Immune checkpoint inhibitor/tyrosine kinase inhibitor (ICI/TKI) combinations are a new standard of care for the initial treatment of metastatic renal cell carcinoma (mRCC). Their efficacy and toxicity beyond the first‐line setting remain poorly defined.We retrospectively reviewed charts for 85 adults with mRCC of any histology receiving combination of ICI/TKI in any line of treatment at two academic centers as of 05/01/2020. We collected clinical, pathological, and treatment‐related variables. Outcomes including objective response rate (ORR), progression‐free survival (PFS), and toxicity were analyzed via descriptive statistics and the Kaplan–Meier method.Patients received pembrolizumab, nivolumab, avelumab, or nivolumab–ipilimumab, with concurrent use of sunitinib, axitinib, pazopanib, lenvatinib, or cabozantinib. Thirty‐three patients received first‐line ICI/TKI therapy, while 52 received ≥ second‐line ICI/TKI. The efficacy of ICI/TKI therapy decreased with increasing lines of treatment . In the ≥ second‐line setting, ICI/TKI was most useful in patients who received ICI only, with an ORR of 50% and a mPFS of 9.1 months. Efficacy was limited in patients who received both TKI and ICI previously, with an ORR of 20% and a mPFS of 5.5 months. Overall, ≥ second‐line ICI/TKI was tolerable with 25 of 52 (52%) patients developing grade ≥3 adverse events.ICI/TKI combination therapy is feasible and safe beyond the first‐line setting. Prior treatment history appears to impact efficacy but has a lesser effect on safety/tolerability. ICI/TKI combination therapy is effective and safe beyond the first‐line setting. Prior treatment history appears to impact efficacy but has a lesser effect on safety/tolerability. Each regimen consists of an immune checkpoint inhibitor (ICI) targeting either the programmed cell death protein 1  or its ligand  in combination with axitinib, a tyrosine kinase inhibitor (TKI) targeting the vascular endothelial growth factor pathway. As such, these regimens represent rationally designed novel therapeutic combinations built upon earlier work showing individual efficacy of each class of drugs in RCC.While ICI/TKI combinations have emerged as new 1L options for mRCC, it remains unclear how such combinations fit into the larger landscape of mRCC management, both in the 1L setting and beyond. Indeed, the most current iteration of the National Comprehensive Cancer Network Guidelines for mRCC lists 10 distinct 1L treatment options, and 14 recommended subsequent‐line options.22.1We retrospectively collected data between 05/01/2015 and 05/01/2020 from The Ohio State University Comprehensive Cancer Center (OSU) and the University of Washington (UW)—Seattle Cancer Care Alliance. The study was approved by the Institutional Review Board and Ethics Committee at both institutions, and patient consent was waived. We collected data on 85 patients with mRCC of any histology who were at least 18 years of age and who had received at least two lines of ICI‐based systemic therapy, consisting of either pembrolizumab, nivolumab, avelumab, or nivolumab–ipilimumab, with concurrent TKI use including one of sunitinib, axitinib, pazopanib, lenvatinib, or cabozantinib. Although our study focused on ≥2L use of ICI/TKI, patients who received 1L ICI/TKI treatment were included as a comparison group. Patients were excluded if they received ICI/TKI therapy elsewhere.2.2Clinical data including patient demographics, histologic subtype, International Metastatic RCC Database Consortium (IMDC) risk group, RCC treatment history, ICI/TKI regimen, and line of therapy were recorded. Measurements of ORR, PFS, and safety/toxicity were used for the assessment of clinical outcomes. ORR was defined as best response by complete response (CR) and partial response (PR), while treatment response was evaluated by medical oncologists based on RECIST 1.1 criteria. Patients without measurable diseases were excluded from ORR analysis. PFS was defined as the time of ICI/TKI therapy initiation to the date of radiographic disease progression or death from any cause. For patients who had not yet progressed but were switched to another therapy , PFS was censored at the date of the last evaluable tumor assessment prior to the treatment change. Safety and tolerability were determined by related descriptions in chart review and were assessed for grade ≥3 treatment‐related adverse events (AEs) based on the Common Terminology Criteria for Adverse Events Version 5.0.2.3Details regarding demographics, disease, treatment, and toxicity characteristics were summarized by descriptive statistics. PFS was estimated using a Kaplan–Meier survival curve. The Cochran‐Armitage trend test and Wilcoxon trend test were performed to assess the association of the lines of ICI/TKI treatment with ORR and PFS outcomes, respectively. All statistical procedures were performed using SAS 9.1 software .33.1N = 69) and the most common histology was clear cell , while 12 (21%) patients had non‐clear cell RCC, including five papillary type, one chromophobe type, one translocation type, and five unclassified type.A total of 85 patients with mRCC were included, of which 65 were from OSU and 20 were from UW. Thirty‐three patients (39%) received 1L ICI/TKI and 52 patients (61%) received ≥2L ICI/TKI. The median follow‐up time was 11.3 months (range 1.7–66.0 months). Clinicopathological characteristics for the cohort are presented in Table N = 64). The three most common metastatic sites were lung (51.8%), lymph node (38.8%), and bone (23.5%).In total, 20 patients (24%) were under favorable risk by IMDC criteria, 46 (54%) were intermediate risk, 17 (20%) were poor risk, and risk category was unknown for two patients (2%) due to missing data. The majority of the patients had undergone prior nephrectomy . We also observed an analogous trend with decreasing PFS with increasing lines of therapy (p trend <0.01) with a mPFS of 15.2, 14.2, 10.1, and 6.8 months, respectively , while 79 93%) patients had measurable diseases for ORR analysis, including 29 patients in the 1L setting and 50 patients in the ≥2L setting. A total of two CRs and 28 PRs were observed in the overall patient population. ICI/TKI had an ORR of 56.7% in the 1L setting, as compared to an ORR of 26.5% in the ≥2L setting. The efficacy of ICI/TKI therapy was negatively associated with the number of lines of treatment, for example the ORRs for 1L, 2L, third‐line, and ≥ fourth line therapies were 56.7%, 37.5%, 21.4%, and 21.0%, respectively , respectively. We then performed stratified analyses based on prior treatment exposure. In TKI‐naïve patients with prior ICI‐only therapy (nivolumab and ipilimumab), the ORR was 50% with a mPFS of 9.1 months . Conversely, in ICI‐naïve patients with prior TKI‐only exposure, the ORR was 20% with a mPFS of 13.3 months . In patients who had both prior TKI and ICI exposure followed by ≥2L TKI/ICI combination, the ORR was 20.8% with a mPFS of 5.5 months   had grade 3 or higher AEs. The most common ≥G3 AEs were anorexia (13.5%), diarrhea and hypertension (11.5% each), and fatigue (9.6%) .As expected, the current cohort demonstrated that the efficacy of ICI/TKI decreased with increasing lines of therapy, which could correlate with the accumulation of treatment‐related resistance by tumor genetic or epigenetic alterations. We observed a dismal PFS curve for ≥ fourth line group which is distinct from the other groups. This observation could be informative in clinical decision‐making, where clinical trials with novel agents should be strongly considered rather than continued FDA‐approved treatments.n = 10) who had previously only received 1L nivolumab–ipilimumab and were therefore TKI naive. These results are consistent with the results of a phase II study of ≥2L pembrolizumab‐lenvatinib in ICI‐pretreated patients, which demonstrated an ORR of 55.8% and a mPFS of 12.2 months.n = 15), at last follow‐up, eight (53.3%) patients had stable disease, resulting in a durable PFS (median = 13.3 months). Our data suggest that the efficacy of ≥2L ICI/TKI was substantially reduced in patients who had previously been exposed to both TKI and ICI therapy with an ORR of only 20.8% and a mPFS of 5.5 months, similar to previously reported efficacy metrics observed with ≥2L TKI monotherapy.Efficacy analyses based on prior treatment exposure are valuable to direct appropriate sequential treatment. For example, it is unclear whether patients will benefit more from upfront ICI/TKI followed by nivolumab–ipilimumab or vice versa. The ICI combination of nivolumab–ipilimumab is a standard 1L option in mRCC with a reported ORR of 42%, and it is also frequently used off‐label in the ≥2L setting. In our cohort, 2L ICI/TKI induced an impressive ORR of 50% with a durable mPFS of 9.1 months in the subgroup of patients (The efficacy of ICI/TKI in patients with non‐clear cell histology is unknown. There were 12 (21%) patients with non‐clear cell RCC in our cohort, and only nine patients were available for measurement of treatment response. Only two patients who received ≥2L ICI/TKI had PRs and both responders had unclassified histology. Although we did not observe objective response in the 1L setting, this sample size is too small to permit any substantive conclusions to be drawn and hence warrants further evaluation.Finally, our study suggested that ≥2L ICI/TKI was safe and tolerable. Fifty‐two percent of the patients experienced ≥G3 AEs during treatment of ≥2L ICI/TKI. The ≥ grade 3 AE rate is lower than the rate of 71.2%–82.4%, which was reported in the phase III trials of 1L ICI/TKI.4.1Our study has several limitations that must be acknowledged. First, it is limited by the inherent biases related to the retrospective study design. The objective response and toxicity assessments were not as rigorous as prospective studies. Nevertheless, the nearly identical response rate and mPFS to those receiving 1L ICI/TKI therapy suggest comparable clinical outcomes as observed in prospective studies. Second, the modest sample size did not allow us to further evaluate the data stratified by different ≥2L ICI/TKI combinations, to perform statistical comparisons among different subgroups, or to determine the impact of confounding factors. Although a trend test for efficacy by line of therapy was performed, our data are largely descriptive. Third, we did not present OS as a study endpoint because such analyses would be heavily biased due to heterogeneity of treatment and the timing of ICI/TKI initiation.4.2Overall, our data suggest that ≥2L ICI/TKI is safe and feasible in the treatment of mRCC that has progressed following initial systemic therapy. The hypothesis generated from our retrospective study was that the clinical benefit of salvage ICI/TKI was most pronounced in patients who had previously received either ICI or TKI treatment. In contrast, the clinical benefit of ≥2L ICI/TKI in patients who had previously received both ICI and TKI treatments was modest. Phase III trials to examine optimal treatment sequences such as PEDIGREE (NCT03793166), CONTACT‐03 (NCT04338269), and TiNivo‐2 (NCT04987203) are currently ongoing.The authors declare no conflict of interest.Ming Yin, Sarah P. Psutka: Conception and design. Ming Yin, Sarah P. Psutka, Anish B. Parikh, Mingjia Li, Katharine Collier, Abdul Miah, Sherry V. Mori, Megan Hinkley, Yuanquan Yang: Acquisition of data. Ming Yin, Sarah P. Psutka, Yuanquan Yang: Analysis and interpretation of data. Ming Yin, Sarah P. Psutka, Yuanquan Yang: Drafting of manuscript. All authors: Critical revision of manuscript. Ming Yin: Statistical analysis. Ming Yin: Supervision.Ethical approval was not sought from an institutional review board or ethics committee prior to commencing this study.


To the Editor,IL2RG mutation in T cells. XSCID is caused by mutations in the gene coding for common gamma chain (γc) of interleukin‐2 receptor (IL2RG), characterized by the lack of T cells and NK cells, and shows hypogammaglobulinemia despite normal B‐cell count [IL2RG mutation in T cells. Although the patient had a milder clinical course with immunoglobulin replacement therapy for at least 20 years due to genetic reversions, questions remained on the longevity of the revertant cells with protective immune function. He consequently developed Epstein‐Barr virus associated lymphoproliferative disorder (EBV‐LPD) at 21 years of age. Here, we report the first case of EBV‐LPD followed by SCID with reversions of an IL2RG mutation in T cells.Kawai et al reported the case of a patient with X‐linked severe combined immunodeficiency (XSCID) having multiple reversions of an ll count . In the ll count , 4, 5, 63/μL). Although bone marrow examination did not show any specific findings, 18F fluorodeoxyglucose‐positron emission tomography/computed tomography (18F‐FDG‐PET/CT) demonstrated abnormal uptake in lymph nodes, spleen, bone marrow, and upper pharynx . Therefore, we planned a re‐biopsy, but it could not be performed because of persistent high fever and severe thrombocytopenia. High fever nonresponsive to antibiotics and increased levels of serum ferritin and lactate dehydrogenase (LDH) were suggestive of hemophagocytic lymphohistiocytosis (HLH). After starting prednisolone (1 mg/kg), recurrent fever and pancytopenia were improved. A diagnostic biopsy of the intraabdominal lymph node revealed large scattered tumor cells positive for CD30, IMP3, EBER, and CD15 , but negative for B‐ and T‐cell markers. A diagnosis of Hodgkin lymphoma (HL)‐like EBV‐LPD (Ann Arbor stage IV) was made based on these findings. High load of EBV‐DNA was found in the peripheral blood sample (650 copies /μg DNA). The proportions of the lymphocyte subsets of CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells in the peripheral blood were 31.9%, 60.2%, 2.3%, and 0.3%, respectively . Expression of common gamma chain  at the time of diagnosis of LPD was compared with the previous level at the age of 9 years  (1.8 mg/kg every 3 weeks) was initiated, owing to presumed intolerance to multiagent chemotherapy due to immunodeficiency and pancytopenia. Follow‐up CT study revealed decreased hepatosplenomegaly and abdominal lymphadenopathy after the first cycle of BV therapy. However, after five cycles, high fever of around 40°C recurred and liver dysfunction and pancytopenia were indicated by laboratory testing. Therefore, we started antibiotics treatment, although CT scans and blood culture revealed no signs of infection. Bone marrow smears suggested the invasion of tumor cells and β and γ genes demonstrated clonal rearrangement in the γ but not in the β gene . In addition, multiplex PCR analysis of both TCR β and γ genes suggested the presence of an oligoclonal T‐cell population. Since originally he was considered to have limited TCR diversity, we assumed that these results did not indicate T‐cell malignancy.Autopsy was performed to determine the extent of tumor involvement, followed by comparison of the pathological diagnosis with the clinical diagnosis. Unexpectedly, LPD was disseminated to whole body including the porta hepatis, lymph node, liver, spleen, lungs, bone marrow, and adrenal gland. Histology of the lymph node showed tumor cells with large nuclei that were positive for CD15, CD30, and EBER, partially positive for PD‐L1, and negative for B, T, and NK cell markers Figure . In addiThe development of EBV‐LPD in the individuals with primary immunodeficiency (PID) disease highlights several critical immune pathways that are needed for protection from viral infection and virus‐induced oncogenicity. Among them, the cytotoxic pathways of CTLs and NK cells are crucial for protection from most viral infections and malignancies . DefectsLPD is a recognized complication of PID, with historically very poor outcome. Reported mortality rates of PID associated with LPD is nearing 70% when the disease is unresponsive to conventional chemotherapy . For patFW and TK wrote and edited the manuscript. SU and MF provided the pathology images, wrote the pathology findings, and revised and edited the manuscript. MN, TM, YH, HS, KI, TY, and ATK revised and edited the manuscript. All the authors have read final version of the manuscript and have approved this submission.The authors declare that they have no conflict of interest.Table S1. Change in number of lymphocytes over time.Click here for additional data file.18F‐FDG‐PET/CT images at the onset of EBV‐LPD. (b) Southern blot analysis of the spleen for EBV‐terminal repeats at autopsy. (c) Southern blot analysis of T cell receptor (TCR) γ gene rearrangement. The arrows indicate monoclonal bands. E: EcoRI digestion, B: BanH‐digestion, K: Kpn I digestionFigure S1. (a) Click here for additional data file.


This report presents a classic case of CMV esophagitis, which may be puzzling to distinguish from other infectious esophageal lesions. Giant (>1 cm) and deep esophageal lesions in immunocompromised patients may suggest CMV esophagitis. A biopsy with immunostaining is needed to confirm the diagnosis. His past medical history was significant for dermatomyositis, for which he was on mycophenolate but had previously received prednisone and methotrexate. The treatment for his dermatomyositis had been complicated by pancytopenia with hemoglobin 6.8 (13.2–16.6 g/dl), platelet 73 (135–317 × 10[9]g/L), and white blood cells 1.9(3.4–9.6 × 10[9]g/L). He underwent esophagogastroduodenoscopy (EGD), which showed a deep esophageal ulcer on the background of diffuse white plaques in the middle third of the esophagus and a non‐bleeding large cratered clean‐based gastric antral ulcer Figure . Barium 2This is a classic presentation of cytomegalovirus (CMV) esophageal ulcer. The biopsy of the ulcer revealed positive CMV immunostaining, with negative immunostaining for Herpes simplex virus and fungus. His quantitative CMV DNA was 20,400 IU/ml (reference range undetected), and his HIV test was negative. CMV predominantly causes opportunistic infection in immunosuppressed patients. It mainly causes gastrointestinal symptoms, with about 12.9% of patients presenting with esophageal disease.Delvise Fogwe, first author, wrote and edited the case report. Matthew Ho, second author, assisted with acquiring images and putting images together. Aditya S. Shah, third author, provided overall mentorship and guidance on the direction of the paper.None.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


The discovery of new oral antidiabetic drugs remains a priority in medicine. This research aimed to evaluate the activity of the flavonoid baicalein and its natural glucuronide baicalin, compared to the antidiabetic drug metformin, as potential antiglycation, anti–radical, and anti-α–glucosidase agents, in order to assess their potential role in counteracting hyperglycemia-induced tissue damage. The study considered: (i) the BSA assay, to detect the formation of advanced glycation end products (AGEs), (ii) the GK peptide–ribose assay, which evaluates the cross–linking between the peptide and ribose, and (iii) the carbonyl content assay to detect the total carbonyl content, as a biomarker of tissue damage. In addition, to obtain a reliable picture of the antiglycation capacity of the investigated compounds, DPPH scavenging and oxygen radical absorbance capacity (ORAC) assays were performed. Furthermore, the anti–α–glucosidase activity of baicalein and baicalin was detected. Furthermore, to estimate cell permeability, preliminarily, the cytotoxicity of baicalein and baicalin was evaluated in HT–29 human colon adenocarcinoma cells using the MTT assay. Successively, the ability of the compounds to pass through the cytoplasmic membranes of HT–29 cells was detected as a permeability screen to predict in vivo absorption, showing that baicalein passes into cells even if it is quickly modified in various metabolites, being its main derivative baicalin. Otherwise, baicalin per se did not pass through cell membranes. Data show that baicalein is the most active compound in reducing glycation, α-glucosidase activity, and free radicals, while baicalin exhibited similar activities, but did not inhibit the enzyme α–glucosidase. Diabetes mellitus (DM) is the fourth leading cause of death in developed countries and is an epidemic in many developing countries. Numerous factors are involved in the aetiology of type 2 diabetes mellitus (T2DM), such as genetic predisposition, lifestyle, and dyslipidemia ,2. In poα–Glucosidase plays a key role in the regulation of blood sugar, and its inhibition reduces glucose absorption from food carbohydrates, reducing the postprandial glycemic peak . Severald–glucuronic acid of 5,6–trihydroxyflavone, Scutellaria baicalensis G.  and in the bark of Oroxylum indicum –2,5 diphenyl tetrazolium bromide (MTT), 1,1–diphenyl–2–picrylhydrazil (DPPH•), α–glucosidase , 6–hydroxy–2,5,7,8–tetramethylchroman–2–carboxylic acid (trolox), metformin, p–nitrophenyl–α–d–glucopyranoside (pNPG) and, generally, all chemicals and solvents were purchased from Merck KGaA, Darmstadt, Germany. Otherwise, the N–acetyl–glycyl–lysine methyl ester (GK) peptide was obtained from Bachem . The purity of the reference standards was 97%, while other chemicals were of at least analytical grade.Acarbose, 2,2′–azobis(2–amidinopropane)–dihydrochloride (AAPH), baicalein , baicalin (7–β––100 µM by adding to substrate solutions and incubation at 37 °C for a time of 5 (ribose) or 7  days. The fluorescence intensity was measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm with a PerkinElmer Victor Nivo microplate reader . Inhibition of AGE formation was calculated as the fluorescence difference between glycation under the control condition and in the presence of each inhibitor. Aminoguanidine  was used as a positive control [Glycated BSA was obtained according to a previously reported method ,60. Brie control .N–acetyl–glycyl–lysine methyl ester (GK) peptide–ribose assay was performed with some modifications to a published method [d–ribose in sodium phosphate buffer (pH 7.4) and test samples were added at final concentrations ranging from 1.0–100 μM. After 48 h of incubation, fluorescence was read at 355 and 460 nm for excitation and emission wavelengths, respectively, with a PerkinElmer Victor Nivo microplate reader . The fluorescence measurement allows the calculation of GK peptide glycation inhibition as a percentage difference between the control condition and glycation in the presence of the inhibitor. Aminoguanidine  was used as a positive control.The d method . Brieflyg rpm, 10 min at 4 °C, to obtain a pellet and the supernatant was removed. DNPH was removed by extracting the pellet twice using a solution of ethyl acetate/ethanol (1:1). Lastly, the pellet was dissolved in guanidine hydrochloride (6 M) before reading the absorbance at 375 nm. The concentration of DNPH derivatized proteins was determined by the molar extinction coefficient of 22,000 M−1 cm−1 [The total protein–bound carbonyl content was determined by derivatization of the carbonyl group with 2,4–dinitrophenylhydrazine (DNPH), which leads to the formation of a stable dinitrophenylhydrazone product , which wM−1 cm−1 . The proM−1 cm−1 , and the• method [•; the mixtures were kept in the dark for 60 min and absorbance was read at 517 nm using a Beckman Coulter DU 8005 spectrophotometer . The radical scavenging capacity was expressed as a percentage effect.The free radical scavenging activity of the samples was measured using the DPPH• method . Sample The radical oxygen absorption capacity (ORAC) assay was performed as previously described . BrieflypNPG as a substrate. The enzyme acts by hydrolyzing pNPG into α–d–glucopyranoside and p–nitrophenol (yellow); the reaction was detected at different inhibitor concentrations by variation in chromatic intensity [pNPG. Absorbance values were detected at 405 nm for 60 min, using a PerkinElmer Victor Nivo microplate reader spectrophotometer . The half–maximal inhibitory concentration (IC50) was estimated by the plot of relative enzyme activity versus inhibitor concentration. The type of enzyme inhibition exerted by baicalein was evaluated from kinetic studies using different substrate concentrations  through the Michaelis–Menten and Lineweaver–Burk plots.The inhibitory activity of baicalein and baicalin was studied using the yeast α–glucosidase assay, using ntensity ,48. AcarCell viability was assessed with the MTT assay . HT–29 cg rpm for 5 min, and added with 500 μL of 80% v/v methanolic solution in water/0,1% v/v acetic acid to extract intracellular content. The samples were kept on ice for 15 min, then sonicated and centrifuged at 20,000× g rpm for 10 min. Precipitated proteins were excluded, while supernatant solutions were collected for chromatographic analysis. The lysates and intracellular solutions were stored at −20 °C for HPLC–DAD analysis  was used. As previously reported [v/v in 12 min , with a flow rate of 1 mL/min. Peaks were detected in the range of 210–400 nm. All samples were filtered with membrane filters  and then injected (10 μL). Standard stock solutions were prepared in methanol. The amount of each compound was calculated using a standard calibration curve. The protein content of the lysate was detected using the Lowry protein assay [Chromatographic analyses were performed with an HPLC instrument  equipped with a 1525 binary HPLC pump and a 2998 photodiode array detector. A Symmetryreported , a lineain assay , to exprp < 0.05.Data are expressed as mean ± SEM of at least three independent experiments. The sigmoid curve fitting and statistical evaluations were performed using GraphPad Prism 9 . Statistical comparisons among three or more groups were performed using one–way ANOVA, followed by Dunnett’s multiple comparison test or Tukey’s test. The level of significance was established at i of 25.89 µM. Natural compounds baicalein and baicalin show favorable antidiabetic properties, supporting their intake through diets or even plant-based preparations.This research shows that both baicalein and baicalin have appreciable antiglycation and antiradical activities comparable to each other. On the contrary, the drug metformin shows only modest antiradical activity, without the capacity to decrease glycation reactions. Furthermore, baicalein, but not baicalin, inhibits α–glucosidase activity. Therefore, the natural compound baicalein exhibits antiglycation, antioxidant and anti–α–glucosidase activities, while baicalin does not cause any enzyme inhibition. Baicalein exhibits a mixed–type reversible inhibition on α–glucosidase, showing a KIn particular, the predicted favorable absorption profile of baicalein makes this natural flavonoid a promising candidate for further investigations about the possibility of developing a formulation of baicalein, finding the appropriate dose and pharmaceutical preparation, to be used in the prevention of hyperglycemia–related diseases.


Statins have anti‐inflammatory and potential immune‐modulatory effects, but their effect on plasmaIn this study, we aimed to evaluate the impact of rosuvastatin on D‐dimer and other inflammatory serum markers in VTE patients.We conducted a prospective, randomized study on 228 patients with VTE. Control group received conventional treatment (warfarin or rivaroxaban), whereas rosuvastatin‐intervention group received rosuvastatin 10 mg daily, in addition to their conventional treatment for 3 months. Serum markers were extracted from both groups at the baseline and 3 months after the beginning of treatment.d‐dimer and mean platelet volume (MPV) but no significant change in neutrophil‐to‐lymphocyte ratio and platelet‐to‐lymphocyte ratio.After 3 months, in patients of the intervention group, there was a statistically significant decrease in levels ofd‐dimer and MPV, which would support a potential role of statins to reduce activated systemic inflammation among VTE patients. Such effects can be used to reduce the rate of recurrent VTE in these patients.Our results showed that a 3‐month treatment with 10 mg rosuvastatin daily can significantly decrease the plasma levels of VTE term comprises of pulmonary embolism (PE) and deep vein thrombosis (DVT), along with an increased rate of mortality and morbidity.d‐dimer, one of the fibrin‐based protein fragments, can be significantly found in plasma when blood clot is forming and its elevated levels is an index for the diagnosis of VTE.Elevated mean platelet volume (MPV) is a sign of increased inflammation, which increases the risk of cardiovascular disorders. Increased MPV levels have been reported in patients with chronic diseases with high mortality and morbidity.Anticoagulants remain the conventional first‐line strategies for the prevention and treatment of VTE, even though they often have their own complications.d‐dimer and coagulation factors such as VII, VIII, and XI.The anti‐inflammatory properties of statins have not been fully understood yet; however, some investigations reported that they are able to mitigate the inflammatory mediators such as interleukin (IL)−1β in neurodegenerative disorders.Based on the beneficial effect of statins and their alternative roles as anti‐inflammatory and antiplatelet, herein we aimed to investigate the effect of 3‐month rosuvastatin consumption on blood‐based biomarkers in patients with VTE in an educational hospital in Tehran, Iran.22.1Diagnosis of provoked VTE.Any history of inflammatory disorders and acute infections.Previous blood‐related disorders affecting neutrophils, lymphocytes, and platelets.Any previous anticoagulants consumption, history of antiplatelet, and statin consumption over a period of 6 months.History of heart failure, hepatic failure, coagulopathy, hyperlipidemia or previous VTE, cancer and hemodialysis.Indication for initiating statin therapy based on 2018 American Heart Association/American College of Cardiology guideline on the management of blood cholesterol.Current clinical trial was carried out on 228 unprovoked VTE patients, aged 20–75 years, who are referred to the hospital and had no definite indication for initiating statin therapy. The exclusion criteria were as follows:All participants provided written informed consent. The protocol and consent forms were approved by the institutional review board at Shahid Beheshti University of Medical Science. Trial protocol has been approved under registration reference IRCT20210416050990N1. Eligible participants were randomized into two groups in a 1:1 ratio: control group and intervention group. A centralized computer based system was used for randomization. They were followed up for 3 months. Follow‐up after hospital discharge was made by telephone calls or visit in clinics.2.2d‐dimer, MPV, neutrophil, lymphocyte, and platelet counts were measured using enzyme‐linked immunosorbent assay, and NLR and PLR were calculated for all the participants. In addition, PE Severity Index (PESI) and Wells scores were calculated.Demographic and clinical data including personal interviews, questionnaires, and medical records were all collected. At the baseline and 3 months after intervention,PESI score is a clinical prognostic score that classifies PE patients into five classes with increasing risk of mortality and is consisted of different variables: age, sex, history of cancer, heart failure, chronic lung disease, altered mental status, respiratory rate, heart rate, systolic blood pressure, arterial oxygen saturation, and temperature. We calculated PESI score as follows:Patient's age in years if more than 80 years old summed with points received from these 10 criteria:Male gender (10 points), history of heart failure (10 points), history of chronic lung disease (10 points), history of malignancy (30 points), altered mental status (60 points), systolic blood pressure < 100 mmHg (30 points), heart rate > 109 beats per minute (20 points), respiratory rate > 29 breaths per minute (20 points), body temperature < 36°C (20 points), and arterial oxygen saturation < 90% (20 points). Then we categorized patients as follows: PESI I (<65 points), PESI II/III (66–105 points), and PESI IV/V (>106 points).d‐dimer following 3‐month treatment with 10 mg rosuvastatin. The secondary endpoints were to evaluate the variation in mean plasma levels of MPV, NLR, and PLR following 3‐month treatment with 10 mg rosuvastatin.Based on sex, age, type of VTE, PESI and Wells scores, and inclusion criteria, patients were divided into two groups, including control group, consisting of only anticoagulant‐receiver group (warfarin or rivaroxaban) and rosuvastatin‐receiver group (warfarin or rivaroxaban + 10 mg rosuvastatin). Blood samples were collected from both groups at the baseline and at 3 months after the beginning of treatment, and the mentioned biomarkers were extracted. The primary endpoint of this study was to evaluate the change in the mean plasma concentration of2.3χ2 test was performed to compare the frequency distribution of nominal variables in the study groups and independent t test was used to compare the mean of the quantitative variable in the study groups. Then, p < .05 was considered significant in all tests.Data were analyzed using SPSS v21 software  and normality of data were assessed by Kolmogorov–Smirnov test. For describing qualitative variables, number and percent were used, and for describing quantitative variables, mean and SD were used. The relationship between variables was measured using Pearson coefficient correlation. In addition, 3n = 114) were women. Approximately 61% of all participants were smokers. In addition, 29.4% of patients had diabetes mellitus and 14% had hypertension (HTN). Table Demographic data indicated that 50% of participants n = 14 were wp > .05) in the frequency of gender, smoking, underlying diseases, mean age and body mass index (BMI) between the control and rosuvastatin groups. Based on PESI score calculation, the control (mean PESI score = 84.19 ± 18.6) and intervention groups (mean PESI score = 92.42 ± 17.57) were both categorized in the same category (PESI II/III = 66–105 points). Both control (Wells score = 4.39 ± 0.90) and intervention groups (Wells score = 4.050 ± 1.07) were considered as likely to develop PE, based on Wells criteria without any statistically significant difference (p > .05).According to our results, there was no significant differences (d‐dimer levels in rosuvastatin group was significantly lower than control group (p < .001). In addition, 3‐month rosuvastatin treatment showed a significant reduction in MPV in intervention group (p < .001). However, other variables including NLR and PLR showed no significant differences between the control and rosuvastatin groups (p > .05) (Table The differences in main variables between rosuvastatin and control groups have been represented in Table 5) Table .4The risk of VTE recurrence increases after the first event. Many studies have indicated that the rate of VTE recurrence would be increased within the first 3 months, despite receiving anticoagulant treatment.Numerous studies have demonstrated that statins can reduce the VTE incidence in high risk patients, as well as recurrent VTE and mortality in patients with a previous VTE.d‐dimer have been reported in patients with residual vein occlusion.d‐dimer will increase the risk of recurrent VTE by 30% for the first 6 months.d‐dimer levels in patients with previous VTE.d‐dimer levels in rosuvastatin group, Sahebkar et al.d‐dimer significantly, whereas lipophilic‐based statins could reduced‐dimer levels after 3 months. A study by Wang et al.d‐dimer levels, in comparison with the control group. However, due to low recruitment rate, they terminated the study earlier than their plan.d‐dimer and MPV were significantly reduced in the intervention group that consumed warfarin or rivaroxaban, and 10 mg rosuvastatin for 3 months.High levels ofIn a meta‐analysis by Ji et al.The statin mediatory effects on lymphocyte levels have been reported previously. Simvastatin at a dose of 20 mg/day has been revealed to reduce plasma lymphocytes after 8 weeks.There are some points in our randomized clinical trial that needs consideration. The patients and the physicians were not blinded to the treatment but the laboratory technicians were blinded to the samples. As a result, it is unlikely that it would affect the outcomes. Also, recurrent VTE was diagnosed by a blinded physician, which reduced the risk of detection bias. In another aspect, there was no significant differences in the frequency of gender, smoking, underlying diseases, mean age, BMI, PESI, and Wells score between control and intervention groups; however, there is still a trivial possibility of residual confounding. Both our control and intervention group consumed anticoagulation, which might influence the effect of rosuvastatin on recurrent VTE. In the end, our results were associated with rosuvastatin and attribution to other statins warrants further studies.5d‐dimer and MPV are elevated during inflammation and hypercoagulable states, decreasing these factors would be a proper goal to decrease VTE recurrence. Conventional treatment of VTE (warfarin or rivaroxaban) combined with 10 mg rosuvastatin daily for 3 months decreasedd‐dimer and MPV levels significantly, in comparison with the patients receiving only anticoagulant therapy. Our data support the use of rosuvastatin in addition to conventional anticoagulant therapy in treatment of VTE. Further studies with bigger study groups are warranted to validate our results and propose underlying mechanisms of this effect.As5.1https://www.irct.ir/trial/55647).The protocol and consent forms were approved by the institutional review board at Shahid Beheshti University of Medical Science. Trial protocol has been approved in Iranian Registry of Clinical Trials (IRCT), which is a primary registry in the World Health Organization registry network set up, under registration reference IRCT20210416050990N1 (5.2d‐dimer levels after VTE propose ongoing hypercoagulability and VTE recurrence, medications that decreased‐dimer levels can decline the rate of recurrence.As prolonged elevation ofd‐dimer and MPV levels significantly among VTE patients, reducing the risk of VTE recurrence.A course of 3‐month rosuvastatin can decreaseThe anti‐inflammatory properties of statins without bleeding complications suggests a complementary treatment for VTE recurrence.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.


Studies on factors affecting weight loss effect after start of dapagliflozin in type 2 diabetes mellitus (T2DM) patients are few. The aim of this study was to identify if there were any patient characteristics that could predict weight loss after starting treatment with dapagliflozin.The study included 200 Korean patients with T2DM who were prescribed dapagliflozin in a family medicine clinic during 2014–2019. We studied patients for 1 year after starting dapagliflozin treatment. Data were collected from medical records. Clinically meaningful weight reduction was defined as ≥3% decrease in body weight and odds ratios (ORs) and 95% confidence intervals (CIs) for succeeding this weight reduction was calculated for different baseline characteristics.In total, 113 (56.5%) patients were male. Weight loss of ≥3% in 1 year treatment with dapagliflozin was achieved in 122 (61%) patients. The likelihood of this level of weight loss was significantly increased with regular exercise , with concomitant metformin treatment , and in patients with normal renal function . Patients receiving sulfonylurea treatment were less likely to achieve ≥3% weight reduction .T2DM patients that performed regular exercise, had normal renal function and were receiving metformin were more likely to have clinically meaningful body weight reduction after one year treatment with dapagliflozin. Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases. Its prevalence is expected to increase to up to 439 million by 2030 . It has Weight loss by lifestyle changes is the first-line therapy for T2DM because of the expected improvement in glycemic control and other associated risk factors . T2DM paThere are many pharmacotherapeutic options available for controlling glycemia in patients with T2DM. Sodium glucose co-transporter 2 (SGLT2) inhibitors are oral glucose-lowering drugs associated with weight reduction . TherefoHowever, studies on the effects of dapagliflozin on weight loss in T2DM patients in primary care settings are still few. In addition, there is a lack of studies on primary care patients who may benefit more from dapagliflozin treatment in terms of its weight loss effects. Thus, this study aimed to investigate to find predictors for ≥3% weight loss during treatment with dapagliflozin among Korean patients with T2DM seeking primary care treatment in Korea.n = 72), those admitted to other departments such as internal medicine (n = 65), and those whose duration of treatment with dapagliflozin was < 1 year (n = 96). Finally, 200 participants (113 men and 87 women) were included in the study.A total of 433 Korean adults who were prescribed dapagliflozin at the Department of Family Medicine in Asan Medical Center between January 1, 2014, and April 30, 2019, were enrolled in the study. The exclusion criteria were as follows: patients with missing data  and analyzed.2. Lifestyle-related factors such as alcohol consumption, smoking, and exercise habits were evaluated using a self-reported questionnaire, which was constructed by the authors. This was completed by the patients on their first visit. Using this questionnaire, the patients’ alcohol consumption was assessed. They were asked whether they drank alcohol, and the frequency of alcohol consumption per week, and the number of alcoholic drinks consumed per day were evaluated. Men who consumed ≥2 drinks per day and women who consumed ≥1 drink per day were referred to as heavy alcohol drinkers [The patients’ age at initiation of dapagliflozin was annotated, and height was measured to the nearest 0.1 cm using a stadiometer . Body weight was measured to the nearest 0.1 kg using a balance scale  with the participants wearing a lightweight gown or underwear. A significant weight loss was defined as ≥3% decrease in body weight after 1 year of dapagliflozin treatment. On the other hand, when the weight reduction was < 3%, we considered it as no change in body weight. An increase in body weight of > 3% was termed as weight gain. Body mass index (BMI) was calculated by dividing the patients’ weight (kg) by the square of their height (m). Next, we divided the participants into BMI quartile groups, and the cutoffs were 26.40, 27.88, and 30.11 kg/mdrinkers . Patient− 1.154 × age− 0.203 (multiplied by 0.742 in women) [2, and normal renal function was defined as eGFR ≥60 mL/min/1.73 m2.Blood samples were obtained after an overnight fast, and serum levels of glucose, HbA1c, and creatinine were measured. The estimated glomerular filtration rate (eGFR) was calculated using the following formula developed in the Modification of Diet in Renal Disease study: eGFR = 175 × serum creatininen women) . Abnormap-value < 0.05 was considered statistically significant.Statistical analyses were conducted using SPSS v. 24.0 . Baseline characteristics of the study participants are presented as mean ± standard deviation for continuous variables and number (percentage) for categorical variables. The values were compared using analysis of variance for continuous variables and chi-squared test for categorical variables. A paired T-test was performed to confirm the change in body weight, BMI, and fasting glucose and HbA1c levels before and after the use of dapagliflozin. We conducted a multivariable logistic regression analysis and calculated the odds ratio (OR) and 95% confidence intervals (CIs), and multivariable logistic regression analysis was performed to evaluate the factors associated with a weight loss of ≥3%. Model 1 was unadjusted, and model 2 was adjusted for age, sex, alcohol consumption, smoking status, physical activity, hypertension, dyslipidemia, CVD, metformin, SU, and renal function. A Table p < 0.001), and BMI decreased from 27.95 to 27.02 kg/m2 (p < 0.001). Additionally, the mean fasting blood glucose and HbA1c levels decreased from 150.88 mg/dL and 7.33% to 136.93 mg/dL and 7.22%, respectively (p < 0.001). The reductions in body weight, BMI, and fasting blood glucose were statistically significant following the use of dapagliflozin in the total population as well as in the male and female subgroups. The reductions in HbA1c were not statistically significant in the male and female subgroups.Table Table Table Our study showed that when starting dapagliflozin treatment, patients that exercised regularly, had normal kidney function or were treated with metformin were more likely to have a clinically meaningful weight reduction after start of dapagliflozin treatment. On the other hand, patients treated with sulfonylurea were less likely to do so. These results could help physicians choose the appropriate DM medication for these patients.Obesity is an independent risk factor for hypertension and dyslipidemia, as well as CVD, which is the major cause of death in patients with T2DM. Weight loss is considered key in T2DM management because it leads to a potential reduction in blood glucose levels. In addition, weight loss can have an impact on other health problems commonly associated with T2DM, thereby reducing the need for medications, not only for hyperglycemia but also for hypertension and hyperlipidemia . In a prMany T2DM patients have problems of weight gain, secondary to intake of drugs such as SUs, insulin, glinides, and thiazolidinediones , 21. MosRegular exercise, renal function, and metformin use were associated with weight loss after dapagliflozin treatment. Exercise contributes to weight loss, improves blood glucose control in T2DM, reduces cardiovascular risk factors, and improves general well-being . The mecOur study had several limitations. First, although the study sample size was determined through studies conducted similar to our study , 40, theWe showed that dapagliflozin can effectively reduce weight in T2DM patients who are regularly exercising, having a normal kidney function, and combining metformin. However, T2DM patients taking SU were less likely to have meaningful weight reduction. In T2DM patients with these conditions, it is possible to consider the preferential use of SGLT2 inhibitors over other antihyperglycemic agents.


Food insecurity combined with chronic disease conditions is a risk factor for Emergency Department (ED) utilization, an indicator of poor quality of care. However, such an association is not certain among school‐age children with chronic conditions. Therefore, we aim to determine the association of food insecurity, chronic conditions, and ED utilization among school‐age children in the United States.N = 5518). MEPS data was released electronically by the Agency for Healthcare Research and Quality (AHRQ). We identified four groups of school‐age children based on the presence of food security and chronic conditions: 1) with food insecurity and chronic conditions; 2) no food insecurity and chronic conditions; 3) with food insecurity and no chronic conditions; and 4) no food insecurity and no chronic conditions. We compared ED utilization among these four groups using incidence rate ratios (IRR) after adjusting children's age, sex, race and ethnicity, household income, insurance coverage, obesity, and geographic region using count data model, specifically multivariable Poison regression. We used SAS 9.4 and STATA 14.2 for all the data analyses.We analyzed the data from the 2017 Medical expenditure panel survey (MEPS) among children aged 6–17 years (p < 0.001). The adjusted IRR of ED utilization among school‐age children with food insecurity and chronic conditions was 1.90  compared with those with food security and chronic conditions.There were unweighted 5518 school‐age children who represented weighted 50,479,419 school‐age children in the final analysis. Overall, 6.0% had food insecurity with chronic conditions. These children had higher ED utilization (19.7%) than the other three groups (13.3%, 8.8%, and 7.2%, One in 16 school‐age children has both food insecurity and chronic conditions. Food insecurity was positively associated with frequent ED visits in the presence of chronic conditions. Therefore, addressing food insecurity may reduce the risk of ED visits. Data were obtained from the 2017 MEPS . MEPS is a nationally representative survey of noninstitutionalized civilian households administered by the Agency for Healthcare Research and Quality (AHRQ). The survey collects information on healthcare services utilization and costs, demographic characteristics, social determinants of health , and health status. Food security information was collected in a separate module in 2017. We limited our study to data collected in the calendar year 2017. As MEPS is a publicly available data set with deidentified data, this study was submitted to regional institution review board and approved as a nonhuman subject research project.2.2N = 5691). Among these children, 173 did not have data on food insecurity. The final study sample included 5518  children that represented 50,479,419 children aged from 6 to 17 years.The study included school‐age children 6–17 years of age  data are not publicly available and are used for editing and imputing household file data.2.3.2Food insecurity and chronic condition categories.In the MEPS, chronic conditions can be derived from several sources, such as directly asking the respondents whether they have been diagnosed with certain priority conditions, conditions for which the respondents sought medical care, conditions that caused the participants to miss school or work or spend more than half a day in bed.MEPS special module includes specific questions about food security that are closely aligned with the United States Department of Agriculture (USDA) 10‐item scale.2.3.3Other variables included children's age , sex , race , and geographic locations . Children's body mass index (BMI) was divided into four categories underweight, normal, overweight, and obese. These categories were determined using CDC age‐ and sex‐specific growth charts for children and teens ages 2–19.2.4The analyses accounted for the complex design of the MEPS by using survey procedures with family weights. Rao‐Scott chi‐square test of independence was used to assess whether food insecurity and chronic conditions  were associated with ED utilization. We calculated unadjusted and adjusted incident rate ratios (IRR) and corresponding 95% confidence intervals (CI) using Poisson regression to determine the association between food insecurity with a chronic condition and the number of ED visits. The adjustment included children's age, sex, race and ethnicity, poverty status, health insurance, and geographical location to further determine the potential confounders. These analyses were performed in SAS 9.4 and STATA 14.2.3n = 2709) female, 49.5% (unweighted n = 1841) White, and 50.8% (unweighted n = 2792) children in the age groupn = 2903) of children reported having FPL less than 200% (low‐income). An overwhelming majority  of children had public or private health insurance. One in five children (20.9%) had a chronic condition, and 8.4% (unweighted n = 410) were categorized “underweight” based on BMI. Overall, 19.7% (unweighted n = 1399) reported having food insecurity; 9.1% (unweighted n = 542) visited an ED during the calendar year.There were 48.8% (unweighted Table p < 0.001). In general, those without chronic conditions had fewer ED visits than those with chronic conditions (Table The unadjusted IRR (U‐IRR) and 95% CIs from the Poisson regression of ED visits are summarized in Table ns Table .p = 0.007). Consistent with unadjusted associations, children with no chronic conditions (with or without food insecurity) had fewer ED visits  children had chronic conditions and lived in households with food insecurity. Children of color, with obesity, and children living in households with a low income had a higher prevalence of food insecurity and chronic conditions  school‐age children had chronic conditions and lived in households with food insecurity. Among all school‐age children with chronic conditions, children with food insecurity had 1.9 times higher ED utilization than those without food insecurity. Our findings suggest that ED visits might be leveraged to screen children at high‐risk for food insecurity.Farheen Ghani: Conceptualization; data curation; resources; validation; writing—original draft; writing—review & editing. Hao Wang: Conceptualization; formal analysis; investigation; methodology; validation; writing – original draft; writing – review & editing. Sydney E. Manning: Conceptualization; formal analysis; methodology; writing—review & editing. Usha Sambamoorthi: Conceptualization; formal analysis; methodology; resources; supervision; validation; writing—review & editing.Dr. Hao Wang is an Editorial Board member of Health Science Reports and a coauthor of this article. To minimize bias, he was excluded from all editorial decision‐making related to the acceptance of this article for publication.The lead author Hao Wang affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained. All authors have read and approved the final version of the manuscript. Hao Wang and Usha Sambamoorthi had full access to all the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.


Hermetia illucens (HI) defatted larvae meal in broiler nutrition has frequently been suggested. For sustainability reasons, however, the larvae fat produced during defatting should also be used and could be particularly beneficial regarding gut health due to its fatty acid composition. To evaluate the suitability of HI larvae as protein and fat source, a 2 × 3 factorial arrangement with two types of protein, i.e. SBM (S) or SBM and 15% of its crude protein replaced by HI larvae meal (L), and three levels of fat sources, namely 0 (0 L), 50% (50 L) or 100% HI larvae fat (100 L) at the expense of soybean oil was applied.The possibility of partially replacing soybean meal (SBM) with In the starter phase, an interaction showed higher body weight (BW), average daily gain (ADG) and improved feed conversion ratio (FCR) if 50% or 100% HI larvae fat was fed with HI larvae meal. Moreover, BW, ADG and FCR improved when feeding HI larvae meal as protein source. Additionally, we observed an increased average daily feed intake in the grower, finisher, and overall phase in the L groups and an improved FCR in 0 L compared to 50 L groups during the overall period. Regarding apparent ileal digestibility, HI larvae meal feeding increased dry matter, organic matter, and fat digestibility. Feeding HI larvae meal as protein source decreased the concentrations of agmatine, spermidine, spermine and ammonia in the caecal digesta, whereas fat source affected agmatine with higher concentrations in 50 L compared to 0 L in the colonic digesta. In contrast, caecal ethanolamine concentrations increased in HI larvae meal groups compared to SBM. Caecal butyric acid concentrations decreased with HI larvae meal feeding. An interaction was found for the jejunal villus area, being higher in L + 100 L compared to S + 100 L. Furthermore, L groups had greater villus width.A partial replacement of SBM with HI larvae meal and soybean oil with HI larvae fat in broiler diets without impairing animal performance or gut health seems possible. Feeding HI larvae meal affected broiler performance positively in the starter phase and improved apparent ileal digestibility. Hermetia illucens, HI) are rich in crude protein (CP) and fat, with levels of about 42% and 15–37% in dry matter (DM), respectively  were collected. Therefore, the intestinal tract was removed and opened. To gain enough digesta, homogenously mixed samples of six animals were pooled per pen, put into narrow mouth bottles and immediately frozen at − 20 °C until further analysis. Two representative broilers per pen , which had already been taken for digesta sampling, were selected to collect intestinal gut tissue samples. For purpose of histological analysis, samples from the jejunum were excised halfway between the duodenum and the Meckel’s diverticulum, and the ileum 3–6 cm above the ileocaecal junction. Samples were flushed thoroughly with ice-cold phosphate buffered saline to remove the entire digesta content, embedded in slotted cassettes and immersed in 4% paraformaldehyde (v/v) for 48 h. Excreta of all birds were collected pen-wise on d 34 for determination of AMEN. For this purpose, excreta samples were frozen immediately at − 20 °C and calculation of AMEN was done according to Kong and Adeola [After 36 d of fattening, all broilers were slaughtered. Directly after slaughtering, digesta were collected from six representative broilers per pen  concentration as described by Leone et al. [Finisher feed and ileal digesta samples were also analysed for titanium dioxide , equipped with a J&W HP-88 GC column  and flame-ionization detector. Hydrogen was used as carrier gas at a constant pressure of 11 psi. The injector temperature was 250 °C and detector temperature 260 °C. The oven temperature was set to 100 °C for 5 min afterwards temperature was raised by 4 °C/min to 240 °C, which was then held for 30 min. Peaks were identified based on commercially available FAME mixtures . The results are expressed as the percentage (%) of total detected FAME.Determination of the fatty acid profile in feed was performed according to the 1-step methylation method of Sukhija and Palmquist . Briefly2O was added and vortexed again. Samples were then shaken  for 1 h, before being centrifuged at room temperature for 10 min at 3215 × g . Storage of the supernatant took place at − 20 °C. Before being analysed, thawed samples were centrifuged at 12,045 × g for 5 min  at room temperature and supernatant was used for analysis of ammonia.Analyses of the concentrations of biogenic amines in caecal and colonic digesta were performed according to Saarinen  using re2O and salicylate-nitroprusside solution) and 0.25 mL of dichloroisocyanurate solution (0.050 g dichloroisocyanurate dissolved in 50 mL double-distilled H2O). To ensure a proper colouring reaction, 1.0 mL of diluted sample extract or standard solution  was added immediately afterwards. Following incubation for 1.5 h at room temperature in the dark, the concentration of ammonia was analysed spectrophotometrically at 660 nm .The ammonia concentration was analysed by preparing a mixture of 0.5 mL of salicylate-nitroprusside colour reagent  after 10 min of incubation. The supernatant was immediately stored at − 20 °C. For analysis, the samples were thawed and centrifuged at 12,045 × g for 20 min . Acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, isocapronic acid, heptanoic acid and hexanoic acid were separated and quantified using a gas chromatographic system  with a C-18 capillary column  by a flame ionisation detection unit.The SCFA concentrations were determined by applying the method of Zhao et al. . The digmuscularis circularis was determined. For all these indices, measurements were taken on six well oriented villi, their crypts, and the underlying tissue layer that were regularly distributed along the entire intestinal surface. The respective means of these six measurements were subsequently used for statistical analysis.For purpose of histological analysis, intestinal gut tissue samples were first dehydrated and embedded in paraffin wax blocks, before being sectioned at 5 μm thickness using a microtome  and mounted onto glass slides . Sections were subsequently stained  following the standard protocol for Alcian blue-periodic acid–Schiff. For visualisation, a light microscope  and the software Leica Application Suite  were used. Morphometric indices examined were: villus height , villus width , villus area  and crypt depth . Using measurement of Vh and Cd, their ratio (Vh:Cd) was calculated. Goblet cells were counted and are expressed as number of cells per 200 μm of villus epithelium. In addition, the thickness of the submucosa and The following equation was used to calculate the apparent ileal digestibility (AID):Yijk is the dependent variable, μ the overall mean, αi the fixed effect of protein source ), βj the fixed effect of fat substitution , bk the random effect of pen  and eijk the residual error term. Statistics of performance parameters, digestibility coefficients, histological measurements and microbial metabolites were subjected to mixed model analysis using the PROC MIXED procedure of SAS v. 9.4 . Priorly, data were analysed for normal distribution using PROC UNIVARIATE. Significance was defined at P < 0.05, a trend at 0.05 ≤ P < 0.1 and results are expressed as least square means. Differences between the least squares means were post-hoc tested using an adjusted Tukey test.The following statistical model was used:Performance data are presented in Table P = 0.03), ADG (P = 0.03) and FCR (P = 0.01). Body weight and ADG increased in L + 100 L compared to S + 100 L. Regarding FCR, S + 50 L was higher than L + 50 L. Concerning the main effect protein, BW and ADG were affected with on average 2% higher BW for L groups compared to S groups. Similarly, FCR improved in L groups compared to S groups (P < 0.001). For fat source, a trend was detected for BW (P = 0.08) with 0 L being higher than 100 L. Average daily feed intake was not affected by interaction, protein or fat source in the starter period.In the starter period, an interaction for the protein and fat source was observed for BW (P = 0.04) and FCR (P = 0.01) was detected. Feeding HI larvae meal as protein source increased ADFI compared to SBM, but impaired FCR. No effects of fat source on performance were detected in the grower period.In the grower period, no interaction effect on performance parameters was observed. Referring to protein source, an effect for ADFI (P = 0.0005) and ADG (P = 0.07) increased or tended to increase in L groups compared to S groups. With regard to the effect of fat source, there was a trend for improved FCR in 0 L groups compared to 50 L groups (P = 0.05). Body weight was not affected in the finisher period.In the finisher period, performance was not affected by the interaction of the experimental factors protein and fat. Concerning the protein source, performance in terms of ADFI (P = 0.005). Fat source tendentially impaired FCR in the overall period in 50 L groups compared to 0 L groups (P = 0.05).In the overall period, animal performance parameter did not show interaction of protein and fat source. For the effect of protein source, ADFI was higher in L groups compared to S groups (P < 0.01) OM (P < 0.01) and fat (P = 0.02), all being increased by HI larvae feeding. Fat source tended (P = 0.08) to affect the AID of CP, showing improved CP digestibility in 100 L compared to 0 L.Table P < 0.01), ethanolamine (P = 0.02), spermidine (P < 0.01), spermine (P < 0.01) and ammonia (P < 0.01) concentrations were affected. This effect manifested itself with higher concentrations in S groups compared to L groups for agmatine, spermidine, spermine and ammonia. In contrast, the ethanolamine concentration was higher for L groups compared to S groups. Moreover, protein source tended to affect methylamine and putrescine concentrations (P = 0.06 each) being lower with HI larvae meal inclusion. Regarding the main effect of fat, a trend was determined for the concentration of spermine being higher in 0 L groups compared to 100 L groups (P = 0.07). No effect was observed for cadaverine, histamine and tyramine.Concentrations of biogenic amines and ammonia in the caecum are given in Table P = 0.03). The fat source tendentially affected the concentration of acetic acid increasing in 100 L groups compared to 50 L groups (P = 0.07). The concentration of propionic, valeric, isobutyric and isovaleric acid were neither affected by an interaction nor by the main effects protein and fat source.Table P = 0.03) when feeding 50% HI larvae fat compared to 0 HI larvae fat.The concentrations of biogenic amines in the colonic digesta are presented in Table P = 0.01), which increased in L +  100 L compared to S + 100 L, as well as increased in 50 L compared to 0 L groups when fed HI larvae meal, but not when fed SBM. Regarding the protein source, Va (P = 0.02) and Vw (P = 0.03) were affected with L groups being higher than S groups. Fat source affected Va (P = 0.03) and tended to affect Vh (P = 0.06) and the ratio of Vh:Cd (P = 0.09). Villus area and Vh were higher in 50 L groups compared to 0 L groups. With respect to the ratio of Vh:Cd, 50 L tended to have a better ratio than 100 L. The variables crypth depth, number of goblet cells, mucosa, submucosa and tunica muscularis were neither affected by an interaction, nor protein or fat in the jejunum. In the ileum, an interaction was observed for Vw (P = 0.03), which was higher in L + 0 L compared to L + 100 L. No effect of protein source was observed. Concerning the fat source, a trend was observed for greater thickness of the submucosa in 50 L compared to 100 L groups. No interaction or main effects were observed for Vh, Va, Vh:Cd, number of goblet cells, mucosa and tunica muscularis in the ileum.The analysis of the intestinal morphometric indices in the jejunum Table  showed aOur study showed that the use of HI larvae meal and fat did not negatively affect the performance of the animals. Remarkably, in the particularly important starter phase, the use of HI larvae meal even had a positive effect on chicken growth as evidenced by increased BW and ADG. For HI larvae fat, this was also true when exclusively fed together with HI larvae meal, which altogether confirms our hypothesis that HI larvae meal and fat can be fed without detrimental effects on performance. Regarding the effect of HI larvae meal as protein source, this coincides with the results of other studies showing that used in small amounts (5–10% of fresh matter in the diet), HI larvae meal leads to similar or even improved animal performance , 25, 40.The substitution of soybean oil with up to 100% of HI larvae fat has previously been shown to allow similar broiler performance , 28. It Tenebrio molitor meal in a cafeteria-type feeding. However, the opposite has previously been observed by Kieronczyk et al. [Furthermore, it was noticeable that ADFI increased in L groups compared to S groups during grower, finisher, and the overall experimental period. This observed increased ADFI could be due to birds’ originally entomophagous food habit. This matches observations of Cullere et al. , who obsk et al. , where bN concentrations. An improved digestibility likely could have exerted an effect on performance if the nutrient supply was limited or in shortage of the requirements. Provided the improved digestibility in the finisher phase can be extrapolated to all feeding phases, this may explain the enhanced performance of birds in the starter phase, in which high nutrient digestibility is especially important due to an immature gut development [Although the higher digestibility of DM, OM and ether extracts in the HI larvae meal diets has to be considered positive, it did only lead to a numerically higher performance in the finisher phase, when digestibility was measured, which is important from an economic point of view. When considering the results, however, it must be borne in mind that the present diets in general fulfilled or even exceeded the nutrient requirements, such as CP, lysine and AMEelopment .In terms of AID of CP, the present results fall neatly into those of previous studies suggesting an equal ileal CP digestibility when HI larvae meal accounted for low amounts (5%) in broiler diets . FeedingThe lack of fat source-related effects on AID was rather surprising, as the different FA composition of HI larvae oil compared to soybean oil would have suggested changes in fat digestibility. On the one hand, HI larvae fat is known to contain MCFA and so the lauric acid content in the present finisher diets increased almost thirtyfold and twelvefold from 0 to 100% HI larvae fat Table . As alreAdditionally, synergistic effects have been reported for the blend of vegetable oils and animal fats improving fat digestion . In the Nutrients escaping the host digestion and absorption in the small intestine are subsequently exposed to microbial fermentation by the caecal microbiota , whose fMerging the observations concerning biogenic amines with the findings related to SCFA analyses, i.e. an interaction of protein and fat on acetate and higher butyrate concentrations in SBM groups, some explanatory approaches arise. The first possible explanation for decreased putrefaction products in digesta of HI larvae fed groups may be chitin, which is found in the exoskeleton of insects and may act as  prebiotic . There iSecondly, the simultaneous observation of a tendency for higher putrescine and ammonia concentrations in SBM groups along with increased butyrate concentrations when feeding solely SBM may indicate a causal relation. Since those measured metabolite concentrations are not only the result of production but also absorption , 52, it Contrary to our hypothesis, we have not seen effects of the fat source on the concentrations of microbial metabolites, apart from a trend for less spermine concentrations in the caecum. This was unexpected, since previous studies indicated an antimicrobial activity of lauric acid . HoweverAlthough the level of potentially harmful microbial fermentation products decreased with HI larvae meal inclusion, it remains to be elucidated why the butyrate level increased in the SBM group, which makes a more in-depth investigation of the microbiome and the metabolic mechanisms related to this effect necessary.With its primary function to digest and subsequently absorb nutrients, the small intestine has a fundamental role for improving animal performance. In the present study, intestinal morphometric indices were analysed in the jejunum and ileum, since both segments are essential for fat digestion, i.e. the jejunum constitutes the major site for fat absorption , althougApart from a slight tendency for the fat source to influence Vh:Cd in the jejunum, there was an absence of differences in Cd and Vh:Cd between treatments in both gut segments. This observation can be considered positive, as these indices are used to evaluate the turnover rate of the intestinal mucosa . Therebytunica muscularis between the experimental groups, it can be assumed that intestinal peristalsis and thus contact of the luminal contents with the mucosa and ultimately nutrient absorption were undiminished [Another important parameter for assessing intestinal integrity are the goblet cells or the mucus secreted by them. The mucus produced serves the epithelium as protective barrier against physical and chemical damage  and operminished . Generalminished , 26.The present study was the first to test the simultaneous use of HI larvae meal and HI larvae fat, administered separately, in broiler feeding. The performance results suggest the possibility of replacing up to 15% CP from SBM with HI larvae meal and up to 100% soybean oil with HI larvae fat without impairments in animal performance. The improved animal performance in terms of increased ADG and BW, observed in the starter period, shows that the use of HI larvae meal and fat is not only an adequate substitute for SBM, but can even have positive effects. This may be attributed to a higher AID, as indicated by improved AID when feeding HI larvae meal as protein source in the finisher phase. In addition, the use of HI larvae protein has partially reduced the concentration of potentially harmful microbial metabolites, which may be regarded as positive in terms of gut health. To confirm the assumption that the improved performance observed in the starter period is attributed to a higher digestibility, it would be useful to explicitly examine the digestibility in the starter phase in the future. Furthermore, assessment of the gut microbiota may help to understand the observed changes in microbial metabolites and should therefore be conducted in future studies.


Current treatment options for diabetic neuralgia are limited and unsatisfactory. Tanezumab, a monoclonal antibody that blocks nerve growth factor (NGF) signaling, has been shown to be effective in relieving the clinical symptoms of osteoarthritis pain, chronic low back pain, cancer pain induced by bone metastasis, and diabetic neuralgia. However, the clinical development of tanezumab has been terminated due to the risk of induction of rapidly progressive osteoarthritis (RPOA), and no other NGF antibodies have been examined for their ability to treat diabetic neuralgia in either animal models or clinical trials. In this study, a humanized high‐affinity NGF monoclonal antibody (mAb), huAb45 that could neutralize the interaction between NGF and its high‐affinity receptor TrkA. In a mouse diabetic neuralgia model, it effectively relieved neuropathic pain. This study may serve as the necessary foundation for future studies of huAb45 to potentially treat diabetic neuralgia. NGF‐directed monoclonal antibody blocks the binding of NGF to its receptor, TrkA. Subsequently this blocks downstream signaling that interrupts diabetic neuralgia signaling. The exaNerve growth factor (NGF) is a neurotrophic factor that regulates the growth and development of peripheral and central neurons, maintains neuronal activities, and is involved in pain perception and processing . There ain vitro. In a mouse model of diabetic neuralgia, huAb45 effectively relieved neuropathic pain. This study may lay the necessary foundation for clinically testing huAb45 on diabetic neuralgia.In this study, a humanized NGF mAb, huAb45 that blocks the interaction between NGF and TrkA was generated, and has different epitopes from tanezumab. The epitope, purity, binding and blocking activities, and stability of huAb45 were confirmed 2. The Cell Counting Kit‐8 (CCK8) was purchased from Dojindo Laboratories . Human antibody germline sequences and primers were synthesized by Sangon Biotech . Tetramethylbenzidine (TMB) substrate was purchased from Thermo Fisher Scientific, PEG400 and thymidine (HAT) were from Sigma‐Aldrich. The human NGF sequences was fused into 6xHis‐tag and cloned into the pCAT1.0 vector (constructed in Darsbio Ltd.), followed by transient transfection of HEK293 cells for protein production. Amino acid sequences of tanezumab light and heavy chains were obtained from the international Immunogenetics information system (IMGT/mAb‐DB), and huAb45 were generated in‐house as described below.TF‐1 cells were purchased from American Tissue Culture Collection , and were cultured with RPMI1640  supplemented with 10% fetal calf serum  at 37 °C and 5% COm hypoxanthine, 0.4 mm aminopterin, and 1.6 mm thymidine (HAT)  selection medium. Enzyme‐linked immunoassay (ELISA) was used to test positive hybridoma cells 12 days after infusion, and then the cells were continually subcloned at least three times with limiting dilution to ensure clonality. Hybridoma cells capable of producing high‐sensitivity monoclonal antibodies against NGF in cell culture supernatants were recognized by ELISA. HuAb45 used in this study was screened out from a pool of neutralizing antibodies as the most potent blocker of NGF binding to TrkA. The variable regions of heavy (VH) and light (VL) chains were cloned into the human IgG2 backbone by “framework shuffling” technology, and then transiently expressed in HEK293F cells.NGF antibodies were generated by the hybridoma technique. BALB/c mice  were immunized by NGF‐6xHis and keyhole limpet hemocyanin. To form hybridomas, immunized mouse spleen cells were fused with myeloma cell SP2/0 at a 5:1 ratio using PEG400  and grown in 100 mELISA  was used to measure the binding affinity of NGF monoclonal antibodies with antigens derived from different species.−1) was coated onto immuno‐plates  at 4 °C overnight. Antibodies were gradiently diluted to three fold series from 100 nm. After 1 h of incubation, horseradish peroxidase (HRP)‐conjugated goat antihuman IgG (H + L) was added and then incubated for another hour at 37 °C. Next, the plate was washed six times with 0.05% phosphate‐buffered saline tween (PBST) and developed with TMB substrate. The SpectraMax M5e microplate reader  was used to measure the absorbance of OD450.NGF‐6xHis (2 µg·mL−1) were immobilized onto Protein A biosensors , and antigens were serially diluted two folds from 100 nm. KD values were calculated by the vendor‐supplied software.BLI was also used to measure the binding affinity of antibodies with antigens derived from different species. The affinities of huAb45 and tanezumab were determined by Fortebio Octet Red 96. All measurements were performed in the sample buffer at room temperature (RT). Antibodies (10 µg·mL−1) was coated onto 96‐well immune plates at 4 °C overnight. Then, three fold serial dilutions of huAb45 and tanezumab were added into each well for 1 h at room temperature. Next, NGF‐6xHis‐biotin (0.5 μg·mL−1) was added into wells for 30 min at room temperature. Finally, the HRP‐conjugated streptavidin was used and the plate was developed with TMB substrate to detect the bounded competitive ligands. The IC50 of antibodies was measured by software on a SpectraMax M5e microplate reader.The ELISA assay was used to investigate the ability of huAb45 to block the NGF binding to TrkA. First, TrkA‐Fc (2 μg·mLm) were loaded onto protein A sensors as the first antibody, followed by washing in 0.05% PBST to reach a baseline. Second, sensors were exposed to 100 nm of human NGF‐6xHis at the binding step, followed by exposure to the second antibody (100 nm) at the association step. Forte Bio’s Data Analysis Software 6.0 was used to process the data.Epitope binning assay was performed on Octet RED96. First, tanezumab and huAb45 , and tested after 72 h in the incubator. Cells were finally counted by the CCK8 kit.TF‐1 cells were seeded into 96‐well cell culture plates at 15,000 cells/well. The antibodies to be tested were diluted with 10% FBS in RPMI‐1640, starting from 450 ng·mLOsteoarthritis in the arthritis model was induced by sub‐plantar injection of complete Freund’s adjuvant (at the dose of 400 µL every 3 days for 3 weeks) into the left hind paw of 6–8 week‐age male BALB/c mice . Three mice were fed in each cage.On the day of the test, 15–30 min before the measurement, the mice were kept in a glass test cage to make them relaxed and stay still.A thermal plantar/Tail Flick instrument  was used to place the infrared light source directly below the middle surface of the mouse's hind paw, and then the start button was pressed. The plantar surface of the hind paw was exposed to the beam of radiant heat through the transparent glass surface .The withdrawal latency of injured and uninjured hind paws was recorded in a balanced sequence. The time (sec) from the start of the thermal stimulus to the paw withdrawal from the heat source was automatically recorded. If the answer was ambiguous, the stimulus was repeated, with the latency to be recorded again. Mice shaking or licking hind paws was also a sign of painful irritation.The thermal stimulus was repeated at least three times at approximately 5‐min intervals, with the latencies recorded, then the average paw withdrawal latencies were calculated.−1) by oral gavage every day for 28 consecutive days. Mice from the test drug groups were injected subcutaneously with the corresponding doses of huAb45 on Day 1 (D1), D8, D15, and D 22, for the first, second, third, and fourth administration, respectively. The 4‐h fasting blood glucose of mice was measured on D6, D13, D20, and D27 of the test, and mice were weighed before fasting. The mechanical pain threshold was measured on D7, D14, D21, and D28 of the test. Before pain measurement, all mice were kept in the test cage to adapt to the environment. A series of von Frey filaments  were used to measure the foot retraction reaction of mice by the up–down method: the middle of the hind paw in each mouse was vertically stimulated with von Frey filaments to make it slightly S‐shaped for 5 s, with observation of the claw withdrawal reaction [To determine the analgesic ability of huAb45, 6–8 week‐age male db/db  mouse models, free of neurodegeneration, were used . Three mreaction . Rapid wMice were fed a standard diet and water and kept in a controlled air‐conditioned environment at 22 ± 2 °C with a relative humidity of 50% ± 10% on a 12‐h/12‐h light/dark cycle. The experiments were performed according to the guidelines outlined by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica, Chinese Academic Science  (IACUC: 2021‐03‐WCH‐05).50EC values of huAb45 and tanezumab were 0.01129 and 0.00934 nm by ELISA, respectively  and size exclusion chromatography (SEC‐HPLC) Fig. . Then weely Fig. , and theyte Fig. . In addiyte Fig. .50 values of 1.626 and 1.671 nm, respectively , huAb45 exhibited better analgesic effects than tanezumab Fig. , and it The analgesic ability of huAb45 was validated in the db/db mouse model of diabetic neuralgia. HuAb45 showed analgesic effects similar to that of gabapentin in the first 3 weeks of the experiment, but surpassed the latter starting from the 4th week Fig. . During There is an increasing demand for medications with better therapeutic efficacy and fewer side effects for diabetic neuralgia , 25. ThiA neutralizing NGF antibody was first discovered from hybridomas and humanized to huAb45, and then its binding and neutralizing abilities were evaluated at the molecular, cellular, and animal model levels. This molecule demonstrated high purity and stability, as well as the capacity to efficiently block the interaction between NGF and it high‐affinity receptor TrkA. More important, huAb45 binds to an antigen epitope different from tanezumab Fig. . In the Tanezumab, an NGF mAb jointly promoted by Pfizer and Eli Lilly, has demonstrated impressive analgesic efficacy in multiple clinical trials , 27. HowThe application of NGF antibodies is a promising strategy for the treatment of diabetic neuralgia. First, there is currently no effective pain‐managing medicines and tanezumab has shown remarkable efficacy in a Phase II clinical trial for diabetic neuralgia. Second, diabetic neuralgia is a non‐OA indication and NSAIDs are not typically used in diabetic patients. Therefore, the risk of causing RPOA in diabetic neuralgia by NGF antibodies is really low . In the Overall, we generated huAb45, a NGF‐neutralizing antibody binding to a novel epitope. It has been demonstrated that huAb45 successfully relieved the pain in an animal model of diabetic neuralgia. More important, its analgesic effects in diabetic neuralgia and arthralgia experiments were superior to that of positive controls. This mAb is expected to substantially minimize the pain caused by diabetes and provide patients with benefit of convenient administration.Xingchen Dong, Yili Chen, Huanhuan Li, Jie Tan, Ganjun Chen, Santai Li, and Zuobin Xie received stipends from Shanghai Mabstone Biotechnology, Co., Ltd.Data are available from the corresponding author upon reasonable request.Conceptualization, Y.L. C. and C.W.; methodology, Y.‐L. C.; validation, X. D., G. L., Y. C, and H. L.; formal analysis, Y.C., X. D., and C.W.; investigation, Y.‐L. C. and C.W.; resources, Y. L. C., C.W..; data curation, S, L. and X. D.; writing—original draft preparation, X. D.; writing—review and editing, Y.‐L. C., X. D and C.W.; visualization, X.D., M.P., and Z.X.; project administration, Y.‐L. C.; funding acquisition, C.W. All authors have read and agreed to the published version of the article.


Multiple system atrophy (MSA) is an incurable neurodegenerative disease. We aimed to investigate the health‐related quality of life (HRQoL) and the determinants of HRQoL in patients with MSA.The five‐level EuroQol five‐dimensional questionnaire (EQ‐5D‐5L) was used to evaluate patients’ HRQoL. The results of HRQoL were indicated by the EQ‐5D‐5L index values and visual analog scale (EQ VAS) scores. Specific scales were used to measure disease severity, cognition, frontal lobe function, anxiety, depression, fatigue, and sleep disorders. The beta mixture model and the linear regression model were used to explore the determinants of HRQoL in patients with MSA.A total of 205 patients with cerebellar variants  and 175 patients with parkinsonian variants  were included in this cross‐sectional study. The mean values of the EQ‐5D‐5L index values and EQ VAS scores were .558 and 59.5, respectively. Problem with mobility was the problem reported by the highest proportion of patients (92.1%), followed by problems with usual activities (88.7%), self‐care (81.3%), anxiety/depression (72.1%), and pain/discomfort (53.9%). The determinants of the lower EQ‐5D‐5L index values in patients with MSA were greater disease severity, fatigue, Parkinson's disease‐related sleep problems (PD‐SP), depressive mood, and anxious mood. Greater disease severity, fatigue, and depressive mood were associated with lower EQ VAS scores.The problem reported most frequently by Chinese individuals with MSA was mobility. In addition to the greater disease severity of MSA, fatigue, PD‐SP, depression, and anxiety were determinants of poor HRQoL. To date, the etiology of MSA is still uncertain, and no cure for MSA has been developed. MSA can be subclassified into a cerebellar variant (MSA‐C) and a parkinsonian variant (MSA‐P) according to the predominant motor symptoms  than healthy controls and patients with Parkinson's disease (PD)   and all the clinical symptoms were included in the multivariate models. Clinical symptoms were included in the two kinds of regression models as dichotomous variables according to the cutoff score of scales as described before; p < .05 was considered statistically significant. SPSS 22.0  and Stata 16.0 (StataCorp) were used to perform data analysis.Spearman's correlation analysis was conducted to explore relationships between the EQ‐5D‐5L index values and scores on the clinical symptoms scale. Since the distribution of the EQ‐5D‐5L utility value was skewed and featuring with truncations  and 175 MSA‐P (46.1%) were included in the study. The clinical characteristics of the patients are displayed in Table 3.2The mean EQ VAS score for MSA was 59.5 SD: 17.81) and the mean EQ‐5D‐5L index value was .558 SD: .276). The EQ‐5D‐5L index values and EQ VAS scores of participants in the different subgroups are displayed in Table 76. The E7.81 and 3.3Spearman's analysis showed a moderate correlation between the EQ VAS scores and total UMSARS, the Fatigue Severity Scale, the Parkinson's Disease Sleep Scale 2nd version, the Hamilton Depression Rating Scale., and the Hamilton Anxiety Rating Scale scores in all MSA patients. The total UMSARS score was strongly correlated with the EQ‐5D‐5L index values. Scores for the frontal assessment battery, MoCA, the Fatigue Severity Scale, the Parkinson's Disease Sleep Scale 2nd version, the Hamilton Depression Rating Scale, and the Hamilton Anxiety Rating Scale had weak to moderate correlations with EQ‐5D‐5L index values for all MSA patients Table .Mixture regression models and linear regression models showed the determinants of EQ‐5D‐5L index values and EQ VAS scores in total MSA and two subtypes of patients. In the total MSA patients, fatigue, PD‐SP, depressive mood, anxiety mood, and greater total UMSARS score were determinants for lower EQ‐5D‐5L index values. In addition, fatigue, depressive mood, and greater total UMSARS score were determinants for the lower EQ VAS scores. Total UMSARS score and PD‐SP were associated with lower EQ‐5D‐5L index values in both MSA‐C and MSA‐P. Total UMSARS score and fatigue were determinants of lower EQ VAS scores in both MSA‐C and MSA‐P. However, some differences existed in the determinants for EQ‐5D‐5L index values and EQ VAS scores between patients with MSA‐C and MSA‐P. For example, fatigue was the determinant for lower EQ‐5D‐5L index values in patients with MSA‐P but not in patients with MSA‐C. More details are displayed in Tables 4Our research measured the HRQoL of patients with MSA using the EQ‐5D‐5L. We found that the mean EQ‐5D‐5L index value and EQ VAS score were .558 and 59.5, respectively. Problem with mobility was the most commonly reported problem, and the problem with pain/discomfort was the least common problem, which was consistent with the previous study using the EQ‐5D‐3L  and the mean EQ VAS score (59.5) of patients with MSA were lower than those of the Chinese healthy population  and those of similarly aged patients with coronary heart disease  and colorectal cancer  but were higher than those of Chinese patients with atrial fibrillation  and hemophilia  (Huang et al., We found that there were some differences between patients with MSA‐P and MSA‐C. A higher frequency of problems with mobility was reported in patients with MSA‐C than in patients with MSA‐P in the current study. However, no difference in the mean scores of motor examination and global disability scale of UMSARS between the two subtypes was found. Our finding suggested that UMSARS has a limited ability to reflect mobility in patients with MSA‐C. In addition, we found that patients with MSA‐P reported more problems with pain/discomfort than patients with MSA‐C. This may be due to the more severe damage in the basal ganglia in MSA‐P than in MSA‐C (Lin, Xu, Hou, Yang, & Shang, Depression was reported to be a determinant of the QoL of patients with MSA (Du et al., In the current study, we found that fatigue was negatively correlated with EQ‐5D‐5L index values and EQ VAS scores of MSA patients, which was consistent with our previous study evaluating the QoL of MSA patients using the PDQ‐39 (Zhang et al., In our previous study, sleep‐related disorders, including PD‐SP, RBD, and excessive daytime sleepiness, were found to be associated with higher disease severity in patients with MSA (Lin et al., This research had several limitations. First, the current study was a cross‐sectional research. We planned to follow up patients included in the current study at 1‐year interval to explore the longitudinal change of HRQoL in patients with MSA (Meissner et al., 5The current research evaluated HRQoL in patients with MSA with the EQ‐5D‐5L. The determinants of lower EQ‐5D‐5L index values were greater total UMSARS, fatigue, PD‐SP, depressive mood, and anxiety symptoms scores, while those of lower EQ VAS scores were greater total UMSARS, fatigue, and depressive symptoms scores. In addition, depression was a determinant of HRQoL in MSA‐C but not in MSA‐P, while anxiety was related to HRQoL in MSA‐P but not in MSA‐C. This research provided important information on the HRQoL and potential determinants of MSA patients, helping guide the development of intervention strategies to improve their health status.Yi Xiao contributed in organization and execution of research project, study design, statistical analysis, writing of the draft, and review and critique of the manuscript. Lingyu Zhang contributed in organization and execution of research project, study design, statistical analysis, and review and critique of the manuscript. Qianqian Wei contributed in organization and execution of research project and design of the statistical analysis. Tianmi Yang, Kuncheng Liu, Ruwei Ou, Yanbing Hou, and Junyu Lin helped in organization and execution of research project. Huifang Shang helped in conception, organization, and execution of research project and review and critique of the manuscript.The authors declare no conflict of interest.https://publons.com/publon/10.1002/brb3.2774.The peer review history for this article is available at: Supplementary Table 1 Data distribution of EQ‐5D‐5L index value in Total MSA and two subtypesSupplementray Table 2 Spearman correlation between EQ VAS score, EQ‐5D‐5L index value and clinical characteristics of patients with MSA.Click here for additional data file.