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Background Primary diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is based on detection of virus RNA in nasopharyngeal swab samples. In addition, analysis of humoral immunity against SARS-CoV-2 has an important role in viral diagnostics and seroprevalence estimates. Methods We developed and optimized an enzyme immunoassays (EIA) using SARS-CoV-2 nucleoprotein (N), S1 and receptor binding domain (RBD) of the viral spike protein, and N proteins from SARS, Middle East respiratory syndrome (MERS), and 4 low-pathogenic human CoVs. Neutralizing antibody activity was compared with SARS-CoV-2 IgG, IgA, and IgM EIA results. Results The sensitivity of EIA for detecting immune response in COVID-19 patients (n = 101) was 77% in the acute phase and 100% in the convalescent phase of SARS-CoV-2 infection when N and RBD were used as antigens in IgG and IgA specific EIAs. SARS-CoV-2 infection significantly increased humoral immune responses against the 229E and NL63 N proteins. S1 and RBD-based EIA results had a strong correlation with microneutralization test results. Conclusions The data indicate a combination of SARS-CoV-2 S1 or RBD and N proteins and analysis of IgG and IgA immunoglobulin classes in sera provide an excellent basis for specific and sensitive serological diagnostics of COVID-19.

Skeletal muscle, the most abundant tissue in the body, plays vital roles in locomotion and metabolism. Understanding the cellular processes that govern regulation of muscle mass and function represents an essential step in the development of therapeutic strategies for muscular disorders. Myostatin, a member of the TGF-β family, has been identified as a negative regulator of muscle development. Indeed, its inhibition induces an extensive skeletal muscle hypertrophy requiring the activation of Smad 1/5/8 and the Insulin/IGF-I signaling pathway, but whether other molecular mechanisms are involved in this process remains to be determined. Using transcriptomic data from various Myostatin inhibition models, we identifiedPak1as a potential mediator of Myostatin action on skeletal muscle mass. Our results show that muscle PAK1 levels are systematically increased in response to Myostatin inhibition, parallel to skeletal muscle mass, regardless of the Myostatin inhibition model. UsingPak1knockout mice, we investigated the role ofPak1in the skeletal muscle hypertrophy induced by different approaches of Myostatin inhibition. Our findings show thatPak1deletion does not impede the skeletal muscle hypertrophy magnitude in response to Myostatin inhibition. Therefore,Pak1is permissive for the skeletal muscle mass increase caused by Myostatin inhibition.

cited By 1 Skeletal muscle wasting is commonly associated with chronic kidney disease (CKD), resulting in increased morbidity and mortality. However, the link between kidney and muscle function remains poorly understood. Here, we took a complementary interorgan approach to investigate skeletal muscle wasting in CKD. We identified increased production and elevated blood levels of soluble pro-cachectic factors, including activin A, directly linking experimental and human CKD to skeletal muscle wasting programs. Single-cell sequencing data identified the expression of activin A in specific kidney cell populations of fibroblasts and cells of the juxtaglomerular apparatus. We propose that persistent and increased kidney production of procachectic factors, combined with a lack of kidney clearance, facilitates a vicious kidney/muscle signaling cycle, leading to exacerbated blood accumulation and, thereby, skeletal muscle wasting. Systemic pharmacological blockade of activin A using soluble activin receptor type IIB ligand trap as well as muscle-specific adeno-associated virus-mediated downregulation of its receptor ACVR2A/B prevented muscle wasting in different mouse models of experimental CKD, suggesting that activin A is a key factor in CKD-induced cachexia. In summary, we uncovered a crosstalk between kidney and muscle and propose modulation of activin signaling as a potential therapeutic strategy for skeletal muscle wasting in CKD.

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes., The soluble bioactive form of the transmembrane protein fibronectin type III domain containing 4 (sFNDC4) has anti-inflammatory effects and improves insulin sensitivity. Here the authors show that liver derived sFNDC4 signals through adipose tissue GPCR GPR116 to promote insulin-mediated glucose uptake.

Blocking of myostatin and activins effectively counteracts muscle atrophy. However, the potential interaction with physical inactivity and fasting in the regulation of muscle protein synthesis is poorly understood. We used blockade of myostatin and activins by recombinant adeno-associated virus (rAAV)-mediated follistatin (FS288) overexpression in mouse tibialis anterior muscle. To investigate the effects on muscle protein synthesis, muscles were collected 7 days after rAAV-injection in the nighttime or in the daytime representing high and low levels of activity and feeding, respectively, or after overnight fasting, refeeding, or ad libitum feeding. Muscle protein synthesis was increased by FS288 independent of the time of the day or the feeding status. However, the activation of mTORC1 signaling by FS288 was attenuated in the daytime and by overnight fasting. FS288 also increased the amount of mTOR colocalized with lysosomes, but did not alter their localization toward the sarcolemma. This study shows that FS288 gene delivery increases muscle protein synthesis largely independent of diurnal fluctuations in physical activity and food intake or feeding status, overriding the physiological signals. This is important for eg cachectic and sarcopenic patients with reduced physical activity and appetite. The FS288-induced increase in mTORC1 signaling and protein synthesis may be in part driven by increased amount of mTOR colocalized with lysosomes, but not by their localization toward sarcolemma.

International audience; Numerous approaches are being developed to promote post-natal muscle growth based on attenuating Myostatin/Activin signalling for clinical uses such as the treatment neuromuscular diseases, cancer cachexia and sarcopenia. However there have been concerns about the effects of inhibiting Activin on tissues other than skeletal muscle. We intraperitoneally injected mice with the Activin ligand trap, sActRIIB, in young, adult and a progeric mouse model. Treatment at any stage in the life of the mouse rapidly increased muscle mass. However at all stages of life the treatment decreased the weights of the testis. Not only were the testis smaller, but they contained fewer sperm compared to untreated mice. We found that the hypertrophic muscle phenotype was lost after the cessation of sActRIIB treatment but abnormal testis phenotype persisted. In summary, attenuation of Myostatin/Activin signalling inhibited testis development. Future use of molecules based on a similar mode of action to promote muscle growth should be carefully profiled for adverse side-effects on the testis. However the effectiveness of sActRIIB as a modulator of Activin function provides a possible therapeutic strategy to alleviate testicular seminoma development.

Aberrant activation of signalling pathways has been postulated to promote age related changes in skeletal muscle. Cell signalling activation requires not only the expression of ligands and receptors but also an appropriate environment that facilitates their interaction. Here we first examined the expression of SULF1/SULF2 and members of RTK and the Wnt family in skeletal muscle of normal and a mouse model of accelerated ageing. We show that SULF1/SULF2 and these signalling components, a feature of early muscle development are barely detectable in early postnatal muscle. Real time qPCR and immunocytochemical analysis showed gradual but progressive up-regulation of SULF1/SULF2 and RTK/Wnt proteins not only in the activated satellite cells but also on muscle fibres that gradually increased with age. Satellite cells on isolated muscle fibres showed spontaneous in vivo satellite cell activation and progressive reduction in proliferative potential and responsiveness to HGF and dysregulated myogenic differentiation with age. Finally, we show that SULF1/SULF2 and RTK/Wnt signalling components are expressed in progeric mouse muscles at earlier stage but their expression is attenuated by an intervention that promotes muscle repair and growth.

Activin A has previously been associated with cancer cachexia and in vitro resistance to platinum-based chemotherapy. We studied circulating activin A concentrations as well as activin B and their antagonists' follistatin/follistatin-like 3 in presurgical patients with non-small-cell lung cancer and malignant pleural mesothelioma. We found that circulating activing A levels were elevated in malignant pleural mesothelioma and associated with cancer cachexia and poor response to platinum-based chemotherapy. Circulating activing A separated non-small-cell lung cancer from benign lung lesion. Background: Previous preclinical studies have shown that activin A is overexpressed in malignant pleural mesothelioma (MPM), associates with cancer cachexia, and is observed in in vitro resistance to platinum-based chemotherapy. We evaluated circulating activin levels and their endogenous antagonists' follistatin/follistatin-like 3 in intrathoracic tumors. Materials and Methods: Patients suspected of thoracic malignancy were recruited prior to surgery. Serum samples were collected from 21 patients with MPM, 59 patients with non-small-cell lung cancer (NSCLC), and 22 patients with benign lung lesions. Circulating activin/follistatin levels were measured using enzymelinked immunosorbent assay and compared with clinicopathologic parameters. Results: Circulating activin A levels were elevated in patients with MPM when compared with patients with NSCLC or benign lung lesion samples (P

Activin A and myostatin, members of the transforming growth factor (TGF)-β superfamily of secreted factors, are potent negative regulators of muscle growth, but their contribution to myocardial ischemia-reperfusion (IR) injury is not known. The aim of this study was to investigate if activin 2B (ACVR2B) receptor ligands contribute to myocardial IR injury. Mice were treated with soluble ACVR2B decoy receptor (ACVR2B-Fc) and subjected to myocardial ischemia followed by reperfusion for 6 or 24 h. Systemic blockade of ACVR2B ligands by ACVR2B-Fc was protective against cardiac IR injury, as evidenced by reduced infarcted area, apoptosis, and autophagy and better preserved LV systolic function following IR. ACVR2B-Fc modified cardiac metabolism, LV mitochondrial respiration, as well as cardiac phenotype toward physiological hypertrophy. Similar to its protective role in IR injury in vivo, ACVR2B-Fc antagonized SMAD2 signaling and cell death in cardiomyocytes that were subjected to hypoxic stress. ACVR2B ligand myostatin was found to exacerbate hypoxic stress. In addition to acute cardioprotection in ischemia, ACVR2B-Fc provided beneficial effects on cardiac function in prolonged cardiac stress in cardiotoxicity model. By blocking myostatin, ACVR2B-Fc potentially reduces cardiomyocyte death and modifies cardiomyocyte metabolism for hypoxic conditions to protect the heart from IR injury., Graphical Abstract, Magga et al. report that systemic blockade of activin receptor type 2 (ACVR2B) ligands protects from cardiac ischemia-reperfusion injury reducing infarct size, apoptosis, and autophagy. Mechanistically, systemic blockade of ACVR2B ligands reduced SMAD2 activation and modified cardiomyocyte metabolism for hypoxic conditions.

Fractures still present a significant burden to patients due to pain and periods of unproductivity. Numerous growth factors have been identified to regulate bone remodeling. However, to date, only the bone morphogenetic proteins (BMPs) are used to enhance fracture healing in clinical settings. Activins are pleiotropic growth factors belonging to the TGF-beta superfamily. We and others have recently shown that treatment with recombinant fusion proteins of activin receptors greatly increases bone mass in different animal models by trapping activins and other ligands thus inhibiting their signaling pathways. However, their effects on fracture healing are less known. Twelve-week old male C57Bl mice were subjected to a standardized, closed tibial fracture model. Animals were divided into control and treatment groups and were administered either PBS control or a soluble activin type IIB receptor (ActRIIB-Fc) intraperitoneally once a week for a duration of two or four weeks. There were no significant differences between the groups at two weeks but we observed a significant increase in callus mineralization in ActRIIB-Fc-treated animals by microcomputed tomography imaging at four weeks. Bone volume per tissue volume was 60%, trabecular number 55% and bone mineral density 60% higher in the 4-week calluses of the ActRIIB-Fc-treated mice (p

Protein homeostasis in cells, proteostasis, is maintained through several integrated processes and pathways and its dysregulation may mediate pathology in many diseases including Duchenne muscular dystrophy (DMD). Oxidative stress, heat shock proteins, endoplasmic reticulum (ER) stress and its response, i.e. unfolded protein response (UPR), play key roles in proteostasis but their involvement in the pathology of DMD are largely unknown. Moreover, exercise and activin receptor IIB blocking are two strategies that may be beneficial to DMD muscle, but studies to examine their effects on these proteostasis pathways are lacking. Therefore, these pathways were examined in the muscle of mdx mice, a model of DMD, under basal conditions and in response to seven weeks of voluntary exercise and/or activin receptor IIB ligand blocking using soluble activin receptor-Fc (sAcvR2B-Fc) administration. In conjunction with reduced muscle strength, mdx muscle displayed greater levels of UPR/ER-pathway indicators including greater protein levels of IREloc, PERK and Atf6b mRNA. Downstream to IREloc and PERK, spliced Xbpl mRNA and phosphorylation of elF2oc, were also increased. Most of the cytoplasmic and ER chaperones and mitochondrial UPR markers were unchanged in mdx muscle. Oxidized glutathione was greater in mdx and was associated with increases in lysine acetylated proteome and phosphorylated sirtuin 1. Exercise increased oxidative stress when performed independently or combined with sAcvR2B-Fc administration. Although neither exercise nor sAcvR2B-Fc administration imparted a clear effect on ER stress/UPR pathways or heat shock proteins, sAcvR2B-Fc administration increased protein expression levels of GRP78/BiP, a triggering factor for ER stress/UPR activation and TxNIP, a redox-regulator of ER stress-induced inflammation. In conclusion, the ER stress and UPR are increased in mdx muscle. However, these processes are not distinctly improved by voluntary exercise or blocking activin receptor IIB ligands and thus do not appear to be optimal therapeutic choices for improving proteostasis in DMD. (C) 2016 Elsevier Inc. All rights reserved.

Objective. Evaluation of circulating tumor markers in ovarian cancer is crucial for optimal patient care. The goal of this study was to verify the most accurate circulating tumor markers for the diagnosis and follow-up of adult-type granulosa cell tumors (AGCT5). Methods. The levels of circulating human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125), together with AGCT markers inhibin B and anti-Mtillerian hormone (AMH), were measured in 135 samples from AGCT patients, 37 epithelial ovarian carcinoma (EOC) patients, and 40 endometrioma (ENDO) patients. The levels were plotted with receiver operating characteristic (ROC) graphs, and the area under the curves (AUC) of the different markers were calculated and compared. Results. HE4 levels were significantly lower in AGCT5 than in EOCs (p

Doxorubicin is a widely used and effective chemotherapy drug. However, cardiac and skeletal muscle toxicity of doxorubicin limits its use. Inhibiting myostatin/activin signalling can prevent muscle atrophy, but its effects in chemotherapy-induced muscle wasting are unknown. In the present study we investigated the effects of doxorubicin administration alone or combined with activin receptor ligand pathway blockade by soluble activin receptor IIB (sACVR2B-Fc). Doxorubicin administration decreased body mass, muscle size and bone mineral density/content in mice. However, these effects were prevented by sACVR2B-Fc administration. Unlike in many other wasting situations, doxorubicin induced muscle atrophy without markedly increasing typical atrogenes or protein degradation pathways. Instead, doxorubicin decreased muscle protein synthesis which was completely restored by sACVR2B-Fc. Doxorubicin administration also resulted in impaired running performance without effects on skeletal muscle mitochondrial capacity/function or capillary density. Running performance and mitochondrial function were unaltered by sACVR2B-Fc administration. Tumour experiment using Lewis lung carcinoma cells demonstrated that sACVR2B-Fc decreased the cachectic effects of chemotherapy without affecting tumour growth. These results demonstrate that blocking ACVR2B signalling may be a promising strategy to counteract chemotherapy-induced muscle wasting without damage to skeletal muscle oxidative capacity or cancer treatment.

Objective: Circulating ANGPTL8 has recently been used as a marker of insulin action. We studied expression and insulin regulation of ANGPTL8 and ANGPTL3 in vivo and in vitro. Design and Methods: Expression of ANGPTL8 and ANGPTL3 was studied in 34 paired samples of human liver and adipose tissue. Effects of insulin on 1) plasma concentrations and adipose tissue expression of ANGPTL8 and ANGPTL3 (in vivo 6-h euglycemic hyperinsulinemia; n = 18), and 2) ANGPTL8 and ANGPTL3 gene and protein expression in immortalized human hepatocytes (IHH) and adipocytes were measured. Effect of ANGPTL3 on secretion of ANGPTL8 in cells stably over-expressing ANGPTL3, -8, or both was determined. Results: ANGPTL3 was only expressed in the liver, whereas ANGPTL8 was expressed in both tissues. In vivo hyperinsulinemia significantly decreased both plasma ANGPTL8 and ANGPTL3 at 3 and 6 hours. Insulin increased ANGPTL8 expression in human adipose tissue 14- and 18-fold at 3 and 6 hours and ANGPTL8 was the most insulin-responsive transcript on microarray. Insulin also increased ANPGTL8 in cultured adipocytes and IHH but the protein mainly remained intracellular. In vitro in IHH, insulin decreased ANGPTL3 gene expression and secretion of ANGPTL3 into growth medium. Overexpression of ANGPTL8 in CHO cells did not result in its release into culture medium while abundant secretion occurred in cells co-expressing ANGPTL3 and -8. Conclusions: Insulin decreases plasma ANGPTL3 by decreasing ANGPTL3 expression in the liver. Insulin markedly increases ANGPTL8 in adipose tissue and the liver but not in plasma. These data show that measurement of plasma ANGPTL3 but not -8 reflects insulin action in target tissues.

Background: Activins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation. Methods: We used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis. Results: Activin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed. Conclusions: The upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF. peerReviewed