Arterial stiffness is evaluated with the measurement of pulse wave velocity (PWV), while overhydration (OH) and nutritional status are evaluated with bioimpedance spectroscopy (BIS). In this study, we investigated the effect of a single dialysis session on arterial stiffness, hydration status, and laboratory parameters. The observational, cross-sectional, cohort study included 71 HD patients with mean age 64 ± 16 yrs. A Complior device was used to perform PWV measurements. The patients were examined immediately before and 15 min after a mid-week hemodialysis session. Body fluids and nutritional status were studied using a Body Composition Monitor (BCM), Fresenius Medical Care. Clinical and laboratory data were also analyzed. Multivariate regression analysis of PWV before HD showed that an OH increase of 1 L relate to a PWV parameter rise before HD of 0.523 m/s. Multivariate regression analysis of PWV after HD showed that a rise of central SBP after HD of 10 mmHg relate to a PWV increase after HD of 0.707 m/s. Our data indicate that hydration status and blood pressure may be major determinants of PWV in HD patients. The mechanisms underpinning increased arterial stiffness in ESRD patients are not fully defined, but major contributors identified include vascular calcification, chronic volume overload, chronic microinflammation, lipid peroxidation, oxidant stress, sympathetic overactivity, and activation of the renin–angiotensin system.In this study, we investigated the effect of a single dialysis session on arterial stiffness, hydration status, and laboratory parameters.The observational, cohort study was carried out in May–July 2015. The 71 patients (mean age 64 ± 16 yrs) who qualified for the study were being hemodialyzed in the Department of Nephrology, Dialysotherapy and Internal Medicine, Medical University of Warsaw, Warsaw, Poland, and the Dialysis Unit of the Infant Jesus Teaching Hospital in Warsaw, Poland. With written and oral instructions, patients were informed about the rules, aims, and benefits of the study and gave their written consent to participation. The inclusion criteria specified that each patient should be tested prior to the midweek HD session, have a stable clinical status, receive standard HD with three dialysis sessions per week, be on HD for at least 3 months prior to study enrollment, be free of overt CVD, and be more than 18 years of age. Excluded from the study were patients who: had atrial fibrillation, since this condition makes pulse wave velocity testing uninterpretable; had an active inflammatory process; had peripheral artery disease; had stage III–IV congestive heart failure according to the New York Heart Association classification; had regular cardiovascular instability on dialysis ; were pregnant; had residual diuresis >500 mL/24 h; had active arteriovenous fistulas on both upper extremities; had metallic implants ; and had undergone amputation of lower or upper limbs.www.clinicaltrials.gov, identifier NCT02443376). The investigation conformed to the principles outlined in the Declaration of Helsinki. PWV and BIS measurements were performed during midweek HD sessions by a trained researcher. Body mass, height, waist circumference, and body mass index (BMI) were assessed. BMI was calculated as body weight (kg) divided by the square of body height (m). Abdominal obesity was defined as a waist circumference of 102 cm in men and 88 cm in women.The study protocol was approved by the local Research Ethics Committee at the Medical University of Warsaw  and registered at the Clinical Trials register (The results of laboratory investigations were measured on the same day as PWV and BIS measurements and were obtained from the patient’s medical records. All laboratory parameters were measured using automated and standardized methods.European Society of Hypertension (ESH). This measurement allowed brachial systolic blood pressure (bSBP) and brachial diastolic blood pressure (bDBP) to be obtained. The values were reported as the mean of two stable readings.Brachial blood pressure was recorded in the dominant or non-fistula arm using a validated oscillometric device , recommended by the t) between the appearance of the pulse wave on the carotid and femoral artery was measured automatically in 10 subsequent cycles (successive beats) and was averaged. Once the result of the measurement of the distance between the sensors (m) was complete, PWV was calculated by the device and expressed in (m/s) according to the equation: PWV = m/t. According to recent recommendation, when measuring PWV the distance between the sensors was automatically multiplied by coefficient 0.8 by software Complior.PWV measurements were performed with the use of a Complior device . Patients were examined in a quiet, air-conditioned room after a 15-min rest in a supine position, directly prior to and 15 min after the commencement of the mid-week HD session. In brief, one of the device’s two sensors was placed on the site of palpable pulse on the carotid artery, and the other on the site of palpable pulse on the femoral artery. The time (2)), Fat Tissue Index (FTI (kg/m2)), Total Body Water (TBW (L)), ECW (L), ICW (L), and OH (L). Two volume ratios were quantified: ECW/ICW and ECW/TBW.,The study of body fluids and nutritional status was performed using a bioimpedance monitor  Body Composition Monitor software version: 3.3.0.1637) that measures 50 different frequencies ranging from 5 to 1000 kHz, thereby analyzing the whole-body bioimpedance. The BCM is commercially available modern device that can classify ESRD subjects in terms of volume status. The bioimpedance measurement was performed directly before the commencement of HD, with the patient in a supine position and with the use of single-use disposable electrodes placed in doubles on the subject’s wrist and ankle on the side opposite to the arteriovenous fistula. The BCM discriminates fluid of the intracellular (ICW) and extracellular (ECW) water content of lean tissue mass (LTM) and overhydration (OH). LTM and OH are obtained from measurements of body weight, height, and whole-body ICW and ECW determined by BIS. OH represents the excess fluid (fluid overload). The following parameters were recorded in each patient: Lean Tissue Index  were presented as quantity (n) and percentage values of the whole group (%), while proportions in groups were assessed with the Chi-squared test. Statistica 12 software  was used in the statistical analysis. p < 0.05 was adopted as the significance level.Results concerning quantitative variables were presented as average values ± standard deviation (SD). In the comparative characteristics of PWV, the n = 27; 38%), followed by diabetes mellitus , hypertensive nephropathy , polycystic kidney disease , and other causes . There were 36 male and 35 female patients. The mean HD time in these patients was 84 ± 71 months. All patients underwent regular dialysis for 4–5 h and were dialyzed with standard bicarbonate HD solutions and synthetic dialyzers with blood flow rates of 200–250 mL/min, aiming at a dialysis dose of Kt/V > 1.2 with mean URR 74.1 ± 4.9%. Mean ultrafiltration (UF) during HD was 2245 ± 659 mL and bodyweight decreased by a mean of 2.3 ± 0.9 kg.Among the causes of ESRD, the most frequent was glomerulonephritis . This testifies to a different arterial elasticity behavior in the patients studied depending on the excess fluid status and that OH is an independent risk factor in arterial distension. The treatment of hypertension and fluid overload are issues of major importance in HD patients.The present study provides new information on the relationship between nutritional status and hydration status, blood pressure, and PWV in HD patients during an intra-dialysis session. The main finding of this study was that in HD patients the PWV correlated with OH, a marker of the fluid status representing body fluid distribution. Our own study findings show that when PWV was increased following HD the mean OH was 2.63 ± 0.9 [L], while when PWV was decreased following HD the mean OH was 1.74 ± 1.1 [L]  are quite low when compared with other studies in HD patient. Too aggressive a rate of ultrafiltration for dialysis lasting 3.5–4.0 h may result in inadequate dialysis, which in turn can lead to OH and the occurrence of volume-dependent hypertension with higher values of PWV. In the present study, the length of dialysis sessions varies on average between 4 and 5 h with low blood flow:- 200–250 mL/h . In the study, after HD if central blood pressure increase, results as increased the PWV, while the decrease, decreased the PWV. PWV increased group have more OH be evocative of paradoxical hypertension. Paradoxical hypertension is defined by BP values during and at the end of the dialysis session exceeding BP values at dialysis onset. It occurs in around 10% of HD patients.p = 0.739). For the second HD session: PWV before HD= 9.58 ± 0.3, PWV after HD = 9.48 ± 0.3 m/s (p = 0.830).In the study by Georgianos et al.The study carried out by Su et al.r = 0.465; p < 0.001) and with after dialysis PWV values . Blood pressure changes during dialysis were weakly correlated with post-dialysis PWV , but not with PWV changes during dialysis.In the study by Di Iorio et al.,p < 0.004). In addition, an HD session significantly reduced AIx from pre-HD 27.9 ± 11.9% to 18.2 ± 18.3% (p < 0.05). With provocative pharmacological use of inhaled salbutamol and sublingual nitroglycerin testing, Covic et al. revealed that an HD session led to a non-significant improvement in the endothelium-dependent vasomotor function, while correcting the endothelium-independent dysfunction to a level comparable to that in a control group of essentially hypertensive patients.The study by Covic et al.p = 0.019). In their study, Yoon et al.Among others, RDW is a relatively new prognostic parameter in heart failure.This is a cross-sectional study in design and cannot provide cause-and-effect associations. The study measurements may be susceptible to biases related to the peridialytic setting of recordings. Data on wave reflection indices were not assessed. To find the most representative time point for PWV measurements during HD, our study design did not include patient evaluation on all three dialysis sessions of the week and inter-dialysis days.Our data indicate that hydration status and blood pressure may be major determinants of PWV in HD patients.


In conclusion, tubular atrophy/interstitial fibrosis and massive proteinuria were poor predictors for complete remission in IgAN, it appears as though patients may have benefited from immunosuppressive treatment but that comparison to a well-matched contemporary control group or, ideally, a randomized controlled clinical trial, would be required to show this.To compare the long-term efficacy of corticosteroids (P) alone or in combination with cyclophosphamide (CTX), leflunomide (LEF), or Angiotensin-convertase inhibitors or angiotensin II receptor blockers (ACEI/ARB) in treatment for IgA nephropathy (IgAN), 311 patients with IgAN were identified. Therapeutic effectiveness  and combined renal endpoint (defined as 30% reduction in eGFR or ESRD) were compared based on different therapies. After immunosuppressive and ACEI/ARB treatment, the levels of eGFR, proteinuria and albumin were significantly improved at the last follow-up, the extent of improvement of eGFR, proteinuria, and albumin was more notable in P + CTX group and P + LEF group. 41%, 52.2%, 55.3% and 55.2% in P + CTX, P + LEF, P and ACEI/ARB group achieved complete remission, respectively. Multivariate regression analysis indicated that only proteinuria (Relative risk (RR) 0.82(0.72–0.94), P = 0.004) and tubular atrophy/interstitial fibrosis (RR 0.26(0.13–0.57), P = 0.001) were predictors for complete remission. The optimal cutoffs of eGFR was 47.085 ml/min/1.73 m IgAN has various clinical and pathological manifestations and corresponding considerable variations in prognosis. It is a relatively benign disease, however, the long term prognosis should not be considered mild, because, after 20 years of disease progression, 20–50% of the patients reach end stage renal disease6. A number of immunosuppressive agents have been used for treatment of IgAN, including calcineurin inhibitors8, azathioprine9, mycophenolate mofetil11, cyclophosphamide12, leflunomide14, corticosteroids17, etc. Many studies have only compared two treatment regimens for a relatively small sample size. However, the protective role of immunosuppressive therapy was still in controversy18. It is essential to find out patients who may benefit from immunosuppressive treatment and the proper choice of immunosuppressive therapy or supportive care3. We therefore retrospectively analyzed 311 patients with IgAN treated with CTX, LEF, P or conservative management alone in our center to identify long-term therapeutic effectiveness and renal outcome, and explore the prognostic factors for renal function recovery in patients with reduced renal function.Immunoglobin A nephropathy (IgAN) is the most frequent primary glomerulonephritis worldwide, especially in China. It accounts for approximately 40–50% of primary glomerulonephritisAll patients with biopsy-proven IgAN in Sichuan Provincial People’s Hospital from 2006 to 2014 were reviewed. The baseline and follow-up data of the patients were obtained from the database of Glomerulonephritis Registry in our department. The diagnosis of IgAN was based on diffuse IgA-dominant depositions in the glomerular mesangial region and mesangial electron dense deposition in electron microscopy. The exclusion criteria included secondary IgAN , crescentic IgAN (defined as ≥50% crescentic glomeruli on kidney biopsy), or minimal change disease with glomerular IgA deposits, with acute kidney injury, aged <18 years at biopsy or with follow-up <6 months. The protocol for this study was approved by medical ethics committee of the Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital, and informed written consent for the treatment they received was obtained from all of the identified patients. All treatment studies were performed in accordance with relevant guidelines and regulations.19. Scr and Upr during follow-up are also collected.Baseline clinical data included gender, age, disease duration, blood pressure (measured at the morning of the biopsy day), gross hematuria at the time of biopsy. Laboratory data included 24-h urinary protein excretion (Upr), serum albumin (Alb), serum creatinine (Scr), uric acid (UA), liver enzymes, and estimated glomerular filtration rate (eGFR) calculated using the equation of CKD-EPI(CKD Epidemiology Collaboration) formula20, and seven pathological variables, namely mesangial hypercellularity (M), endocapillary proliferation (E), segmental sclerosis or adhesion (S), tubular atrophy/interstitial fibrosis (T), crescents (C), glomerulus necrosis (N), and vascular changes were assessed. Tubulointerstitial lesions, including tubular atrophy, interstitial inflammation, and interstitial fibrosis, were semi-quantitatively sorted into the following categories: none , mild , moderate , and severe 21.The scoring sheet was based on the Oxford classification of IgAN2 received steroid therapy with or without ACEI/ARB. Those with moderate(1–3 g/day) to severe(>3 g/day) proteinuria, sever acute tubulointerstitial or acute glomerular injury received immunosuppressive agents including LEF or CTX. P was given at a dosage of 0.8–1.0 mg/kg/day (The maximum dosage of 60 mg per day) for 8 weeks, then gradually tapered to stop within 6–9 months. Intravenous CTX was given twice a month for 6 months and the dosage was 0.5–0.75 g/m2 body surface area per month according to a modified National Institutes of Health (NIH) protocol22. LEF was orally administered with 50 mg/day for 3 days, reduced to 20 mg/day for 3–6 months, and subsequently tapered24. All the patients who were treated with CTX or LEF also received prednisone followed the previous protocol. ACEI/ARB were used in a dosage titration method until they reached the maximum tolerable dosage. Major adverse events included elevated liver enzymes, leucopenia, respiratory infection, and steroid diabetes were retrospective reviewed of outpatient and inpatient electronic medical record system.Most patients in the immunosuppressant groups also received ACEI/ARB therapy. Patients with persistent proteinuria (more than 1 g/day) and a eGFR of more than 50 ml/min per 1.73 m2, initiation of dialysis for more than 3 months or transplantation)25. Therapeutic effectiveness included progression, partial remission and complete remission based on proteinuria and renal function26, assessed after six months of treatment. Progression was defined as proteinuria ≥1 g/day and declining eGFR, partial remission was defined as proteinuria ≤1 g/day and stabilization of eGFR, complete remission was defined as a reduction of proteinuria to <0.3 g/day and renal function recovery. Normal range Scr was defined as Scr <104 umol/L. Renal function recovery was defined as improvement of eGFR by at least 20% or back to normal range.ESRD was defined as eGFR <15 mL/min/1.73 mP < 0.10. ROCs of the above significant risk factors by logistic regression were drawn to determine the optimal cutoffs predicting recovery of renal function. The function to calculate the probability of this event was as follows:All data were analyzed by using statistical software SPSS 19.0. Normally distributed variables were expressed as mean ± SD. Non-parametric variables were expressed as median and interquartile range. Categorical variables were expressed in percentages. All parameters were compared by Chi-square test, Fisher test for categorical data and the Student’s t-test, one-way analysis of variance (ANOVA) analysis or Kruskal-Wallis test for continuous data. Survival rate was estimated with the Kaplan–Meier method. The outcome event was defined as a combined renal endpoint: 30% reduction in eGFR or ESRD. Univariate analysis followed by multivariate logistic regression analysis was used to evaluate the cumulative probability of complete remission in the whole group and renal function recovery in patients with reduced renal function in subgroups. All baseline variables were entered in the initial model and maintained if P < 0.001) and a lower level of eGFR (P < 0.001) than other groups. LEF group, steroid group and especially P + CTX group had more severe hypoproteinemia (P < 0.001) and heavier proteinuria than ACEI/ARB group. ACEI/ARB group had a significantly lower percentage of nephrotic proteinuria than other three groups  were females and 119 (38.3%) were males, with an average age of 36.4 ± 11.0 years old. P + CTX group had a much higher level of Scr (P < 0.001), highest proportion of global and segmental glomerulosclerosis (P < 0.001), most severe tubular atrophy/interstitial fibrosis lesions (P < 0.001), most severe interstitial lymphocyte and monocyte infiltration and most prominent vascular lesions (P < 0.001) than other three groups. While the ACEI/ARB group had the least prominent glomerular, tubular, interstitial and vascular lesions than other three groups (P < 0.001)  (1) Table .Table 2PP < 0.001) The ACEI/ARB group had the highest level of eGFR than other groups. P + CTX group had higher annual increase rate and value of eGFR, higher proportion of 30% and 50% rise in eGFR than ACEI/ARB group , 5 patients (1.6%) of the 311 cases entered ESRD and 22 patients (7.1%) reached the combined endpoint. 17 patients (5.5%) had ≥40% reduction in eGFR and 13 patients (4.2%) had ≥50% reduction in eGFR.  had impaired renal function, after immunosuppressants and ACEI/ARB treatment, 75 patients (58.6%) achieved renal function recovery, and 57 patients’ (44.5%) Scr concentration returned to normal range. We afterwards respectively explored the risk factors for renal recovery in patients with reduced renal function in each group.After a complete or partial remission, four patients in P + CTX group had a relapse of nephrotic proteinuria and achieved a complete or partial remission after P + LEF therapy ,five patients in P + LEF relapsed, two of them were treated with prednisone at a dose of 0.8–1.0 mg/kg/day, and three were treated with P + LEF, two patients in P group relapsed and were re-induced with 0.8–1.0 mg/kg/day dose of prednisone.The cumulative dosage of CTX in P + CTX group was 7.5 ± 0.3 g. 53 cases in P + CTX group had impaired renal function, in which 21 patients’ (39.6%) Scr concentration returned to normal range and 31 cases (58.5%) achieved renal function recovery. At the time of biopsy, univariate logistic regression indicated that gross hematuria , eGFR  and proteinuria  were predictors for renal function recovery in CTX group. Multivariate regression analysis indicated that only eGFR  was independently associated with renal function recovery Table .2   was 0.742 , indicating that the initial eGFR at renal biopsy had high predictive accuracy for renal function recovery for IgAN patients with P + CTX treatment. The optimal cutoffs of eGFR was 47.085 ml/min/1.73 m2 = 0.999, P = < 0.001) .We used the strongest predictive factor, the initial eGFR as the variable, and demonstrated that the association between the probability of renal function recovery and eGFR fitted a cubic shape, we calculated the model for the prediction of renal function recovery as follows:2, the cumulative probability of renal function recovery was above 80%  achieved renal function recovery during follow-up.When the initial eGFR was higher than 51.2 ml/min/1.73 m80% Fig. . In the 31 cases in P + LEF group had impaired renal function, in which 15 patients’ (48.4%) Scr concentration returned to normal range and 18 cases (58.1%) achieved renal function recovery. At the time of biopsy, univariate logistic regression indicated that gross hematuria , and Scr concentration  were predictors for renal function recovery in P + LEF group. Multivariate regression analysis indicated that only gross hematuria  was an independent predictor of poor response to P + LEF therapy Table .23 cases in P group had impaired renal function, in which 12 patients’ (52.2%) Scr concentration returned to normal range and 15 cases (65.2%) achieved renal function recovery. At the time of biopsy, univariate and multivariate logistic regression indicated that crescents  predicted poor response to corticosteroid therapy Table Elevated liver enzymes, leucopenia and respiratory infection occurred in 4 cases (4.7%), 4 cases (4.7%) and 2 cases (2.4%) in P + CTX group. Elevated liver enzymes, alopecia and respiratory infection occurred in 2 cases (2.7%), 2 cases (2.7%) and 1 case (1.4%) in P + LEF group. Elevated liver enzymes and steroid diabetes occurred in 3 cases (3.8%), 3 cases (3.8%) in P group. No significant difference was found of the incidence of adverse effects among the three groups. None of the patients in either group developed incident diabetes, hyperkalemia or serious infections that would cause drug withdrawal or termination. After symptomatic treatment, those with liver function injury, leucopenia and infection recovered and continued the therapy.30. The therapeutic measures range from no therapy with the only need of regular follow-up, to supportive therapy eventually associated with low dose immunosuppression, to immunosuppressive treatment in the attempt to avoid the evolution to end stage renal disease31. There is an ongoing debate over the efficacy and safety of immunosuppressive agents other than ACEI/ARB monotherapy in IgAN. However, the current evidence about the different therapies remains to be elucidated31. Here we included four most widely applied therapies in our center to explore the treatment response, predictors for complete remission and adverse effects of immunosuppressive agents in IgAN.IgA nephropathy has remained the most common form of primary glomerulonephritis leading to chronic kidney disease in developed countries. ESRD occurs in 30% to 40% of patients 20 to 30 years after the first clinical presentation. Identification of high-risk patients and who may benefit from immunosuppressive treatment is importantACEI/ARB were given in more than 90% but not all of patients in three suppressants groups. Though ACEI/ARB group had higher incidence of complete remission, those with cytoxic therapy had more severe renal injury and pathological features before treatment.36. Due to insufficient evidence and uncertain risk-benefit balance, a possible role is suggested by the guidelines only for patients with crescentic IgAN and rapidly decreasing renal function, analogous to the treatment of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis34.The efficiency of CTX with corticosteroids has been principally examined in small retrospective studies concerning patients with progressive renal deterioration or with crescentic IgANOur study found that patients who received CTX therapy presented with more proteinuria, microscopic hematuria and hypoalbuminemia and severe pathologic lesions than those with P + LEF, P and ACEI/ARB, however, P + CTX was effective in reducing urinary protein levels and conserve kidney function in patients with IgAN, although we excluded crescentic IgAN. 44.7% and 28.2% had more than 30% and 50% rise in eGFR. 58.5% of 53 cases in P + CTX group with impaired renal function achieved renal recovery during follow-up.2, supporting that P + CTX should be considered promising therapeutic agents for the treatment of IgAN with reduced renal function. Using logistic curves, we developed a simple model based on eGFR to predict renal recovery of IgAN treated with P + CTX. In this model, a plot of the association between the probability of renal recovery and the initial eGFR formed a cubic-shaped curve, the probability of recovery in kidney function goes down if the GFR is less than 70 ml/min, and when the initial eGFR was higher than 51.2 ml/min/1.73 m2, the cumulative probability of renal function recovery was above 80%., indicating that cyclophosphamide is useful if eGFR is more than 47 ml/min, CTX therapy may not achieve a promising effect when eGFR is less than 47 ml/min, this finding needs large, prospective studies to confirm.Multiple regression in our study revealed that eGFR was an independent risk factor for renal recovery in IgAN patients with P + CTX treatment. We further used eGFR as variable to draw ROC and demonstrated that the optimal cutoff for predicting renal recovery was eGFR 47.1 ml/min/1.73 m14 is an immunosuppressive agent inhibiting T- and B-cell functions which has long been used in rheumatology. Its mechanism of action involves inhibition of dihydroorotate dehydrogenase38, as well as a number of tyrosine kinase signaling molecules involved with immune function. Leflunomide appears to be a promising treatment for lupus nephritis39, but there are limited data supporting the use of leflunomide as an investigational therapy for progressive IgA40. Our study demonstrated that LEF can effectively reduce proteinuria, as compared with the steroid therapy, similar to previous studies41. In addition, P + LEF group in our cohort had higher annual increase rate and value of eGFR, higher proportion of 30% and 50% rise in eGFR than P group, with almost identical baseline clinicopathological features. Gross hematuria was identified to be an independent predictor of poor response to P + LEF therapy. Our results revealed that more than half of patients with reduced renal function in P + LEF group achieved renal recovery after treatment. Therefore, P + LEF may be considered promising therapeutic agents for the treatment of IgAN41, especially in those without gross hematuria, but this conclusion is limited by lack of a control group of untreated patients, and should be investigated further in large sample size, high-quality studies.Leflunomide46, while other studies did not confirm a steroid related beneficial effects47 or highlight the problem of corticosteroid side effects48. STOP-IgA12 study showed that the addition of immunosuppressive therapy to intensive supportive care did not significantly improve the outcome and may increase adverse effects. Our study suggested that adding corticosteroid and immunosuppressants to RAS blockers resulted in a better reduction of proteinuria, a higher rate of renal function recovery and a higher incidence of side effects in comparison with administering RAS blockers alone, even P group in our cohort had heavier proteinuria and severe hypoproteinemia than ACEI/ARB group.Several studies have been performed to prove the effectiveness of corticosteroid therapy in IgANThere were some limitations to this study. Firstly, this was a single-center retrospective observational analysis. It was difficult to control for all factors that may affect renal survivorship. Secondly, treatment failure and previous renal biopsy data, which could possibly influence the outcomes, was not included because many records were lost or incomplete. Thirdly, we did not add different treatments into our multivariate regression analysis because the treatment assignment was not blinded and unified, which was based on the patients’ financial conditions and different physicians’ clinical experiences. Fourthly, ACEI/ARB and P group had a shorter period of follow-up compared to the P + LEF and P + CTX group, this could be attributed to less severe baseline clinicopathological manifestations and better prognosis than other groups, resulting in greater loss to follow-up. In addition, as a retrospective study, we didn’t include a control group of untreated patients to compare with treated groups, thus it is not known what would have happened for these patients who have not been treatment. Any improvements in renal function or proteinuria may have been due to ACE/ARB treatment, BP control, spontaneous improvement or other factors and is not necessary due to the immunosuppressive treatment. The retrospective design also limited our study with regard to adverse events. We could only include major adverse events that were mentioned in the outpatient and inpatient hospital electronic records.In conclusion, tubular atrophy/interstitial fibrosis on renal biopsy and massive proteinuria were poor predictors for complete remission in IgAN. Reduced eGFR, gross hematuria and crescents were predictors for a lower rate of renal function recovery with CTX, LEF and corticosteroid therapy, respectively. It appears as though patients may have benefited from immunosuppressive treatment but that comparison to a well-matched contemporary control group or, ideally, a randomized controlled clinical trial, would be required to show this.supplementary material


Candida albicans. This antifungal has moderate water solubility and exhibits a nonlinear pharmacokinetic (PK) profile. By entrapping VCZ into liposomes, it is possible to circumvent certain downsides of the currently available product such as a reduction in the rate of its metabolization into an inactive form, avoidance of the toxicity of the sulfobutyl ether-beta-cyclodextrin (SBECD), vehicle used to increase its solubility. PKs and biodistribution of VCZ modified by encapsulation into liposomes resulted in improved antifungal activity, due to increased specificity and tissue penetration. In this work, liposomal VCZ resulted in AUC0–24/MIC ratio of 53.51 ± 11.12, whereas VFEND® resulted in a 2.5-fold lower AUC0–24/MIC ratio (21.51 ± 2.88), indicating favorable antimicrobial systemic activity. VCZ accumulation in the liver and kidneys was significantly higher when the liposomal form was used. Protection of the drug from biological degradation and reduced rate of metabolism leads to a 30% reduction of AUC of the inactive metabolite voriconazole-N-oxide (VNO) when the liposomal drug was administered. Liposomal VCZ presents an alternative therapeutic platform, leading to a safe and effective treatment against systemic fungal infections.Voriconazole (VCZ), a triazole with a large spectrum of action is one of the most recommended antifungal agents as the first line therapy against several clinically important systemic fungal infections, including those by  The whole world needs new medicines against infectious diseases. Thanks to antimicrobials, an increase in life expectancy, especially of immunosuppressed individuals , as well as of those living in areas lacking basic sanitation infrastructure and infection prevention programs became possible , a second-generation triazole with a large spectrum of action is one of the most recommended systemic antifungal agents as the first line therapy against several types of systemic mycoses, including  Dupont, . VCZ exh Dupont, . In addi Dupont, .®) for injection contains sulfobutyl ether-beta-cyclodextrin (SBECD) to increase drug apparent solubility in water. Similar to other cyclodextrins, SBECD can cause several clinically relevant issues, such as nephrotoxicity, hemolysis, blood vessels congestion, and hepatic and renal toxicity due to potential accumulation  parameters are highly dependent on the physical-chemical characteristics of the drug, once encapsulated into liposomes, these parameters can be modulated. Different blood circulation times, tissue distribution, and the prevention of biological degradation of the antifungal might influence the efficacy of the drug  was conducted as well as in vivo efficacy evaluation of LVCZ using a mouse model of systemic candidiasis.In this study, we prepared and characterized a novel formulation for the antifungal VCZ entrapped into liposomes (LVCZ) for intravenous delivery. In addition, PK and tissue distribution profiles were measured. Cholesterol and soybean phosphatidylcholine (PC) were purchased from Avanti Polar Lipids . Voriconazole (VOR) was purchased from Hangzhou Dayangchem Co., Limited . and voriconazole-N-oxide (VNO) was purchased from Genone Biotechnologies  Alpha-tocopherol and Sabouraud’s dextrose agar were purchased from Sigma Aldrich . Acetonitrile was purchased from J. T. Baker . Water was purified using a Milli-Q system  with a 0.22 µm pore end-filter. All other chemicals and reagents were of analytical grade or superior.in vitro susceptibility assay, a total of eight fungal strains were tested as following: two American Type Culture Collection (ATCC) strains, C. albicans ATCC 90028 and Candida parapsilosis ATCC 22019, and six clinical isolates, including two C. albicans (77 and 11 U) and four Aspergillus sp.  strains. The clinical isolates were obtained from the fungal library of the Laboratory of Mycology from Federal University of Goiás and originated from samples of patients with onychomycosis and hematologic malignancies from works previously performed at the Hospital das Clínicas da Universidade Federal de Goiás and Hospital Araújo Jorge/Associação de Combate ao Câncer em Goiás (ACCG) of Goiânia-GO. C. albicans ATCC 90028 strain was used to establish the mouse model of systemic candidiasis. All strains were aerobically cultured on Sabouraud agar and maintained on Sabouraud 4%-glucose agar at 37 °C overnight before the assay.For the in vivo studies were carried out on Balb/c male mice , obtained from the Animal Facilities of the Institute of Tropical Pathology and Public Health of Federal University of Goiás . Before beginning the experiments, the animals were acclimatized for a week in the laboratory. Considering that animal experimentation in infectious diseases research remains essential to understand the fundamental mechanisms of antifungal drugs as well as to meet regulatory requirements, the in vivo analyses conducted in this study were focused on animal welfare. The experimental design considered the minimum number of animals without reducing the scientific integrity of data generated. In additions, procedures such as reduced total amount of blood collected, small volume of injections, anesthesia, and analgesia were observed.The ad libitum. At the end of each experiment, the mice were anesthetized with a solution containing xylazine (10 mg/kg) and ketamine hydrochloride (100 mg/kg) administered intraperitoneally and euthanized by cervical dislocation  cycles and controlled temperature (23 ± 2 °C). Water and food (standard granulated chow) were provided ® Extruder device  under nitrogen pressure.A liposomal formulation was prepared by lipid-film hydration followed by extrusion. PC, cholesterol, VCZ, and alpha-tocopherol, were dissolved in chloroform . Solvent was evaporated at 25 °C and 200 rpm under reduced pressure leading to the formation of a thin layer on the inner side of a round-bottomed flask. Lipid film was hydrated with water for a final volume reaching the concentration of 2 mg/mL of VCZ. Hydrated vesicles were reduced in size by extrusion through 200-nm-pore polycarbonate filters followed by 100-nm-pore filters, in a LipexFormulations were lyophilized using sucrose as cryoprotectant (4:1 of molar ratio sucrose:PC) and reconstituted in sterile saline immediately before use. Particle size and polydispersity index (PdI) were evaluated by dynamic light scattering (DLS) in a Zetasizer Nano S  and the morphology by Transmission Electron Microscopy (TEM) examined in a Jeol JEM-2100 Electron Microscope  operating at 100 kV, following negative staining. Drug-free liposomes were prepared as controls.via electrophoretic mobility in a ZetaPlus . Liposome Entrapment Efficiency (EE%) was determined after ultrafiltration  by the following equation: VCZ was quantified by High Performance Liquid Chromatography (HPLC) with UV detection at 255 nm. Zeta potential was assessed In vitro susceptibility of yeast and filamentous fungi was performed using the microdilution method in broth following M27-A3, M27-S4 , and M38-A2 (2008), proposed by the Clinical and Laboratory Standards Institute (CLSI). All the strains used were diluted in saline at the concentration of 0.5−2.5 × 103 and 0.4−5×104 CFU/mL for Candida and Aspergillus, respectively. VFEND® and LVCZ were diluted in the range of 0.0312−32 μg/mL.Aspergillus species.Plates were incubated at 35 °C for 48 h for Candida species and for 72 h for C. parapsilosis ATCC 22019 was used as control of MIC, according to CLSI 2008  was determined as the lower concentration of the compound capable of inhibiting the total growth of the microorganism. The experiment was performed in triplicate, and Aspergillus species. MFC was defined as the lowest concentration of antifungal that resulted in the growth of up to two colonies, which represent the death of more than 99% of the initial inoculum  determination, 10 μL of antifungal MIC and four immediately higher concentrations were seeded in Petri dishes containing Sabouraud dextrose agar (SDA). Plates were incubated at 35 °C for 48 h for Candida species and for 72 h for um John, .n = 12 for each group) according to the formulation received. Lyophilized powders of LVCZ and VFEND® were reconstituted with sterile saline to 2 mg/mL. An intravenous (I.V) bolus injection of 10 mg/kg of each formulation/group was administered via tail vein. Using a non-mortality model  randomly assigned to two groups , composed of a quaternary pump, an auto-sampler, a column oven, with a hyphenated API 3200 triple quadrupole mass spectrometer (MS/MS) detector . Drugs and internal standard (ketoconazole) were partitioned through liquid–liquid extraction using methyl tert-butyl ether. Concentrations of VCZ and VNO in blood were determined simultaneously from blood samples collected at 0.5 and 1 h following injection.® V.15.12.3 and the add in: PKSolver . After 2 h of infection, the animals were treated with 150 μL of either sterile saline, VFEND® or LVCZ, both at 10 mg/kg of VCZ, for 3 d. Treatment groups (n = 5/group) were distributed as follows: Group I – infected and treated with placebo ; Group II – infected and treated with VFEND® (IT-VFEND® group); Group III – infected and treated with LVCZ (IT-LVCZ group). On day 7, after 24 h of treatment, mice were previously anesthetized for blood collection by cardiac puncture. After that, animals were euthanized and kidneys and liver were removed for fungal burden and histopathology analyzes. For the fungal burden evaluation, liver and kidneys sections were macerated in 3 and 5 mL of sterile saline, respectively, using a glass tissue macerator. Liver and kidneys homogenates were then diluted at 1/100 and 1/1000, respectively, and 100 μL were plated on sterile Petri dishes containing SDA. After incubation for 48 h at 35 °C, colony forming units were counted. The experimental schedule of mouse model of systemic candidiasis is shown in Supplementary data 1.Infection model was based on protocols previously described  at room temperature for the histopathological analysis. In brief, two sagittal macroscopic cross sections were dehydrated in graded ethanol (70–100%), cleared in xylene and then paraffin-embedded. After that, paraffin-embedded tissue sections of organs (5 μm thickness) were obtained using a microtome . After mounting, the slides were dewaxed in xylene, hydrated using graded ethanol and stained with hematoxylin and eosin (HE) or periodic acid-Schiff (PAS). The slides were observed using a light microscope  using a 40× objective. The images were taken using an AxioCam MRc Carl Zeiss camera and AxioVs40 version 4.7.2.0 Carl Zeis software .t-test or one-way Analysis of Variance (ANOVA) followed by Dunnett’s test. p <.05 was considered statistically significant.Data were reported as mean ± SD. Statistical analysis was performed using GraphPad Prism version 5.01 software for Windows . The intergroup variation was carried out by the Student’s C. albicans systemic infection by lipid-film hydration followed by extrusion. LVCZ exhibited a diameter of 95.3 ± 1.27 nm, very narrow size distribution (PdI =0.09 ± 0.01) and a relatively neutral charge. Entrapment efficiency was approximately 80%. Even though VCZ water solubility is low (<100 μg/mL), it is also only moderately lipophilic (log p ∼1.8). Drugs with intermediate partition coefficients (1.7 < log p < 4.0) can partition between the lipid membrane and the aqueous phase. .Intravenous liposomal formulation containing VCZ constituted by PC and cholesterol, herein called LVCZ, was developed for the treatment of ® and LVCZ against Candida sp. and Aspergillus sp. yeasts were carried out after 48 and 72 h of incubation, respectively, using the broth microdilution method. MIC and MFC values are summarized in Candida sp. strains were more susceptible than Aspergillus sp. strains for both formulations. For Candida sp. strains, VFEND® and LVCZ have similar potency, although LVCZ triggered higher fungicide activity against C. albicans 77 U strain since the MFC value (0.12 μg/mL) was about 4-fold lower than that found for VFEND® (0.5 μg/mL). Among Aspergillus sp. strains, LVCZ showed higher inhibitory/fungicide activity against Aspergillus Flavus. For the A. Flavus V2 strain, MIC and MCF values were 2- and 4-fold lower (both at 0.25 μg/mL), respectively, than those found for VFEND® (MIC =0.5 μg/mL and MCF =1.0 μg/mL); while for V1F1 strain these values were 2-fold lower  in comparison to VFEND® values (MIC =1.0 μg/mL and MCF =2.0 μg/mL).Activities of VFENDThe bioanalytical quantitation method by HPLC-MS/MS was sensitive and selective enough to allow the simultaneous quantification of the antifungal VCZ, and its inactive metabolite VNO, even from tissue homogenates or in the whole blood samples from the animals. Chromatograms, mass spectra, and calibration curves of the above-mentioned substances are given in ®, liposomes reduced VCZ clearance by half; increased AUC0–24 by a 2.5-fold and reduced the drug volume of distribution. Main PK parameters and data for predicting the magnitude of the best PK/PD index, which were calculated by the ratio AUC0–24/MIC and Cmax/MIC also are shown in Differences in PK parameters of VCZ from both formulations were observed. When compared to VFEND® or LVCZ formulations in Balb/c mice are shown in ® at any time point. 0–1 h was 0.49 µg/mL.h for VFEND® and 0.35 µg/mL.h for LVCZ.Blood concentration profiles following I.V injections of VFENDIn order to avoid bias caused by the occurrence of hysteresis , the quantification of VCZ in several tissues was performed.Tissue distribution of VCZ from both formulations was evaluated in various tissues, including several organs of the reticuloendothelial system .® was able to maintain concentrations above MIC for only 8 h after a single bolus dose.Our PK assays using healthy animals have shown that blood concentrations of VCZ remained for 12 h above the MIC, following the administration of 10 mg/kg of the liposomal formulation. Conversely, VFEND® formulations showed inhibitory activity against fungal growth  exhibited 7.03 ± 0.29 log10 CFU/g. Livers of the animals from both groups were cleared from fungal cells ), strikingly evident for the placebo group.All animals presented different degrees of weight loss , may leave gaps for microbial resistance , might be a mechanism of improving the existing drug therapy  remained for approximately 12 h above the MIC (i.e. a T > MIC =12 h), following the administration of 10 mg/kg of the LVCZ, ensuring the maximization of antifungal exposure and also avoiding toxicity by exposure to unnecessary high concentrations of the drug . In clinical trials using azole antimicrobials for the treatment of invasive candidiasis, when this AUC/MIC ratio is >25, treatment success is at least 91%   of VCZ  shows the decay of basal leukocyte levels, over cyclophosphamide dosing period, confirming leukopenia of animals).Improvements in PK s and a tissue distribution more selective to the organs most affected by the infection might increase the efficacy of LVCZ, when compared directly with VFENDSince the efficacy of antifungals cannot be estimated based merely on their blood concentration, it is important to correlate this with tissue distribution, which relates to the VCZ/fungal burden colocalization (“drug and bug”) . The extravasation of the liposomes to infected tissues was probably increased due the more permeable endothelium found in the infection sites (Maeda et al., ®.A high AUC/MIC correlation obtained in our PK/PD assays, suggests a more pronounced antifungal response when the liposomal formulation was used, highlighting a gain in the efficacy of the LVCZ formulation over VFENDNevertheless, limitations of murine models using this triazole may hinder further clinical correlation. Accelerated clearance and even genetic polymorphism in humans require thorough investigations involving different animal species or even patients of different age, gender, stage of disease, or ethnicity. Previous literature reports suggest the oral use of grapefruit concomitant with VCZ or the use of guinea pigs lineages instead of mice in order to reduce enzymatic activity that can lead to irrelevant serum VCZ concentrations due to their extremely rapid conversion to the inactive metabolite, VNO (Sugar & Liu, C. albicans infection in a murine model, compared to current therapy.Liposomal formulation entrapping VCZ is an alternative formulation for intravenous antifungal treatment, avoiding the use of potentially toxic adjuvants and showing significant improvements in antifungal activity to target systemic The ability of VCZ to exert fungicidal activity might be enhanced by its entrapment within liposomes. Liposomal encapsulation showed a certain degree of protection from premature metabolism, contributing to more effective antimicrobial blood concentrations, allowing for deeper and more selective penetration and accumulation into the tissues more severely affected by the pathogen.We have successfully obtained an effective, biocompatible, biodegradable, targeted and safe antifungal liposomal formulation for VCZ. Liposomal VCZ can be used for intravenous delivery of the drug, resulting in improved PK s parameters with positive impact in the antifungal activity of VCZ.Our contribution encourages the development of an alternative formulation to the commercially available VCZ for intravenous delivery. PK data also reinforce the evidence that, by encapsulating VCZ in liposomes, it is possible to slow down the metabolic depletion of the antifungal. Additionally, a more selective tissue accumulation may be an effective strategy to deliver the antifungal into deeper regions of organs severely affected by the fungal infection.


JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.Glucocorticoids (GCs) have unparalleled anti‐inflammatory and immunosuppressive properties, which accounts for their widespread prescription and use. Unfortunately, a limitation to GC therapy is a wide range of negative side effects including Cushing's syndrome, a disease characterized by metabolic abnormalities including muscle wasting and osteoporosis. GC‐induced osteoporosis occurs in 30% to 50% of patients on GC therapy and thus, represents an important area of study. Herein, we characterize the molecular and physiologic effects of GC‐induced osteoporosis using the Cushing's mouse model, the corticotropin releasing hormone (CRH) transgenic mouse (CRH‐Tg). The humeri, femurs, and tibias from wild‐type (WT) and CRH‐Tg male mice, aged 13 to 14 weeks old were subjected to multiple bone tests including, micro–computed tomography (μCT), static and dynamic histomorphometry, strength testing, and gene expression analyses. The CRH‐Tg mice had a 38% decrease in cortical bone area, a 35% decrease in cortical thickness, a 16% decrease in trabecular thickness, a sixfold increase in bone adiposity, a 27% reduction in osteoid width, a 75% increase in bone‐resorbing osteoclast number/bone surface, a 34% decrease in bone formation rate, and a 40% decrease in bone strength compared to WT mice. At the gene expression level, CRH‐Tg bone showed significantly increased osteoclast markers and decreased osteoblast markers, whereas CRH‐Tg muscle had increased muscle atrophy gene markers compared to WT mice. Overall, the CRH‐Tg mouse model aged to 14 weeks recapitulated many features of osteoporosis in Cushing's syndrome and thus, represents a useful model to study GC‐induced osteoporosis and interventions that target the effects of GCs on the skeleton. © 2017 The Authors.  It is estimated that between 0.5% to 1.2% of the global population is currently being treated with glucocorticoid (GC) drugs.GC‐induced osteoporosis (GIO) is characterized by an initial stage of rapid bone loss, followed by a slower, but consistent decrease in bone mass. GIO results in bone fractures in 30% to 50% of patients, which can lead to increased morbidity, mortality, and reduced quality of life.The mouse model of Cushing's syndrome used in this study was developed in 1992 and is transgenic for the rat corticotropin releasing hormone (CRH) gene downstream of the metallothionein promoter.CRH‐Tg mice were obtained by reconstitution of cryopreserved embryos from the Jackson Laboratory 227.1Pbl/J; Stock #003210). The cryo‐recovered CRH‐Tg (hemizygous) males were bred two to three generations with C57Bl/6 females from our colony to generate sufficient CRH males, and corresponding WT littermate controls for the experiment. Verification of the C57Bl/6 background of the CRH litters was carried out by the Jackson Laboratory Genome Scanning Service. Results showed that the generated CRH litters and C57Bl/6 female breeders showed a 99% similarity in background. Litters were genotyped following the Jackson Laboratory protocol for the CRH transgene.ad libitum. Male, 13‐week‐old to 14‐week old, CRH‐Tg (n = 6) and WT (n = 9) littermate control mice were euthanized by decapitation within 6 hours after the start of the light cycle. The gastrocnemius muscle and humeri bone samples were flash frozen in liquid nitrogen. Trunk blood was collected in tubes containing 5 μL of 0.5M EDTA and plasma was separated by centrifugation. Dynamic histomorphometry was measured in a separate cohort of male 14‐week‐old mice that had been injected with 30 mg/kg calcein green  7 and 2 days before euthanasia. These mice served as control groups for a separate experiment and were all injected s.c. with 30 μL of 5% ethanol in sesame oil twice daily for 6 weeks. All animal experiments performed were approved by the Faculty of Medicine and Pharmacy Animal Care Committee at the University of Toronto in accordance with the guidelines from the Canadian Council on Animal Care. Although the genotype of the mice was apparent at euthanasia (because of the thinning of the fur for the CRH‐Tg mice), once the bones were removed and cleaned for further processing, all samples were only identified by number. The μCT, three‐point bending and histomorphometry analyses were performed in a blinded manner.Mice were housed in a pathogen‐free, temperature‐controlled facility with a 10‐hour/14‐hour light/dark cycle. All mice were fed the Teklad 2016 global diet  For RNA analysis, humeri were flash frozen in liquid nitrogen without removal of the bone marrow. Tissues were homogenized in RNA STAT‐60  to extract total RNA and reverse transcribed into cDNA with random hexamer primers using the High Capacity Reverse Transcription System . Real‐time quantitative PCR (qPCR) reactions were performed in 384‐well plates containing 12.5 ng cDNA, 150 nM of each primer and 5 μL 2× SYBR Green Master Mix in a 10‐μL total volume. The comparative threshold cycle (Ct) method was used to determine relative mRNA levels normalized to Cyclophilin or Gapdh mRNA, as indicated. The qPCR primers used are listed in Table The right tibias were fixed immediately after euthanasia in 70% ethanol at 4°C, followed by acetone dehydration, infiltration with ascending concentrations of Spurr resin, and embedding in Spurr resin. A Leica RM2265 microtome  was used to cut 5‐μm‐thick sections with a tungsten carbide knife. The undecalcified 5‐μm sections of the right tibia were stained with Goldner's Trichrome stain and imaged with magnification ×200 for analysis.The left tibias were fixed in 10% neutral buffered formalin. The bones were then decalcified in 0.5M EDTA, pH 7.4, at 4°C. After decalcification, the specimens were processed in a graded series of ethanol and xylene, and embedded in paraffin wax. Five‐μm‐thick (5‐μm‐thick) sections were cut using a Reichart‐Jung 2030 microtome . The decalcified sections were incubated in 0.2M Tris buffer  for 1 hour and then stained for tartrate‐resistant acid phosphatase (TRAP) using the Acid Phosphatase Leukocyte kit (Sigma‐Aldrich), following the manufacturer's instructions. The TRAP slides were imaged under magnification ×100 for analysis. Undecalcified 7‐μm‐thick sections were used for dynamic calcein histomorphometry to measure bone formation.Histomorphometric data was quantified using Bioquant Osteo v13.2 MIR software . The regions of interest (ROIs) for analysis of trabecular bone were measured 150 μm under the growth plate in the secondary spongiosa of the proximal tibial metaphysis. In undecalcified sections, the parameters measured were the following: percent bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), osteoid surface per bone surface (OS/BS), osteoid volume per bone volume (OV/BV), osteoid width (O.Wi), number of osteoblasts per bone surface (N.Ob/BS), adipocyte volume per tissue volume (Ad.V/TV), mineralizing surface per bone surface (MS/BS), bone formation rate per bone surface (BFR/BS), and mineral apposition rate (MAR). In decalcified sections, the following were measured: number of osteoclasts per bone surface (N.Oc/BS) and number of osteoclasts per osteoclast surface (N.Oc/Oc.S). All histomorphometric measurements were expressed following the guidelines of the American Society of Bone and Mineral Research (ASBMR).Left femurs were scanned in saline‐soaked gauze using a Skyscan 1174 compact desktop micro‐computed tomography  to measure the volumetric bone mineral density (vBMD) and microarchitecture of the femurs. A set of two hydroxyapatite phantoms were also scanned for BMD calibration. For trabecular bone assessment, the distal femur metaphysis was scanned at a voxel size of 6.1 μm and for cortical bone assessment, the femur mid‐diaphysis was scanned at a voxel size of 11.6 μm. The acquisition of images was performed at a 50 kV voltage, with 800 μA and a 0.25‐mm aluminum filter to reduce image noise. Image reconstruction was performed using the Skyscan NRecon software (version 1.6.3.2) and then analyzed by the Skyscan CT analyzer (version 1.10.0.1). For trabecular bone analysis, a volume of interest (VOI) was calculated 200 sections proximally from the formation of the first cartilage bridge, with a VOI of 100 sections (0.6 mm). Cortical bone analysis was performed on the femur mid‐diaphysis in order to obtain an 86‐section‐thick (1 mm) VOI. The parameters measured by μCT were as follows: BV/TV, Tb.Th, Tb.N, Tb.Sp, trabecular vBMD, mean total cross‐sectional bone area (B.Ar), cross‐sectional thickness (Cs.Th), cortical volumetric bone mineral density , mean mediolateral diameter (ML), mean anteroposterior diameter (AP), and mean cortical thickness (Ct.Th).The left femurs were subjected to a three‐point bending test with the use of the Instron 4465 machine . Initially, the femur lengths were measured with digital calipers to acquire their mid‐point, and other geometrical properties were obtained from the μCT imaging. The specimens were thawed to room temperature, rehydrated with saline, and then placed on two supports at a distance of 6 mm. A load of 100 N was applied to the mid‐point of the femurs at a rate of 0.5 mm/min until failure. The load‐deformation data was recorded using the LabVIEW 5.0 Acquisition Software  and normalized parameters were computed using the necessary femoral geometry data acquired from μCT analyses. The resulting structural parameters are ultimate load, energy to fail, and stiffness, and the material parameters are ultimate stress, toughness, and modulus.ELISA kits were used to measure osteocalcin  and TRAP‐5b  from plasma. Corticosterone was measured by radioimmunoassay .t test with Welch's correction . Outliers were detected by the ROUT method and removed. Outliers were detected in Fig.  2 and Table p < 0.05 was considered statistically significant.Data are presented as mean ± SE. GraphPad PRISM was used to perform the A). There was no difference between genotypes in total body weight  and liver weight . In contrast, epididymal white adipose tissue (WAT) weights and overall abdominal adiposity were significantly increased in CRH‐Tg mice compared to WT mice . Adrenal weights were also significantly increased in CRH‐Tg mice, likely due to chronic ACTH stimulation in response to CRH overexpression . Spleen weights were decreased in CRH‐Tg mice, as expected, because corticosterone is known to promote lymphoid organ atrophy .At 13 to 14 weeks of age, male CRH‐Tg mice had fivefold increased plasma corticosterone levels compared to WT mice . However, there was a trend toward a decrease in percent bone volume  and a 16% decrease in trabecular thickness  in CRH‐Tg mice compared to WT. In contrast, trabecular number was unchanged between CRH‐Tg and WT mice . Trabecular separation, a measure of the distance between the trabeculae, was also unchanged between genotypes . Representative images of cortical bone from WT and CRH‐Tg femurs are shown in Fig. G. The vBMD of the cortical bone from CRH‐Tg mice was significantly lower than that of WT mice . Consistent with this, cortical bone from CRH‐Tg mice had a 38% reduction in bone area , a 42% reduction in cross‐sectional thickness , and a 35% reduction in cortical thickness compared to WT mice . Anteroposterior diameter was unchanged , but mediolateral diameter was decreased in CRH‐Tg mice , suggesting minor changes to the cortical shape by chronic GC exposure. Together, these data show that although parameters in both the trabecular and cortical compartments are significantly impacted by chronic GCs, the cortical compartment is more significantly affected by chronic GC exposure in this model.Cortical and trabecular parameters of the femur were measured by μCT. Representative images of trabecular bone from WT and CRH‐Tg mice are shown in Fig. s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D s p = 0.0; Fig. D p = 0.0006; Fig. A). Osteoid volume to bone volume was decreased 47% in CRH‐Tg mice compared to WT . Osteoid surface to bone surface showed a trend toward a reduction in CRH‐Tg mice , whereas osteoid width was decreased by 27% in CRH‐Tg mice relative to WT mice . In contrast, the number of osteoblasts per bone surface was not different between the two genotypes . Representative trichrome stained tibial sections are shown in Fig. F. Next, we stained for osteoclasts (OCs) using the OC marker TRAP. TRAP staining revealed a 75% increase in OC number per bone surface  and a 61% increase in OC surface per bone surface in CRH‐Tg mice compared to WT mice . A representative example of TRAP staining in WT and CRH‐Tg is shown in Fig. C. Together, these results show that the CRH‐Tg mice have increased bone adiposity and increased osteoclasts, with an overall decrease in osteoblast‐associated osteoid width and volume, reflecting a decrease in bone formation.In order to elucidate the cellular mechanisms behind the changes in bone parameters, static histomorphometry of proximal tibial metaphyses was performed. Remarkably, adipocyte volume was increased sixfold in the bones of CRH‐Tg mice when compared to WT mice  and 34% decrease in BFR  was observed in CRH‐Tg mice compared to WT mice. The MAR tended to be lower in CRH‐Tg mice relative to WT mice but was not statistically significant .To quantitate the bone formation, dynamic histomorphometric analysis was performed in 14‐week‐old mice after calcein double labeling spaced 5 days apart. A 23% decrease in MS/BS , Oscar (osteoclast associated receptor), and Ctsk (Cathepsin K) were significantly increased in CRH‐Tg mice compared to WT mice . In agreement with the gene expression data for Acp5, plasma TRAP5b levels were increased 6.5‐fold in CRH‐Tg mice compared to WT . These data suggest the CRH‐Tg mice have increased bone turnover relative to WT mice, a finding consistent with increased osteoclastogenesis by GCs.To explore how changes observed may be mediated at the cellular level, gene expression was measured by qPCR in the humeri of WT and CRH‐Tg mice. The osteoclast associated markers TRAP . The transcription factor Runx2, on the other hand, is responsible for driving mesenchymal cells toward the osteoblast lineage, and expression of this factor tended to be decreased . These data suggest that there is an increase in the transcription of factors that drive adipocyte differentiation, which complements the increase in adipocytes observed by histomorphometry.To better understand the mechanism of increased adiposity of the bone in CRH‐Tg mice compared to WT mice, we measured the expression of two transcription factors that contribute to adipocyte versus osteoblast differentiation. Tnfsf11) is a cytokine expressed by osteoblasts that acts on osteoclasts to increase their differentiation. Osteoprotegerin  is also secreted by osteoblasts and binds to RANKL, thus blocking RANKL signaling in osteoclasts. We measured the Tnfsf11/Tnfrsf11b ratio (RANKL/OPG mRNA ratio) and found no significant difference between WT and CRH‐Tg mice . In contrast, the expression of osteoblast‐derived gene products important for bone formation was reduced 48% for Col1a1  and 77% for the gene encoding osteocalcin  in the CRH‐Tg mice compared to WT mice. At the protein level, plasma osteocalcin measured by ELISA was decreased 50% in the CRH‐Tg mice relative to WT mice . Together, these findings are consistent with the decreased bone formation, as shown by our static and dynamic histomorphometric data.Receptor activator of nuclear factor κ‐B ligand  and Fbxo32 (Mafbx), were both increased 1.9‐fold in CRH‐Tg mice compared to WT mice . In contrast, there was no significant change in myostatin (Mstn), the negative growth differentiation factor . MuRF1 and MAFbx are ubiquitin ligases that signal proteins for proteasomal degradation and are potently upregulated by glucocorticoids. Increased expression of these genes is associated with increased muscle breakdown.Foxo1, a transcription factor that binds to the upstream promoters of Trim63 and Fbxo32, was increased fourfold in CRH‐Tg mice compared to WT mice .Muscle atrophy is another common side effect of chronic GC exposure, and muscle loss can contribute to increased fracture risk.tor Fig. C. MuRF1In the present study, we performed phenotypic characterization of the bones of CRH‐Tg mice and determined that they exhibit several properties consistent with GIO in humans with Cushing's syndrome. The CRH transgenic mouse, developed in 1992,Tnfsf11), and decreased expression of its decoy receptor OPG (Tnfrsf11b) by osteoblasts.Cushing's syndrome patients present with both structural and functional impairments of the musculoskeletal system. Chronic GC exposure in Cushing's syndrome leads to a decreased function of osteoblasts, with a shift in the differentiation of bone marrow stromal cells away from osteoblasts and toward the adipocyte lineage in the bone.Baseline parameters in our CRH‐Tg mice derived from reconstituted embryos were similar to previous reports of the non‐cryopreserved animals. Despite having overall body weights that were similar, epididymal WAT weights were significantly increased in Cushing's mice relative to WT mice, consistent with what has been shown.We next measured both the trabecular and cortical compartments by μCT in order to characterize the effects of chronic GC exposure on bone microarchitecture in this mouse model. vBMD in the cortical bone of the femur was significantly reduced in the CRH‐Tg mice. In contrast, vBMD of the trabecular compartment did not decrease despite a decrease in trabecular thickness and percent bone volume. This finding was interesting because, as aforementioned, GIO in humans generally results in more pronounced changes in the BMD of trabecular bone compared to cortical bone. However, in line with the CRH‐Tg model, several studies have shown significant decreases in cortical thickness with Cushing's syndromeThe effect of exogenous GC drugs on the bones of mice have been investigated using different background strains and ages while varying the duration of GC treatment, resulting in disparate effects on trabecular versus cortical bone.Acp5, Oscar, and Ctsk) were consistent with the increased osteoclast numbers from the histomorphometry data. Although these markers suggest an increase in osteoclast activity, this idea would be strengthened by the measurement of a bone resorption marker downstream of the osteoclast, such as CTX. The decreased expression of osteoblast markers in CRH‐Tg mice, including Bglap and the osteoblastogenic transcription factor Runx2, did not correlate with the lack of significant change in osteoblast numbers by histomorphometry, suggesting that the osteoblasts which were present were less functional. The adipogenic transcription factor, Pparγ2, was increased and was consistent with the increased adipocytes in the bone marrow. Overall, these results highlight that chronic exposure to GCs in this model result in increased adipocytes and osteoclasts and a decrease in the normal function of osteoblasts in the bone.We examined changes at the cellular level through static and dynamic histomorphometry. Adipocyte volume was significantly increased in the CRH‐Tg mice, which is expected with chronic GC exposure.Many reports of Cushing's syndrome highlight the dramatic decrease in osteocalcin (a bone formation marker) with conflicting reports of changes in bone resorption markers (reviewed in Toth and GrossmanMechanical testing of the femurs by three‐point bending revealed significant reductions in the structural properties of bone; however, the material properties were not significantly reduced. The decreased structural properties were likely due to reduced femur cortical thickness and not because of inherent material properties of the bone. These results suggest that CRH‐Tg mice would be more prone to fracture, and are consistent with the high fracture rates observed in Cushing's patients.Last, muscle atrophy markers were measured to evaluate the effects of chronic GC exposure on muscle, which can indirectly affect fracture risk. Because we found that muscle atrophy markers were increased in the CRH‐Tg mice, these results suggest that GC‐induced muscle atrophy can be induced at the gene expression level in this mouse model.In conclusion, our results indicate that the CRH‐Tg mouse model has many features that are similar to that of Cushing's syndrome patients, including loss of trabecular bone volume, trabecular thinning, cortical thinning, and decreased osteoblast and increased osteoclast activity markers at the gene expression and cellular level. Together, these data indicate that 14‐week‐old CRH‐Tg mice are an appropriate model to study GIO and test new therapeutic interventions to mitigate bone loss.All authors state that they have no conflicts of interest.


Real‐world data (RWD) on health‐related outcomes in persons with haemophilia A (PwHA) provide insights into patient needs and can guide clinical study design. A global, prospective, non‐interventional study  collected detailed RWD on bleeding outcomes, health‐related quality of life (HRQoL) and health status in PwHA treated per local routine clinical practice.To report HRQoL and health status in the adult/adolescent PwHA with inhibitors cohort in the NIS.This cohort enrolled PwHA aged ≥12 years with high‐titre factor VIII inhibitor history. Participants remained on their usual treatment (no protocol‐specified interventions). Health‐related outcomes: Haemophilia Quality of Life Questionnaire for Adults (Haem‐A‐QoL), Haemophilia‐specific Quality of Life Questionnaire for Children Short Form (Haemo‐QoL SF), EuroQol 5‐Dimensions 5‐Levels (EQ‐5D‐5L) index utility score (IUS) and visual analogue scale (EQ‐VAS).One hundred three participants were enrolled on episodic (n = 75) or prophylactic treatment (n = 28); median (range) age, 31 (12‐75) years; median (range) observation time, 26 (4‐70) weeks. Haem‐A‐QoL scores indicated impairments in HRQoL aspects; comparable between episodic/prophylactic regimens and relatively consistent over time. Haemo‐QoL SF scores with both regimens varied over time, and appeared poorer with episodic than prophylactic treatment. IUS and EQ‐VAS were comparable between regimens, stable over time and lower on bleeding days. Mean proportions of missed work and school days were 16% and 23%, respectively; mean (standard deviation) number of days hospitalized was 3.2 (8.8) (comparable between groups).These RWD demonstrate that PwHA with inhibitors have impaired HRQoL, despite standard treatment, and that more effective treatment options are needed. Hoffmann‐La Roche, Basel, Switzerland), as soon as it was opened for enrolment at their treatment centre . Emicizumab is a subcutaneously administered recombinant humanized bispecific monoclonal antibody recently approved in several countries for prophylaxis in PwHA with inhibitors of all ages.An international non‐interventional study (NIS) was conducted to prospectively collect detailed RWD in PwHA, with and without inhibitors, treated according to local routine clinical practice. As long as they met respective eligibility criteria, participants from this cohort who were compliant with requirements of the NIS  could start rolling over into the phase III HAVEN 1 study of emicizumab . The data cut‐off was 31 March 2017 for the final analysis. The study was conducted in accordance with the International Conference on Harmonisation Guidelines for Good Clinical Practice, informed consent guidelines and the Declaration of Helsinki,2.2In cohort A, eligible participants with congenital haemophilia A (any severity) were aged ≥12 years; had a documented history of high‐titre FVIII inhibitors (≥5 Bethesda units/mL); documented episodic or prophylactic treatment for bleeds for ≥6 months prior to enrolment; and had experienced ≥6 bleeds on episodic treatment or ≥2 bleeds on prophylaxis in the prior 6 months. Participants receiving ITI with FVIII were ineligible.2.3The target participant number for each treatment type (episodic or prophylactic) enrolled into each cohort was estimated based on the approximate number of participants initially planned for the interventional emicizumab phase III pivotal study NCT02622321).22321.26 2.42.4.1HRQoL data were collected in adults using the Haemophilia Quality of Life Questionnaire for Adults (Haem‐A‐QoL), and in adolescents using the Haemophilia‐specific Quality of Life Questionnaire for Children Short Form (Haemo‐QoL SF).The Haem‐A‐QoL is a validated measure designed to assess aspects of HRQoL in adult PwHA aged ≥18 years,The Haemo‐QoL SF, a 35‐item measure designed to assess aspects of HRQoL in children and adolescents aged 8‐17 years, is composed of 9 domains ,2.4.2Health status was assessed in all participants using the EuroQol 5‐Dimensions 5‐Level (EQ‐5D‐5L) questionnaire,2.4.3Every 4 weeks, participants were asked to report the number of missed work or school days during the previous 4 weeks, and the number of days they expected to attend. The proportion of missed days was calculated for each participant by dividing the number of missed days by the number of days they should have attended. The mean proportion of missed days for each group was calculated from the sum of the proportions for each participant, divided by the number of participants.2.4.4Hospitalizations during the study period were recorded in the serious adverse events page of the electronic case report forms (eCRFs) by investigators every 4 weeks.2.5Clinicians collected demographic data and medical histories from participants' medical records onto the eCRFs.All other outcomes, except hospitalizations (described above), were recorded by participants using an electronic handheld device provided at enrolment. Participants completed all HRQoL and health status questionnaires on a 4‐weekly basis for ~6 months. Patients completed an additional EQ‐5D‐5L and EQ‐VAS on days when bleeding occurred.Summary statistics for the HRQoL questionnaires and the EQ‐5D‐5L IUS and EQ‐VAS were calculated at each scheduled assessment; no statistical testing was performed. The analysis of IUS and EQ‐VAS when a bleed occurred (unscheduled assessments) versus when no bleed occurred (monthly scheduled assessments) included only participants with at least one scheduled and one unscheduled assessment.The completion rates for Haem‐A‐QoL, Haemo‐QoL SF and scheduled EQ‐5D‐5L and EQ‐VAS were calculated by dividing the number of completed questionnaires by the number of questionnaires expected at each time point (determined from the number of eligible participants on study at the time). Completion rates for unscheduled EQ‐5D‐5L and EQ‐VAS were calculated as the number of unscheduled questionnaires completed, divided by number of bleeding days.The proportion of days away from work/school in the past 4 weeks were reported separately, and were determined based on the expected number of days of attendance and absence, as documented by the participant.All analyses were regarded as exploratory and descriptive; the study was not designed to make confirmatory claims.33.1Cohort A included 103 PwHA with inhibitors on episodic (n = 75) or prophylactic (n = 28) treatment with a median (range) age of 31 (12‐75) years (Table Completion rates at the first assessment were 75% (61/81) for Haem‐A‐QoL and 88% (15/17) for Haemo‐QoL SF. Rates remained high for Haem‐A‐QoL (>81%) and Haemo‐QoL SF (>73%) through week 25. Completion rates for the EQ‐5D‐5L were comparable: 85% (88/103) at the first assessment and >88% through week 25.3.2All Haem‐A‐QoL, Haemo‐QoL SF, EQ‐5D‐5L IUS and EQ‐VAS scores, starting with week 1, reflect the HRQoL and health status of participants treated according to local clinical practice; treatment was initiated before enrolment in the NIS and continued according to local standards thereafter.3.2.1Among participants who completed the Haem‐A‐QoL at week 1, mean (standard deviation [SD]) Haem‐A‐QoL “Total” score was 47.3 (14.8) in the episodic group n = 52) and 44.5 (16.7) in the prophylaxis group (n = 14). Mean Haem‐A‐QoL domain and total scores were generally comparable between episodic and prophylaxis groups and relatively consistent through week 25  Haemo‐QoL SF “Total” scores in the episodic (n = 8) and prophylaxis (n = 7) groups, respectively, were 49.5 18.0) and 35.6 (10.9) at week 1, and 57.1 (21.0) and 22.1 (7.0) at week 25. The domains with greatest impairments  at week 1 were “Sports & School” and “Family” in the episodic group and “Sports & School” and “View of Yourself” in the prophylaxis group  EQ‐5D‐5L IUS scores were 0.72 0.23) and 0.69 (0.24), and mean (SD) EQ‐VAS scores were 73.5 (20.1) and 65.0 (25.0). Although mean baseline EQ‐5D‐5L IUS and EQ‐VAS scores were higher in the episodic group than the prophylaxis group, there was no consistent difference between the groups and no systematic change in either value during the study  participants in the episodic group and 12 of 28 (43%) participants in the prophylaxis group. One participant in each treatment group did not complete the questionnaire, while the remaining participants were not working . The mean  proportion of days away from work for all patients was 16% (10%‐22%) and was comparable between groups .Assessment of school absenteeism included 35 of 75 (47%) participants in the episodic group and 16 of 28 (57%) participants treated with prophylaxis. One participant in each treatment group did not complete the questionnaire, while the remaining participants were not in school . The mean (95% CI) proportion of days away from school for all patients was 23% (14%‐32%) and was comparable between groups .The mean (SD) number of days hospitalized was comparable between groups , and the median number of days hospitalized in each group was zero . The number of days hospitalized ranged from 0 to 50 in the episodic group and from 0 to 26 in the prophylaxis group.4This prospective NIS collected detailed RWD on bleeding and health‐related outcomes in PwHA with inhibitors aged ≥12 years (cohort A) treated according to local routine clinical practice. The results presented here show these individuals experienced impairments  in HRQoL despite standard treatment, with mean scores in the majority of HRQoL domains in adults and adolescents indicative of impairments reported, on average, to occur “sometimes” to “often.”These health‐related outcomes may result from a combination of poor bleed control and treatment burden. Bleeding outcomes for this NIS cohort have been published,In adults, mean Haem‐A‐QoL scores were similar at each analysis time point. Mean Haemo‐QoL SF scores among adolescents appeared to vary over time, but this should be interpreted with caution given the small number of adolescent participants. Because participants remained on their usual regimen, substantial changes in HRQoL and health status were not expected during the NIS. Haem‐A‐QoL and Haemo‐QoL SF total and domain scores were comparable with other reports for PwHA with inhibitors treated with standard of care.Adult PwHA with inhibitors reported greatest impairments (highest scores) in the “Sports & Leisure” domain of the Haem‐A‐QoL, reflecting reports that they often had to refrain from participating in these activities. The high scores for the “Physical Health” and “Sports & Leisure” domains, as well as the high “Total” score, are not unexpected given the high bleeding rates reported for this cohort. Similarly, on the Haemo‐QoL SF (adolescents), the “Sports & School” domain showed greatest impairment.There was no systematic change in EQ‐5D‐5L IUS or EQ‐VAS throughout the study. At week 1, mean EQ‐VAS was lower in the prophylaxis than the episodic group, but the scores were comparable at most other time points. Mean EQ‐5D‐5L IUS scores in PwHA with inhibitors in the present study  were lower than those reported for PwHA with inhibitors in the US Centers for Disease Control and Prevention Universal Data Collection system (0.75),The impact of haemophilia A and associated bleeding events on daily activities in PwHA with inhibitors is not well documented. In the NIS cohort A, the overall mean proportions (95% CI) of missed work or school days were 16% (10%‐22%) and 23% (14%‐32%), respectively, and were comparable in the episodic and prophylaxis groups. In the Dosing Observation Study in Haemophilia, PwHA with inhibitors receiving BPAs and their caregivers missed many full days  from work/school.The limitations of the study included the small number of adolescent participants, potential selectivity of the sample, and participant attrition rate. The latter limitation is reflected in the fact that a number of data sets at each time point were incomplete, particularly later in the study. There are many possible reasons for this, including participant rollover into the phase III HAVEN 1 emicizumab clinical trial.5These RWD from cohort A of the NIS demonstrate that adolescent and adult PwHA with inhibitors experience substantially impaired health‐related outcomes while receiving standard care, revealing an unmet need for more effective treatment options.J. Mahlangu has received research grants from Bayer, Biogen, BioMarin, CSL Behring, Novo Nordisk, Sobi, Roche and Unique Pharmaceuticals; been a member of scientific advisory committees for Amgen, Bayer, Biotest, Biogen, Baxalta, CSL Behring, Catalyst Biosciences, Novo Nordisk, Roche and Spark Therapeutics; and been a speaker bureau member for Alnylam, Bayer, Biotest, Biogen, Novo Nordisk, Pfizer, Sobi, Shire, Roche, ISTH and WFH. J. Oldenburg has received reimbursement for attending symposia/congresses and/or honoraria for speaking/consulting and/or funds for research from Bayer, Biogen, Biotest, Chugai Pharmaceutical Co., CSL Behring, Grifols, Novo Nordisk, Octapharma, Pfizer, Roche, Shire and Sobi. M.U. Callaghan has been a paid consultant and speaker for Roche/Genentech; a paid speaker for Bayer, Novo Nordisk and Shire; has been a paid consultant for Bayer, Bioverativ, Grifols, HEMA Biologics, Pfizer and Shire; and owns stock in Alnylam. M. Shima is a board member of the Feiba and Advate Safety Board in Japan organized by Baxalta; has received payment for consultancy meetings with Baxalta, Bayer, Biogen, Chugai Pharmaceutical Co., CSL Behring, Kaketsuken, Novo Nordisk and Pfizer; and has received unrestricted grants supporting research from Baxalta, Bayer, Chugai Pharmaceutical Co., CSL Behring, Kaketsuken, Novo Nordisk and Pfizer. M. E. Mancuso has acted as a speaker for Baxalta/Shire, Bayer, Biotest, CSL Behring, Novo Nordisk, Octapharma, Pfizer and Roche; and been a paid consultant for Baxalta/Shire, Bayer, CSL Behring, Kedrion, Novo Nordisk, Pfizer and Roche. P. Trask is an employee of Genentech, Inc. M. Recht has received grant/research support from Bioverativ, Genentech, Novo Nordisk and Shire, and is a paid consultant for Bioverativ, CSL Behring, Genentech, Kedrion, Novo Nordisk, Pfizer, Shire and uniQure. C. Garcia has received reimbursement for attending symposia for Janssen and Novo Nordisk; and received speaking fees from Janssen, Novartis, Novo Nordisk, Pfizer and Roche. R. Yang has nothing to disclose. M. Lehle, H. Macharia and E. Asikanius are employees of F. Hoffmann‐La Roche, Ltd. G. G. Levy is an employee of Genentech, Inc and owns Roche stock. Kruse‐Jarres has acted as a paid consultant to Grifols, Novo Nordisk, Pfizer, Roche and Shire; and has received research funding from CSL Behring, Pfizer and Roche. S. von Mackensen is a consultant for Roche.


Endometrial cancer is one of the most common gynecological malignant tumors. The roles of competing endogenous RNAs (ceRNAs) in this disease, however, remain unclear. In this study, we constructed a ceRNA network to reveal the core ceRNAs in endometrial cancer. Differentially expressed genes were summarized from The Cancer Genome Atlas database, whereupon 140 genes were identified for building the network. Further correlation, survival, and enrichment analyses suggested that these genes may help towards elucidating the molecular mechanisms of endometrial cancer. After validation of the findings with the GSE17025 data set, LINC00958, microRNA‐761, and DOLPP1 were highlighted as the critical genes in the ceRNA network. Our work suggests that LINC00958 may regulate DOLPP1 by “sponging” miR‐761 in endometrial cancer. The high complexity and share of ncRNAs in the genome suggest that they play a significant influence in diseases, including cancer.Among the ncRNAs, the microRNAs  and long noncoding RNAs  have been the most focussed upon.Uterine corpus endometrial carcinoma (UCEC) is the fourth most common cancer amongst women worldwide, and its incidence has increased steadily in the past decades.Given the lack of comprehensive investigations on ceRNAs in endometrial cancer, we used available ncRNA and mRNA expression and single‐nucleotide polymorphism array data of UCEC from The Cancer Genome Atlas (TCGA) and constructed a ceRNA‐ceRNA interaction network for endometrial cancer. We were mainly interested in the ceRNAs involved in cancer processes. Finally, one set of ceRNAs  was proven to be a potential prognostic signature in endometrial cancer.22.1The workflow of this study is shown in Figure 2.2P‐value of < .05 and log2|Fold‐change| of > 1 were set up as the criteria.To analyze the differential expression of mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and miRNAs (DEmiRNAs) between tumor and normal tissues in UCEC, the bioconductor package GDCRNATools v1.2.02.3P‐value of < .01 were retained. Sponged miRNAs with lncRNAs and mRNAs were identified to construct the ceRNA network, which was then visualized using Cystoscape v3.5.1.To identify potential pairs among DEmRNAs, DElncRNAs, and DEmiRNAs in endometrial cancer, data from several databases were retrieved. StarBase v2.0,P‐value of < .01 and an R‐value of > 0.30.Using the number of target interactions, a hypergeometric test was conducted with the R package GDCRNATools for determining the significance of the shared miRNAs. Regulation similarity and sensitivity correlation were used to measure the regulation pattern. The noneliminated DEmRNA‐DElncRNA pairs were tested by Pearson's correlation analysis with a 2.4P < .05.Kaplan‐Meier survival analysis was used to evaluate the association between the expression levels of nodes in the ceRNA network and the overall survival of the patients. For Cox regression analysis, data from the Gene Expression Profiling Interactive Analysis (GEPIA) database were retrieved for the pairs satisfying 2.5P < .05 were significantly enriched.We performed Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Disease Ontology (DO) functional enrichment analyses on the DEmRNAs, DElncRNAs, and DEmiRNAs. KEGG pathways, GO terms, and DO terms with 2.6P < .05.All statistical analyses were performed using SPSS v20.0 . Differences were considered statistically significant at 33.1P‐value < .05 and log2|Fold‐change| > 1, we detected 1630 DEmRNAs, 51 DElncRNAs, and 88 DEmiRNAs in endometrial cancer compared with the normal controls, and these differentially expressed genes were visualized more intuitively  and correlation test (P < .01). Consequently, 171 nodes (RNAs) and 520 edges were included in the ceRNA network from the computational prediction  with both the hypergeometric test , MAGI2AS3‐EDIL3 (R = 0.61), and MAGI2AS3‐NOVA1 (R = 0.581) were the ones with the highest R‐values.Owing to the unique “sponge” function of lncRNAs, it is believed that they should have the same expression patterns as mRNAs. Therefore, 133 DElncRNA‐DEmRNA pairs were tested by Pearson's correlation analysis with P < .0010), UST (P = .0014), and MECP2 (P = .0026), and seven miRNAs . As showed in Table 4Given the yearly increases in the incidence and mortality rate of endometrial cancer, it is of great importance to unravel the molecular mechanisms of this disease.The enrichment analysis results were inconsistent with those of a previous report by Chen et al.The network provides a straightforward representation of interactions between ceRNAs. The selected lncRNA (LINC00958) and mRNA (DOLPP1) were both expressed at a higher level in endometrial cancer tissue than in healthy tissue samples. Interestingly, a few reports have shown that the silencing of LINC00958 in bladder cancer promotes tumor invasion, which echoes the function of miR‐761 in inducing aggressive phenotypes in breast cancer.Collectively, our work has identified the DOLPP1‐LINC00958‐miR‐761 ceRNA set as a good prognostic signature in UCEC. We expect that this systematic analysis of ceRNA interactions will contribute toward our understanding of the molecular pathogenesis of endometrial cancer.The authors declare that there are no conflict of interests.


PD1 monoclonal antibodies (mAbs), including all the patients who developed pneumonitis after anti‐PD‐1 mAbs treatment in the Cancer Center of the Chinese People's Liberation Army from September 2015 to September 2017. Two controls per case were matched according to a propensity‐score matching algorithm to account for confounding effects caused by individual baseline variables. Demographic and clinical information was obtained from medical records. In total, 55 cases and 110 controls were included in the study. No association was observed between smoking status or primary lung cancer and risk of pneumonitis. Significant risk factors for pneumonitis related to anti‐PD‐1 mAbs were prior thoracic radiotherapy, prior lung disease and combination therapy with odds ratios of 3.34 (1.51‐7.39), 2.86 (1.45‐5.64) and 2.73 (1.40‐5.31), respectively. The associations remained significant in the multivariable logistic regression model. The risk of pneumonitis induced by anti‐PD‐1 mAbs is associated with prior thoracic radiotherapy, prior lung disease, and combination therapy. Clinicians should monitor these features in patients receiving anti‐PD‐1 therapy to optimize clinical safety and efficacy.Immune checkpoint blockade‐related pneumonitis is a rare but potentially life‐threatening adverse effect, but its risk factors are not completely understood. This case‐control study was conducted to identify pneumonitis risk factors in patients treated with anti‐ Checkpoint blockade disrupts normal immune homeostasis and results in immune‐related adverse events (irAEs), including dermatologic, hepatic, endocrine, gastrointestinal, and pulmonary complications as well as other less common side effects.In a case report, 3 of 25 patients who received thoracic radiotherapy and anti‐PD‐1 therapy developed pneumonitis, suggesting a possible association between pneumonitis and prior thoracic radiotherapy.22.1Eligible patients included those with advanced tumor who developed pneumonitis after anti‐PD‐1 mAbs treatment in the Cancer Center of the Chinese People's Liberation Army from September 2015 to September 2017. Pneumonitis was diagnosed by a radiologist according to computed tomography scans. Patients were excluded if there was a clear alternative etiology, such as proven malignant lung infiltration or active lung infection. Pneumonitis grading was performed by the treating investigators in real time using Common Toxicity Criteria for Adverse Events (version 4.0). Controls were defined as advanced tumor patients treated with anti‐PD‐1 mAbs who did not develop pneumonitis. This study was approved by the Ethics Committee of Chinese People's Liberation Army General Hospital.2.2For all the patients, the following pretreatment demographic and clinical information was obtained from medical records: age, sex, smoking status, weight, metastatic sites, baseline lactic dehydrogenase, previous anticancer treatments, treatment agents with anti‐PD‐1 mAbs, the number of anti‐PD‐1 mAbs treatment cycles, primary tumor type, and prior lung disease. Smoking status was classified as no history of smoking or a positive history. Combination therapy was defined as anti‐PD‐1 treatment with chemotherapy, targeted therapy or CTLA‐4 blockade. Previous anticancer treatment (6 months prior) was classified as chest surgery, thoracic radiotherapy, targeted therapy or chemotherapy. Prior lung disease included pneumothorax, pleural effusion, and pneumonitis before anti‐PD‐1 mAbs therapy. In patients with pneumonitis, the clinical features of pneumonitis were collected retrospectively from medical records. The severity of the pneumonitis was described using the criteria for interstitial lung diseases.2.3t tests or the Mann‐Whitney U test. Categorical variables were compared by Chi‐square test or Fisher's exact test. Propensity‐score matching was used to create case and control groups with similar baseline characteristics. Matching with a ratio of 1:2 was performed using the optimal‐matching algorithm in MatchIt R package 3.4.2.P = .05 was set as the level of significance. All the reported P‐values were 2‐sided.Statistical analyses were performed using SPSS statistical software . Continuous variables were described with the median (range) and were compared by 2‐sided 33.1Overall, 55 cases and 110 controls were included in this analysis. The characteristics of cases and controls are described in Table The clinical features of pneumonitis are summarized in Table Treatment was performed on 45.5% (25 of 55) patients, and 25.5% (14 of 55) patients received oral or intravenous steroid therapy. Pneumonitis was improved, resolved, or unchanged in 61.8% (34 of 55) of cases, and it was improved or resolved in 12 of 14 patients (85.7%) who received steroid therapy. Two patients died during the course of pneumonitis treatment.3.2P < .01). The odds ratios were 3.34 (1.51‐7.39), 2.86 (1.45‐5.64) and 2.73 (1.40‐5.31), respectively  might vary due to distinct pharmacological mechanisms. However, the sample size was not adequate to address each combined therapy individually. Third, drug‐induced pneumonitis remains a diagnosis of exclusion and requires the consideration of competing diagnoses. Fourth, we did not include patients treated with anti‐PD‐L1 mAbs who may share similar risk factors for pneumonitis with anti‐PD‐1 mAbs. Fifth, prior lung disease did not include asthma, chronic obstructive pulmonary disease or interstitial lung disease because this information was not provided. Other limitations common to case‐control studies are the risk of observational bias and confounding bias, which was minimized by applying the propensity‐score matching protocol.In conclusion, we identified prior thoracic radiotherapy, prior lung disease and combination therapy as risk factors for the life‐threatening pneumonitis observed in patients receiving anti‐PD‐1 immunotherapy. Although the detailed role and mechanism underlying these risk factors for the development of pneumonitis requires further investigation, patients with these characteristics require intensive care in cases of anti‐PD‐1 mAbs‐related pneumonitis. To the best of our knowledge, this is the first case‐control study systematically seeking to identify risk factors for anti‐PD‐1 mAbs‐related pneumonitis development in a Chinese population of multiple types of advanced cancer. This study fills an important gap in the literature and supplements the limited number of published reports on anti‐PD‐1 pneumonitis.The authors have declared no conflict of interests.