This study aimed to quantify the dosimetric differences between the planned and delivered dose to tumor and normal organs in locally advanced non-small cell lung cancer (LANSCLC) treated with hypofractionated radiotherapy (HRT), and to explore the necessity and identify optimal candidates for adaptive radiotherapy (ART).Twenty-seven patients with stage III NSCLC were enrolled. Planned radiation dose was 51Gy in 17 fractions with cone-beam CT (CBCT) acquired at each fraction. Virtual CT was generated by deformable image registration (DIR) of the planning CT to CBCT for dose calculation and accumulation. Dosimetric parameters were compared between original and accumulated plans using Wilcoxon signed rank test. Correlations between dosimetric differences and clinical variables were analyzed using Mann-Whitney U test or Chi-square test.51 of planning target volume for GTV (PTV-GTV) was similar between original and accumulated plans . Only 11.1% of patients had above 5% relative decrease in V51 of PTV-GTV in accumulated plans. Compared to the original plan, limited increase (median relative increase < 5%) was observed in doses of total lung , esophagus  and heart  in accumulated plans. Less than 30% of patients had above 5% relative increase of lung or heart doses. Patients with quick tumor regression or baseline obstructive pneumonitis showed more notable increase in doses to normal structures. Patients with baseline obstructive atelectasis showed notable decrease (10.3%) in dose coverage of PTV-GTV.Patients had varied gross tumor volume (GTV) reduction by HRT . The VLANSCLC patients treated with HRT had sufficient tumor dose coverage and acceptable normal tissue dose deviation. ART should be applied in patients with quick tumor regression and baseline obstructive pneumonitis/atelectasis to spare more normal structures.Supplementary information accompanies this paper at 10.1186/s12885-020-07617-3. Definitive radiotherapy combined with chemotherapy remains the main part of treatment for patients with unresectable locally advanced non-small cell lung cancer (LANSCLC) , 2. HypoImage-guided adaptive radiotherapy (ART) has been developed to account for the anatomic variations. It has been proposed that ART may be beneficial in reducing toxicities and allowing safe dose escalation in HRT. However, it is not widely used in clinic due to its time-consuming process and low cost-effectiveness. Therefore, it is essential to quantify the dosimetric differences between delivered and planned dose to tumor as well as OAR during HRT, in order to provide more information for proper patient selection. Cone beam CT (CBCT) has been routinely used for position verification and monitoring anatomical changes during radiotherapy for NSCLC patients . HoweverIn this study, CBCT images and DIR technique were used to investigate the impact of inter-fractional anatomic changes on the dose delivered to tumor and normal structures in LANSCLC treated with HRT. By comparing the planned and delivered treatment dose, we aimed to explore the necessity and identify optimal candidates for ART.Twenty-seven patients with stage III NSCLC were enrolled from a prospective clinical trial (NCT 02573506). Patients were immobilized and simulated according to the standard protocol for lung cancer . A simulThe study was reviewed and approved by Ethics Committee of our center. All patients provided signed informed consent for scientific usage of clinical data.Patients underwent daily kilovoltage (kv)-based CBCT scan for set-up correction prior to the delivery of each radiation fraction. The CBCT scans were obtained using an Elekta VersaHD system . All scans were acquired under free breathing with patients in treatment position, which has accounted for internal movement because of the blurring of respiratory motion. The maximum scan length was 26.7 cm with a slice thickness of 3 mm. Other scanning parameters included 120 kV tube potential, 40 mA tuber current, 40 ms exposure time, 640 frames and 360° data collection. CBCT images obtained at fractions 1, 5, 9, 13, and 17 of treatment were selected for analyses in this study . First, two sets of images were manually aligned using bonny structures as reference. Then, rigid registration was performed between planning CT and daily CBCT. After that, a correction procedure was performed to apply a fade correction and enhance low signal regions of the CBCT prior to the following registration. DIR was performed using modified B-spline deformable registration with mutual information-based matching . The defThe treatment beams from the original plan were copied and applied to the isocenters of virtual CT. Doses delivered by the original plan was recalculated on virtual CT images. It was noted that all beam parameters were consistent with the original treatment plan, including isocenter, control points and monitor units.sum = (Dfrac1 + Dfrac5 + Dfrac9 + Dfrac13) × 4 + Dfrac17. The following dosimetric parameters were recorded from the original and accumulated plan respectively: PTV-GTV: volume receiving ≥51 Gy (V51), dose covering at least 95% volume (D95), mean dose; Total lung: mean dose, volume receiving ≥20 Gy (V20) and 30 Gy (V30); Esophagus: maximum dose (Dmax) and mean dose; Heart: mean dose, volume receiving ≥30 Gy (V30) and 40 Gy (V40).Delivered dose was calculated for fraction 1, 5, 9, 13 and 17 using the virtual CTs respectively. The calculated dose on the virtual CT was then deformed back to the planning CT for dose accumulation using DVF obtained in the DIR between planning CT and daily CBCT. The cumulative dose was calculated as follows: DDosimetric parameters were presented as mean ± SD. Relative dosimetric difference between original and accumulated plan was calculated as: /original plan, and was presented as median value with range. In this study, a relative change ≥5% in dosimetric parameters was considered clinically meaningful. Tumor volume and dosimetric parameters were compared between original and accumulated plans using Wilcoxon signed rank test. Correlations between dosimetric differences and clinical variables, including sex, age, tumor location, presence of bulky mediastinal lymph node, cTNM stage, presence of obstructive pneumonitis/atelectasis and tumor regression, were analyzed using Mann-Whitney U test or Chi-square test. All tests were two-sided, and p<0.05 was considered statistically significant. All the analysis was performed with SPSS (Version 22.0).The clinical characteristics of 27 patients were summarized in Table 3 at fraction 1 to 117.9 cm3 at fraction 17, and the difference was statistically significant (p<0.001). The median relative GTV reduction was 2.1, 6.2, 10.6, and 11.1% from start to fraction 5, 9, 13 and 17 of therapy, respectively. Greater tumor shrinkage was achieved from fraction 5 to 13, compared with that from fraction 1 to 5 or fraction 13 to 17 (8.5% vs. 2.1% vs. 0.5%). Detailed information about GTV volume change was shown in Table As shown in Fig. 51 of PTV-GTV were 88.2 and 87.6% in original and accumulated plans (p = 0.452), with average D95 of 48.1 Gy and 48.0 Gy (p = 0.781), respectively. In accumulated plans, only 3 patients (11.1%) showed a relative reduction in V51 above 5% when compared with original plans.As demonstrated in Table 95 and V51 of PTV-GTV were compared between fraction plans  and original plan, which was shown in Supplementary Table 95 was higher at fraction 5 compared with that at fraction 9, 13 or 17 ; the percentage of patients obtaining a reduction above 5% in V51 was higher at fraction 5 compared with that at fraction 9, 13 or 17 ; the percentage of patients obtaining a reduction above 5% in V51 was higher at fraction 9 compared with that at fraction 13 or 17 .Besides, the D20 increased from 29.5% ± 4.2% in original plans to 30.1% ± 4.1% in accumulated plans (p = 0.001), with a median relative increase of 1.6% . The total lung V30 increased from 19.9% ± 4.4% in original plans to 20.4% ± 4.4% in accumulated plans (p = 0.004), with a median relative increase of 1.9% . Significant difference in the mean lung dose was also observed in accumulated plans , with a median relative increase of 1.4% . In accumulated plans, there are 3 (11.1%) and 6 (22.2%) patients obtained a relative increase above 5% in the lung V20 or V30 when compared with original plans.Slight, although statistically significant differences were observed in the lungs’ dosage between original and accumulated plans. The total lung Vp = 0.002) and maximal doses  in accumulated plans, with median relative increase of 1.4%  and 4.1% , respectively. Ten patients (37.0%) obtained a relative increase above 5% in maximal dose.For the esophagus, an increase was observed in mean , V30  or V40  between original and accumulated plans. Seven patients (25.9%) achieved above 5% relative increase in the heart V30 and 8 (29.6%) achieved that in V40.There was no significant difference in the heart mean dose , V30  and mean dose  in accumulated plans than those who did not. Besides, in quick tumor regression group, more patients were observed to have a relative increase above 5% in the lung V20  and V30 . Tumor regression also correlated with the differences of the esophagus mean dose , but not maximal dose . No correlation was found between tumor regression and heart doses change , otherwise it was defined as slow. Patients with quick tumor regression showed more notable relative increase in the total lung Vp = 0.023), lung V30  and esophagus mean dose  was observed compared to those without. Greater increase was also observed in the total lung V20 , heart mean dose , heart V30  and heart V40 , despite none of them was statistically significant. Furthermore, in the group with baseline obstructive pneumonitis/atelectasis, more patients were observed to gain above 5% relative increase in heart V30  and heart mean dose  , except for maximal esophagus dose with a median relative increase of 4.1%. Considering that a 5% relative difference in dosimetric parameters would be clinically meaningful, we analyzed the number of patients who obtained above 5% relative reduction in PTV-GTV coverage or 5% relative increase in normal organs’ dosage. Overall, only 11.1% of patients were observed to have above 5% relative decrease in V51 of PTV-GTV. Meanwhile, less than 30% of patients achieved above 5% relative increase in lung or heart doses. According the results, non-ART strategy was acceptable for most patients treated by HRT, and only a small proportion of patients might be in need of ART during the treatment.Previous studies showed that HRT with a dedicated technique allows safety dose escalation, minimizing the effect of tumor repopulation and improving local control –18. Comp95 and V51 of PTV-GTV when comparing the fraction plan at different time point of treatment course with the original plan. The results showed that the percentage of patients obtaining a reduction above 5% in D95 and V51 of PTV-GTV were higher at fraction 5 compared with that at fraction 9, 13, or 17. Besides, a higher percentage of patients obtaining >5% reduction in V51 of PTV-GTV was also observed at fraction 9 compared with that at fraction 13 or 17. Taking together the changes in GTV volume, D95 and V51 of PTV-GTV at different time points, we recommended the fraction 9 in the course of radiotherapy as the time point for re-planning.For these reasons, the changes of GTV during treatment course need to be investigated. Previous studies found that the mean GTV reduction was about 19–51.1% in patients treated by conventional fractionated radiotherapy –25. A leFor lung cancer patients with obstructive pneumonitis or atelectasis before radiotherapy, the volume and position of lung tissue could progressively change with treatment . PreviouOther than the change of lung volume and position, mediastinal shift remained crucial for dose deviation of OARs for patients with baseline obstructive pneumonitis/atelectasis. Mediastinal shift could occur as the regression of tumor and resolution of pneumonitis/atelectasis during radiotherapy. In fact, it is difficult to directly quantify the movement of mediastinum, since it consisted of multiple organs and structures. Esophagus, as a longitudinal organ located in the middle of mediastinum, could largely reflect the motion of mediastinum. In previous study, we quantified the interfractional motion of esophagus during fractionated radiotherapy for 36 patients with locally advanced non-small cell lung cancer . Oral baThe limitation of the study is that the dose accumulation was not based on single delivered dose of each fraction, which may have an impact on the accuracy of dose accumulation. We chose fractionated dose at five time-points  for dose calculation and accumulation, which can largely reflect the actual dose that patient has received. Tissue appearance and disappearance (TAD) (such as the tumor regression) might also hamper the accuracy of DIR between CBCT and reference CT . While iIn summary, most patients with LANSCLC had sufficient tumor dose coverage and acceptable normal tissue dose deviation without ART in the treatment of modestly HRT. Quick tumor regression, baseline obstructive pneumonitis/atelectasis during treatment are potential indicators for the need of ART. Daily CBCT scans prior to treatment was essential for patients treated by HRT, which enabled the timely detection of anatomical change for tumors and normal structures. To assess the advantage of ART based on the above factors in prospective studies are warranted.Additional file 1: Supplementary table 1. The comparison of PTV-GTV D95 and V51 between the fraction plans at different time point of treatment course with the original plan.


The objective of this study was to analyze the possible relationship between the glycemic control and the prevalence of apical periodontitis in type 2 diabetic patients. The null hypothesis was that apical periodontitis is not associated with glycemic control.In a cross-sectional design, the radiographic records of 216 type 2 diabetic patients (65.0 ± 10.7 years), 117 men (54.2%) and women (45.8%), were examined. Glycated hemoglobin (HbA1c) was used to assess glycemic control, considering an HbA1c level < 6.5% as well-controlled diabetes. Apical periodontitis was diagnosed as radiolucent periapical lesions using the periapical index score. The Student t test, chi-square test, and logistic regression analysis were used in the statistical analysis. p = 0.31).The average HbA1c value was 7.0 ± 2.2%. Forty seven (21.8%) had HbA1c levels under 6.5% (mean ± SD = 6.0 ± 2.2%), being considered well-controlled patients, and 169 (78.2%) had an HbA1c level ≥ 6.5% (mean ± SD = 7.8 ± 2.24%), being considered poor controlled patients. Forty four per cent of diabetics had apical periodontitis, 12.5% had root-filled teeth, and 52.3% had root filled teeth with radiolucent periapical lesions. No significant differences were observed in any of these three variables between patients with good or poor glycemic control. In the multivariate logistic regression analysis the presence of radiolucent periapical lesions in at least one tooth did not correlate significantly with HbA1c levels (OR = 1.4; 95% C.I. = 0.70 – 3.09; The results reveal no association of glycemic control with the prevalence of apical periodontitis or root canal treatment in diabetic patients. Key words:Apical periodontitis, diabetes mellitus, endodontic medicine, glycated haemoglobin. Diabetes mellitus (DM) is a group of metabolic diseases affecting the metabolism of carbohydrates, lipids and proteins, with hyperglycemia, as a result of a deficiency in insulin secretion, lack of insulin action or both . ChronicApical periodontitis (AP) is an inflammatory lesion around the root apex consecutive to bacterial infection of the pulp canal system . A periaSeveral studies have reported results supporting a relationship between the prevalence AP and diabetes -17. MoreThe aim of this study was to analyze the possible relationship between the prevalence of AP, diagnosed as a periapical radiolucent lesion (PRL), and the glycemic control, assessed by HbA1c levels, of type 2 diabetic patients. The null hypothesis was that AP is not associated with glycemic control in diabetic patients.An observational descriptive cross-sectional study was designed. The study was approved by the ethical committee of the Dental Clinic of the University of Barcelona , reference number 10/31/2018. The investigation was conducted following the World Medical Association-Declaration of Helsinki. All participants signed the written informed consent.-PatientsParticipants were recruited among patients presenting consecutively seeking routine dental care (not emergency care) at the Dental Hospital  and from a primary health center , during the years 2013 through 2016. All type 2 diabetic patients, diagnosed according to the current criteria for the diagnosis of diabetes , were aset al.  by day, and periodontal status assessed using the criteria of Machtei et al. .-Assessment of glycemic control The status of the metabolic control of the diabetic patients was achieved by checking the values of the glycosylated hemoglobin (HbA1c). The blood test had to be performed less than a month prior to the study visit. Adequate glycemic control was defined as HbA1c < 6.5% .-Radiological examination and periapical status assessmentRadiographic periapical status was diagnosed on the basis of examination of digital panoramic radiographs of the jaws. Two qualified radiologic technicians, with more than 10 years of experience in their field, performed the OPG using a digital orthopantomogram .All teeth, excluding third molars, were recorded. Teeth were categorized as root filled teeth if they had been filled with a radiopaque material in the root canal(s). For each subject the number of teeth present, the number and location of root-filled teeth, and the number and location of teeth having identifiable radiolucent periapical lesions were recorded.The periapical status was assessed using ‘Periapical Index’ (PAI) score . A score-Observers’ calibrationet al. 1986). Each tooth was assigned to one of the PAI scores by using visual references  for the 5 categories within the scale. After scoring the teeth, the results were compared to a “gold standard atlas”, and a Cohen Kappa was calculated (0.78 – 0.89).Three observers with a broad clinical experience in endodontics assessed the radiographs ; disagreements were weighted by JSE and JMG. Before evaluation, the observers participated in a calibration course for PAI system, which consisted of 100 radiographic images of teeth, some root-filled and some not, kindly provided by Dr. Ørstavik . Then, one month after this first examination, the observer was recalibrated in the PAI system and repeated the scoring of the radiographs of the same 20 patients. The intra-observer agreement test on PAI scores on the 20 patients produced a Cohen’s Kappa ranging 0.80 - 0.91.Intra-observers reproducibility was also determined comparing the PAI scores on the 20 radiographs provided by each observer. The agreement test produced a Cohen’s Kappa ranging 0.78 - 0.94. The Cohen’s Kappa for inter-observers variability ranged 0.83 - 0.90. The consensus radiographic standard was the simultaneous interpretation by the three examiners of the panoramic radiograph of each patient.-Statistical analysisMinimal sample size was calculated using “Sample size and power calculator”, v7.12 software (https://www.imim.cat/ofertadeserveis/software-public/granmo/). Accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test, 204 subjects are necessary in the observed group to recognize a difference greater than or equal to 0.1 units. A proportion in the reference group has been estimated to be 0.35. It has been anticipated a drop-out rate of 10%. Post-hoc power analysis has been performed using the same software.p value <0.05 was considered to be statistically significant.The primary data was introduced into an Excel spreadsheet . SPSS  was used for the statistical analysis. Data is presented as media ± standard deviation. A t-test and X2 test were used to determine the differences between groups. A logistic regression analysis was performed to measure the strength of the association between the HbA1c levels and the presence of periapical radiolucencies, adjusting the presence of co-variables. A The characteristics and dental status of the type 2 diabetic patients are described in p > 0.05). Forty four per cent of diabetic patients showed at least 1 radiolucent periapical lesion (RPL), 12.5% had at least 1 RFT, and 52.3% had at least 1 root-filled tooth with RPL (p = 0.10). The percentage of good-controlled patients with at least 1 root-filled tooth with RPL was 34.0%, whereas amongst poor-controlled patients this percentage was 47.3% (p > 0.05). The number of RFT , and the number of RFT with periapical lesions  was not associated to HbA1c levesl.Diabetic patients were classified dichotomously according to their glycemic control . Forty swith RPL . There wp = 0.31).To further investigate whether HbA1c levels were related to endodontic variables, multivariate logistic regressions were run with age, gender, smoking habits, alcohol consumption, number of teeth, number of RFT, number of RFT with AP, periodontal status, and periapical status as independent explanatory variables, and HbA1c levels as dependent variable  . In the p = 0.31). Nor has an association been found between the level of HbA1c and the prevalence of RCT.In this observational cross-sectional study, the possible association between the prevalence of RPL and glycemic control in diabetic patients has been investigated. The results reveal no higher prevalence of RPL in poor controlled type 2 diabetic patients, with HbA1c levels ≥ 6.5% . However, other studies have also found no association between these variables and glycemic control in diabetic patients  had well-controlled HbA1c levels (< 6.5%). Sanchez-Domínguez et al.  found a et al. ,40. Thiset al. ,23,40. Ip = 0.10). The high percentage of poorly controlled diabetics have decreased the power of the study. Accepting an alpha risk of 0.05 in a two-sided test with 169 poor-controlled patients and 47 good-controlled diabetics, the statistical power is only 37.4% to recognize as statistically significant the difference from 47% of prevalence of RPL in the first group to 34% in the second group.The present results show that 44.4% of diabetic patients had AP, diagnosed as RPL, in one or more teeth. Some previous studies have found higher prevalence of periapical lesions in diabetics ,28,38, bp = 0.049), calculated through multivariate logistic regression analysis, concluding that HbA1c levels of diabetic patients are associated with periapical status (p = 0.001). Moreover, poor controlled diabetic showed higher prevalence of RFT with AP (32.0%) compared to well-controlled patients (21.8%) (p = 0.02), concluding that a poor glycemic control may be associated with a higher prevalence of AP and increased rate of endodontic failures. The third study analysed only the periapical status of RFT . However, the total prevalence of RPL associated to endodontically treated teeth was high (52.3%). Other previous studies have found similar percentages (p = 0.006) (p = 0.0001)   and that 0.0001) .The possible relationship between glycemic control and endodontic infection has been also investigated in animal models. It has been demonstrated that diabetes enhance the development of periradicular lesions in rats , and hypThe results of these animal studies, together with those of human epidemiological studies ,23,40 suThe results reveal no association of glycemic control with the prevalence of apical periodontitis or root canal treatment in diabetic patients.


IntroductionPulmonary hypertension (PH) is a known complication that occurs in patients of end-stage renal disease (ESRD) that have an arteriovenous fistula (AVF) for hemodialysis (HD). It is defined as pulmonary artery pressure (PAP) of greater than 30 mmHg on echocardiography. The presence of PH in ESRD is an independent risk factor and decreases the survival likelihood among HD patients. Unexplained PH is frequently seen in ESRD following AVF.Obesity can lead to various complications, such as sleep apnea, cardiac complications, pulmonary hypertension, and mortality. Data on the prevalence of coexisting PH and obesity are scarce.Obese patients often have increased albumin excretion rates (AER) that can lead to early renal impairment and an increase in intraglomerular pressure, which may increase the risk of cardiovascular (CV) morbidity and mortality.Therefore, the study aimed to evaluate and compare the associated PH and obesity separately and collectively among ESRD patients.MethodsThis comparative cross-sectional study was conducted in a tertiary care public sector hospital with the approval of the medical ethics review board committee.2 from April 2017 till March 2019, who presented to our facility. These patients underwent dialysis twice or thrice a week, each session lasting three to four hours approximately. On initial encounter, trans-thoracic echocardiography (TTE) was done by the cardiologist to diagnose pulmonary hypertension.The study enrolled all consecutive patients with ESRD as defined by having an estimated glomerular filtration rate (GFR) of <15 mL/min/1.7 3 mIn addition, body mass index (BMI) was calculated for all patients, and the patients were categorized into underweight, normal, overweight, or obese. All patients underwent post-dialysis TTE at one hour or when patients were at the optimal dry weight. Systolic PAP and ejection fraction were measured, and pulmonary hypertension was defined as a PAP of 30 mmHg or greater on TTE.ESRD patients that were diagnosed with PH prior to hemodialysis or had primary PH were excluded from the study. Only ESRD patients developing secondary PH after hemodialysis were included in the study.The chi-square test was used to see the correlation of gender, ambulation status, smoking status, obesity, pulmonary hypertension, body mass index (BMI), and pulmonary hypertension and obesity combined on the final outcome. A p-value of 0.05 was considered significant. Odds ratio (OR) and relative risk (RR) were calculated for pulmonary hypertension and obesity combined, obesity, and pulmonary hypertension in the final outcome.Results2 (±13.29SD), 52 (25.5%) patients were underweight, 40 (19.6%) had a normal BMI, 29 (14.2%) were overweight, and 83 (40.7%) patients were obese.The study enrolled 204 patients with a mean age of 46.23 (±20.45 SD) having higher female participation of 108 (52.9%), whereas 96 (47.1%) were males. The average weight of the cohort was 66.78 kg (±22.98 SD) with a mean BMI of 29.91 kg/mPulmonary hypertension and obesity combined were observed in 48 (23.5%) of the cases and there was a 4.60 relative risk of death among these individuals, with an odds ratio of 13.35 and a p-value of 0.00.ConclusionThe study shows a strong synergistic effect of pulmonary hypertension and obesity towards the final survival outcome in ESRD patients who are on hemodialysis. Pulmonary hypertension (PH) is a known complication that occurs in patients with end-stage renal disease (ESRD) who have an arteriovenous fistula (AVF) for hemodialysis (HD). PH is defined as pulmonary artery pressure (PAP) of greater than 30 mmHg on echocardiography . It is aTherefore, the World Health Organization (WHO) has categorized PH into five diagnostic groups . Group 12 had moderate or severe pulmonary hypertension on echocardiography [Obesity can lead to various complications, such as sleep apnea, cardiac complications, and pulmonary hypertension, as described by Group 3 of the WHO Classification for PH, and can also result in mortality of individuals -12. Dataiography .Obesity is a potential risk factor for kidney disease and is associated with abnormal renal parameters . Obese pTherefore, the study aimed to evaluate and compare the associated PH and obesity separately and collectively among ESRD patients.Study demographicsThe study was conducted in a tertiary care public sector hospital with the approval of the medical ethics review board committee, and all participants signed informed consent before entering the study.Study designThis was a comparative cross-sectional study done to evaluate the combined effects of pulmonary hypertension and obesity on the mortality of ESRD patients undergoing hemodialysis.Study populationThe study enrolled all consecutive patients with ESRD as defined by having an estimated GFR of <15 mL/min/1.7 3m2 from April 2017 till March 2019 who presented to our facility. These patients underwent dialysis twice or thrice a week, each session lasting three to four hours approximately.Study protocolOn initial encounter, trans-thoracic echocardiography (TTE) was done by the cardiologist to diagnose pulmonary hypertension.Inclusion and Exclusion CriteriaESRD patients who were diagnosed with PH prior to hemodialysis or had primary PH were excluded from the study. Only ESRD patients developing secondary PH after hemodialysis were included in the study.2, normal weight with a BMI between 18.5 and 24.9 kg/m2, overweight if BMI was 25-29.9 kg/m2, and obesity was defined as BMI of 30 kg/m2 or greater according to the WHO guidelines. All patients underwent post-dialysis TTE at one hour or when patients were at the optimal dry weight. Systolic PAP and ejection fraction were measured and pulmonary hypertension was defined as PAP of 30 mmHg or greater on TTE.In addition, BMI was calculated for all patients and patients who were categorized into underweight - less than 18.5 kg/mAge, gender, smoking status, etiology of renal failure, ambulation status, weight, duration of treatment, and final outcome  were also recorded. Patients were also categorized as having obesity, developed pulmonary hypertension, both (pulmonary hypertension and obesity), or not.Final outcome The final outcome was mortality.Statistical analysisData were anonymized, and the analysis was conducted on SPSS version 22 . Frequencies and percentages were calculated for ordinal data, whereas for numerical data, mean and standard deviations were obtained from the average values of the age, weight, BMI, and PAP from the first HD session till the study period or the final outcome of death.The chi-square test was used to see the correlation of gender, ambulation status, smoking status, obesity, pulmonary hypertension, BMI, and pulmonary hypertension and obesity combined on the final outcome. A p-value of 0.05 was considered significant. Odds ratio (OR) was calculated for pulmonary hypertension and obesity combined, obesity, and pulmonary hypertension on the final outcome.2 (±13.29SD), 52 (25.5%) patients were underweight, 40 (19.6%) had a normal BMI, 29 (14.2%) were overweight, and 83 (40.7%) patients were obese.The study enrolled 204 patients, with a mean age of 46.23 (±20.45 SD) having higher female participation of 108 (52.9%), whereas 96 (47.1%) were males. The average weight of the cohort was 66.78 kg (±22.98 SD) with a mean BMI of 29.91 kg/mFor the cause of dialysis, diabetics were 41 (20.1%), liver failure 33 (16.2%), obstetric sepsis 33 (16.2%), hypertension 32 (15.7%), lupus nephritis 17 (8.3%), heart failure 13 (6.4%), glomerulonephritis 12 (5.9%), lung failure 10 (4.9%), chronic pyelonephritis seven (3.4%), and other cause six (2.9%). Overall, 146 (71.6%) people were alive and 58 (28.4%) died. Table Furthermore, 66 (32.4%) patients developed pulmonary hypertension during hemodialysis, whereas 138 (67.6%) required assisted ambulation. The mean PAP recorded on TTE was 34.89 (±16.89 SD). Sixty-three (30.9%) of the population were smokers.Pulmonary hypertension and obesity combined was observed in 48 23.5%) of the cases and there was a 4.60 relative risk of death among these individuals with an odds ratio of 13.35 and a p-value of 0.00. Table 3.5% of tSeveral isolated studies have been conducted on PH and obesity in ESRD and the effect they have on mortality in ESRD -18. ThisPulmonary hypertension (PH) is commonly seen in patients of CKD and ESRD -20. O’LeIn another study, Navaneethan et al., in his multivariate study, reported a 21% prevalence of PH, with a high rate of PH associated with poor kidney function, and PH and high tricuspid regurgitant velocity and PAP were associated with adverse outcomes in CKD. In addition, Navaneethan et al. also established that PH was independently associated with increased risk for death (hazard ratio 1.38) .Our study, on the other hand, only includes cohorts with secondary PH developed after the initiation of HD and tries to fill this void, even though a better model is required to study the cause and effect of PH on the mortality of ESRD patients.Over the past decade, the prevalence of obesity has increased among adults with ESRD to 10% in the Netherlands . ObesityIn our cohort, 40.6% were obese, out of which 17.64% died. This inference is related to the previous studies conducted to see the association of mortality and obesity in ESRD.When we studied the combined effect of PH and obesity, we found that 16.6% ESRD patients had died, which is less than the independent effect of PH and obesity but the OR was 13.3 as compared to 9.04 and 3.44 for PH and obesity, respectively. However, we are unable to identify the reason for this but we hypothesize that obesity is pro-inflammatory and leads to endothelial dysregulation, which can ultimately lead to PH and can aggravate cardiac events . FurtherThe limitations of this study were that it was a single-center study and the sample size was small as compared to large trials and previous studies. Second, we did not address the obesity paradox in this study and did not include the body fat percentage; This may have different results on the final outcome as per the previous literature dictates .The study shows a strong synergistic effect of pulmonary hypertension and obesity towards the final survival outcome in ESRD patients who are on hemodialysis. The odds of mortality increase if pulmonary hypertension and obesity co-exist in patients who are on hemodialysis at the end stage of renal failure.


Background: The most important series devoted to antithyroid drug-induced severe neutropenia and agranulocytosis are Japanese studies, almost specifically in relation to the intake of methimazole. The clinical data of 30 Caucasian patients followed up for antithyroid drug-induced neutropenia at a third-level hospital are reported. Methods: The data of 30 patients with idiosyncratic antithyroid drug-induced neutropenia and agranulocytosis from a cohort study on drug-induced neutropenia and agranulocytosis conducted at the University Hospital of Strasbourg (France) were retrospectively reviewed. Results: The mean patient age was 61.7 years old (range: 20–87), and the gender ratio (F/M) was 4. Several comorbidities were reported in 23 patients (76.7%), with the mean Charlson comorbidity index of 1. The causative drugs were carbimazole and benzylthiouracil, in 28 (93.3%) and 2 cases, respectively, prescribed primarily for multi-hetero-nodular goiter or thyroid nodule to 18 patients (60%). Sore throat and acute tonsillitis (40%), isolated fever (20%), septicemia (13.3%), documented pneumonia (6.7%), and septic shock (6.7%) were the main clinical features upon admission. The mean neutrophil count at nadir was 0.02 and 0 × 109/L (range: 0–0.3). Regarding the patients’ hospital course: 13 cases (43.3%) worsened during hospitalization, severe sepsis was found in 26.7%, systemic inflammatory response syndrome—in 13.3%, and septic shock—in 3.3% of the cases, respectively. Broad-spectrum antibiotics were indicated for all the patients, and 21 (73.3%) of them received hematopoietic growth factors. Hematological recovery (neutrophil count ≥ 1.5 × 109/L) was seen at 8.3 days (range: 2–24), but faster in those receiving hematopoietic growth factors . Two patients died during hospitalization, and the rest had a favorable clinical outcome. Conclusions: Antithyroid drug-induced neutropenia represents a serious complication resulting from the rates of severe infections especially in those cases severe neutropenia. In this setting, an established procedure for the management of patients seems useful or even indispensable in view of potential mortality. They include carbimazole, methimazole, thiamazole (active form of carbimazole), benzylthiouracil, and propylthiouracil . These mectively . In thisectively ,3,4. To The present article shows the results of the follow-up of 30 patients with ATD-related (mostly carbimazole) neutropenia and agranulocytosis at a university hospital.All non-chemotherapy-related neutropenia and agranulocytosis cases identified in patients hospitalized at the University Hospitals of Strasbourg (Hôpitaux Universitaires de Strasbourg), France, were included into a register since the 1990′s . Patien9/L, fever, and any kind or degree of clinical infection, as well as the standardized criteria defined by Benichou et al. [https://www.cancer.gov/). This classification proposed by the National Cancer Institute includes four categories ranging from Grade 1 to 4. Grade 1: absolute neutrophil count from 1.5 to 1 × 109/L, Grade 2—from 1 to 0.5 × 109/L, Grade 3—from 0.5 to 0.1 × 109/L, and Grade 4 ≤ 0.1 × 109/L. Severe neutropenia, also known as agranulocytosis, is biologically characterized by a severe decrease or an absolute lack of circulating granulocytes, resulting in an absolute neutrophil count of ≤ 0.5 × 109/L with fever or absolute neutrophil count ≤ 0.1 × 109/L (®), methimazole (Thyrozol®), benzylthiouracil (Basdène®), and propylthiouracil . The active form of carbimazole, thiamazole, is not available in France.The inclusion criteria included the neutrophil count ≤ 0.5 × 10u et al. ,8. Patie × 109/L . In the 9/L , length of hospitalization, outcome, transfer to intensive care unit, and mortality were collected with special regard to the use of hematopoietic growth factors (HGFs).This study was aimed at describing the clinical features, management, and outcome of all ATD-related neutropenia and agranulocytosis cases, with a focus on carbimazole, the main ATD used in France and Europe. The data were obtained from the medical records. The medical record of every patient was assessed by two members of the monitoring committee, and the following variables were recorded (when available): age, sex, reason for hospitalization, associated comorbidities, and Charlson comorbidity index. We collected information on the underlying thyroid disease and the synthetic antithyroid drug used . The clinical features upon diagnosis and the disease course were evaluated. Complete white blood cell counts, neutrophil counts, hemoglobin and platelet counts  were collected as the bone marrow aspects. The delay to reach a neutrophil count > 1.5 × 10t-test were used for paired data. Quantitative variables distribution was graphically assessed through the Shapiro–Wilk test. The nonparametric Mann–Whitney test was used for in between-group comparison of quantitative variables. The qualitative variables were presented in absolute numbers and percentages and were analyzed using the Pearson’s chi-squared or the Fisher’s exact test. To assess the factors associated with mortality and/or intensive care unit admission, a univariate analysis was performed. The results were considered statistically significant with the p-value < 0.05. We used the R software (version 3.2.2) for the statistical analyses.The data were expressed as the mean and the standard deviation (SD). The Mann–Whitney test and the Student’s https://www.rfcrpv.fr/) and were subject to the French National Data Protection Act  (CNIL) (https://www.cnil.fr/professionnel). The local ethics committee approved the present study. Ethical approval code: CEFM_1996_3, Date of approval: 4 March 1996. Written informed consent for publication was obtained from the participating patients.The data collected were registered in the French National Database of drug side effects  . In the same study period, 177 patients were hospitalized for neutropenia related to other drugs . One female patient had two episodes of drug-induced agranulocytosis three years apart, the first one under carbimazole (daily dose of 40 mg) and the second one under benzylthiouracil treatment (daily dose of 100 mg), respectively.All the patients had a Caucasian background. In the sample, the mean and the median age was 61.7 and 66 years, respectively; 7 patients (23.3%) were aged < 50 years, and 14 (4.7%)—< 65 years. Only 30 percent (9) of the patients were older than 75 years. There was a female predominance (sex ratio 4). In 23 patients (76.7%), the coexistence of multiple medical conditions was observed, consisting mainly of arterial hypertension ; atrial fibrillation ; chronic renal failure ; cardiac disorders  , arteritis of lower limbs ; diabetes mellitus ; stroke ; and rheumatoid arthritis . Moreover, 2 patients (6.7%) were followed up for a non-progressing and treated Waldenström’s disease. The mean Charlson comorbidity index was 1 (range: 0–4).In all but two cases (6.7%), a synthetic ATD was found to be “causative” or “likely causative”. In these two cases, other “likely causative” drugs  were related to the biological picture. Carbimazole in 28 patients (93.3%) and benzylthiouracil in 2 cases were identified as the causative drugs. These drugs were discontinued during the first 48 h after hospital admission in 67.9% of the patients. The mean and the median daily doses of carbimazole and benzylthiouracil were 48 and 40 mg (40–100) and 150 and 100 mg, respectively. The mean and the median treatment duration for carbimazole was 42.2 days and 35 days (18–120), respectively. Regarding benzylthiouracil, the patient receiving 150 mg was treated for 3 days, and the other one (100 mg)— 8 days.The underlying thyroid disorders were: multi-hetero-nodular goiter or thyroid nodule in 18 patients (60%); administration of Cordarone for atrial fibrillation in 9 patients (30%); and Basedow‘s (Graves’) disease in 3 cases (10%). Twenty-two patients (73.3%) were under treatment with at least one drug (mean number of drugs: 5). In eight patients (26.7%), no other medication except the causative drug was identified within the 10 days prior to the detection of neutropenia.p > 0.05). In 6 patients (20%), microbiology results were contributive: gram-negative bacilli (n = 4), Staphylococcus aureus and Streptococcus pneumoniae.Fever of unknown origin was the clinical sign leading to neutropenia detection in 15 patients (50%). Neutropenia was found in 13 patients (43.3%) in the absence of any symptoms as part of routine monitoring of their blood counts. Only 2 patients (6.7%) had a documented infection with pneumonia and septic shock. Sore throat and acute tonsillitis in 12 patients (40%) and isolated fever in 6 patients (20%) were the principal clinical findings on admission. During hospitalization, clinical worsening was noted in 13 patients (43.3%) ranging from severe sepsis , systemic inflammatory response syndrome (SIRS)  to septic shock . The worsening of the clinical condition was initially observed in 62.5% of patients over 75 years of age compared to 36.4% of other patients. Two patients (6.6%) had to be admitted to intensive care due to visceral failures related to septic shock.9/L (range: 0–0.5) upon admission. Twenty patients (66.7%) had the absolute neutrophil level ≤ 0.1 × 109/L (Grade 4 neutropenia). All the remaining patients had Grade 3 neutropenia. In this setting, 90% of the patients presented with a mild clinical picture of isolated fever or acute tonsillitis. Concerning neutrophil nadir, the mean and the median neutrophil counts were 0.02 and 0 × 109/L (range: 0–0.3), respectively. Ninety percent of the patients (n = 27) had the neutrophil level ≤ 0.1 × 109/L (Grade 4). Isolated neutropenia without any other blood abnormalities was found in 26 patients (86.7%). The mean and the median hemoglobin levels were 113 and 110 g/L, respectively. Anemia (Hb < 120 g/L) was found in 16 patients (53.3%). Regarding the platelet counts, the mean value was 281, and the median level was 278 × 109. Thrombocytopenia was not detected in any patient. The bone marrow study was available in 13 cases: primarily detected a partial disappearance or even aplasia in 7 patients (53.8%) and myeloid hypocellularity in 4 other patients (30.8%). The bone marrow was described as normal in 2 cases (15.4%).The mean and the median absolute neutrophil counts were 0.08 and 0.02 × 109/L) was 8.3 and 6 days (the mean and the median), respectively. As for achieving the neutrophil count ≥ 0.5 × 109/L, these values were 8.1 and 6 days.The hematological recovery . Of note, HGFs were started immediately after diagnosis, except in 1 patient with isolated fever. The duration of this treatment administration was 5.9 and 5 days (the mean and the median). In patients receiving this treatment, the mean time to hematological recovery dropped to 4.9 days, from 12.2 to 7.2 days (p = 0.046).HGFs were administered in 21 cases (73.3%), especially in those presenting with the neutrophil count ≤ 0.1 × 10p > 0.005).Because of the severity of neutropenia, all the patients were hospitalized. In 19 patients (63.3%), the incriminated drugs were withdrawn within the first 48 h. In case of fever (> 38 °C) or any signs of infection, broad-spectrum parenteral antibiotherapy, mainly an association of cephalosporins and aminoglycosides, was initiated if no ß-lactam allergy was present. Subsequently, this treatment was adapted to the microbiology results, and other antibiotics, such as fluoroquinolone, glycopeptides and penems, were used. Antibiotics were administered for 11.7 and 12 days (the mean and the median), and the hospital stay length was 17.8 and 12 days (the mean and the median), respectively. Antibiotics and hospital stay length were not influenced by the use of G-CSF therapy  for cardiothyreosis related to Cordarone intake; the second one was also under carbimazole (daily dose of 40 mg) for hyperthyroidism related to Cordarone intake. Of note, HGFs were used in these two patients.During the hospitalization, 2 patients (6.6%) were admitted to intensive care due to multiple organ failure related to septic shock. The outcome was favorable in 28 patients (93.3%), whereas 2 subjects (6.7%) died of either uncontrolled septicemia due to This is the first study of well-documented neutropenia related to ATDs (primarily carbimazole) focusing on Grade 3, Grade 4 neutropenia and agranulocytosis and reporting a detailed clinical picture for each patient, and managed using the same procedure. To date, the most important series devoted to ATD-induced neutropenia are primarily local or regional epidemiological studies ,10. They9/L at the neutrophil nadir; and 90% had the level ≤ 0.1 × 109/L (Grade 4 neutropenia) at nadir. In all cases except for the two patients who died, the principal diagnostic criterion was fulfilled with a full hematological recovery once the causative drug was discontinued [Our patients met the definition of idiosyncratic drug-induced neutropenia and agranulocytosis ,11 Tabl. All casontinued . Consideontinued . A synth9/L) is relatively common with ATDs and is often included in other series [In our study, the clinical features differ from those described in other series of ATD-induced neutropenia ,4,5, andr series . In thisr series ,13. Thisr series ,14. In tr series ,12,15. I9/L were found to be poor prognostic factors leading to infections in the study carried out by Julia et al. [The mortality rate was 6.6% in relation to Grade 3 and Grande 4 neutropenia and agranulocytosis, with one case of uncontrolled septicemia complicated by pyelonephritis, and one other case of septic shock in two old females. In this setting, the literature related to ATD-induced severe neutropenia and agranulocytosis for the last 30 years shows a progressive decrease in mortality with each decade. A Swedish study conducted in 1966–1975 reported 5 deaths among 29 cases of agranulocytosis induced by ATDs 17%); the risk appeared similar for carbimazole, methimazole, and propylthiouracil . The Coo%; the ria et al. . Age . Thisp = 0.046), but no improvement concerning antibiotherapy and hospital stay length. Our results are similar to those described in other studies [p = 0.046), and also for hospitalization duration . As no fatal cases were observed, the benefit on mortality could not be studied. To date, there are two other published clinical studies specifically dedicated to ATD-induced neutropenia and agranulocytosis treated with HGFs [Hematological improvement was rapidly observed in the HGF group: -4.9 days . However, this is the first study mainly evaluating carbimazole-induced neutropenia in Caucasian patients. It is important to highlight the presence of Grade 4 neutropenia in 90% of the patients during the biological course. The patients were managed by the physicians experienced in this field and in a single center, which makes it difficult to extrapolate these results. The sample size is small, and the statistical analysis could not take into consideration any confounding factors. A large-scale study including different centers could be of help to confirm our findings.Idiosyncratic ATD-induced neutropenia and agranulocytosis are serious medical conditions as severe sepsis was found in half of the patients and the mortality rate was 6.7%. Updated management including immediate antibiotherapy and HGFs in frail patients may reduce infection-related mortality. A specific protocol of care seems to be indispensable in view of the mortality rate. Although ATD-induced neutropenia is a known adverse drug reaction, many questions remain unanswered. The studies available include rare low-number series, non-European populations, and treatment with methimazole, a drug not used in France and only rarely used in Europe. Our study is one of the first to investigate a mainly carbimazole-induced Grade 4 neutropenia and documented agranulocytosis in Caucasian patients. Physicians must be aware of the most commonly implicated drugs as suspicion is a key element in the diagnosis. Early ATD-induced neutropenia detection may decrease the severity and mortality when the causative drug is withdrawn.


Further, 56.8% received inappropriate prescriptions, with a significantly lower probability in subjects with at least a nephrologist visit (Adj OR 0.54 (95% CI 0.36–0.81)) and a greater probability in patients treated with more active substances (1.10 (1.08–1.12)), affected by more comorbidities (1.14 (1.06–1.230)), or with serious CKD (G4/G5 21.28 (7.36–61.57)). Nonsteroidal anti-inflammatory drugs (NSAIDs) were the most used contraindicated drugs (48.5%), while acetylsalicylic acid was the most inappropriately prescribed (39.5%). Our results highlight the inappropriate prescriptions for CKD authorized by GPs and underline the need of strategies to improve prescribing patterns.Nephrotoxic drugs prescriptions are often prescribed inappropriately by general practitioners (GPs), increasing the risk of chronic kidney disease (CKD). The aim of this study was to detect inappropriate prescriptions in patients with CKD and to identify their predictive factors. A retrospective study on patients with creatinine values recorded in the period 2014–2016 followed by 10 GPs was performed. The estimated glomerular filtration rate (eGFR) was used to identify CKD patients. The demographic and clinical characteristics and drugs prescriptions were collected. A descriptive analysis was conducted to compare the characteristics and logistic regression models to estimate the predictive factors of inappropriate prescriptions. Of 4098 patients with creatinine values recorded, 21.9% had an eGFR <60 mL/min/1.73 m GFR values <60 mL/min/1.73 m2 indicate a CKD classified as G3a, while values of GFR <15 mL/min/1.73 m2 define a kidney failure (category G5) [Chronic kidney disease (CKD) is one of the most widespread diseases and it is deemed to be a real public health problem worldwide . CKD is The Global Burden of Disease study data showed that the incidence and the prevalence of CKD globally increased by 89% and by 87% from 1900 to 2016, respectively . In more2 [2 and with at least one of the following conditions: progressive worsening of renal function (eGFR <15% in 3 months), diabetes mellitus, or aged <70 years [Epidemiological data available in the last decades has sparked interest in CKD from health authorities, nephrologists, and general practitioners (GPs). GPs are certainly the first category of physicians able to identify CKD early in patients, taking into account risk factors that could induce a renal failure. They can also take measures timely to slow the progression of the disease and promptly send the patient to a nephrologist ,11. The 70 years . GPs’ de70 years . Unfortu70 years .Furthermore, the presence of CKD could be associated with several chronic diseases including hypertension, diabetes, chronic respiratory disorder, cardiovascular diseases, and depression ,16,17. PGuidelines strongly recommend to avoid the prescription of drug-induced nephrotoxicity principally in patients with several conditions . HoweverA retrospective observational study was carried out by the Department of Clinical and Experimental Medicine of the University of Messina with a group of 10 GPs of Messina. All patients of each GP aged ≥18 years in the three-year period from 2014–2016 were recruited. Of them, subjects with registered serum creatinine levels in the GPs’ medical records during the considered period were selected.Encrypted patient code, age, sex, weight, height, and body mass index (BMI);Information on lifestyles, such as alcohol use and smoking;Diagnostic instrumental and laboratory exams, of which serum creatinine, blood count, plasma glucose (FPG), glycated hemoglobin (HbA1c), lipid profile, hepatic cytolysis indexes, electrolytes, and albuminuria were examined;All drugs prescriptions registered in the GPs’ medical records and classified according to the Anatomical Therapeutic and Chemical (ATC) Classification System;Registered diagnosis of CKD and comorbidities codified using the International Classification of Diseases code, 9th revision (ICD-9);Requests for specialist visits.In detail, the following data concerning patients’ clinical and demographic characteristics were collected:The Charlson comorbidity index score (CH index) was adopted to evaluate the severity of the disease for each patient. The total number of drugs prescriptions as well as the number of different active substances used for any disease by patients during the study period were considered to assess the drugs exposure.2 calculated on consecutive registered serum creatinine values at least after 90 days were identified as CKD-affected patients and classified into stages G3a–G5 according to the last recorded eGFR value. In particular, stage G3a (eGFR 45–59 mL/min/1.73 m2), G3b (eGFR 30–44 mL/min/1.73 m2), G4 (15–29 mL/min/1.73 m2), and G5 (eGFR <15 mL/min/1.73 m2) were considered [2.Regardless of the GPs’ recorded diagnosis of CKD, patients were classified according to the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines based on GFR values estimated with a CKD-epidemiology collaboration (CKD-EPI) formula (eGFR). Patients with an eGFR <60 mL/min/1.73 mA patient encrypted code has been used to maintain anonymity. The study protocol was approved by the local Ethical Committee of Messina University Hospital.For the identification of nephrotoxic drugs, literature reviews were performed using the Medical Subject Headings (MeSH) terms “nephrotoxic drug” and “drug-induced renal failure”. Moreover, an evaluation of the cautionary notes for prescriptions in people with CKD was executed . Specifi2 vs. patients with an eGFR ≥60 mL/min/1.73 m2.A descriptive analysis was performed to compare all characteristics of the study population  between patients with an eGFR <60 mL/min/1.73 mAbsolute and relative frequencies with corresponding 95% confidence intervals (CIs) were evaluated for the categorical variables, while medians with an interquartile range (Q1–Q3) were calculated for continuous variables. The Kolmogorov–Smirnov test for normality was performed to evaluate the normal distribution. Since some of the numerical variables were not normally distributed, a non-parametric approach was used. The Mann–Whitney U test for independent samples was applied for the continuous variables and a two-tailed Pearson chi-squared test for the categorical variables.p-value < 0.05 was considered statistically significant. The statistical analysis was performed with SPSS version 23.0 .To identify the predictors of inappropriate prescriptions in patients affected by a stage of CKD ≥G3a, a univariate logistic regression model, using patients without inappropriate prescriptions as comparators, was carried out to assess the possible influence of sex, age, number of nephrologist visits, CKD diagnosis, CH index, stage of CKD, number of comorbidities, number of active substances used, and number of total prescriptions for each patient. Moreover, all predictors were included in a stepwise multivariate logistic regression model . Odds ratios (ORs) with 95% CIs were calculated for each covariate of interest in the univariate (crude OR) and multivariate (adjusted OR) models. The goodness of fit of the regression model was assessed by the Hosmer–Lemeshow test for adequacy. A 2, defined as CKD patients, were 896 in number (21.9%). However, only in 29.6% of them did GPs record the diagnosis of CKD during the study period, and only 25.5% of CKD patients were referred to a nephrologist. The frequency of nephrologist visits as well as GPs’ diagnosis of CKD (G3a–G5) increased at the increasing CKD stage  had at least a registered serum creatinine value in the last three years with a high variability among each single GP (from 12.3% to 48.5%). eGFR values were calculated by using the CKD-EPI formula: 3202 subjects (78.1%) had an eGFR of 60 mL/min or more. Patients with an eGFR <60 mL/min/1.73 mtage see .2 were mainly females, older, and affected by more serious diseases, evaluated by the CH index (p < 0.001). Furthermore, subjects with an eGFR <60 mL/min/1.73 m2 had a greater number of total drugs prescriptions, as well as more different active substances prescribed per patient compared with subjects with an eGFR ≥60 mL/min/1.73 m2 (p < 0.001) . Moreover, patients with an eGFR <60 mL/min/1.73 m2 were mostly affected by hypertension (87.6%), dyslipidemia (60.0%), arthritis and arthrosis (56.1%), chronic pulmonary disease (47.2%), and osteoporosis (45.6%)  . All com (45.6%) .An increased number of prescriptions was found with an increased stage of CKD (G3a–G5): G3a: 113 (60–187); G3b: 152 (91–229); G4/G5: 196 (117–65), as well as the number of different active substances prescribed: G3a: 18 (12–25); G3b: 22 (15–29); G4/G5: 25 (20–33). The frequency of comorbidities stratified by the severity of CKD are shown in Of 4098 patients with registered serum creatinine values in the last three years, 4018 (98.1%) had at least one drug prescription. Among them, the drugs for acid related disorders, agents acting on RAS, lipid modifying agents, antithrombotic agents, and drugs used in diabetes were the five most frequently prescribed classes.2 received at least one drug prescription with a total of 128,996 prescriptions during the three-year study period. More than half of the patients with an eGFR <60 mL/min/1.73 m2  received at least one prescription of contraindicated drugs for a total of 4818 (3.7%) inappropriate prescriptions. The median (Q1–Q3) number of contraindicated prescribed medications per patient was 5 (2–14). The number of patients with inappropriate prescriptions increased with the increased CKD G3a–G5 stages, specifically, 47.5% in stage G3a, 64.9% in G3b, and 95.4% in G4/G5. Recording the diagnosis of CKD did not significantly affect the percentage of patients with inadequate prescriptions in every stage of CKD (All 896 patients with an eGFR <60 mL/min/1.73 me of CKD .2 was significantly lower in subjects with at least a nephrologist visit (Adj OR 0.54 (95% CI 0.36–0.81), p = 0.003). Furthermore, a greater probability of receiving an inappropriate prescription was observed in patients treated with more different drugs (Adj OR 1.10 (95% CI 1.08–1.12), p < 0.001) or affected by a higher number of comorbidities (Adj OR 1.14 (95% CI 1.06–1.23), p = 0.001). The severity of CKD was also an independent predictive factor of inappropriate prescriptions (Adj OR G3b 1.84 (95% CI 1.29–2.62), p = 0.001; Adj OR G4/G5 21.28 (95% CI 7.36–61.57), p < 0.001). Nevertheless, sex, age, CH index, the number of prescriptions, and the presence of a registered diagnosis of CKD were not predictive factors of prescriptive inappropriateness  and 161 (18.4%) patients, respectively. Moreover, 2026 (52.8%) of all prescriptions related to anti-inflammatory drugs and 1917 (19.1%) referring to antithrombotic drugs were contraindicated  followed by diclofenac (17.7%). The contraindicated drugs most commonly prescribed per patient and the related contraindicated prescriptions are shown in Regarding single active substances, overall, 31.1% and 19.4% of patients with an eGFR <60 mL/min/1.73 mThis is the first study concerning the evaluation of the predictive factors of contraindicated drug prescriptions in patients with CKD in a general practice setting. To the best of our knowledge, a previous study specifically identified the predictors of NSAIDs use and only recently did a few studies focusing on inappropriate drug prescriptions in patients followed by nephrologists become available ,24,26,272 instead of the 29.6% in our sample. Regarding the registered diagnosis of CKD in patients with an eGFR <60 mL/min/1.73 m2, our analysis showed a lower focus of CKD, especially in subjects in stage G3a, with a percentage that raised from 18.1% to 74.7% with the increasing severity of CKD. A similar trend has also been observed with regard to nephrological visits in agreement with previous studies [Our results confirmed the poor attention in monitoring renal function and inadequate awareness of CKD management by GPs. However, these results bode better than those observed in other studies, which showed at least one registration of a serum creatinine dosage in only 17% of patients compared with 29.3% in our sample, as well as a correct diagnosis of CKD in only 15.2% and 23.2% of patients with an eGFR <60 mL/min/1.73 m studies ,28.2 were mainly females, older, with more comorbidities, in particular affected by hypertension, dyslipidemia, arthritis and arthrosis, chronic pulmonary disease, and osteoporosis. In addition, an increased age and frequency of chronic diseases such as cerebrovascular disease, diabetes mellitus, heart failure, gout, and metabolism disorders have been found with the increased severity of CKD stages (see 2 compared with patients with an eGFR ≥60 mL/min/1.73 m2. This could be explained by the comorbidities data described above. Indeed, the presence of more chronic diseases could lead to polytherapy with a great number of drugs used for each patient [In our setting, patients with an eGFR <60 mL/min/1.73 mages see . In the  patient . More th patient . In addi patient . However patient .As previously observed, the diagnosis of CKD by GPs did not reduce the probability of receiving at least one prescription of nephrotoxic drugs along with the new diagnosis . Once agThe use of inappropriate prescriptions related to the severity of CKD was contradictory. In line with other findings in general practices , in our 2 [Anti-inflammatory and antirheumatic products and antithrombotics were the most widely inappropriately prescribed drugs in our study. In more detail, one-third of patients with CKD had at least one contraindicated prescription of diclofenac; additionally, 19.4% and 14.0% of subjects received at least one contraindicated prescription of acetylsalicylic acid and etoricoxib, respectively. Furthermore, acetylsalicylic acid was the most inappropriately prescribed medication of all contraindicated prescriptions followed by diclofenac, etoricoxib, and metformin. A higher use of acetylsalicylic acid, diclofenac, and coxib in patients with CKD was observed in other studies ,23,37. I2 .The present study has several main strengths and limitations. The major strength is that we conducted the first overview of the predictive factors of contraindicated drugs prescriptions in a large cohort of patients affected by CKD, in a general practice setting, and in a long-time study period. Alternatively, other studies focused on nephrology departments or on a single drug class ,23,26. HOur findings suggest that CKD is not optimally framed in general practices. GPs have a crucial role regarding prescriptions for people with CKD. Nevertheless, the number of contraindicated prescriptions is very high in these subjects, despite the awareness of the disease by GPs. The use of nephrotoxic drugs in patients with CKD may result in increased renal damage with the worsening and progression of CKD that could lead to serious consequences for the sufferer’s health. For these reasons, the dissemination of information and guidelines as well as specific training courses aimed to properly diagnose CKD should be implemented. Moreover, more efficient drug information is needed in order to avoid contraindicated prescriptions in patients with CKD, especially when therapeutic alternatives are available. Lastly, a closer collaboration with nephrologists, not only in the late stages of CKD, might be effective in implementing preventive measures aimed to slow the disease progression and to improve drug management in CKD.


The aim was to describe the population of patients with moderate rheumatoid arthritis (RA) in the United Kingdom and the burden of disease from the perspectives of the patient, caregiver, and health service.In this descriptive study, retrospective patient-level data were extracted from hospital medical records to assess healthcare resource utilisation and validated outcome measures were administered via questionnaire to patients with moderate RA (Disease Activity Score [DAS28] between 3.2 and 5.1) from eight secondary care centres, and their caregivers. Patient-reported outcome instruments were scored according to licensed manuals.n = 26), a mean 29% (SD 26%) reduction in work productivity was recorded. Patients experienced significant fatigue as a result of their RA . Over 50% of caregivers reported providing > 7 h of support care per week to the patient with RA, and 16 and 11% took paid/unpaid leave or reduced working hours, respectively. Mean Caregiver Reaction Assessment subscale scores were 1.9 (SD 0.9) for finance, 1.7 (SD 0.8) for health, 2.3 (SD 1.0) for schedule disruption, and 1.9 (SD 0.8) for family support. Patients had a mean 5.5 (SD 4.1) outpatient attendances and a median 9.0 (IQR 2.0–20.0) diagnostic and monitoring tests in the 12 months prior to enrolment.Outcome measures were completed by 102 patients and 38 caregivers. The mean EuroQoL-5 dimension-5 level crosswalk index value for patients was 0.62 (SD 0.24) compared to an England population norm of 0.82. Mean pain VAS score was 37.7 (SD 24.0) and mean Health Assessment Questionnaire Disability Index was 1.1 (SD 0.8). In employed patients who completed the Work Productivity and Activity Impairment questionnaire (This study shows that moderate RA has a considerable impact on healthcare resources and on patients’ and caregivers’ lives. There is scope to improve the management of patients with moderate RA.The online version contains supplementary material available at 10.1186/s12891-021-04110-1. Rheumatoid arthritis (RA) is an autoimmune disorder that affects more than 400,000 adults (approximately 1%) in the United Kingdom (UK) –4.RA is associated with physical and psychological disabilities and poor quality of life and poses a substantial burden on patients and healthcare resources , 6. DeprThe National Institute for Health and Care Excellence (NICE) recommends first-line treatment with conventional disease-modifying antirheumatic drug (cDMARD) monotherapy using oral methotrexate, leflunomide, or sulfasalazine. Additional cDMARDs are recommended as combination therapy when the treatment target (remission or low disease activity [LDA]) has not been achieved despite dose escalation. Short-term bridging treatment with glucocorticoids should be considered when a new cDMARD is started . BiologiThe European League Against Rheumatism (EULAR), as well as NICE, endorse the ‘treat-to-target’ principle for RA and recommends that the primary treatment goal in RA should be clinical remission (i.e. absence of signs and symptoms of significant inflammatory disease activity) or LDA in order to maximise long-term health-related quality of life (HRQoL) , 17. StuThere is a paucity of evidence from the UK on burden of disease in patients who are not eligible for advanced therapies based on current access restrictions (hereafter referred to as those with moderate RA disease activity). This information is important to help understand unmet needs in this population and to guide future clinical management. Thus, we aimed to describe this population of patients with moderate RA in the UK and the burden of disease from the perspectives of the patient, caregiver, and health service.A multi-centre study was conducted at eight secondary care centres in the UK, including seven in England and one in Scotland. The study comprised a series of cross-sectional patient-reported outcome (PRO) questionnaires and matched caregiver questionnaires and a retrospective review of patient-level data from hospital medical records.Eligible patients were identified either prospectively (selected consecutively at a routine clinic visit) or retrospectively  by members of the direct care team between June 2018 and March 2019. Patients who met the following criteria were included in the study: ≥18 years of age with a DAS28 score between 3.2 and 5.1 at the time of study enrolment (or at the most recent recording up to 8 weeks prior to enrolment), confirmed diagnosis of RA at least 2 years prior to enrolment, and who received a cDMARD in the 24 months prior to enrolment. For the purposes of this study, moderate RA was defined as a DAS28 > 3.2 ≤ 5.1. The exclusion criteria included DAS28 score > 5.1 recorded at any point during the 12 months prior to enrolment, previous biologic therapy or Janus kinase inhibitor for any condition, current or previous participation (≤5 years) in an interventional clinical trial for RA, patient under the care of the rheumatology department at the participating centre for less than 12 months prior to enrolment, and/or unable or unwilling to give consent for study participation. A sample size of 100 patients was deemed sufficient to enable the estimation of endpoints with adequate precision and also pragmatic based on the number of eligible patients expected for the study to give a representative sample of patients with moderate RA.Caregivers were included if they were aged ≥18 years, met the definition of an informal caregiver  at the time of study enrolment, and were the patient’s primary caregiver (defined as the person who provides the most unpaid assistance to the patient for their RA). Caregivers themselves unable or unwilling to give consent for completion of study questionnaires or those who cared for patients unable or unwilling to give consent were excluded from the study.Data extracted from hospital medical records included patient demographics and clinical characteristics, disease severity, comorbidities, disease and treatment history, and data on healthcare resource utilisation (HCRU) for a minimum of 12 months and up to 24 months prior to study enrolment (where data were available). The date of RA diagnosis and number of cDMARDs received may have preceded this time period but were still captured in this study even if the date of diagnosis was more than 24 months prior to enrolment. Source data verification (SDV) was performed on a 15% random sample of patients across all participating centres to ensure data accuracy.Questionnaires were either administered to the patient at their routine clinic visit or by post. Validated questionnaires comprised the following: 5-dimensional EuroQoL (EQ-5D-5L), Health Assessment Questionnaire Disability Index (HAQ-DI), Pain Visual Analogue Scale (VAS), Functional Assessment of Chronic Illness Therapy - Fatigue (FACIT-F), Morning Joint Stiffness Scale (MJS), and Work Productivity and Activity Impairment (WPAI) questionnaire.These questionnaires have been shown to have excellent psychometric properties, including validity and reliability, for use in RA –27.The EQ-5D-5L measures HRQoL  and comprises the following five dimensions: mobility, self-care, usual activities, pain/ discomfort, and anxiety/depression . In addiCaregivers were also asked to complete the EQ-5D-5L to assess the impact of providing care on caregiver HRQoL and the caregiver-specific WPAI to assess the impact looking after the RA patient had on caregiver work productivity. In addition, the validated Caregiver Reaction Assessment (CRA), which comprises five subscales , was used to assess the impact of caring on the caregiver . The totStatistical analyses were performed using Stata version 14.2 . Initial data processing tasks  and tabulation of results were conducted in Microsoft Excel. All analyses were performed with the available data with missing data reported. Quantitative variables approximating a normal distribution and variables describing hospital resource utilisation are presented as mean (standard deviation [SD]); other quantitative variables are presented as medians (interquartile range [IQR]). Categorical variables are described with frequencies and percentages. Distributions are presented as appropriate to the variable reported. The cost of HCRU was calculated using National Health Service (NHS) national schedule of reference costs (2017/18) . The lown = 108) and 5.1 (IQR 2.3–10.9) years at current cDMARD initiation (n = 102). All patients had a DAS28 score between 3.2 and 5.1 at their visit closest to enrolment (mean 3.8 [SD 0.5]) indicating moderate disease activity at the time of recruitment. Only 10% (7/70) and 17% (12/70) of DAS28 scores in the 12 months prior to enrolment indicated remission or LDA, respectively; 73% (51/70) indicated moderate disease. Patients had more tender joints (median 3.0 [IQR 2.0 to 5.0]) than swollen joints (median 1.0 [IQR 1.0 to 3.0]). Patients had a median of 2.0 (IQR 1.0 to 2.0) comorbidities at enrolment; 15% (n = 17) had no comorbidities. The most common comorbidity was cardiovascular disease , followed by respiratory disease , osteoporosis , depression , and diabetes . Half of patients received two cDMARDs (mean 2.1 [SD 0.8]) between RA diagnosis and the start of the observation period (24 months prior to enrolment). The mean number of cDMARDs received between RA diagnosis and enrolment was 2.4 (SD 0.9).A total of 114 patients were included in the analyses. A flow diagram of patient selection is presented in Fig. Of 114 patients included in the study, 102 (89%) completed PRO questionnaires. The PRO results are presented in Table n = 22) of patients reported no change in employment status due to moderate RA. In those who were still employed and completed the WPAI (n = 26), a mean 29% (SD 26%) reduction in work productivity was recorded. The median FACIT-F overall score for patients with moderate RA was 97.0 (IQR 83.6–104.6). The violin plots presented in Fig. Most patients reported some level of impairment (i.e. levels 2–5) for the EQ-5D domains of pain/discomfort , usual activities 73/102, 72%), and mobility , usual activities , anxiety/depression , and mobility ; however, 71% (n = 27) reported slight or moderate pain or discomfort. Median scores for caregivers across the WPAI domains were 0% except for activity impairment which was 10% (IQR 0–45%). Mean CRA subscale scores for finance (1.9 [SD 0.9]), health (1.7 [SD 0.8]), schedule disruption (2.3 [SD 1.0]), and family support (1.9 [SD 0.8]) indicate mild to moderate negative impact on the caregiver. Fifty-six percent (20/36) of caregivers provided > 7 h of support care per week to the patient with RA, while 8% (3/36) provided > 35 h of care per week. Sixty-one percent of caregivers (11/18) reported no change in their employment status as a result of caring for the person with moderate RA; however, five caregivers (28%) took paid and/or unpaid leave and three (17%) stopped work or reduced working hours.Thirty-eight caregivers completed questionnaires  outpatient attendances in the 12 months prior to enrolment Table ; routinen = 93, 82%), hydroxychloroquine , sulfasalazine , leflunomide , and azathioprine , and other RA treatments including corticosteroids , other painkillers , nonsteroidal anti-inflammatory drugs , and other . The total median HCRU cost per patient was £567 (IQR £361–1031) during the 12-month observation period. The highest median HCRU cost per patient in the 12-month period prior to enrolment was for outpatient attendances, costing £336 (IQR £243–516) per patient . Fifty-four percent (61/114) of patients had no new cDMARD initiated or dose change to ongoing cDMARD in the 12 months prior to enrolment. Patients had a mean of 3.0 (SD 1.3) RA treatments (including cDMARDs and other RA treatments) in the 12 months prior to enrolment. Ongoing treatments at enrolment included cDMARDS such as methotrexate  . The EQ-The FACIT fatigue subscale score for this study (17.2) was lower than those reported by Pavelka et al., Smolen et al. (mean DAS28 4.4), and Žagar et al.  indicating that patients in this study experienced significantly more fatigue as a result of their RA despite a lower mean DAS28 , 43, 44.Patients in our study experienced moderate disability (mean HAQ-DI score 1.1). Mean HAQ-DIs of 1.2, 1.1, 1.2, and 1.0 were recorded in other studies with higher mean DAS28 scores of 4.4, 4.4, 5.0, and 4.5, respectively , 44, 45.Total work productivity loss (mean 29% in this study versus 33.5 and 43.2%) and activity impairment (mean 37% versus 48.1 and 56.2%) were lower than that reported in other studies which included patients with more severe disease , 47. HowData from this study also show a significant impact on caregivers looking after patients with moderate RA. Over 50% of caregivers provided > 7 h of support care per week to the patient with RA, mean CRA subscale scores indicate mild to moderate negative impact on the caregiver, and 16 and 11% took paid and/or unpaid leave or reduced working hours, respectively. There are little data available on caregiver burden in RA. Here we show that the burden of moderate RA extends beyond the patient.Data on HCRU for patients with moderate RA are sparse within the literature. Existing data show similar levels of day case admissions (mean 0.2 in the literature versus 0.1 in the 12 months prior to enrolment in our study) and cDMARD utilisation (mean 1.43 in the literature versus 1.8 in our study) . It shouThis study represents one of the most comprehensive attempts to measure burden of disease in a population of UK patients with moderate RA. We used a range of validated PRO measures and also considered caregiver and health service perspectives to give an all-round better picture of the burden of moderate RA in the UK. Some limitations however also need consideration. First, the sample may not be representative. The consent rate was 63%, and the characteristics of the patients who consented may not be representative of the wider population of patients with moderate RA. Patients included in this study were under the care of physicians and study sites that were willing to participate and therefore these centres may not be representative of current wider UK secondary care clinical practice. Another possible limitation is that the interpretation of data collected retrospectively is dependent on the completeness and quality of the medical records and the reliability of data abstraction. We employed SDV in this study to identify and correct abstraction errors. Furthermore, patient-reported data are subject to recall bias; however, this likely had little impact on the results as only short recall was required. Finally, due to only 6% of patients having ongoing treatment with corticosteroids at enrolment, the results may not be generalisable to other non-UK cohorts where steroid use is more prevalent.In conclusion, we show that moderate RA requires frequent hospital visits for clinic appointments and has a considerable impact on the lives of patients (and their caregivers) who are not eligible for advanced therapies based on current access restrictions. The impact  is comparable to that seen in patients on the more severe end of the RA disease spectrum. This suggests that there is substantial opportunity for patients within this disease severity spectrum. The information presented here will aid decision making around the cost benefit of different therapeutic strategies for this often overlooked RA subpopulation.Additional file 1: Supplementary Table e1. on “Caregiver-reported outcomes amongst caregivers looking after patients with moderate RA” provided in Word .doc format.Additional file 2: Supplementary Table e2. on “Costs for healthcare resource use in the 12 months prior to enrolment” provided in Word .doc format.


However, some of the mutations preclude development of animal models, leaving cell-based models as the only options. Mouse embryonic stem cells (mESCs) are attractive because of the well-established technologies for introducing disease-associated mutations and the feasibility of investigating the abnormalities during different stages of neurogenesis. Importantly, such transgenic mESCs enable large-scale screening and identification of the most promising small molecules and/or drug candidates before undertaking expensive animal studies. Although neuronal differentiation from mESCs is one of the earliest methods to be developed, we observed that the published as well as publicly available methods did not yield neurons consistently. Here, we describe a 16-day differentiation protocol that consistently induced differentiation of mESCs into neurons. This step-wise protocol enables monitoring of the neuronal differentiation process at different stages as well as characterization using the markers for immature and mature neurons by using immunocytochemistry and quantitative real-time PCRs.  Specifications tablehttps://www.who.int/ceh/capacity/neurodevelopmental.pdf). Although the exact cause is not known in many cases, a number of etiological factors have been recognized of which several studies emphasized on genetic factors Neurodevelopmental disorders constitute a diverse group of health conditions affecting ≥ 15% of the world population  Gelatin-coated dishes were prepared as described in 2.2 in 2 ml ES medium into 6-well dishes. The cells were incubated in a CO2 incubator at 37°C.Gelatin was aspirated and the ES cells were seeded at a density of ~50,000 cells/ cm3.On day 2, ~90% of the cells were attached. The cells were replaced with fresh medium every day.4.By day 3, the ESCs started growing as colonies A.Fig. 1DDays 1-3B. Generation of Embryoid BodiesDay 45.By day 4 the colonies had grown in size and covered most of the culture area (~80-90% confluence). The medium was replaced with 2 ml of IX PBS  and wash6.0.5 ml of 1X Trypsin-EDTA  was adde7.The cell suspension was transferred to a 15 ml centrifuge tube and centrifuged at 1,000 rpm for 5min. Without disturbing the pellet, the medium was aspirated.8.The cell pellet was resuspended in 1 ml of ES medium and counted using a hemocytometer as described in Step1. For further culturing, the cells were seeded onto a gelatin-coated T-25 or T-75 flask(s).9.2 were plated in 2 ml of EB medium (For generation of embryoid bodies (EBs), ~50,000 ES cells/cmB medium  onto non10.On day 5, EBs had formed after overnight incubation and appeared as spherical aggregates of cells. The EBs were cultured for another day (day 6) in EB medium B.11.Six-well dishes with or without coverslips were coated with 0.2% gelatin as described above.Day 5C. Neuronal Differentiation12.The EBs were collected in suspension from the culture dish, transferred to a 15 ml centrifuge tube and left undisturbed for 15 min to allow the EBs to settle down.13.The medium was aspirated without disturbing the EBs.14.The settled EBs were resuspended in 1 ml of neuronal differentiation medium.15.2 in 2ml of neuronal differentiation medium onto 0.2% gelatin coated dishes.The EBs were plated at a density of ~5 EBs per/cm16.The EBs were uniformly spread over the culture area by rocking the plate in horizontal and vertical motions. Swirling of the EBs was avoided as it would result in settling of all the EBs in the middle of the well. Larger dishes could not allow uniform spreading of the EBs and were therefore avoided.17.2 incubator and were left undisturbed for 48 h.The EBs, once evenly spread on the culture surface, were gently placed in the CODays 6-718.On day 8, most of the EBs were attached C. The meDay 819.Half of the medium was continued to be replenished every day without disturbing the differentiating cells.20.From day 9 onwards, cell death concomitant with neural differentiation was observed.21.From day 10 onwards, small projections were observed D.22.On day 12, rosette-like formation of cells was observed with elongated projections resembling neurites E.Days 9-1223.By days 15-16, most of the cells differentiated with extensive neurite formation and branching F. The neDay 13-16A. Monitoring differentiation by quantitative real-time PCRsProcedure1.Ⓡ.RNA samples were prepared from different cells using the RNeasy kit as per manufacturer's instructions and quantified using NanoDrop2.5 µg each of the RNA samples were converted to cDNA using Superscript IV cDNA synthesis kit as per manufacturer's instructions.3.~50 ng each of the cDNA samples were then mixed with gene-specific primer mix (0.2 µM each) , 2x SYBR4.Gapdh was used as endogenous reference control and fold-changes were calculated by the 2−ΔΔCt method The reactions were used for real-time PCR analyses . p <0.05 was taken as statistically significant.All the experiments were done with at least three biological and two technical replicates. The expression levels of the different transcripts from EBs and neurons were compared with those from ESCs by the student's two-tailed t-test and Oct4 and Nanog) were significantly downregulated during the differentiation into EBs as well as neurons (Brachyury (mesoderm marker) and Pax6 (ectoderm marker) in EBs and neurons (Gata6 (endoderm marker) was observed to be decreased in both EBs and neurons. Importantly, we also observed increased transcript levels of Nestin  in EBs and neurons  and Map2 (mature neurons). Among the glial cell markers, Mbp (oligodendrocytes) showed significantly increased transcript levels in neurons whereas Gfap (astrocytes) and Itgam (microglia) were downregulated. These results suggested that this method of differentiation resulted in an efficient neuronal differentiation into immature and mature neurons.Analysis of transcript levels of neuronal markers showed significantly increased levels of Analysis of transcript levels of markers for different neurons indicated the presence of dopaminergic, cholinergic, GABAergic, glutamatergic and serotonergic neurons upon differentiation. As expected, these markers expressed at significantly higher levels in neurons than ESCs. Data on three biological replicates showed a batch to batch variation with a standard error of ~12.1 % D. These B. ImmunocytochemistryProcedure1.Cells plated on coverslips were used for these experiments. When the cells are ready for the experiments, the medium was aspirated and the cells were washed thrice with 2 ml of warm 1X PBS  by gently rocking the dish manually.2.The cells were fixed with 1ml of 4% paraformaldehyde for 15 min at room temperature.3.The fixative was aspirated and the cells were again washed thrice with 2 ml of 1X PBS for 5 min each time at room temperature. The washes were performed by placing the dishes on a rocker at low speed.4.The cells were blocked in 1 ml of blocking buffer for 1 h at room temperature by placing the dishes on a rocker at low speed.5.During the blocking step, the primary antibodies were diluted in 0.5 ml of antibody dilution buffer, as per the dilutions given above. The diluted antibodies were mixed properly for at least 15 min by placing the tube on a rocker at low speed.6.After one hour, the blocking buffer was replaced with diluted primary antibodies and incubated overnight at 4°C by placing the dishes on a rocker at low speed.7.After incubation, the antibody solution was removed and the cells were washed thrice with 1X PBS as described above. The cells were then incubated with 1 ml of diluted fluorochrome-conjugated secondary antibodies for two hours at room temperature in dark by placing on a rocker at low speed.8.The cells were washed thrice with 1X PBS as described above and counterstained with 1 ml of DAPI solution for 5 min at room temperature in dark.9.The DAPI solution was aspirated and the cells were washed thrice with 1X PBS as described above and used for mounting.10.10-20 µl of mounting medium was placed in the middle of a clean and dry glass slide. The coverslips containing stained cells were then picked up gently using sterile forceps and placed on the mounting medium, avoiding any air bubbles. The edges of the coverslips were sealed using a transparent nail polish.11.The slides were left undisturbed for 3 to 4 h at room temperature in dark, before using them for confocal microscopy. For long-term storage, the slides were stored at 4°C in dark and in flat position.12.Images were captured and analyzed using LasX Life science software platform (Leica).Note: Immunocytochemistry experiments with ~50 EBs each were performed in 1.5 ml transparent centrifuge tubes in 200 µl volumes. For each step, mixing was done by gentle pipetting followed by centrifugation at 1000 rpm for 30 s. At the end of all steps, the EBs were mixed in 20 µl of mounting medium, covered with coverslips and processed as described above.We studied the expression of pluripotency (OCT4), germ layer , neural progenitor (NESTIN) and neuronal markers (β-III TUBULIN and MAP2) by immunocytochemistry of the relevant cell types . OCT4 leThese results suggest that the method of differentiation described here results in efficient and reproducible differentiation of pluripotent ESCs into mature neurons in about 16 days.Troubleshooting:The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Glycosaminoglycan plays an important role in the maintenance of glomerular charge selectivity of diabetic nephropathy. Sulodexide, a mixture of naturally occurring glycosaminoglycan polysaccharide components, has shown a nephroprotective effect in an experimental model of diabetic nephropathy. Although sulodexide reduced albuminuria in patients with type 1 and type 2 diabetes, long-term effects in patients with type 2 diabetes with significant proteinuria have not been established.  The study was aimed at investigating the effects of sulodexide on proteinuria and renal function in patients with type 2 diabetes and nephropathy.  Fifty-two patients with proteinuria between 500 and 3000 mg/day received sulodexide 200 mg/day for 12 months, while 56 matched patients with type 2 diabetes constituted the control group. All patients received standard metabolic and blood pressure controls. Primary outcome was evaluated as percentage of reduced proteinuria compared with the control group. Renal function was assessed using estimated glomerular filtration rate (GFR). P = 0.001], whereas it remained stable in the sulodexide group . At 12 months, proteinuria was higher by 19.4% (IQR 10.3 to 37.6) in the control group while proteinuria was lower by -17.7% (IQR -53.1 to 3.2) in the sulodexide group with a significant difference between groups (P = 0.001). Renal function was noted as a change of estimated GFR, and serum creatinine decreased significantly during the study in both groups but did not significantly differ between groups. No significant changes in the blood pressure, fasting plasma glucose, and hemoglobin A1C were reported.  Proteinuria significantly increased in the control group [0.9 (IQR 0.3 to 1.78) to 1.16 (IQR 0.44 to 2.23) g/gCr,  In addition to standard treatment, sulodexide is efficient in maintaining proteinuria in patients with type 2 diabetes with nonnephrotic range proteinuria, but it did not provide an additional benefit concerning renal disease progression. Diabetic nephropathy is the main cause of end stage renal disease (ESRD), and the prevalence of nephropathy increased in direct proportion to the prevalence of type 2 diabetes , 2. NoveProteinuria is known to be an independent risk factor of renal disease progression and ESRDe in the general population and type 2 diabetes , 8. ShorThis 12-month retrospective cohort study was conducted in patients with type 2 diabetes at the outpatient clinic, Phramongkutklao Hospital. The study was approved by the Institutional Review Board of Phramongkutklao Hospital. Recruitment began in August 2010 and was completed in January 2015. Inclusion criteria comprised age, 18 years or older, and patients with type 2 diabetes with proteinuria between 500 and 3000 mg/day or nonnephrotic range proteinuria. Only patients with proven regular follow-up based on medical reports and prescriptions recorded in our hospital electronic database for a period of 12 months before the study were recruited for the screening visit. Exclusion criteria included type 1 diabetes; pregnancy; active malignancy; severe heart, lung, or liver disease; stroke; chronic infection within one year of starting the study; and any immunological disorders.N = 52) and the control group without sulodexide treatment (N = 56). The prescriptions were made by the nephrologists monitoring the patients in the period intending to maintain renal function. The main outcome was to evaluate differences in urine protein and GFR rates between the treatment and control groups.All patients with type 2 diabetes on standard care were followed in nephrology clinics every three months. According to the medical history records, eligible patients were divided into two groups based on their regimen 12 months before the study as described below. The treatment group was supplemented with sulodexide 200 mg/day  and percentage. The paired 2, respectively. All patients received standard treatment with average systolic blood pressure (139.3 ± 16.7 mmHg), diastolic blood pressure (75.5 ± 11.3 mmHg), HbA1c (7.2 ± 1.3%), and prescribed medications including ACEIs/ARBs/ARB (85.4%) and statins (70.2%). Baseline characteristics of the selected patients are reported in A total of 144 patients were screened for possible study enrollment. One hundred eight patients were eligible according to the entry criteria, and 52 patients received sulodexide treatment. In the sulodexide and control groups, average age was 64.2 ± 10.7 and 66.5 ± 12.8 years, respectively; male prevalence was 61.5 and 66.1%, respectively; and estimated GFR was 49.3 ± 27.5 and 46.3 ± 29.4 mL/min/1.73 m2, P = 0.001) and control  groups exhibited a significant change in estimated GFR levels from baseline during the study (P < 0.001), but no significant difference was observed in the rate of change in estimated GFR between the two groups (P = 0.201) (P = 0.001), but no significant difference in mean change in BUN was observed between the two groups (Both sulodexide (49.3 ± 27.5 to 43.5 ± 28.5 mL/min/1.73 mhe study . GFR lev= 0.201) . Similaro groups .P = 0.001), but no significant difference was observed in the sulodexide group   g/gCr at baseline to 1.16 (IQR 0.44 to 2.23) g/gCr at 12 months  vs. control: 4.5% (IQR -1.8 to 10.5),  to 5.4 vThe study was a retrospective clinical trial of sulodexide treatment on renal outcomes in patients with type 2 diabetes. Sulodexide subjects could decrease the percentage of proteinuric progression compared with the control subjects. However, other changes of renal injury parameters including serum creatinine and estimated GFR did not significantly differ in the control group.The beneficial effects of sulodexide treatment on renal function were limited in our study. The results showed a significant GFR decline over 12 months in patients with diabetes- and nephropathy-prescribed sulodexide therapy, and no significant difference was observed in the rate of GFR decline between the groups. Our results were similar to the results from the randomized, double-blind, placebo-controlled, sulodexide macroalbuminuria (Sun-MACRO) trial. No significant differences were observed in the renal composite end points including doubling of baseline serum creatinine, development of ESRD, or serum creatinine ≥ 6.0 mg/dL between the sulodexide and placebo groups in patients with type 2 diabetes and macroalbuminuria . CurrentOvert diabetic nephropathy exhibits progressive albuminuria and renal deterioration over time . Renal oSeveral limitations were associated with the study. First, the patient selection in our study was not random and could have contained selection bias including compliance to other medications, other nephroprotective therapies, and sodium restriction. However, factors affecting renal disease progression were included and analyzed at baseline of the study, and the confounding factors were similar. Secondly, the present study included a relatively small number of patients and short follow-up time. The relatively short follow-up time may explain the nonsignificant differences in main renal outcomes including renal replacement therapy and end stage renal disease. Thirdly, urine protein and renal function were assessed using urine protein creatinine ratio and estimated GFR equation in our study. They were less accurate than standard 24-hour urine protein collection and radioisotope renal clearance. Finally, as a retrospective study using medical electronic databases, we were unable to confirm whether patients actually took the dispensed medications.In conclusion, the study indicated that sulodexide treatment prevents rising proteinuria levels in type 2 diabetic nephropathy with significant proteinuria but provided no effect on preservation of renal function. Therefore, long-term clinical trials on a larger scale are warranted to elucidate the benefits that sulodexide affords renal protection.