dysplasia . Strong risk factor for ad may be obesity and the absence of helicobacter pylori infection . Smoking and acid reflux from the stomach into the esophagus increases the risk of ad ., this type of esophagus cancer dramatically increases in iran because of obesity and overweight epidemic associated with acid reflux from the stomach into the esophagus and reducing the amount of helicobacter pylori infection . Improving the cancer registration system may be one of the reasons of increasing the cancer . A study on the trend of skin cancer incidence performed in italy declared that advances in diagnostic techniques and the development of cancer registration lead to a large proportion of the increase in the incidence of skin cancer . According to the report of iranian ministry of health, the cancer registry in the country hence, in interpreting the results of studies regarding all cancers, the problems and of cancer registration should be considered . In other words, the share of the development of cancer registration and high proportion of the most difficult areas should be distinguished from each other . The lack of population - based data regarding the prevalence and mortality is one of the major problems in esophageal cancer . In some low - income countries, there are the primary barriers such sanctions and racial and cultural beliefs, except for the diagnosis and effective management of cancer . Researchers believe that advances in cancer treatment, even in countries with limited resources, can helpful because early detection increases the effectiveness of treatment . According to this study, the trend of asir of esophagus cancer in iran is rising . Hence to prevent and control this cancer, it is necessary to investigate related risk factors and implement prevention programs in iran.
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Amyotrophic lateral sclerosis (als) is a rapidly progressive degenerative neurological disease . About 5%-10% of als is familial, with the remaining 90% of people diagnosed with als being classified as having sporadic disease (1). Although several environmental risk factors have been considered, the causes of als are largely unknown . The association between als and exposure to neurotoxic chemicals, such as solvents, pesticides and metals, has been investigated in several epidemiologic studies with inconsistent results (2, 3). Pesticides are known to be important risk factors for als and other neurodegenerative diseases such as parkinson's and alzheimer's (4). Although the biologic mechanisms contributing to risk of als associated with exposure to pesticides are unknown, many pesticides are considered as potential neurotoxins in various ways (5). In addition, an increased als risk with pesticide exposure was also reported from alteration of paraoxonase 1 function, which detoxifies organophosphates (6). Recently, a case of als was reported in korea involving a worker at a waste disposal site who had crushed glass pesticide bottles for for 15 yr (7). Previous epidemiologic studies, however, have produced inconsistent results when examining the association of pesticides and als . Exposure to pesticides has been reported to be associated with als risk in some investigations (8, 9, 10), but others have found no relationship (3, 11, 12, 13, 14). One possible explanation for these inconsistencies may be low statistical power since the number of cases available for study is typically limited in the case of rare diseases such as als . Therefore, two meta - analyses with regard to pesticide exposure and als have been conducted to date (15, 16). (15) showed that occupational exposure to pesticides as a group significantly increased (about two - fold) the risk for als . (16) also reported a roughly two - fold increase in risk of als among men, but not among women . Previous meta - analyses, however, only included studies of pesticide exposure but excluded studies based on job title such as a farmer or those based on living on a farm, both of which would be used as important surrogate indices for pesticide exposure . Since rural residence or aspects of agricultural activity other than pesticide use may also serve as a potential risk factor for als (13, 17, 18, 19, 20), it is important to investigate the risk of als with overall environments, from residence in rural area to pesticide exposure . The objectives of this meta - analysis, therefore, were to investigate the overall scope of exposure to pesticides and rural environments with the risk of als by including studies for broad categories of exposure assessment categories such as rural residence, farmers, and pesticide exposure . We conducted systematic reviews for rural residence, farmers, and pesticide exposure according to the moose guidelines (21). A search was performed in ovid medline and embase up to september 2013 using the medical subject headings (mesh). The search terms for als included' motor neurone disease',' amyotrophic lateral sclerosis',' lou gehrig disease',' charcot disease' . These were combined with search terms for the exposure which included' agrochemicals',' pesticide',' organophosphorus compounds',' insecticides',' cholinesterase inhibitors',' herbicides',' paraquat',' gramoxone',' fungicide',' agriculture',' occupational exposure',' farmer',' farmworker',' rural residence',' rural environment' . Studies included in our analysis were selected based on the following inclusion criteria: 1) peer - reviewed cohort or case - control studies; 2) studies which investigated the association between rural residence, farmers or pesticide exposure and amyotrophic lateral sclerosis; 3) reported outcome measures with odds ratio (or) or relative risk (rr) for als, or that provided the number of individuals; and 4) written in english . Review articles, case reports, case - series, letters to editors, commentaries, proceedings, laboratory science studies, and any non - relevant tudies were excluded from analysis . As shown in fig . 1, a total of 1,720 articles were obtained after searching databases and references and performing a hand - search . After excluding the duplicates (n=434), the remaining articles were reviewed (n=1,286) and 1,220 articles were excluded for not meeting the selection criteria . The remaining 66 articles were selected for review of their entire content . Among them, 44 were excluded for the following reasons: 25 were not case - control or cohort studies, 17 provided insufficient data; no control group (n=7), no eligible exposure for rural living, farmer, pesticide (n=4), and no eligible outcome for als (n=6), and two were duplicate articles . Therefore, a total of 22 studies were included in our meta - analysis (3, 8, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31). When the study samples were overlapped in two or more articles, we selected the article with the most comprehensive population . Based on the predetermined selection criteria, two of the authors independently selected all trials retrieved from the databases and bibliographies . Disagreements between evaluators were resolved through discussion or in consultation with a third author . In the case of insufficient or missing data, they were derived either from the text or tables or, when possible, calculated from the relevant data within the study . Standardized data extraction forms were used to extract the following data from the studies included in the final analysis: name of the first author with year of publication, journal name, country where the study was conducted, study design, diagnostic criteria, definition of rural residence, farmer, and pesticide exposure, adjusted factors, number of cases / controls or cohort participants, and rr or or with 95% confidence intervals (cis). Case ascertainment was based on using or not using el escorial criteria for diagnosis of als . The world federation of neurology developed the el escorial diagnostic criteria for als, which have proven to be accurate in diagnosis of als using pathology as a' gold standard' and represents them as universal guidelines for the diagnosis of als (32). Exposure assessment methods were summarized into two categories (i.e., self - reported vs. expert judgement). Overall pooled estimates and their corresponding 95% ci were obtained using dersimonian and laird random effects models (33). If the article reported stratified estimates, the strata were combined and the crude or was recalculated (22, 26, 27, 31). Available raw data were used in a 22 table to calculate the or and 95% ci in a study (20). As the incidence of als is low (i.e., 1 - 3 per 100,000 persons per year), we assumed odds ratio to be equal to relative risk . We conducted meta - analyses stratified by rural residence, farmers, and pesticide exposure separately . Subgroup analyses were performed according to the following characteristics: 1) study design, 2) region (europe, the usa, and others including australia and india), 3) gender, 4) case ascertainment (el escorial criteria or not), 5) exposure assessment (self - reported or expert judgment). Between - study q - statistic p value of <0.1 was considered statistically significant and i of 25, 50, or 75 indicates low, medium, or high heterogeneity, respectively (34). We estimated publication bias by using begg's funnel plot (35) and egger's test (36). In addition, contour - enhanced funnel plots were performed in order to aid the interpretation of the funnel plot . Although publication bias for pesticide exposure was not significant for egger's test (p=0.09), asymmetry in the funnel plot was observed and trim and fill analyses were therefore performed (37). We used the stata se version 12.0 software package for statistical analysis (statacorp, college station, tx, usa). We conducted systematic reviews for rural residence, farmers, and pesticide exposure according to the moose guidelines (21). A search was performed in ovid medline and embase up to september 2013 using the medical subject headings (mesh). The search terms for als included' motor neurone disease',' amyotrophic lateral sclerosis',' lou gehrig disease',' charcot disease' . These were combined with search terms for the exposure which included' agrochemicals',' pesticide',' organophosphorus compounds',' insecticides',' cholinesterase inhibitors',' herbicides',' paraquat',' gramoxone',' fungicide',' agriculture',' occupational exposure',' farmer',' farmworker',' rural residence',' rural environment' . Studies included in our analysis were selected based on the following inclusion criteria: 1) peer - reviewed cohort or case - control studies; 2) studies which investigated the association between rural residence, farmers or pesticide exposure and amyotrophic lateral sclerosis; 3) reported outcome measures with odds ratio (or) or relative risk (rr) for als, or that provided the number of individuals; and 4) written in english . Review articles, case reports, case - series, letters to editors, commentaries, proceedings, laboratory science studies, and any non - relevant tudies were excluded from analysis . 1, a total of 1,720 articles were obtained after searching databases and references and performing a hand - search . After excluding the duplicates (n=434), the remaining articles were reviewed (n=1,286) and 1,220 articles were excluded for not meeting the selection criteria . The remaining 66 articles were selected for review of their entire content . Among them, 44 were excluded for the following reasons: 25 were not case - control or cohort studies, 17 provided insufficient data; no control group (n=7), no eligible exposure for rural living, farmer, pesticide (n=4), and no eligible outcome for als (n=6), and two were duplicate articles . Therefore, a total of 22 studies were included in our meta - analysis (3, 8, 9, 11, 12, 13, 14, 15, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31). When the study samples were overlapped in two or more articles, we selected the article with the most comprehensive population . Based on the predetermined selection criteria, two of the authors independently selected all trials retrieved from the databases and bibliographies . Disagreements between evaluators were resolved through discussion or in consultation with a third author . In the case of insufficient or missing data, they were derived either from the text or tables or, when possible, calculated from the relevant data within the study . Standardized data extraction forms were used to extract the following data from the studies included in the final analysis: name of the first author with year of publication, journal name, country where the study was conducted, study design, diagnostic criteria, definition of rural residence, farmer, and pesticide exposure, adjusted factors, number of cases / controls or cohort participants, and rr or or with 95% confidence intervals (cis). Case ascertainment was based on using or not using el escorial criteria for diagnosis of als . The world federation of neurology developed the el escorial diagnostic criteria for als, which have proven to be accurate in diagnosis of als using pathology as a' gold standard' and represents them as universal guidelines for the diagnosis of als (32). Exposure assessment methods were summarized into two categories (i.e., self - reported vs. expert judgement). Overall pooled estimates and their corresponding 95% ci were obtained using dersimonian and laird random effects models (33). If the article reported stratified estimates, the strata were combined and the crude or was recalculated (22, 26, 27, 31). Available raw data were used in a 22 table to calculate the or and 95% ci in a study (20). As the incidence of als is low (i.e., 1 - 3 per 100,000 persons per year), we assumed odds ratio to be equal to relative risk . We conducted meta - analyses stratified by rural residence, farmers, and pesticide exposure separately . Subgroup analyses were performed according to the following characteristics: 1) study design, 2) region (europe, the usa, and others including australia and india), 3) gender, 4) case ascertainment (el escorial criteria or not), 5) exposure assessment (self - reported or expert judgment). Between - study q - statistic p value of <0.1 was considered statistically significant and i of 25, 50, or 75 indicates low, medium, or high heterogeneity, respectively (34). We estimated publication bias by using begg's funnel plot (35) and egger's test (36). In addition, contour - enhanced funnel plots were performed in order to aid the interpretation of the funnel plot . Although publication bias for pesticide exposure was not significant for egger's test (p=0.09), asymmetry in the funnel plot was observed and trim and fill analyses were therefore performed (37). We used the stata se version 12.0 software package for statistical analysis (statacorp, college station, tx, usa). The study included a total of 19 case - control studies (8, 9, 11, 12, 13, 14, 17, 18, 19, 20, 22, 23, 25, 26, 27, 28, 29, 30, 31) and three cohort studies (3, 15, 24) (table 1). The studies were conducted mainly in europe or the usa, with the exception of one indian and one australian study . Among the total studies, three (13, 17, 18) had data for rural residence, farming occupation, and pesticide exposure and two (19, 26) included data for farming occupation and pesticide exposure . They include five case - control studies for rural residence (13, 17, 18, 23, 25), ten case - control studies for farming occupation (13, 17, 18, 19, 20, 26, 27, 29, 30, 31), and 15 studies for pesticide exposure; three cohorts (3, 15, 24) and 12 case - control studies (8, 9, 11, 12, 13, 14, 17, 18, 19, 22, 26, 28). El escorial criteria were used in six studies (8, 17, 18, 22, 26, 27) and pesticide exposure was defined by expert judgment in four studies (8, 9, 19, 24). The risk of als was significantly increased with pesticide exposure (or, 1.44; 95% ci, 1.22 - 1.70) and with farming occupation (or, 1.42; 95% ci, 1.17 - 1.73), but was not significant for rural residence (table 2). Individual estimates from 22 studies and their overall pooled ors for rural residence, farming occupation, and pesticide exposure are presented in the forest plot separately in fig . Pesticide exposure showed a significantly increased risk of als for studies with case - control design (or, 1.49), among males (or, 1.96) and with applied expert judgment exposure assessment (or, 2.04). Results of the q test and i2 statistics were significantly heterogeneous for total studies but not significant when sub - group analyses were conducted by region or gender . No evidence of publication bias was observed for all three exposure indices, but the plot was a slightly asymmetric in contour - enhanced funnel plot at pesticide exposure index (fig . Ors for pesticide exposure were still significant (or, 1.40; 95% ci, 1.10 - 1.79) (data not shown). Our findings from the meta - analysis support an association between pesticide exposure and als . The increased risks of als were consistent by study deign, country, gender, als definition, and type of exposure assessment . The estimates for als had a tendency to be significant as the order of accuracy for pesticide exposure indicators, and the risks were higher in men, in studies using el escorial criteria and in those using expert judgment, compared to their counterparts . Our results were consistent with previously published meta - analyses, which reported an association between pesticide exposure and als (15, 16). However, our increased risk both men and women were different with a previous study (16) which reported that the significant association was found only among men . In addition, our results showed that the ors for als became significant in the order of rural residence, farmer, and pesticide exposure, which was the order of accuracy for pesticide exposure indicators, and statistical significance was found both for farming occupation and pesticide exposure . The information on rural residence is a crude measure of pesticide exposure because not all rural residents are farmers nor are exposed to farming, and not all farmers actually use pesticides (38). Rural residents may also be exposed to physical, chemical or biological factors other than pesticides . Since rural residence or farmer are a wider category of exposure than pesticide, using rural living or farmer as an indicator for pesticide exposure may underestimate the risk of association with pesticide exposure . Similarly, no significant association between childhood leukemia and parental occupational exposure was observed when farming / agricultural work was used as a surrogate for pesticide exposure, whereas significantly increased risks were observed when specific use of pesticides by the parents was considered (39). When using el escorial criteria for case ascertainment, ors for als tend to be higher than when not used . This may be explained by the assertion that clarifying case ascertainment by using el escorial criteria allows greater precision in the diagnosis of als which may impact these effects . Similarly, the or for als was higher when expert judgement was used for exposure assessment compared to self - reported interviews or questionnaires . Although self - reported information may provide detailed data at the individual level, exposure misclassification from recall bias or reliability issues are always of concern . Expert judgment by using job title and occupational history creates greater precision in exposure assessment, despite the fact that it might also result in non - differential exposure misclassification (40). Therefore, our findings may stress the importance of using more objective information for defining disease and exposure in epidemiologic studies . Men had a higher risk for als than women in regard to pesticide exposure, although the confidence intervals were overlapped . This discrepancy by gender was also observed in a previous meta - analysis (16) and a study from india (17). This gender discrepancy included in these studies may partly be explained as men having different features of occupational exposure to pesticides, for example their being exposed to pesticide more frequently or in larger amounts when they use pesticide, or being influenced by sex - related factors . The possible factors leading to the different risks of als and pesticide exposure among countries may include differences in the amounts, pattern, and methods of pesticide use, as well as by genetics . We have considered all pesticides as a single exposure category although pesticides include many different chemicals . However, few studies investigated individual pesticides, their exposure duration or intensity; therefore, we were not able to do subgroup analysis on these issues . Possible interaction among pesticides and genetic factors may also potentiate the different results from countries . Future studies with more detailed information on pesticide use and other potential risk factors are needed to clarify this issue . Further studies for non - occupational pesticide exposure are also needed to describe the full scope of association with pesticide exposure and als . First, the original studies included in this meta - analysis had adjusted for limited potential confounding factors . However, majority of studies included cases and controls of similar demographic characteristics, thus the association between als and exposures would not be expected substantially to change by uncontrolled confounding factors . Subgroup analyses by confounding factors were also limited due to few studies adjusted for same factors . However, the present analysis did not appear to be hampered by publication bias, since there was no evidence of publication bias as observed by begg's funnel plot and egger's test . Duval and tweedie's trim and fill analyses also provided an adjusted estimate of the effect of pesticide exposure on als and revealed that there was still significant risk of als with pesticide exposure (37). Heterogeneity may be inevitable in meta - analysis, but sub - group analyses showed consistent positive associations with pesticide exposure and the risk of als . Despite some limitation in terms of detailed information on pesticide exposure, our findings from the meta - analysis of 19 case - control and three cohort studies support an association between pesticide exposure and als, but not for rural residence . These meta - analyses present overall scopes of categories for exposure to pesticide and/or rural environment assessment such as rural residence, farming occupation, and pesticide exposure, which help us to more comprehensively understand the relation between als and exposure to pesticides . Considering that farmers are commonly frequently exposed to pesticides at a high level, it is important to recommend lowering exposure to pesticides in order to reduce the risk of development of als.
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The qualitative process used for reflection on and assessment of this particular participatory method is also provided . Ten students in the grade 912 cohort were trained as researchers and conducted interviews with students in grades 212 at the klemi dene school and students in grades 26 at the neighbouring kaw tay whee school (fig . 1). Ethical permission to conduct the research was obtained through the health research ethics board (panel b) of the university of alberta . In addition, a northwest territories (nwt) scientific research licence was obtained through the aurora research institute . Research training was done by members of the research team (including academic, community and government members) and school staff (teacher and principal). Information was provided on the research process, from concept through to analysis and interpretation . This included the fundamentals of research ethics, creating interview questions, conducting interviews (including providing information to participants and obtaining consent) and using the digital recording equipment . The class collectively developed the script for the initial interview on smoking knowledge and behaviours and the briefing for the photovoice method . The high school students then conducted practice interviews with the researchers and teachers to make all participants more comfortable with the process and equipment . At the start of the interview, the student researchers described the project for the participants and informed them that the interviews would be audio recorded, how their confidentiality would be maintained and how the information would be used . Even though parental / guardian permission was obtained, further oral assent to participate in the research was sought from the students . The interview questions, as developed by the youth researchers, are shown in table i. interview questions designed by youth researchers have you smoked in the past?yes: when did you quit? How much money do you spend on smokes? Where do you get the money for smokes? A total of 48 students ranging in age from 7 to 19 years were interviewed . Most of the students were dene, although a few students were of other ethnicity . The students all spoke english: although dogrib is taught in the school, few if any of the students are fluent in this language . All students attending school during the 2 days in which interviews were conducted and who assented to being part of the project participated in the research . At the end of the interview they were given the general assignment to take pictures of tobacco use in their community . The photovoice briefing involved ensuring that ethical issues related to the use of photovoice were addressed (30) and was based on the following instructions to the researchers about advising the participants of specific considerations: ask people's permission before you take their picture . Make sure you know their name in case we need to ask them later if we can use the picture in a presentation or report.do not put yourself and anyone else in danger to take a picture . For example, do not stand on a wobbly chair to take a picture.do not take pictures that might embarrass someone or make them feel bad . Make sure you know their name in case we need to ask them later if we can use the picture in a presentation or report . Do not put yourself and anyone else in danger to take a picture . For example, do not stand on a wobbly chair to take a picture . Do not take pictures that might embarrass someone or make them feel bad . Of the 48 cameras distributed, the student researchers again collectively decided on the semistructured guide for the photovoice interviews (table ii). Some very innovative questions for coaxing shyer younger students to discuss their pictures were developed by the student researchers, including can you give me 2 words to describe this picture? Photovoice interview questions designed by youth researchers why did you take this picture?? Would looking at this picture make you want to smoke? What does this _ _ _ _ _ _ _ _ _ _ _ mean to you? Give me 2 words to describe this picture? (can you describe this picture?) Can you tell me a story about this picture?? Is there something you wanted to take a picture of but couldn't? The pictures and accompanying words were assembled and presented back to the student researchers for discussion and analysis . Relevant themes were determined through iterative discussions between the students, teachers and researchers on the meaning and relevance of the photographs . It was collectively decided to assemble a book to showcase the student pictures and words . One student precociously pointed out that all of the pictures involved negative aspects of each theme and that perhaps the final portrayal of the results should pair the negative photos with positive depictions related to that theme . For example, pictures for the theme smoking is unhealthy were paired with pictures on healthy activities (fig . 2). Based on their experiences and understanding (both as members of an aboriginal community and as youth), the students also decided that 2 further themes were needed for the book: one showing the traditional use of tobacco and one on why students choose to smoke or not to smoke . The additional pictures required for the positive portrayals and for the new themes were either taken by the student researchers or obtained from school photograph galleries . All decisions on the photographs to be used in the book were made jointly between the academic researcher and the student researchers . An example of the pairing of a negative theme and positive theme in the results booklet youth voices on tobacco . The final book of pictures with selected accompanying words was produced as an ibook on an apple computer . A student researcher worked with the academic researcher to select the pictures for the book and decide how these should be portrayed . At least 1 picture from every student who participated in the photovoice project was included . The book was distributed to all students of the klem dene school and the kaw tay whee school . Books were also distributed to every family in the communities of ndilo and dettah through a regular band council mail out . The methods used for research evaluation must be appropriate to the research paradigm (31). Traditional quantitative conceptualizations of evaluation are generally acknowledged to be unsuitable for the assessment of qualitative research (32). Instead, qualitative evaluations are more aptly based on established qualitative methods such as reflection, observation and interpretation of meaning . Within a participatory research process, this type of evaluation must be both an individual and cooperative undertaking: while the assessment may be subject to analysis and interpretation by the researcher, it must also remain true to the information provided by and understanding of the other research participants (33). The assessment of the benefits and limitations of conducting community - based participatory research using youth as researchers was based on this type of qualitative evaluation approach . The researcher's reflections on the process were based on observations of the student researchers, student participants, teachers and community members . These observations were evaluated using member checking, whereby students, teachers and community partners were given the opportunity to agree or disagree with the researcher's interpretation through informal one - on - one and class discussions . It is considered analogous to an evaluation of internal validity in quantitative assessment, only instead of seeking to establish confidence in the truth of the findings, the intent is to focus on the degree to which findings make sense (34). The student researchers and teachers were further asked to informally share their own reflections and observations on this process, thus making the assessment process reciprocal and dialogic . In this assessment, comparisons are inevitably made against assumptions of an alternative process (i.e. If the academic researchers had planned and conducted the research without student involvement). Although it is obviously impossible to know definitively what would have happened if, it is possible to use experiential knowledge to make reasonable assumptions about the potential outcomes under this alternate scenario . These assumptions were generally shared by the research participants in discussions about benefits and limitations of this process, adding legitimacy to the comparisons and conclusions . Ten students in the grade 912 cohort were trained as researchers and conducted interviews with students in grades 212 at the klemi dene school and students in grades 26 at the neighbouring kaw tay whee school (fig . 1). Ethical permission to conduct the research was obtained through the health research ethics board (panel b) of the university of alberta . In addition, a northwest territories (nwt) scientific research licence was obtained through the aurora research institute . Research training was done by members of the research team (including academic, community and government members) and school staff (teacher and principal). Information was provided on the research process, from concept through to analysis and interpretation . This included the fundamentals of research ethics, creating interview questions, conducting interviews (including providing information to participants and obtaining consent) and using the digital recording equipment . The class collectively developed the script for the initial interview on smoking knowledge and behaviours and the briefing for the photovoice method . The high school students then conducted practice interviews with the researchers and teachers to make all participants more comfortable with the process and equipment . At the start of the interview, the student researchers described the project for the participants and informed them that the interviews would be audio recorded, how their confidentiality would be maintained and how the information would be used . Even though parental / guardian permission was obtained, further oral assent to participate in the research was sought from the students . The interview questions, as developed by the youth researchers, are shown in table i. interview questions designed by youth researchers have you smoked in the past?yes: when did you quit? How much money do you spend on smokes? Where do you get the money for smokes? A total of 48 students ranging in age from 7 to 19 years were interviewed . Most of the students were dene, although a few students were of other ethnicity . The students all spoke english: although dogrib is taught in the school, few if any of the students are fluent in this language . All students attending school during the 2 days in which interviews were conducted and who assented to being part of the project participated in the research . At the end of the interview, participants were briefed on the photovoice method and provided with disposable cameras . They were given the general assignment to take pictures of tobacco use in their community . The photovoice briefing involved ensuring that ethical issues related to the use of photovoice were addressed (30) and was based on the following instructions to the researchers about advising the participants of specific considerations: ask people's permission before you take their picture . Make sure you know their name in case we need to ask them later if we can use the picture in a presentation or report.do not put yourself and anyone else in danger to take a picture . For example, do not stand on a wobbly chair to take a picture.do not take pictures that might embarrass someone or make them feel bad . Ask people's permission before you take their picture . Make sure you know their name in case we need to ask them later if we can use the picture in a presentation or report . Do not put yourself and anyone else in danger to take a picture . Do not take pictures that might embarrass someone or make them feel bad . Of the 48 cameras distributed, the student researchers again collectively decided on the semistructured guide for the photovoice interviews (table ii). Some very innovative questions for coaxing shyer younger students to discuss their pictures were developed by the student researchers, including can you give me 2 words to describe this picture? Photovoice interview questions designed by youth researchers why did you take this picture? _ _ _ _ _ _ _ _ _ _ mean to you? Give me 2 words to describe this picture? What do you think is going to happen next? Is there something you wanted to take a picture of but couldn't? The pictures and accompanying words were assembled and presented back to the student researchers for discussion and analysis . Relevant themes were determined through iterative discussions between the students, teachers and researchers on the meaning and relevance of the photographs . It was collectively decided to assemble a book to showcase the student pictures and words . One student precociously pointed out that all of the pictures involved negative aspects of each theme and that perhaps the final portrayal of the results should pair the negative photos with positive depictions related to that theme . For example, pictures for the theme smoking is unhealthy were paired with pictures on healthy activities (fig . 2). Based on their experiences and understanding (both as members of an aboriginal community and as youth), the students also decided that 2 further themes were needed for the book: one showing the traditional use of tobacco and one on why students choose to smoke or not to smoke . The additional pictures required for the positive portrayals and for the new themes were either taken by the student researchers or obtained from school photograph galleries . All decisions on the photographs to be used in the book were made jointly between the academic researcher and the student researchers . An example of the pairing of a negative theme and positive theme in the results booklet youth voices on tobacco . The final book of pictures with selected accompanying words was produced as an ibook on an apple computer . A student researcher worked with the academic researcher to select the pictures for the book and decide how these should be portrayed . At least 1 picture from every student who participated in the photovoice project was included . The book was distributed to all students of the klem dene school and the kaw tay whee school . Books were also distributed to every family in the communities of ndilo and dettah through a regular band council mail out . The methods used for research evaluation must be appropriate to the research paradigm (31). Traditional quantitative conceptualizations of evaluation are generally acknowledged to be unsuitable for the assessment of qualitative research (32). Instead, qualitative evaluations are more aptly based on established qualitative methods such as reflection, observation and interpretation of meaning . Within a participatory research process, this type of evaluation must be both an individual and cooperative undertaking: while the assessment may be subject to analysis and interpretation by the researcher, it must also remain true to the information provided by and understanding of the other research participants (33). The assessment of the benefits and limitations of conducting community - based participatory research using youth as researchers was based on this type of qualitative evaluation approach . The researcher's reflections on the process were based on observations of the student researchers, student participants, teachers and community members . These observations were evaluated using member checking, whereby students, teachers and community partners were given the opportunity to agree or disagree with the researcher's interpretation through informal one - on - one and class discussions . It is considered analogous to an evaluation of internal validity in quantitative assessment, only instead of seeking to establish confidence in the truth of the findings, the intent is to focus on the degree to which findings make sense (34). The student researchers and teachers were further asked to informally share their own reflections and observations on this process, thus making the assessment process reciprocal and dialogic . In this assessment, comparisons are inevitably made against assumptions of an alternative process (i.e. If the academic researchers had planned and conducted the research without student involvement). Although it is obviously impossible to know definitively what would have happened if, it is possible to use experiential knowledge to make reasonable assumptions about the potential outcomes under this alternate scenario . These assumptions were generally shared by the research participants in discussions about benefits and limitations of this process, adding legitimacy to the comparisons and conclusions . Using youth to research other youth within a participatory research framework had many benefits for the quality of the research, the youth researchers and the community . From a research perspective, more valid and credible results were obtained . Students were obviously more comfortable talking with older, familiar students instead of adult outsider researchers, and were consequently more candid about talking about smoking behaviours without the anticipated disapproval of an authority figure . Although this result is obviously very difficult to prove empirically (particularly in a qualitative study), it is substantiated by comments made by the student researchers and through observations made by the academic researchers in reviewing the audio transcripts . The research design and implementation benefited greatly from the collective approach between research team members and youth researchers . As a result of the overall participatory process, one of the community research team members even quit smoking to act as a role model for the project . The research instruments designed by the youth researchers provided unique interview approaches and questions that were better suited to both the age and cognitive processing of the participants . The final book on youth voices on tobacco was significantly enhanced by the youth researchers ideas and the additional photographs they provided . Students, teachers and community partners commented that pairing the negative pictures with positive depictions of how things should be greatly increased the impact of the book by providing a constructive and optimistic perspective on each theme . Psychological theories of knowledge, attitudes and behaviours support the concept that the favourability of messages, as determined through past experiences and worldviews, may affect the type and tendency of cognitive processing used to determine thoughts and actions (35,36). Negative messages that elicit unfavourable thinking, while sometimes necessary to increase knowledge and consideration of tobacco issues, may actually decrease persuasion to change smoking behaviours or community conditions, even if the message is understood (37). Coupling of negative and positive messages thus provides both information and affective cues that are more likely to result in desired attitude and/or behaviour changes related to smoking . In addition, a book based on youth - generated pictures with obvious youth input was of more compelling interest for the community . Many people commented that viewing the stories created by their youth and situated in their own environment resulted in a very relevant and powerful message . Everyone involved described the research as a positive experience . It has led to pursuing options for additional collaborative research in this area, using a new cohort of students as researchers . Additionally, as a result of the youth researchers interacting within their community, it has raised awareness of tobacco use and helped both the youth researchers and the community to consider possible steps towards changing to healthier lifestyle choices . Youth researchers benefited from exposure to the research process and the wonders of exploration and learning . They developed research skills and leadership abilities, which empowered several of them in terms of their future success 3 of the 4 student researchers who graduated that year returned to work with the school the next year as teacher aides . As true participants in the research, the students exhibited a definite sense of ownership of both the research process and the final research product . These qualities have the potential for encouraging sustained interest in contributing to changes in community tobacco use individual skill building, participation and empowerment were shown to facilitate active youth involvement in a long - term, tobacco - related community change in another study (38). Finally, there was evidence from the class discussions of the pictures and words that participation in this research encouraged student critical appraisal of an apparent shared cognitive dissonance (39) between smoking knowledge and smoking behaviours (while all students knew and understood the health implications of smoking, the majority of the grade 912 cohort participating as youth researchers smoked). This is the first step in evoking personal self - examination of motivations and awareness of individual power to make positive health choices, which may in turn lead to changed behaviours and community action . For the community, viewing smoking through their children's eyes was stated by many to be a much more powerful and potentially influential message than a report generated by outside researchers . The photographs and words produced by the youth are known to have garnered community interest . These visual messages will hopefully result in raised awareness of the community conditions that are contributing to the high prevalence of smoking amongst youth, as has been demonstrated in other youth photovoice projects (8). In general, it was found that smoking was ubiquitous in the community and that most students had family members and friends who smoked . The long standing cycle of addiction related to smoking is very strong in many northern communities: if youth see everyone around them smoking, they are more likely to view smoking as a normal behaviour . This research project and the resulting book of photographs is the first step in prompting community recognition that a prevalent smoking environment will only lead to the negative outcome of more and younger new smokers (40). Despite the fact that having youth researchers undoubtedly increased the accuracy of the results, there was still an obvious response bias in the research, with many students providing the correct response that they do not smoke regardless of their actual behaviour . While only 6 of the participants indicated they currently smoke in the interviews, at least 9 of 12 students in the grade 912 cohort this discrepancy may be related to the desire to provide the researchers with the acceptable answer that would meet with the approval of the teachers . To accommodate school scheduling, students were given 12 days to take pictures, thereby limiting opportunities and possible creativity . The younger participants tended to take pictures that were convenient; it was amusing to see swarms of children taking pictures of cigarette butts on the ground in the area around the school on the day the cameras were distributed . This is consistent with behaviours of young participants in other photovoice projects (8,17,19). Time restrictions also limited relationship building with the youth researchers and the academic research team, which would have led to more open and honest opportunities to discuss some complex and sensitive issues in more depth . Based on their experiences with a related youth photovoice project on childhood obesity, findholt et al . (8) recommend a thorough project orientation that goes beyond a discussion of cameras and photographic ethics and helps youth to understand how community context can affect health . This type of discussion might have been useful in this study to further explore the interpretation of the pictures and the possibilities for community action . Even though the research was deemed a success by everyone, it did fail to completely address the research study objective related to student decision making on smoking or not smoking . This objective had arisen from a comment made by a yellowknife teacher on how does it go from being gross and yucky to students in grade 4, to having students in grade 5 smoking in the schoolyard? While it had been thought this theme might arise naturally in the choice of pictures taken by the participants, it was not directly apparent in the original set of pictures . Although the youth researchers decided to explore this theme specifically for the book with additional pictures, it still proved difficult to fully understand motivations for smoking and not smoking . In hindsight, participants should have been provided with more specific instructions to take pictures related to this theme . Research involving children and youth raises specific ethical issues, primarily related to concerns regarding competence, autonomy and vulnerability . Although the school had obtained blanket parental consent for school activities (allowing all students to participate in the research), specific consent was required to use the research results . Obtaining written consent is problematic in many northern aboriginal communities as it may be seen as contrary to respecting aboriginal approaches to research initiatives (42, p. 21). Determining who is responsible for granting consent for minors is also difficult in communities where current guardianship is often not formally recognized . Information sheets and consent forms were sent home with all student participants, and written consent was obtained from approximately one - third of the parents / guardians . The teacher then phoned all other parents / guardians to explain the study and obtain verbal consent for the remaining students . Verbal assent was also obtained from each student participant prior to conducting the interviews, as the researchers strongly believed that this was necessary to respect the rights of the individual . It had originally been planned to use digital cameras that could be deployed successively amongst the students . The teacher made the very compelling case that the research should foster feelings of success in the students; if a valuable camera was lost or stolen, requiring an investigation and possible blame, success could be quickly compromised and the positive experience of the research negated . A final ethical concern involved representation . It was considered very important that all participants should be able to see themselves in the final research product . Care was thus taken that at least 1 photograph from each participant was included in the final book youth voices on tobacco . Using youth to research other youth within a participatory research framework had many benefits for the quality of the research, the youth researchers and the community . From a research perspective, more valid and credible results were obtained . Students were obviously more comfortable talking with older, familiar students instead of adult outsider researchers, and were consequently more candid about talking about smoking behaviours without the anticipated disapproval of an authority figure . Although this result is obviously very difficult to prove empirically (particularly in a qualitative study), it is substantiated by comments made by the student researchers and through observations made by the academic researchers in reviewing the audio transcripts . The research design and implementation benefited greatly from the collective approach between research team members and youth researchers . As a result of the overall participatory process, one of the community research team members even quit smoking to act as a role model for the project . The research instruments designed by the youth researchers provided unique interview approaches and questions that were better suited to both the age and cognitive processing of the participants . The final book on youth voices on tobacco was significantly enhanced by the youth researchers ideas and the additional photographs they provided . Students, teachers and community partners commented that pairing the negative pictures with positive depictions of how things should be greatly increased the impact of the book by providing a constructive and optimistic perspective on each theme . Psychological theories of knowledge, attitudes and behaviours support the concept that the favourability of messages, as determined through past experiences and worldviews, may affect the type and tendency of cognitive processing used to determine thoughts and actions (35,36). Negative messages that elicit unfavourable thinking, while sometimes necessary to increase knowledge and consideration of tobacco issues, may actually decrease persuasion to change smoking behaviours or community conditions, even if the message is understood (37). Coupling of negative and positive messages thus provides both information and affective cues that are more likely to result in desired attitude and/or behaviour changes related to smoking . In addition, a book based on youth - generated pictures with obvious youth input was of more compelling interest for the community . Many people commented that viewing the stories created by their youth and situated in their own environment resulted in a very relevant and powerful message . Everyone involved described the research as a positive experience . It has led to pursuing options for additional collaborative research in this area, using a new cohort of students as researchers . Additionally, as a result of the youth researchers interacting within their community, it has raised awareness of tobacco use and helped both the youth researchers and the community to consider possible steps towards changing to healthier lifestyle choices . Youth researchers benefited from exposure to the research process and the wonders of exploration and learning . They developed research skills and leadership abilities, which empowered several of them in terms of their future success 3 of the 4 student researchers who graduated that year returned to work with the school the next year as teacher aides . As true participants in the research, the students exhibited a definite sense of ownership of both the research process and the final research product . These qualities have the potential for encouraging sustained interest in contributing to changes in community tobacco use individual skill building, participation and empowerment were shown to facilitate active youth involvement in a long - term, tobacco - related community change in another study (38). Finally, there was evidence from the class discussions of the pictures and words that participation in this research encouraged student critical appraisal of an apparent shared cognitive dissonance (39) between smoking knowledge and smoking behaviours (while all students knew and understood the health implications of smoking, the majority of the grade 912 cohort participating as youth researchers smoked). This is the first step in evoking personal self - examination of motivations and awareness of individual power to make positive health choices, which may in turn lead to changed behaviours and community action . For the community, viewing smoking through their children's eyes was stated by many to be a much more powerful and potentially influential message than a report generated by outside researchers . The photographs and words produced by the youth are known to have garnered community interest . These visual messages will hopefully result in raised awareness of the community conditions that are contributing to the high prevalence of smoking amongst youth, as has been demonstrated in other youth photovoice projects (8). In general, it was found that smoking was ubiquitous in the community and that most students had family members and friends who smoked . The long standing cycle of addiction related to smoking is very strong in many northern communities: if youth see everyone around them smoking, they are more likely to view smoking as a normal behaviour . This research project and the resulting book of photographs is the first step in prompting community recognition that a prevalent smoking environment will only lead to the negative outcome of more and younger new smokers (40). Despite the fact that having youth researchers undoubtedly increased the accuracy of the results, there was still an obvious response bias in the research, with many students providing the correct response that they do not smoke regardless of their actual behaviour . While only 6 of the participants indicated they currently smoke in the interviews, at least 9 of 12 students in the grade 912 cohort this discrepancy may be related to the desire to provide the researchers with the acceptable answer that would meet with the approval of the teachers . To accommodate school scheduling, students were given 12 days to take pictures, thereby limiting opportunities and possible creativity . The younger participants tended to take pictures that were convenient; it was amusing to see swarms of children taking pictures of cigarette butts on the ground in the area around the school on the day the cameras were distributed . This is consistent with behaviours of young participants in other photovoice projects (8,17,19). Time restrictions also limited relationship building with the youth researchers and the academic research team, which would have led to more open and honest opportunities to discuss some complex and sensitive issues in more depth . Based on their experiences with a related youth photovoice project on childhood obesity, findholt et al . (8) recommend a thorough project orientation that goes beyond a discussion of cameras and photographic ethics and helps youth to understand how community context can affect health . This type of discussion might have been useful in this study to further explore the interpretation of the pictures and the possibilities for community action . Even though the research was deemed a success by everyone, it did fail to completely address the research study objective related to student decision making on smoking or not smoking . This objective had arisen from a comment made by a yellowknife teacher on how does it go from being gross and yucky to students in grade 4, to having students in grade 5 smoking in the schoolyard? While it had been thought this theme might arise naturally in the choice of pictures taken by the participants, it was not directly apparent in the original set of pictures . Although the youth researchers decided to explore this theme specifically for the book with additional pictures, it still proved difficult to fully understand motivations for smoking and not smoking . In hindsight, participants should have been provided with more specific instructions to take pictures related to this theme . Research involving children and youth raises specific ethical issues, primarily related to concerns regarding competence, autonomy and vulnerability . Although the school had obtained blanket parental consent for school activities (allowing all students to participate in the research), specific consent was required to use the research results . Obtaining written consent is problematic in many northern aboriginal communities as it may be seen as contrary to respecting aboriginal approaches to research initiatives (42, p. 21). Determining who is responsible for granting consent for minors is also difficult in communities where current guardianship is often not formally recognized . Information sheets and consent forms were sent home with all student participants, and written consent was obtained from approximately one - third of the parents / guardians . The teacher then phoned all other parents / guardians to explain the study and obtain verbal consent for the remaining students . Verbal assent was also obtained from each student participant prior to conducting the interviews, as the researchers strongly believed that this was necessary to respect the rights of the individual . It had originally been planned to use digital cameras that could be deployed successively amongst the students . However, at the request of the teacher, disposable cameras were used instead . The teacher made the very compelling case that the research should foster feelings of success in the students; if a valuable camera was lost or stolen, requiring an investigation and possible blame, success could be quickly compromised and the positive experience of the research negated . A final ethical concern involved representation . It was considered very important that all participants should be able to see themselves in the final research product . Care was thus taken that at least 1 photograph from each participant was included in the final book youth voices on tobacco . Using youth to research youth proved to be a successful strategy for exploring youth knowledge of the health concerns related to smoking and the role of tobacco use in their community . Perhaps more importantly, the research proved valuable in providing the community with a lens through which to view how youth see smoking in their environment . Unfortunately, the traditional use of tobacco for spiritual purposes has been radically changed to the modern addiction to tobacco, which has caused negative health issues for many youth and community members (43). This research methodology was thus an effective way to reach the community, have them reflect on the changes in tobacco use and promote awareness of the need for environmental change, while simultaneously engendering development of youth research skills and abilities (8). The research approach was also important in promoting the view of youth as resources as opposed to the commonly portrayed image of youth as problems (44). This asset - based perspective has to be strengthened as a methodology to work with youth if any changes are to continue . If the adult expectations of youth (which are inevitably reflected in youth behaviours) can be changed, youth may come to believe in themselves as agents of change rather than troublemakers (43). However, realizing this vision will require a commitment to creating openings, opportunities and obligations for continued youth participation in the issues that affect their lives (4). It must also be accompanied by a continued focus on enabling youth action through involvement in decisions and policy making (45). Having youth actively involved in research that affects their lives and opens their eyes to tobacco - related health issues provides them with potential opportunities to pursue real community change . As the future generation, they are also the carriers of the traditions and culture and need to bring forth healthy attitudes towards tobacco's spiritual purposes, not its negative effects related to addictions and adverse health outcomes (43). Finally, this approach successfully met the specific requirements of community - based participatory research (46). The youth researchers learned research skills, while the research team partners learned valuable lessons on structuring research and dissemination products that were more appropriate, effective and powerful for the student participants and community . Shared decision - making was an integral part of the research, ranging from initial decisions on the research concept and design to the final decisions on dissemination . Based on this research, youth assuming a partnership role in research activities presents fruitful ground for continued advances in making positive health changes in northern aboriginal communities . The authors have not received any personal funding or benefits from industry or elsewhere to conduct this research.
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Treatment of severe sepsis and septic shock remains a major challenge in the critically ill, and it is still one of the leading causes of death worldwide . Despite increased awareness of the importance of early resuscitation, mortality in north america and europe ranges between 28 and 41% . Based on a consensus agreement sepsis is defined as infection in the presence of systemic inflammatory response syndrome (sirs). However, the signs of sirs are nonspecific and can often be seen in several (none septic) critically ill conditions . Fever, tachycardia, or leukocytosis on their own has low sensitivity and specificity [4, 5]. Detailed microbiological results are often only available after 24 hours or later, and negative results do not necessarily rule out infection . Nevertheless, early diagnosis of infection in critically ill patients is of utmost importance, and delay in starting appropriate antibiotic therapy may lead to lethal events . However, giving antibiotics unnecessarily to every acutely ill patient is an unacceptable practice for several reasons . Therefore, fast reacting biomarkers of infection have been used for almost 50 years to help the clinician, of which c - reactive protein (crp) and procalcitonin (pct) are the most often used and studied . Procalcitonin is a fast reacting biomarker with a half - life of around 24 hours . Its sensitivity and specificity for bacterial infection seem to be superior compared to crp [10, 11]. However, it must be considered that the same absolute values of pct cannot be used in all circumstances . It has been reported that pct levels are higher in surgical compared to medical patients, and elevated pct can also be present without infection, in conditions such as trauma and surgery or after cardiac arrest . There is some evidence that evaluating pct kinetics may be superior to absolute values [12, 16]. In this study, our aim was to investigate whether the absolute value of pct measured in critically ill patients on the day when infection was suspected, or the change in pct (delta - pct) from the day before to the day when infection was suspected, was a better indicator of infection . This prospective observational study was part of the early procalcitonin kinetics (eprok) study, which was undertaken between october 2012 and october 2013 and approved by the regional and institutional human medical biological research ethics committee, university of szeged, hungary (who-3005; 19.04.2012, chairperson professor t. wittmann). The investigation was performed at the university of szeged (szeged, hungary), albert szent - gyrgyi health center in four tertiary intensive care units . Written informed consent was obtained from all subjects or from their relatives . In the eprok study all patients over 18 years with suspected infection on admission or during their stay on the intensive care unit were screened for eligibility . Patients were enrolled, when the attending intensive care specialist suspected infection, based on (1) suspected source which could be identified, (2) new onset organ dysfunction, and (3) body temperature, pct, crp, and the decision to start empirical antibiotic therapy . Once the original eprok study was completed, in a post hoc analysis those patients in whom pct and crp values were available from the previous day (t1) were included in the current analysis . Exclusion criteria included patients younger than 18 years, who had received antibiotic therapy in the previous 48 hours, and those who received acute renal replacement therapy 24 hours before enrollment . Patients were also excluded following cardiopulmonary resuscitation and with end stage diseases with a do not resuscitate order . Immunocompromised patients (human immunodeficiency virus infection, bone marrow transplantation, malignant haematological disorders, and chemotherapy) were also excluded . Diagnosis of infection was based on a post hoc analysis of mainly microbiological results but also clinical parameters and biochemical results which were evaluated by two experts (infectologist, eh, and an intensivist, fj) blinded for the pct data apart from the first pct measurement (t0, see below). The experts also took into consideration the recommendations of international guidelines [18, 19]. Based on these results, patients were grouped into infection- (i-) and noninfection- (ni-) groups . For subgroup analysis the medical - group represented patients who had had no surgical intervention before and during the study period and for source control did not require surgery . In the surgical - group infection either was related to an operation or required surgery for source control . Whenever infection was suspected by the attending physician, the signs of infection and the suspected source were recorded, which included high / low body temperature (<36c;> 38c), high / low white blood cell count (<4,000;> 12,000 million / ml), acute worsening of the clinical picture (hemodynamic instability, worsening pao2/fio2 ratio, and deterioration in mental status or any other clinical sign indicating infection). Microbiological specimens were collected from all suspected sources immediately before the administration of the first dose of antibiotics (t0). After enrollment, demographic data, signs of infection, the suspected source of infection, and corresponding microbiological samples were registered . The length of intensive care unit and hospital stay, 28 days, and the overall mortality were also documented . It is common practice in our icu to measure pct daily in critically ill patients . Procalcitonin levels were documented from the previous day of enrollment (t1) and immediately before the initiation of abs (t0). Core temperature, c - reactive protein (crp), and white blood cell count (wbc) were also recorded with every pct measure . Serum pct levels were measured with cobas 6000 analyzer (hitachi high - technologies corporation, tokyo, japan). Analyzer reagents (elecsys brahms pct assay) were developed in collaboration with brahms corporation (hennigsdorf, germany) and roche diagnostics (mannheim, germany). Procalcitonin was determined by electrochemiluminescence immunoassay (eclia) serum on the automated roche elecsys and cobas immunoassay analyzers . Microbiological tests were performed and sent at t0, before the first antibiotic dose was administered and if needed they were repeated on the following days, to identify infection . Data were analyzed using ibm spss statistics version 20 (armonk, ny, usa) and systat software inc . Demographic data were analyzed between groups with student's t - test or nonparametric data with the mann - whitney u test as appropriate . Biomarkers were analyzed by using two - way repeated measures analysis of variances (all pairwise multiple comparison procedures: holm - sidak method). Logistic regression, receiver operating characteristic (roc) curve, and the respective areas under the curves (auc) were calculated for pct, crp, body temperature, and white blood cell count levels . The best cut - off values were determined using the youden index (j = max[sens + spec 1]). The test parameters (sensitivity, specificity, positive, and negative predictive values) were compared by their 95% confidence intervals . Logistic regression analysis was used to determine the best combination of parameters and cut - offs for predicting infection . Data are given as mean standard deviation or median (interquartile range) as appropriate . The delta was considered as the changes in the absolute values (subtracting t1 from t0); the percentage values were calculated as [(t0/t1) 100 100]. Over the one - year study period all icu patients were screened for eligibility and 209 patients were recruited into the eprok study . Out of the 209 patients in the current post hoc analysis demography and outcomes characteristics for the entire cohort are summarised in table 1 . Out of the 114 patients, 85 (75%) patients were identified as having proven infection and in 29 (25%) patients the presence of infection was highly unlikely . Disease severity scores and outcomes were similar in the two groups, but the ni - group required less organ support . The clinical and laboratory signs of infection on which the clinicians suspected infection at the time of inclusion (t0) are summarised in table 2 . Although all indices were higher in the i - group, but only the altered level of consciousness, hemodynamic instability, and the pct was significantly different between the two groups . Regarding the suspected source of infection, generally there was nonsignificant difference between the groups, but significantly more patients were suspected of having abdominal related infection in the ni - group . Detailed data on the isolated pathogens and their sources are summarised in the supplemental digital content tables s57 (see supplementary material available online at http://dx.doi.org/10.1155/2016/3530752). Measurement results at t1 and t0 in the i- and ni - groups are shown in figure 2 . Pct absolute values were similar at t1, but by t0 in the i - group levels were significantly higher compared to the ni - group and there was also a significant increase from t1, while there was no such change in the ni - group . There was no significant difference in crp and wbc count between the two groups nor could we find significant changes from t1 to t0 . There was no difference between the groups for body temperature but there was a statistically significant increase in the ni - group by t0 . Measurement results in medical (n = 80) and surgical (n = 34) patients are summarised in table 3 . In the surgical subgroup pct absolute values were significantly higher than in the medical cohort, but the pattern of change was similar . In the ni - group there was a slight, but statistically significant increase in medical patients from t1 to t0, while there was no significant change in surgical patients, where levels actually decreased slightly . However, in the i - group there was an almost 3-fold increase in the pct levels . Regarding the crp, body temperature, and wbc count, there was no significant changes over time and no differences between medical and surgical patients . The predictive value for infection for the absolute values of pct, crp, temperature, and wbc count can be seen in figure 3 and is summarised in table 4 . Only pct had a significant predictive value, but with a poor auc (figure 3). However, regarding the percentage and delta changes crp, temperature and wbc counts diagnostic value did not change, while pct's auc for both percentage and delta changes had a significantly better performance for predicting infection . The best cut - off values were defined for pct only as there was no significant predictive value for the other parameters, as determined by the youden index . For the pct absolute value it was 0.84 ng / ml with a sensitivity of 61% (95% ci: 5072) and specificity 72% (5387) to indicate infection in the icu . Regarding the percentage change a pct increase of> 88% from t1 to t0 had a sensitivity of 75% (6584) and specificity of 79% (6092) and a pct delta change of> 0.76 ng / ml had a sensitivity of 80% (7088) and specificity of 86% (6896) to indicate infection . Data were also analyzed using the logistic regression model for finding the best combination of these four parameters together to predict infection in the icu . However, none of the combinations tested improved the performance for predicting infection (data not shown). The main finding of this observational study was an increase in pct levels from the day before (t1) to the day when infection was suspected (t0) predicted infection, while in patients with no proven infection pct remained unchanged . Furthermore, regarding the conventional indicators of infection such as wbc, body temperature, and crp, neither the absolute values nor their change from t1 to t0 could predict infection . Diagnosing infection in the critically ill is challenging . Appropriate decision making has paramount importance as any delay in adequate antibiotic treatment of sepsis and septic shock evokes worsening morbidity and mortality results [6, 20]. On the other hand unnecessary antibiotic administration in patients without infection has led to the emergence of multidrug - resistant bacteria [21, 22], complications related to the side effects of the antibiotics themselves and an increased burden of healthcare expenses . Despite its importance, there is no gold standard for diagnosing / proving infection in the critical care setting . In our study 75% of patients had proven infection . This complex post hoc analysis of all results is fundamentally different from labelling patients as septic, based solely on the surviving sepsis guideline criteria at the time of initial assessment as seen in several studies [24, 25]. Although our method also has some uncertainties, it provides a more robust approach utilising all data, clinical, biochemical, and microbiology alike, to aid in the diagnosis of patients with bacterial infection . However, it is also important to acknowledge that there is no gold standard to diagnose infection; therefore despite all our efforts, some patients in the ni - group may have had culture negative infection . In our investigation it was found that conventional indicators of infection such as body temperature and white cell count had less value in diagnosing infection . Levels of wbc count remained elevated on both days and there was no significant change over time . This phenomenon can be explained by the nonspecific activation of the immune cascade as often seen in icu patients . Although there was a statistically significant increase in body temperature in the ni - group, levels largely remained below 38c in almost all patients . These results are in accordance with recent findings that increased temperature alone does not predict infection . Although microbiology remains the gold standard for confirming pathogens, results only come back at least 2448 hours after sampling . Furthermore, in several cases results remained negative, despite obvious signs of infection . In order to help the diagnostic process however, all biomarkers share the same limitations that one size will not fit all, due to the complex pathomechanism and the heterogeneity of patients . The two most commonly used markers in infection / sepsis diagnostics are pct and crp . Procalcitonin is detectable in the serum a few (24) hours after the onset of bacterial infection . It reaches its peak within 24 hours and then starts to decline with adequate treatment by around a 50% daily decrease according to its half - life . It reaches its maximum value usually after 48 hours of an insult and in general it lags behind the actual events of the inflammatory and clinical process . Furthermore, crp levels are generally elevated in most icu patients regardless of the aetiology . In our study neither the absolute values of crp nor its delta changes were able to indicate new onset infection . Patients had elevated crp values with a median of almost 200 mmol / l for the whole cohort, which makes interpretation very difficult . Therefore, our results question the place of crp measurements for diagnosing infection on the icu . The most important finding of the current study was to show the superiority of pct kinetics over the absolute values to indicate new onset infection in the icu . However, this requires at least daily measurements of pct, which has been common practice in our icu in critically ill patients in whom infection cannot be excluded . They also measured pct daily and observed a twofold increase of pct levels from the day before to the day when there was a sudden onset of fever in patients with proven infection, but no change in pct was found in patients without infection . They concluded that, in patients treated chronically in the icu, pct values on the day of fever onset must be compared to values measured the previous day in order to define whether this rise in temperature was due to infection or not . An important difference between their and our study is that in our patients body temperature merely reached 38c; in fact most of these patients were apyrexial, despite 75% having proven infection . Therefore, we recommend to evaluate pct kinetics not only in the onset of fever, but whenever infection is suspected on the icu . Based on the current results, the best cut - off values were also determined for change in pct, which were> 88% and> 0.76 ng / ml delta change from t1 to t0 . The reasons why a given absolute value of any biomarker, not just pct, may be of limited value as compared to its changes can be explained by the pathomechanism of systemic inflammation . It was a very important discovery that after trauma, burns, ischemia - reperfusion, pancreatitis, major surgery, and so forth, the same or similar molecules are released predominantly from the mitochondria, as after an infectious insult . Based on aetiology these are called damage - associated molecular patterns (damp), or pathogen associated molecular patterns (pamp). Once similar mediators / proteins are released they act on the same receptors of monocytes inflicting a similar inflammatory response, including pct release and subsequent organ dysfunction [28, 29]. Indeed, pct levels were found to be severalfold higher in surgical compared to medical patients in septic shock despite the similar clinical manifestation and severity of the clinical picture . This explains why pct levels were elevated in our surgical patient population without proven infection, with median values of around 3.5 (ni - group) and 3.8 (i - group) ng / ml at t1 . The corresponding pct values in medical patients were substantially lower (0.26 and 0.89 ng / ml, resp . ). Although levels were higher in the i - group at t1, this difference did not reach statistical significance while there was a severalfold increase in the i - group in both medical and surgical patients with no change in kinetics in the ni - groups . In two large recent multicenter trials the authors could not show any benefit from a pct - based approach in antibiotic management in the icu [30, 31]. However, in both studies the threshold for intervention was a pct of> 1 ng / ml . As 40% of the patients in both trials were surgical, in whom this threshold for intervention may be too low, one cannot exclude that these patients may have had received antibiotics unnecessarily . This overuse of antibiotics may be one of the reasons for the worse outcome in the pct - guided group in both studies . Our study provides further evidence that changes or kinetics of pct may be superior to absolute values . Firstly, one may argue that there was a selection bias; in other words, physicians suspected infection more often when they observed a pct increase in a patient . Although this cannot be excluded completely, at the time when the study was performed, pct collection was not the routine practice within the department, and delta - pct was not included among the criteria of inclusion either . The whole idea of retrieving pct data from the day before came after we analyzed the original eprok database . Secondly, despite all our efforts of allocating patients into the i- and ni - groups, this took place in a post hoc fashion . The available clinical results were analyzed in a blinded manner for delta - pct (apart from pct values at t0) and thoroughly by our experts; however, one cannot exclude the possibility of inappropriate judgment during the decision making . The lack of gold standard for diagnosing infection is aggravated by this obscurity when configuring groups . Furthermore, the sample size was generally small, especially to be able to draw firm conclusions regarding the medical, surgical subgroups, although the trend in our results was certainly promising . Finally, it remains uncertain why pct values were measured on the previous day before starting empiric antibiotic therapy in more than 50% of the 209 patients of the eprok study . The median day of inclusion into the study from icu admission was 1 day, indicating that 50% of patients had pct measurements on the ward / accident and emergency unit, before admission . The main finding of this observational study was that an increase in pct levels from the day before (t1) to the day when infection was suspected (t0) predicted infection, while in patients with no proven infection pct remained unchanged . Based on the data presented a single pct measurement may not be adequate to differentiate between an infectious and noninfectious inflammatory response . This means that the kinetics of procalcitonin values based on daily measurements are superior to absolute values in diagnosing infection on the icu and absolute values of procalcitonin may be of limited use . Both absolute values and kinetics of c - reactive protein are poor indicators of infection; furthermore, conventional indicators of infection such as white cell count and body temperature have limited use for predicting infection in the icu . The clinical implication of these results is that daily pct measurements in patients at high risk of infection allow the opportunity to evaluate pct kinetics, which may improve diagnostic accuracy and rationalise antibiotic therapy on the icu and improve outcome.
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Short - term atmospheric blocking over greenland contributes to melt episodesassociated temperature anomalies are equally important for the meltduration and strength of blocking events contribute to surface melt intensity short - term atmospheric blocking over greenland contributes to melt episodes associated temperature anomalies are equally important for the melt duration and strength of blocking events contribute to surface melt intensity recently, a data record of the clear - sky ice surface temperature (ist) of the greenland ice sheet (gis) was developed using moderate resolution imaging spectroradiometer (modis) data from the terra and aqua satellites [hall et al ., the record extends from march 2000 through the present, providing daily and monthly average ist, and melt maps at 6.25 6.25 km resolution . Based on this modis ist record, years experiencing major melt (defined as melt covering 80% or more of the ice sheet surface) have occurred twice since 2000 [hall et al ., the most unusual melt event occurred on 1112 july 2012 and was unprecedented during this and the previous century, covering 99% of the ice sheet surface including areas> 3000 m at summit station (figure 1a) according to data from multiple satellite sensors [nghiem et al ., 2012]. Melt this extensive had not occurred since 1889 (+ /1 year) according to ice core records [nghiem et al ., 2012; clausen et al ., another large melt event occurred on 29 july 2012, where 79% of the surface experienced some melt according to data from multiple satellite sensors [nghiem et al ., the cumulative melt during the 2002 melt season covered> 87% of the ice sheet surface according to modis ist clear - sky data [hall et al ., extent of melt on the greenland ice sheet for (a) 1 january to 31 december 2012 (days 1366) and (b) 1 january to 30 august 2013 (days 1243) as determined from modis - derived melt maps . A maximum of 95% of the ice sheet surface (shaded red) experienced some melt in 2012 and only 49% of the ice sheet surface experienced some melt in 2013 . White represents no melting (according to modis), and green represent non - ice covered land areas . Elevation contours are shown at 1500, 2000, 2500, and 3000 m. the negative phase of the north atlantic oscillation (nao), with a high - pressure anomaly over the gis, has previously been implicated [mote, 1998] in enhancing melting of the surface of the gis . Also, the 2012 melt event was associated with a high - pressure ridge over the gis [nghiem et al ., 2012; tedesco et al ., 2013; hanna et al ., a high - pressure ridge brings relatively warm southerly winds over the western flank of the ice sheet causing widespread surface melting [nghiem et al ., a high - pressure ridge also represents atmospheric blocking, which is a long - lived (5 days or longer) atmospheric circulation system with strong meridional flows embedded within the latitude belt of westerlies [tibaldi and molteni, 1990; tibaldi et al ., 1997] (definition also shown in the supporting information). Blocking in the north atlantic sector is usually associated with the negative phase of the nao, and known as a greenland blocking episode (gbe) [fang, 2004; woollings et al ., gbes have a continuum of behavior, from being relatively frequent but weak events, to longer and stronger events that better conform to the conventional interpretation (and definition) of midlatitude blocking . Here we are also interested in shorter than 5 day blocking activity, because we anticipate that even a 2 day burst of warm subtropical air over the gis could lead to melting . These short events are called local and instantaneous blocking (lib), if on any day a longitude is blocked based on the reversal of the gradient in the 500 hpa geopotential height field [tibaldi and molteni, 1990; tyrlis and hoskins, 2008]. Some of the libs belong to gbes if they are spatially stationary for 5 days or more . We focus here on daily variability of melt and atmospheric conditions instead of seasonal variability . Both libs and gbes are accompanied by warm air temperatures and we will show that are both capable of initiating ice sheet melt . We will also show that daily air temperature at about 5 km height, about 2 km above the ice sheet, varies in - phase with modis ist in june and july, the months most likely to have intense melt events . Finally, we will discuss the relationship between gis melt and blocking and associated temperature variability . We use the modis clear - sky ist data record (20002013) to calculate gis melt . For the retrieval of modis clear - sky ist, a split - window technique is used, where split - window refers to the brightness - temperature difference in the 1112 m atmospheric window . The technique was first used to determine ist in the arctic with advanced very high resolution radiometer (avhrr) data on noaa polar - orbiting satellites [key and haefliger, 1992] and later adapted for use with modis . Using modis ist we quantify number of melt days and areal extent of melt for each year of the study (figure 1). Melt cloud cover is determined from the standard modis cloud mask of ackerman et al . . To partly compensate for the effects of cloud cover, for this work we employ a cloud - gap filling algorithm (see supporting information) to minimize the impact of cloud cover . As in previous work [hall et al ., 2012, 2013], we also classify an ist grid cell as melt if the surface temperature is 1c . This temperature has been found to be representative of melting conditions over the gis, in consideration of modis ist measurement uncertainty of + /1c at the high (near 0c) values of ist over ice [hall et al ., we utilize the national centers for environmental prediction / national center for atmospheric research (ncep / ncar) reanalysis data [kalnay et al ., 1996] to analyze daily average 500 hpa geopotential height (z500) and temperature (t500), and daily average air temperature at 2 m (t2 m), all of which are derived from 6-hourly data . The reanalysis data resolution is 2.5 2.5. to compute area - average temperatures, t500 and t2 m were interpolated to the modis ist grid of 6.25 km and constrained by the modis ice sheet mask . Blocking is computed by searching reversals of the gradient in the daily (average) 500 hpa geopotential height at each grid longitude and latitude in a region 20w60w, 50n85n instead of over fixed latitudes [tibaldi and molteni, 1990] (supporting information). The maximum area that experienced at least 1 day of melt during the melt period in 2012 and 2013 is shown in figures 1a and 1b, as determined from the modis ist product . The contrast in the extent of melt between 2012 and 2013 is striking, with modis - derived melt covering 95% of the ice sheet surface in 2012 and 49% in 2013 . (by combining modis with microwave sensors that can detect melt through cloud cover, the 2012 melt extent actually covered 99% of the ice sheet surface [nghiem et al ., 2012] as discussed previously .) Using available atmospheric ncep / ncar reanalysis fields, we can illuminate the differences in the synoptic regime between these two consecutive years and illustrate the relationship between atmospheric patterns and melt over the entire 20002013 melt season modis ist record on the daily time scale . The modis - derived daily melt area is shown in figure 2 for june and july . Modis melt data are not plotted if the gap - filled cloudiness is> 11% of the ice sheet area . (this cutoff of 11% was determined by visual scanning of the cloudiness data and thus appeared to be a logical cutoff value . Though it is a subjective choice, the results do not change significantly if we select a cutoff number between 10 and 15% .) Daily percent melt fractions do not reach as high as quoted above for the cumulative area of melt because the melt location varies day - to - day . The 14 year june - july mean daily melt percentage is 13% and the standard deviation (sd) is 8% (based on 834 daily values excluding modis data gaps). The maximum 1 day percent melt of 71% (clear - sky) occurred on 13 july 2012 . The only other days with melt percentage over 40% occurred on 29 june 2002 (46%), and 1112 july (49% and 62%), and 1416 july (63%, 54%, and 41%) 2012 . Low melt years in the modis record are the following: 2000 (potentially 2001; based on atmospheric temperatures, shown later), 2003, 2008, 2010, and 2013 . In each of the low melt years, the average june - july melt fraction was less than or equal to the 14 year june - july mean . The low melt years 2000 (2001), 2003, 2008, and 2010 occurred during a negative or neutral nao phase (table 1 and the nao index in figure s1 in the supporting information), which should have favored increased melt . The low melt in 2013, however, was associated with a positive nao index as expected . Wrong sign is the extensive melt year 2002, which was dominated by a positive phase of the nao (table 1). Thus, the gis melt - nao relationship is not consistent during the modis years, and when using daily nao index data, the nao index explains only 15% of the gis melt variance . Total melt area percentage over the area of the greenland ice sheet (y axis,%) for june and july for each year, 20002013, derived from daily modis ist data . Nao index versus the total gis melt percentage from modisa modis data updated from hall et al . ; nao index from noaa / climate prediction center . Minimal melt years are in italics . To identify the atmospheric pattern associated with the intense melt events in the modis record on a daily time scale, we analyzed daily june - july z500 and t500 fields for 20002013 . We composited these fields based on the daily anomaly in the modis melting fraction as an index time series . The daily modis melt anomalies were derived, and the modis melt index was formed, by normalizing the anomaly time series by its sd: we selected z500 and t500 fields from the days when the melt index exceeded 1 sd (in absolute terms). This approach groups the selected z500 and t500 fields to positive melt index anomaly days (120 fields) and negative melt index anomaly days (113 fields). The composited fields show that the large melt events are associated with a meander in the z500 field resembling an omega - block (pattern, see figures 3a and 3b) over greenland, with lows flanking the high - pressure domain . Minimal melt anomalies are associated with a more - or - less zonal flow over the ice sheet (figures 3c and 3d). The warmest intrusion of the subtropical air masses envelopes both western and eastern flanks of the gis south of about 75n . However, the influence of blocking on temperatures extends far outside this core region as a subtropical regime has moved northward pushing cold air masses far into the arctic . The 20002013 composite of 500 hpa (a, c, and e) geopotential heights (meters) and (b, d, and f) temperatures (c) when the modis melt (shown in figure 2) anomaly is stronger than + 1 standard deviation (figures 3a and 3b), and less than 1 standard deviation (figures 3c and 3d). 3a and 3c, z500 = 5600 m is drawn as a white contour; also, in figure 3a, maximum z500 = 5647 m is marked . While it is obvious that the composited patterns corresponding to high and low melt events are significantly different, we show also the difference fields of z500 and t500 with cross hatching for areas significant at the 99% level (figures 3e and 3f). The z500 difference field has a strong positive anomaly over greenland but lacks a strong negative height field anomaly at the midlatitudes across the north atlantic, typical of the negative nao [barnston and livezey, 1987]. This is consistent with a weak relationship between modis melt area variations and the nao index . Comparing the blocked flow with the zonal flow (figures 3c and 3d), the upper air temperature anomaly over greenland reaches almost 10c during these subtropical air mass intrusions . Another approach to show the impact of the subtropical intrusions is to analyze the daily area - average modis ist, t2 m, and t500 variability . We created an anomaly time series for each of these quantities and then normalized them by their sd (anomaly and sd with respect to 20002013 june - july average, sd based on all days in each time series) giving sds of 2.5c, 2.1c, and 2.9c for ist, t2 m, and t500, respectively . The normalized area - average modis ist, t2 m, and t500 are displayed in figure 4 for june - july of each year . The daily temperature variations from the ice sheet surface to the upper atmosphere, about 2 km higher than summit station, vary in - phase and in most years with similar normalized amplitude . Previously, box and cohen showed this relationship between surface and tropospheric temperatures on seasonal and annual time scales from the coastal radiosonde measurements . (in their data, the relationship was not as strong in the stations on the eastern side of greenland .) The tight coupling between the surface and upper atmosphere shows that during the period of high insolation, the only preconditioning necessary for melt is an intrusion of subtropical air . Here we note that this relationship is the strongest for june - july; adding august to the time series does not affect the timing of the variations but changes slightly the relationship between surface and upper air temperatures by increasing the differences in amplitudes (not shown). Area - average modis ist (black), t2 m (pale blue), and t500 (pink) temperature anomalies and modis melt anomaly (in orange) (referenced to 14 year june - july averages), which are then normalized by their individual standard deviation (y axis measures sds). During 2012 summer normalized melt anomaly reached 7 standard deviations (beyond the y axis upper limit). Blocking days (green bars) and all days when the 500 hpa geopotential height gradient is reversed (blocking + wave breaking events, blue bars) are shown for june the blocking and wave breaking events do not always start and end at the same day, because not all gradient reversals occurred at the same locations on the ice sheet (and lasting 5 days). In figure 4 we have also identified days of libs (there is at least one longitude that is blocked at least 1 day), of which some belong to gbes, if the condition of z500 gradient reversal existed at least in one grid point for 5 days or more . Considering first the temperature anomalies above 1.5 sds, these warm events occur when they overlap with libs and/or gbes with the spatial pattern depicted in figures 3a and 3b . There are two exceptions: on 1618 july 2004 and 1315 june 2011, with gis melt reaching 2831% (2 sds above 14 year june - july mean of 13%; sd = 8%). In both cases the z500 gradient reversal occurred but the westerlies north of the blocking high were too weak to classify them as libs by our definition; however, the circulation associated with their z500 pattern favored bringing subtropical air masses over greenland (figure s2). In the case of weaker warming events with 11.5 sds, the warming - blocking relationship also holds with three exceptions23 july 2003, 78 july 2011, and 1315 july 2011 . During these three events, the gis melt percentage either did not reach above 1 sd (15% in 2003) or did so marginally (22% in 2011). These three marginal warming / melting events are not classifiable by our blocking definition because they had complex patterns, involving a cutoff low with or without a ridge over the gis (figure s3). Only a few libs / gbes occurring in the first half of june will result in a large melt event . If melt occurs, the temperature anomaly, whether surface or upper air, has to be about 1.5 sds above the summer average (e.g., in 2002, 2005, and 2012). Furthermore, we can estimate that area - average temperature anomalies of almost 2 sds above the summer average are needed to reach a melt fraction over 40% as happened in 2002 and 2012 (figures 2 and 4). Once temperature anomalies reach 2 sds (56c), the melt amplifies almost exponentially (figure s4). Even if the blocking activity does not lead to a melt event figure 4 shows that june - july 2007 had the most blocking days but did not have the largest melt, although 2007 has been identified as a large melt year in a seasonal sense [mote, 2007; tedesco, 2007]. The melt was largely confined to the southern part of the ice sheet, but figure 4 also shows that the 2007 area - average temperature anomalies barely reached 1.5 sds (only once in the end of june). If compared especially to 2012 with a long - lasting anomaly of 22.5 sds at the peak melt 1015 july, the potential of 2007 to be an extreme melt year evaporated due to the weak temperature anomalies in the subtropical air masses . We conclude that the total number of days with blocking over greenland does not necessarily correlate with the most melt area, but the associated temperature anomalies are equally, if not more, important . Previously studies have shown that blocking can be associated with increased greenland ice sheet melt on seasonal time scales [e.g., fettweis et al ., 2013], and here we show, using modis and atmospheric reanalysis (ncep / ncar) data, that the relationship holds also on a daily time scale . Our analysis of all 14 melt seasons (20002013) shows that blocking that occurs in a range of time scales, from short - term blocking activity (<5 days) (libs) to full - fledged gbes (blocking for 5 days or longer), can bring warm subtropical air masses over the gis to instigate melt . Despite the overall close relationship between gbes / libs and ice sheet melt, not all blocking activity leads to melt as exemplified in the summer 2007 which had the most june - july blocking days during the period 20002013 . In addition, the surface and upper air temperature anomalies, computed here from area - average temperatures, play an important role during the largest melt years such that temperature anomalies have to reach almost 2 standard deviations to result in a melt area above 40% as happened in 2002 and 2012 . In the case of summer 2007, the temperature anomalies barely reached 1.5 standard deviations; hence, the summer 2007 melt did not achieve extreme melt status.
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Abnormal amounts of maternal circulating glucocorticoids (gcs), which can be induced by conditions such as chronic maternal stress, may produce protracted neurobehavioral alterations in the offspring . It has been shown that maternal prenatal (but not postnatal) emotional stress is linked to abnormal infant affective reactivity at 4 mo of age (1) and reductions in gray matter density in various brain regions, including the prefrontal, premotor, temporal, and cerebellar cortices, at 69 yr of age (2). Similar results have been reported in animal models of prenatal stress, with offspring showing long - term anxiety - like behaviors and delayed dendritic morphology in prefrontal (3), hippocampal (4), and cerebellar purkinje cells (5). Because stressful conditions produce elevated levels of circulating gcs, neurobehavioral impairments have been attributed to the toxic effects of excessive circulating prenatal gcs (6,7,8). In our laboratory, we have systematically studied the effect of controlled prenatal synthetic gc (sgc) administration (betamethasone, bet) to the mother on behavioral and neuronal development in the offspring . We have observed that administering bet to pregnant rats at a therapeutic dose equivalent to that administered to women who are at risk of preterm delivery produces a significant decrease in dendritic arborization in both dentate granule and cerebellar purkinje cells (9, 10). However, it is unknown whether similar neuronal changes to pyramidal cortical cells occur during the early and late postnatal periods . Thus, in the current study, we analyzed the impact of prenatal bet administration (0.17 mg / kg) (11,12,13) on the dendritic growth of layer ii / iii pyramidal cells using the golgi - stained procedure along with histochemical staining of the dendritic marker microtubule - associated protein 2 (map2) in infant (postnatal day 22, p22), adolescent (p52), and young adult (p82) rats . Eleven pregnant multiparous sprague - dawley rats were housed under controlled environmental conditions (temperature, 20 1 c; 12h:12h light - dark cycle) with food and water available ad libitum . Pregnant animals were placed in individual cages (45 cm 25 cm 20 cm), and gestational day 0 (g0) was determined by the presence of sperm detected in vaginal smears . Rats were randomly classified into the following two groups: control - saline (con, n = 6) and betamethasone - treated (bet, n = 5). Bet - treated rats were given a single course of betamethasone phosphate subcutaneously (0.17 mg / kg of body weight in the dorsal neck region; cidoten, schering - plough, inc ., santiago, chile) on gestational day 20 (g20), with the doses separated by an 8-hour interval . Importantly, this rodent developmental stage corresponds approximately to a human fetus at 2836 gestational wk (14). Furthermore, according to the rat s bet pharmacokinetics and pharmacodynamics, the dose used in the current and previous studies was equivalent to that administered to a woman who is at risk of preterm delivery (11, 13). After the pre - weaning period (postnatal day 22, p22), the con (n: 23) and bet (n: 28) males were weaned and rearranged to house 23 animals per cage . All procedures involving animals were approved by the local animal ethics committee and were in accordance with the guide for the care and use of laboratory animals (institute for laboratory animal research, national research council, washington dc, 2011). All male animals were deeply anesthetized with pentobarbital (50 mg / kg of body weight; sigma - aldrich, co., 3050 saint louis, misuri, usa), and intracardiac perfusion was performed with 0.9% nacl followed by 4% paraformaldehyde (sigma - aldrich). Each brain was carefully dissected under a stereoscopic lens and freshly weighed (g) on a digital analytical balance (sartorious, entries 224). Additionally, body weight (g) was assessed using a classical balance (radwag - wtb200). Parietal slices were golgi - stained (see 15 for further details), stabilized with collodion solution (fluka, sigma - aldrich), and sliced into 120-m thick sections . Pyramidal cells were selected according to the following criteria: (1) having a well - defined pyramidal shape, (2) having a single apical dendrite oriented perpendicularly to the pial surface, (3) demonstrating an adequate staining of the soma and dendrites, (4) having no extensive processes overlapping neighboring neurons, and (5) located in a cortical strip at 350600 m under the pial surface (cortical layers ii / iii were delimited by the stereotaxic coordinates as described in paxinos and watson, 1998) (16). Selected neurons were then photographed (400) and digitized, and the basilar dendritic length (m) per neuron was quantified . The number of pyramidal cells assessed at each stage was as follows: con - p22: 178; bet - p22: 135; con - p52: 120; bet - p52: 160; con - p82: 160; and bet - p82: 160 . The contralateral hemisphere of each animal was post - fixed for one hour and stored in 30% sucrose at 4 c for 7 d (cryoprotection). For the immunohistochemical procedure, the parietal zone was sliced into 20-m - thick sections with a cryostat thermo scientific microm hm525 (walldorf baden - wurtemberg, germany), 46 sections per rat . Sections that had been previously attached to a coded slide were washed twice in phosphate - buffered saline (pbs) for 10 min per wash at 90 rpm and then incubated with 0.5% h2o2 (merck) for 30 min at room temperature . After two additional washes in pbs, the sections were blocked for 1 h with 3% bovine serum albumin (bsa; sigma - aldrich) and 0.4% triton x-100 (sigma - aldrich). The primary antibody used was monoclonal anti - map2 (m4403, sigma - aldrich). The sections were incubated with the primary antibody in blocking solution overnight at room temperature and under agitation (40 rpm). The tissue was then washed three times with pbs and incubated in 1.5% bsa and 0.2% triton x-100 for 2 h at room temperature and under agitation (40 rpm). The secondary antibody used was conjugated goat anti - affine pure rabbit igg (h + l) for map2 incubation (1:500) diluted in 1.5% bsa and triton x-100 at 0.2% for 2 h at room temperature, without agitation . To visualize the labeled parietal map2 expression, an avidin - biotin peroxidase complex (vectastain elite abc kit; vector laboratories) was prepared in 1.5% bsa and triton x-100 and incubated for one hour prior to addition to the substrate coupled with diaminobenzidine (dab) for 20 min without stirring (immpact dab peroxidase substrate; vector laboratories). The sections were finally washed in distilled water for 10 sec, attached to coded slides, air - dried, enclosed with entellan (merck), and coverslipped . Images from the parietal cortex coronal sections were captured with a bioblue model, bb.1153.pli, euromex microscope, considering the same cortical region used in the golgi - stained cortical tissue (located at 350600 m under the pial surface, corresponding to approximately layers ii / iii). Map2 immunoreactivity was evaluated with imagej software (nih, bethesda, md, usa) using grayscale images (% of controls, arbitrary values). The number of brain sections assessed per animal was as follows: con - p22, n: 4; bet - p22, n: 3; con - p52, n: 4; bet - p52, n: 4; con - p82, n: 3; and bet - p82, n: 3 . A one - way analysis of variance (anova) and scheff post - hoc test were used to analyze the basal dendritic length per neuron . For immunohistochemical data, we used the kruskal - wallis (kw) non - parametric test . Eleven pregnant multiparous sprague - dawley rats were housed under controlled environmental conditions (temperature, 20 1 c; 12h:12h light - dark cycle) with food and water available ad libitum . Pregnant animals were placed in individual cages (45 cm 25 cm 20 cm), and gestational day 0 (g0) was determined by the presence of sperm detected in vaginal smears . Rats were randomly classified into the following two groups: control - saline (con, n = 6) and betamethasone - treated (bet, n = 5). Bet - treated rats were given a single course of betamethasone phosphate subcutaneously (0.17 mg / kg of body weight in the dorsal neck region; cidoten, schering - plough, inc ., santiago, chile) on gestational day 20 (g20), with the doses separated by an 8-hour interval . Importantly, this rodent developmental stage corresponds approximately to a human fetus at 2836 gestational wk (14). Furthermore, according to the rat s bet pharmacokinetics and pharmacodynamics, the dose used in the current and previous studies was equivalent to that administered to a woman who is at risk of preterm delivery (11, 13). After the pre - weaning period (postnatal day 22, p22), the con (n: 23) and bet (n: 28) males were weaned and rearranged to house 23 animals per cage . All procedures involving animals were approved by the local animal ethics committee and were in accordance with the guide for the care and use of laboratory animals (institute for laboratory animal research, national research council, washington dc, 2011). All male animals were deeply anesthetized with pentobarbital (50 mg / kg of body weight; sigma - aldrich, co., 3050 saint louis, misuri, usa), and intracardiac perfusion was performed with 0.9% nacl followed by 4% paraformaldehyde (sigma - aldrich). Each brain was carefully dissected under a stereoscopic lens and freshly weighed (g) on a digital analytical balance (sartorious, entries 224). Additionally, body weight (g) was assessed using a classical balance (radwag - wtb200). Parietal slices were golgi - stained (see 15 for further details), stabilized with collodion solution (fluka, sigma - aldrich), and sliced into 120-m thick sections . Pyramidal cells were selected according to the following criteria: (1) having a well - defined pyramidal shape, (2) having a single apical dendrite oriented perpendicularly to the pial surface, (3) demonstrating an adequate staining of the soma and dendrites, (4) having no extensive processes overlapping neighboring neurons, and (5) located in a cortical strip at 350600 m under the pial surface (cortical layers ii / iii were delimited by the stereotaxic coordinates as described in paxinos and watson, 1998) (16). Selected neurons were then photographed (400) and digitized, and the basilar dendritic length (m) per neuron was quantified . The number of pyramidal cells assessed at each stage was as follows: con - p22: 178; bet - p22: 135; con - p52: 120; bet - p52: 160; con - p82: 160; and bet - p82: 160 . The contralateral hemisphere of each animal was post - fixed for one hour and stored in 30% sucrose at 4 c for 7 d (cryoprotection). For the immunohistochemical procedure, the parietal zone was sliced into 20-m - thick sections with a cryostat thermo scientific microm hm525 (walldorf baden - wurtemberg, germany), 46 sections per rat . Sections that had been previously attached to a coded slide were washed twice in phosphate - buffered saline (pbs) for 10 min per wash at 90 rpm and then incubated with 0.5% h2o2 (merck) for 30 min at room temperature . After two additional washes in pbs, the sections were blocked for 1 h with 3% bovine serum albumin (bsa; sigma - aldrich) and 0.4% triton x-100 (sigma - aldrich). The primary antibody used was monoclonal anti - map2 (m4403, sigma - aldrich). The sections were incubated with the primary antibody in blocking solution overnight at room temperature and under agitation (40 rpm). The tissue was then washed three times with pbs and incubated in 1.5% bsa and 0.2% triton x-100 for 2 h at room temperature and under agitation (40 rpm). The secondary antibody used was conjugated goat anti - affine pure rabbit igg (h + l) for map2 incubation (1:500) diluted in 1.5% bsa and triton x-100 at 0.2% for 2 h at room temperature, without agitation . To visualize the labeled parietal map2 expression, an avidin - biotin peroxidase complex (vectastain elite abc kit; vector laboratories) was prepared in 1.5% bsa and triton x-100 and incubated for one hour prior to addition to the substrate coupled with diaminobenzidine (dab) for 20 min without stirring (immpact dab peroxidase substrate; vector laboratories). The sections were finally washed in distilled water for 10 sec, attached to coded slides, air - dried, enclosed with entellan (merck), and coverslipped . Images from the parietal cortex coronal sections were captured with a bioblue model, bb.1153.pli, euromex microscope, considering the same cortical region used in the golgi - stained cortical tissue (located at 350600 m under the pial surface, corresponding to approximately layers ii / iii). Map2 immunoreactivity was evaluated with imagej software (nih, bethesda, md, usa) using grayscale images (% of controls, arbitrary values). The number of brain sections assessed per animal was as follows: con - p22, n: 4; bet - p22, n: 3; con - p52, n: 4; bet - p52, n: 4; con - p82, n: 3; and bet - p82, n: 3 . A one - way analysis of variance (anova) and scheff post - hoc test were used to analyze the basal dendritic length per neuron . For immunohistochemical data, we used the kruskal - wallis (kw) non - parametric test . Golgi - stained layer ii / iii pyramidal cell basal dendrites located in the parietal cortex of animals treated prenatally with bet showed a significant reduction in length compared with the age - matched con animals at both p52 and p82 (fig . 1. (a) mean basilar dendritic length per neuron in golgi - stained superficial (layer ii / iii) pyramidal cells . P22, p52, p82: postnatal days 22, 52, and 82, respectively . The data are presented as the mean sem (* * p<0.01, * * * p<0.001, one - way analysis of variance). (b) representative photomicrographs of golgi - stained pyramidal cells (layers ii / iii) from con and bet animals . Bar: 40 m . ; * * p<0.01, * * * p<0.001, anova). 1b shows representative layer ii / iii pyramidal cells from each condition and age . (a) mean basilar dendritic length per neuron in golgi - stained superficial (layer ii / iii) pyramidal cells . P22, p52, p82: postnatal days 22, 52, and 82, respectively . The data are presented as the mean sem (* * p<0.01, * * * p<0.001, one - way analysis of variance). (b) representative photomicrographs of golgi - stained pyramidal cells (layers ii / iii) from con and bet animals . However, animals exposed to prenatal bet showed a significant reduction in map2 immunohistochemical expression at both p22 and p52 but not at later ages (p82) (fig . 2afig . Con: control group; bet: betamethasone group; p22, p52, p82: postnatal days 22, 52, and 82, respectively . The data are shown as mean sem and are presented as a percentage of the control value (* p<0.05, kruskal - wallis). (b) representative photomicrographs of the parietal cortical tissue stained with anti - map2 antibodies from con and bet animals . 2b shows representative neocortical (parietal) micrographs from con and bet offspring with the immunohistochemical map2 labeling of superficial (layers ii / iii) pyramidal cells at p22, p52, and p82 . Con: control group; bet: betamethasone group; p22, p52, p82: postnatal days 22, 52, and 82, respectively . The data are shown as mean sem and are presented as a percentage of the control value (* p<0.05, kruskal - wallis). (b) representative photomicrographs of the parietal cortical tissue stained with anti - map2 antibodies from con and bet animals . In the current study, we showed that a single course of prenatal bet causes a reduction in basilar dendritic length per neuron in layer ii / iii neocortical pyramidal cells and a transient reduction in neocortical histochemical map2 immunoreactivity . Consistent with our previous studies in the rat cerebellar cortex, adolescent (p52) and young adult (p82) animals exposed prenatally to a single course of bet showed a significant reduction in basal dendritic trees in the superficial neocortical pyramidal neurons (layers ii / iii). The long - term morphological alteration of dendritic development is the most consistent result observed in our studies . For example, dentate granule cells of adolescent rats (p52) treated with prenatal bet (g20; 0.17 mg / kg) showed a significant reduction (35%) in total dendritic length compared with age - matched controls (9). A similar reduction in dendritic domain (39%) has been observed in cerebellar purkinje cells at p52 in offspring treated antenatally with bet (10). In both studies, the neuronal changes were related to significant impairments in spatial memory tasks along with anxiety - like behaviors . Although we did not evaluate functional variables, it is possible that the reduced dendritic arborization could alter pyramidal cell function because the majority of synaptic connections, transmission, and signal integration significantly depend on the number, length, and complexity of the cell s dendritic branching (17). Moreover, since the glucocorticoid receptor (gr) is highly expressed in layers ii / iii cerebrocortical neocortical neurons (18), the morphological changes in pyramidal cells reported here are probably due to long - term effects of prenatal sgc administration on cortical neuronal maturation . The results of the current study in conjunction with previous studies on dendritic morphometry indicate that prenatal sgc administration could produce subtle microstructural changes that may be related to the long - term behavioral and cortical findings described in children whose mothers experienced stressful prenatal conditions (1, 2). Moreover, map2 is the most predominant cytoskeletal protein isoform present in dendritic branches; it contributes to dendritic growth and plasticity (19, 20). Regarding the results obtained in the present study, although we observed that the reduction of map2 in the neocortex of animals exposed prenatally to bet is protracted (infant and adolescent rats), we did not observe significant differences when the animals reached young adulthood (p82). This transient reduction in map2 is consistent with our observation in previous studies carried out in cerebellar neurons at the same ontogenetic age (p52, 56%) (12). It should be noted that the mere transience of the reduction in immunohistochemical expression of map2 does not rule out the possibility that map2 underexpression changes the course of neuronal dendritic maturation and causes permanent structural dendritic developmental impairments, as observed in the current golgi - stained layer ii / iii pyramidal cells . However, because immunohistochemical map2 staining is an indirect measure of protein expression, it is difficult to determine whether the transient map2 reduction observed in bet - treated animals clearly reflects quantitative changes in map2 protein expression . To make this differentiation, it is necessary to complement the immunohistochemical approach used in the current work with other quantitative methods such as western blot analysis . In addition, since our previous studies carried out in the cerebellar cortex indicated significant changes in the immunohistochemical expression of bdnf and its trkb receptor, along with map2, in the purkinje cells of animals exposed to prenatal bet, it is not possible to rule out that the changes observed in the present study also involve changes in these neurotrophic variables . In conclusion, the present data indicate that administration of prenatal bet to pregnant dams at g20 is associated with a long - term reduction in the basilar dendritic length of cortical pyramidal cells and a transient reduction of the immunohistochemical expression of map2 in superficial ii / iii neocortical cells of the offspring.
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Derangements of the condyle - disc complex arise from a breakdown of the normal rotational movement of the disc on the condyle . The thinning of the posterior border of the disc can cause the disc to be displaced in a more posterior position . With the condyle resting on a more posterior portion of the disc or retrodiscal tissues, an abnormal translatory shift of the condyle over the posterior border of the disc can occur during the opening . A click is associated with the abnormal condyle - disc movement and may be initially felt just during opening (single click) but later may be felt during opening and closing of the mouth (reciprocal clicking).1 molinari et al.2 reported that occasionally a second clicking sound is heard during mouth closure (reciprocal click), because the posterior band of the disc slips forward off the condyle . Other clicking sounds can also be produced by irregularities or defects in the surface of the disc or by changes in the convexity of the condylar and/or articular eminence . They are also found at the same point of the temporomandubular joint (tmj) traslator movement rather than at different points, as occurs with reciprocal clicking . Clicking and crepitation should be considered signs of morphological alterations, being indicative of anterior disk displacement with reduction3 and arthrosis, respectively . Electrovibratographic records and macroscopic examinations of articulations of corpses showed that 20% of the tmjs with clicking had the disk displaced anteriorly and 22% of the tmjs with crepitation had arthrosis or disk perforation.4 later recapture of the disk causes clicking at the end of mouth opening and indicates that the bilaminar zone is more affected.5 the microscopic aspects of the disk surface can also be altered.6 qualitative and semi - quantitative methods have been developed for tmj sound classification, but the criteria presented are completely inhomogeneous.7 - 12 thus, to develop more objective criteria for defining tmj sounds, electroacoustical systems have been developed.7 - 9, 11 - 15 we used joint vibration analysis (jva) in the biopak system (bioresearch inc ., milwaukee, usa) as the electrovibratography, and jaw tracker (jt)-3 device in the biopak system (bioresearch inc ., milwaukee, usa). Using jt-3 deivce allowed the computer to estimate where a joint vibration occurs in the open / close cycle and let us distinguish tooth contact from joint sound precisely . Ishigaki et al.17 reported a disc displacement with reduction generates a " click " in the lower frequencies (under 300 hz) and a degenerative condition generates " crepitus " in the higher frequencies (over 300 hz). In the previous study, we found that in an integral> 300 hz / <300 hz ratio it is conceivable that the higher the integral> 300 hz / <300 hz ratio number, a more advanced degenerative condition exists . Gallo et al.16 reported that tmj clicking was subjectively and objectively stable over a period or 10 days . We found few studies about long term follow - up based on the frequency spectrum patterns associated with the integral> 300 hz / <300 hz ratio . The aim of this study was to examine the tmj sounds with repect to frequency spectra patterns and the integral> 300 hz / <300 hz ratios via six - months follow - up . Twenty dental school students (18 males and 2 females: age range 25 - 34 years old; mean age = 22.4 years old) participated in the before (control group) and after (experimental group) the six - months joint sound recordings . Group i (8 males and 2 females) was composed of the subjects that showed anterior disk displacement with reduction . They were selected by means of clinical examinations . As inclusion criteria, all subjects had clicking in both tmjs upon mouth opening and/or closing and a normal range of jaw movement during opening and/or pain at palpation (any of the masseter, temporalis, pterygoid, digastric muscles) and jaw movement during chewing . Group ii (10 males) was composed of subjects that showed a normal state of tmj . They showed absence of tmj noises, pain at palpation (any of the masseter, temporalis, pterygoid, digastric muscles) and jaw movement or chewing . In each subject, a magnet was attached to the labial surface of mandibular incisors of the subjects in order to bring the midline of the magnet to the labial frenum and to locate the groove of the magnet to the left side of the subjects . If the subject tended to have a deep bite so that it is impossible to attach the magnet, it was attached to the labial gingival surface or lingual tooth surface . One transducer was placed on the skin over the right tmj, and the other over the left tmj . Once the horizontal and vertical standard points were set, we controlled them to fit with the subjects'heads . The bar of the front side was kept parallel to the interauditory axis and the lateral side to the frankfort horizontal plane . The accessory bar for approaching the magnet was fixed temporally and operated in order to set the exact midline . As the subject performed metronome - guided maximun active opening / closing with the jva, the condyles rubbed against the various surfaces in the joint, creating characteristic vibrations which are then, in turn, detected by the accelerometers, which convert those specific vibrations into an electronic signal . The signal from the accelerometers is amplified by a small, light - weight amplifier which is placed around the patient's neck . The amplified signals are then transmitted to a pc computer where they are recorded and analyzed with a software program, then displayed on a crt . After the best recording was selected from three, vibrations showing the highest amplitude were screened priorly . When we excluded tooth contact precisely, reproducible joint sound was analyzed for each opening & closing cycle . After subject selection, the largest vibration amplitude consistently occurring in each joint recording was used to calculate frequency spectrum computed by the fast fourier transform (fft) algorithm . The numeric values that are calculated and displayed in the jva summary view are based on the absolute frequency spectra . The frequency spectra view plots amplitude (vertical axis) versus frequency (horizontal axis). The height of the curve is directly proportional to the energy of the spectrum at each frequency . Two spectra are plotted for each side: the smaller of the two represents the absolute magnitude of the vibrations'spectra as recorded (n / m), the larger one has been scaled to the maximum range (at the recorded amplification) and is known as the relative plot . The relative plot accentuates features that may not be visible in the absolute plot (fig . The integral> 300 / <300 ratios in group i and group ii listed in table i and ii show variations before and after the six - months recordings respectively . In group ii, 17 - 20 showed integral> 300 / <300 ratios disappeared six - months later . The integral> 300 / <300 ratios and the frequency spectra were analyzed in all subjects . The frequency spectra in some subjects showed similar patterns while the others showed varied patterns . By the comparative study between the integral> 300 / <300 ratios and the frequency spectrums, it was conceivable that the frequency spectrums showed similar patterns at the same location that the joint sound occurred between before and after the six - months recordings . While the frequency spectra showed varied patterns at the different location that the joint sound occurred in before and after the six - months recordings (fig . 2, 3). Disc displacement is characterized by a normal range of jaw movement during opening and eccentric movements . When reciprocal clicking is present, the two clicks normally occur at different degrees of mouth opening, with the closing click usually occuring near the intercuspal position.1 although physiological changes occur in the disc, its ability to remodel is lower than that of other tissues of the tmj, such as the capsule, capsular ligaments, and retrodiscal tissues . Decreased vascularity and extensive fibrous transformation have been reported in the retrodiscal tissue for continuous compression and shear . These adaptative changes can also have mechanical implications on the behavior of the articular disc . However, as long as the system preserves the ability to adapt to the new functional status, the altered mechanical loading is compensated for by the structural modeling of the tmj . Although the coordination of the disc - condyle complex may be lost in this stage, the patient is usually asymptomatic.2 garcia et al.19 reported that some patients present alterations in the structure of the arcitular disk located in several areas . Small vibrations in the position of the condyle may induce unstable areas with production of articular vibrations . The frequencies (in hertz), as well as the amplitude of the vibration can be expressed mathematically . The precise moment of the sound generated in the opening and closing cycles . In this study group i and group ii showed varied integral> 300 / <300 ratios before and the after six - months recordings . Also, by the comparative study between the integral> 300 / <300 ratios and the frequency spectrums, it was conceivable that the frequency spectrums showed similar patterns at the same location that the joint sound occurred before and after the six - months recordings . While the frequency spectrums showed varied patterns at the different locations that the joint sound occurred before and after the six - months recordings, it would possibly be due to the differences in the degree of internal derangement and/or in the shape of the disc . It is suggested that clinicians consider the integral> 300 / <300 ratios as well as the frequency spectrums to decide the starting - point of the treatment for tmj sounds . Therefore jva will provide the clinician with the visible patterns of tmj sounds for patient management.
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Corneal collagen cross - linking (cxl) is the only conservative therapy for keratoconus that has been demonstrated to stiffen the cornea and halt the progression of the ectasia . Cxl results in an increase in tensile strength of the cornea as a result of an interaction between riboflavin photosensitizer and ultraviolet light, which results in an increase in covalent bonding within or between collagen fibers that make up the anterior stromal lamellae . Requires debridement of the central 9 mm of the corneal epithelium to facilitate diffusion of a solution containing 0.1% riboflavin with 20% dextran t500 to the corneal stroma . Epithelium - on cxl with modified technique has been proposed to reduce the risk of complications associated with epithelial removal [3, 4]. Provided that sufficient effect is obtained, transepithelial cxl is highly desirable from both the patient's and the ophthalmologist's perspective because ideally this approach avoids the pain, risk of infection, transient visual impairment, and all other consequences and potential complications of epithelial debridement . A number of modified riboflavin formulations have been introduced to facilitate diffusion through the corneal epithelium . To our knowledge, to date, there has been no comparison of transepithelial formulations to evaluate whether these goals of transepithelial cxl are met . The purpose of this short - term study is to evaluate and compare the impact of various transepithelial riboflavin delivery protocols on the corneal epithelium in regard to pain and epithelial integrity in the early postoperative period . One hundred and sixty - six eyes of 104 subjects affected by progressive keratoconus underwent transepithelial cxl between 05/2011 and 12/2013 at the center for refractive surgery, st . Inclusion criteria included keratoconus i iii according to the amsler - krumeich classification with documented progression in the previous 12 months, defined as an increase in maximum keratometry (k max) or subjective cylinder of 1.00 diopter (d) or more or subjective deterioration of visual acuity . Exclusion criteria included endothelial decompensation, central corneal opacities, history of herpetic keratitis, active corneal infection, aphakia, concomitant ocular or systemic autoimmune disease, pregnancy, and breastfeeding . All eyes were evaluated by slit lamp examination to assess the presence or absence of any epithelial defects on each postoperative day until the eye was quiet and the epithelium was unremarkable . Visibly loose epithelium was considered as defective . On the first postoperative day, all patients were queried if they had experienced ocular pain of any level since transepithelial cxl . At every following visit the patients were again asked if they had experienced any ocular pain since the last visit . Optical coherence tomography (oct) was used to qualitatively assess riboflavin diffusion postoperatively in some patients . Riboflavin application procedure was determined by a stepwise optimization protocol using one of 6 treatment regimens . In all cases, riboflavin application and subsequent uva irradiation were performed according to manufacturer recommendations for the use of the riboflavin formulation and recommended parameters for uva irradiation . The riboflavin formulations used are presented in table 1, with the corresponding uva delivery device used for the study treatments . In all treatments, the corneal epithelium was left intact, and riboflavin application and uva treatment were performed according to one of six regimens described below and summarized in table 2 . Postoperative care included the use of a soft bandage contact lens in all of the eyes in groups 46 . No bandage contact lens was used in group 1 and no bandage contact lens was used in the first 5 of eyes of groups 2 and 3, respectively . The use of bscl was introduced after observing epithelial defects in the first 5 eyes of groups 2 and 3 in order to minimize stress on the epithelium by lid movements . In group 1, ricrolin te (sooft, italy) was applied at a rate of 1 drop every 2 minutes for approximately 30 minutes . Riboflavin was not rinsed from the cornea, and 3 mw / cm of irradiance was applied to the cornea for 30 minutes, for a total energy dose of 5.4 j / cm . During illumination the cornea was kept moist by further application of ricrolin te at a rate of 1 drop every 2 minutes . In group 2, medio - cross te (peschke meditrade gmbh, germany) was applied at a rate of 1 drop every 2 minutes for approximately 30 minutes . Riboflavin was not rinsed from the cornea, and 3 mw / cm of irradiance was applied to the cornea for 30 minutes, for a total energy dose of 5.4 j / cm . During illumination the cornea was kept moist by further application of medio - cross te at a rate of 1 drop every 2 minutes . In group 3, paracel (avedro inc ., usa) was applied at a rate of 1 drop every 60 seconds for approximately 15 minutes . Riboflavin was rinsed from the cornea using bss, and 45 mw / cm of irradiance was applied to the cornea for 2 minutes and 40 seconds, for a total energy dose of 7.2 j / cm . No further paracel was applied during illumination . In group 4, ricrolin+ (sooft, italy) was administered after applying preservative - free anesthetic eye drops 10 minutes, 5 minutes, and immediately before, while only one application of anesthetic eye drops was used in all other groups as recommended by the respective manufacturers . A circular reservoir with a surrounding annular suction ring was affixed to the cornea during the procedure . A stainless steel grid inside this reservoir served as the cathode at a minimal distance from the cornea, and an anode was affixed to the subjects' forehead . The generator was used to apply a constant current of 1 ma for a period of 5 min . After the 5-minute impregnation period, 10 mw / cm of irradiance was applied to the cornea for 9 minutes for a total energy dose of 5.4 j / cm . In group 5, a two - stage application procedure for paracel and vibex xtra (avedro inc ., paracel was applied at a rate of 1 drop every 90 seconds for 3 minutes . Additional vibex xtra was applied at a rate of 1 drop every 60 seconds for 7 minutes . A total riboflavin forty five mw / cm of irradiance was continuously applied to the cornea for 2 minutes and 40 seconds, for a total energy dose of 7.2 j / cm . In group 6, the same two - stage application procedure for paracel and vibex xtra was used as in group 5 . However, the irradiance was applied in a pulsed mode in which the uv light was alternately turned on for one second and turned off for one second . One hundred sixty - six eyes were treated with transepithelial cxl according to 6 treatment regimens, with 110 eyes in group 1, 8 eyes in group 2, 12 eyes in group 3, 10 eyes in group 4, 13 eyes in group 5, and 13 eyes in group 6 . Minimum corneal thickness was 335 m in group 1, 396 m in group 2, 367 m in group 3, 442 m in group 4, 377 m in group 5, and 460 m in group 6, respectively . There was no serious complication except for one eye in treatment protocol 2 that had a corneal infection associated with an epithelial defect . After 18 months, central visual acuity was fully restored; however, a paracentral subepithelial opacification was still visible (figure 1). No other adverse event including endothelial decompensation or endothelial damage was observed in any eye, except for epithelial damages . The incidence of postoperative epithelial defects according to treatment protocol is presented in figure 2 . Often the complete illuminated epithelium was affected leading to a detachment as an intact sheet similar to a lasek flap (figure 3). In some eyes, however, parts of it were loose and mobile over the corneal stroma leading to pain perception . The incidence of reported postoperative pain is shown in figure 4 . In all groups, reported pain was the greatest in the 24 hours following the procedure, resolved by complete epithelial healing after 14 days . Oct revealed limited or superficial hyperreflectivity in eyes treated according to the protocol for group 1 . Oct evaluation was comparable between the remaining groups, with deeper reaching hyperreflectivity observed in the corneal stroma in the postoperative period in groups 26 . Standard riboflavin formulations containing 0.1% riboflavin and 20% dextran show minimal penetration through intact or partially disrupted epithelium [6, 7]. The optimal approach for transepithelial cxl must minimize the impact on the corneal epithelium while permitting a sufficient amount of riboflavin to diffuse into the stromal tissue where cross - linking occurs . Epithelial disruption without full debridement leaves the cornea vulnerable to early postoperative infection and delays the return to gas permeable contact lens wear and visual recovery . The results of this study reveal variability in postoperative recovery following transepithelial cxl with different treatment regimens . The use of ricrolin te resulted in the least disruption of the corneal epithelium, with no epithelial defects reported in any case and minimal postoperative discomfort . However, some epithelial disruption is necessary to allow diffusion of riboflavin to the corneal stroma . Reports assessing the diffusion of ricrolin te revealed a shallow penetration of the riboflavin which may be insufficient for cross - linking [5, 8, 9]. Qualitative evaluation of the depth of the riboflavin penetration with oct revealed deeper penetration to the stroma following the remaining protocols in this study . Eyes treated with ricrolin+ and iontophoresis showed epithelial defects in 20% of eyes and pain in 50% of eyes . Based on our observation of eyes with apparently loose epithelium that leads to pain perception in the absence of an epithelial defect, we hypothesize that eyes experienced pain more often than they had epithelial defects because of subtle epithelial disruptions which were not detectable at slit lamp exam . Fifty percent of eyes in the paracel (alone) group and greater than 50% of eyes in the medio - cross te group presented with epithelial defects in the first postoperative day . Both the paracel and medio - cross te formulations contain benzalkonium chloride, which acts as an epithelial permeability enhancer . The disruptive effects of bac are both duration and concentration dependent, and therefore it is logical that reduction of the duration of exposure to bac might reduce the incidence of epithelial defects . This was the rationale for the development of the two - stage riboflavin application method employing sequential application of 0.25% riboflavin with bac (paracel) and 0.25% riboflavin without bac (vibex xtra). According to a theoretical model proposed by avedro, inc ., the initial soak with the riboflavin and bac solution is sufficient to open the epithelial junctions and to provide the initial dose of riboflavin . Once the junctions have been sufficiently loosened, further exposure to bac is not thought to provide any additional benefit, and it is flushed away . The remainder of the presoak time is completed using a bac - free, dextran - free riboflavin solution . The two - stage application appeared to be a near optimal protocol with respect to epithelial integrity, resulting in zero incidences of postoperative epithelial defects in group 5 and a reduction in the percentage of eyes experiencing postoperative pain (0%) as compared to the use of paracel alone (83%). However, when pulsed, illumination was introduced to the treatment protocol of group 5; that is, in group 6, greater pain perception was observed . We may speculate that the prolonged treatment time may lead to desiccation of the ocular surface adding to the epithelial trauma . While oct evaluation of the depth of riboflavin penetration provides evidence of the efficacy of the two - stage application protocols, a clinical means of quantifying the concentration of riboflavin in the stroma as a function of depth would have added to this study . To our knowledge therefore, longer term follow - up is necessary to evaluate the relative efficacy of these cross - linking protocols in regard to stabilization of the progression of keratoconus . In conclusion, the findings of this study suggest that different transepithelial cross - linking protocols have varying impacts on epithelial integrity . At present, it may be desirable to minimize discomfort and accept a less than maximum efficacy as the procedure may be repeated later on . In contrast, in very thin corneas, it may be an option to use an aggressive protocol to maximize efficacy even if the epithelium sloughs off postoperatively in order to have the epithelium as a protective spacer to the endothelium . Longer term outcomes of these various treatment protocols will follow and will provide insight into the selection of an appropriate treatment protocol for each of these patient scenarios.
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