A novel estrogen receptor 1: sphingomyelin phosphodiesterase acid-like 3B pathway mediates rituximab response in myositis patients.

OBJECTIVES: The B-cell depleting biologic, rituximab, is used to treat refractory autoimmune myositis. However, the beneficial effects of rituximab appear to outweigh the known contribution of B cells in myositis. We aimed to elucidate how myositis patients respond differently to rituximab and possible alternative mechanisms of action. METHODS: Here we have: (i) comprehensively investigated concurrent mRNA and microRNA expression in muscle biopsies taken at baseline and 16 weeks post treatment in 10 patients who were part of the rituximab in myositis (RIM) trial; and (ii) investigated the beneficial effect of rituximab on myositis muscle cells. RESULTS: Our analyses identified an increased number of changes in gene expression in biopsies from patients who had a clinical response to rituximab (n = 5) compared with non-responders (n = 5). The two groups had completely different changes in microRNA and mRNA expression following rituximab therapy, with the exception of one mRNA, BHMT2. Networks of mRNA and microRNA with opposite direction of expression changes highlighted ESR1 as upregulated in responders. We confirmed ESR1 upregulation upon rituximab treatment of immortalized myotubes and primary human dermatomyositis muscle cells in vitro, demonstrating a direct effect of rituximab on muscle cells. Notably, despite showing a response to rituximab, human dermatomyositis primary muscle cells did not express the rituximab target, CD20. However, these cells expressed a possible alternative target of rituximab, sphingomyelinase-like phosphodiesterase 3 b (SMPDL3B). CONCLUSION: In addition to B-cell depletion, rituximab may be beneficial in myositis due to increased ESR1 signalling mediated by rituximab binding to SMPDL3B on skeletal muscle cells.

Acute serum protein and cytokine response of single dose of prednisone in adult volunteers.

Pharmacological glucocorticoids are the most prescribed anti-inflammatory medications, and are chemical variants of cortisol, the circadian and stress hormone. Both endogenous and pharmacological glucocorticoids bind the glucocorticoid receptor (NR3C1) with high affinity, and both then bind downstream gene promoter elements (GRE) to drive positive gene transcription of many proteins. Glucocorticoid/GR complexes also bind distinct negative gene promoter elements (nGRE) to inhibit expression of genes involved in NF-κB innate immunity signaling. We sought to define the acute response of a single dose of prednisone (0.2 mg/kg) in young adult volunteers, with blood samples taken at baseline, 2, 3, 4 and 6 h post-oral dose. To control for circadian morning cortisol hitting the same molecular pathways, a day of blood draws was done without oral prednisone (same time of day), one day prior to drug day. Serum samples were processed for steroid hormone profiles (mass spectrometry; 9 steroidal hormones), proteomics (SOMAscan aptamer panels, 1,305 proteins), and inflammatory markers (Meso Scale Discovery; 10 pro-inflammatory cytokines). The pharmacological effect of the prednisone dose was shown by significant declines of adrenal steroids by 3 h after dosing. IL-10 showed drug-related increase to 4 hrs, then decrease to 6 hrs. IL-8 showed drug-related decrease in serum by 4 h, consistent with direct negative action of GR/ligand on IL-8 gene promoter. Proteomics data showed beta-2 microglobulin, TNFSF15, TSH, CST3, NBL1 to show time-related decreases with prednisone, while CXCL13 showed increases, although these require validation. In summary, a single low dose of prednisone leads to broad suppression of the adrenal axis within 3 h, and down-regulation of inflammatory serum proteins by 6 h.

A Dystrophin Exon-52 Deleted Miniature Pig Model of Duchenne Muscular Dystrophy and Evaluation of Exon Skipping.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the DMD gene and the subsequent lack of dystrophin protein. Recently, phosphorodiamidate morpholino oligomer (PMO)-antisense oligonucleotides (ASOs) targeting exon 51 or 53 to reestablish the DMD reading frame have received regulatory approval as commercially available drugs. However, their applicability and efficacy remain limited to particular patients. Large animal models and exon skipping evaluation are essential to facilitate ASO development together with a deeper understanding of dystrophinopathies. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, we generated a Yucatan miniature pig model of DMD with an exon 52 deletion mutation equivalent to one of the most common mutations seen in patients. Exon 52-deleted mRNA expression and dystrophin deficiency were confirmed in the skeletal and cardiac muscles of DMD pigs. Accordingly, dystrophin-associated proteins failed to be recruited to the sarcolemma. The DMD pigs manifested early disease onset with severe bodywide skeletal muscle degeneration and with poor growth accompanied by a physical abnormality, but with no obvious cardiac phenotype. We also demonstrated that in primary DMD pig skeletal muscle cells, the genetically engineered exon-52 deleted pig DMD gene enables the evaluation of exon 51 or 53 skipping with PMO and its advanced technology, peptide-conjugated PMO. The results show that the DMD pigs developed here can be an appropriate large animal model for evaluating in vivo exon skipping efficacy.

Human muscle stem cells are refractory to aging.

Age-related loss of muscle mass and strength is widely attributed to limitation in the capacity of muscle resident satellite cells to perform their myogenic function. This idea contains two notions that have not been comprehensively evaluated by experiment. First, it entails the idea that we damage and lose substantial amounts of muscle in the course of our normal daily activities. Second, it suggests that mechanisms of muscle repair are in some way exhausted, thus limiting muscle regeneration. A third potential option is that the aged environment becomes inimical to the conduct of muscle regeneration. In the present study, we used our established model of human muscle xenografting to test whether muscle samples taken from cadavers, of a range of ages, maintained their myogenic potential after being transplanted into immunodeficient mice. We find no measurable difference in regeneration across the range of ages investigated up to 78 years of age. Moreover, we report that satellite cells maintained their myogenic capacity even when muscles were grafted 11 days postmortem in our model. We conclude that the loss of muscle mass with increasing age is not attributable to any intrinsic loss of myogenicity and is most likely a reflection of progressive and detrimental changes in the muscle microenvironment such as to disfavor the myogenic function of these cells.

Genetic modifiers of respiratory function in Duchenne muscular dystrophy.

OBJECTIVE: Respiratory insufficiency is a major complication of Duchenne muscular dystrophy (DMD). Its progression shows considerable interindividual variability, which has been less thoroughly characterized and understood than in skeletal muscle. We collected pulmonary function testing (PFT) data from a large retrospective cohort followed at Centers collaborating in the Italian DMD Network. Furthermore, we analyzed PFT associations with different DMD mutation types, and with genetic variants in SPP1, LTBP4, CD40, and ACTN3, known to modify skeletal muscle weakness in DMD. Genetic association findings were independently validated in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG-DNHS). METHODS AND RESULTS: Generalized estimating equation analysis of 1852 PFTs from 327 Italian DMD patients, over an average follow-up time of 4.5 years, estimated that forced vital capacity (FVC) declined yearly by -4.2%, forced expiratory volume in 1 sec by -5.0%, and peak expiratory flow (PEF) by -2.9%. Glucocorticoid (GC) treatment was associated with higher values of all PFT measures (approximately + 15% across disease stages). Mutations situated 3' of DMD intron 44, thus predicted to alter the expression of short dystrophin isoforms, were associated with lower (approximately -6%) PFT values, a finding independently validated in the CINRG-DNHS. Deletions amenable to skipping of exon 51 and 53 were independently associated with worse PFT outcomes. A meta-analysis of the two cohorts identified detrimental effects of SPP1 rs28357094 and CD40 rs1883832 minor alleles on both FVC and PEF. INTERPRETATION: These findings support GC efficacy in delaying respiratory insufficiency, and will be useful for the design and interpretation of clinical trials focused on respiratory endpoints in DMD.

MicroRNA Profiling in Adipose Before and After Weight Loss Highlights the Role of miR-223-3p and the NLRP3 Inflammasome.

OBJECTIVE: Adipose tissue plays a key role in obesity-related metabolic dysfunction. MicroRNA (miRNA) are gene regulatory molecules involved in intercellular and inter-organ communication. It was hypothesized that miRNA levels in adipose tissue would change after gastric bypass surgery and that this would provide insights into their role in obesity-induced metabolic dysregulation. METHODS: miRNA profiling (Affymetrix GeneChip miRNA 2.0 Array) of omental and subcutaneous adipose (n = 15 females) before and after gastric bypass surgery was performed. RESULTS: One omental and thirteen subcutaneous adipose miRNAs were significantly differentially expressed after gastric bypass, including downregulation of miR-223-3p and its antisense relative miR-223-5p in both adipose tissues. mRNA levels of miR-223-3p targets NLRP3 and GLUT4 were decreased and increased, respectively, following gastric bypass in both adipose tissues. Significantly more NLRP3 protein was observed in omental adipose after gastric bypass (P = 0.02). Significant hypomethlyation of NLRP3 and hypermethylation of miR-223 were observed in both adipose tissues after gastric bypass. In subcutaneous adipose, significant correlations were observed between both miR-223-3p and miR-223-5p and glucose and between NLRP3 mRNA and protein levels and blood lipids. CONCLUSIONS: This is the first report detailing genome-wide miRNA profiling of omental adipose before and after gastric bypass, and it further highlights the association of miR-223-3p and the NLRP3 inflammasome with obesity.

Muscle Weakness in Myositis: MicroRNA-Mediated Dystrophin Reduction in a Myositis Mouse Model and Human Muscle Biopsies.

OBJECTIVE: Muscle inflammation is a feature in myositis and Duchenne muscular dystrophy (DMD). Autoimmune mechanisms are thought to contribute to muscle weakness in patients with myositis. However, a lack of correlation between the extent of inflammatory cell infiltration and muscle weakness indicates that nonimmune pathologic mechanisms may play a role. The present study focused on 2 microRNA (miRNA) sets previously identified as being elevated in the muscle of patients with DMD-an "inflammatory" miRNA set that is dampened with glucocorticoids, and a "dystrophin-targeting" miRNA set that inhibits dystrophin translation-to test the hypothesis that these miRNAs are similarly dysregulated in the muscle of patients with myositis, and could contribute to muscle weakness and disease severity. METHODS: A major histocompatibility complex class I-transgenic mouse model of myositis was utilized to study gene and miRNA expression and histologic features in the muscle tissue, with the findings validated in human muscle biopsy tissue from 6 patients with myositis. Mice were classified as having mild or severe myositis based on transgene expression, body weight, histologic disease severity, and muscle strength/weakness. RESULTS: In mice with severe myositis, muscle tissue showed mononuclear cell infiltration along with elevated expression of type I interferon and NF-κB-regulated genes, including Tlr7 (3.8-fold increase, P < 0.05). Furthermore, mice with severe myositis showed elevated expression of inflammatory miRNAs (miR-146a, miR-142-3p, miR-142-5p, miR-455-3p, and miR-455-5p; ~3-40-fold increase, P < 0.05) and dystrophin-targeting miRNAs (miR-146a, miR-146b, miR-31, and miR-223; ~3-38-fold increase, P < 0.05). Bioinformatics analyses of chromatin immunoprecipitation sequencing (ChIP-seq) data identified at least one NF-κB consensus element within the promoter/enhancer regions of these miRNAs. Western blotting and immunofluorescence analyses of the muscle tissue from mice with severe myositis demonstrated reduced levels of dystrophin. In addition, elevated levels of NF-κB-regulated genes, TLR7, and miRNAs along with reduced dystrophin levels were observed in muscle biopsy tissue from patients with histologically severe myositis. CONCLUSION: These data demonstrate that an acquired dystrophin deficiency may occur through NF-κB-regulated miRNAs in myositis, thereby suggesting a unifying theme in which muscle injury, inflammation, and weakness are perpetuated both in myositis and in DMD.

Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM).

The need for a reliable and accurate method to quantify dystrophin proteins in human skeletal muscle biopsies has become crucial in order to assess the efficacy of dystrophin replacement therapies in Duchenne muscular dystrophy as well as to gain insight into the relationship between dystrophin levels and disease severity in Becker's muscular dystrophy. Current methods to measure dystrophin such as western blot and immunofluorescence, while straightforward and simple, lack precision and sometimes specificity. Here, we standardized a targeted mass spectrometry method to determine the absolute amount of dystrophin in ng/mg of muscle using full-length 13 C6-Arg- and 13 C6,15 N2-Lys-labeled dystrophin and parallel reaction monitoring (PRM). The method was found to be reproducible with a limit of quantification as low as 30 pg of dystrophin protein per mg of total muscle proteins. The method was then tested to measure levels of dystrophin in muscle biopsies from a healthy donor and from Duchenne and Becker's muscular dystrophy patients.

Orthogonal analysis of dystrophin protein and mRNA as a surrogate outcome for drug development.

Aim: Detection of drug-induced dystrophin in patient muscle biopsy is a surrogate outcome measure for Duchenne muscular dystrophy. We sought to establish and validate an orthogonal approach to measurement of dystrophin protein and RNA in muscle biopsies. Materials & methods: Validated methods were developed for dystrophin western blotting, mass spectrometry, immunostaining and reverse transcriptase PCR of biopsy mRNA using muscle biopsy standards. Results: Both western blotting and mass spectrometry validated methods demonstrated good linearity, and acceptable precision and accuracy with a lower limit of quantitation at 1%. Immunostaining and reverse transcriptase PCR methods were shown to be reliable. Conclusion: The described orthogonal approach is sufficient to support measures of dystrophin as a surrogate outcome in a clinical trial.

Asymmetric independence modeling identifies novel gene-environment interactions.

Most genetic or environmental factors work together in determining complex disease risk. Detecting gene-environment interactions may allow us to elucidate novel and targetable molecular mechanisms on how environmental exposures modify genetic effects. Unfortunately, standard logistic regression (LR) assumes a convenient mathematical structure for the null hypothesis that however results in both poor detection power and type 1 error, and is also susceptible to missing factor, imperfect surrogate, and disease heterogeneity confounding effects. Here we describe a new baseline framework, the asymmetric independence model (AIM) in case-control studies, and provide mathematical proofs and simulation studies verifying its validity across a wide range of conditions. We show that AIM mathematically preserves the asymmetric nature of maintaining health versus acquiring a disease, unlike LR, and thus is more powerful and robust to detect synergistic interactions. We present examples from four clinically discrete domains where AIM identified interactions that were previously either inconsistent or recognized with less statistical certainty.

Vamorolone targets dual nuclear receptors to treat inflammation and dystrophic cardiomyopathy.

Cardiomyopathy is a leading cause of death for Duchenne muscular dystrophy. Here, we find that the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) can share common ligands but play distinct roles in dystrophic heart and skeletal muscle pathophysiology. Comparisons of their ligand structures indicate that the Δ9,11 modification of the first-in-class drug vamorolone enables it to avoid interaction with a conserved receptor residue (N770/N564), which would otherwise activate transcription factor properties of both receptors. Reporter assays show that vamorolone and eplerenone are MR antagonists, whereas prednisolone is an MR agonist. Macrophages, cardiomyocytes, and CRISPR knockout myoblasts show vamorolone is also a dissociative GR ligand that inhibits inflammation with improved safety over prednisone and GR-specific deflazacort. In mice, hyperaldosteronism activates MR-driven hypertension and kidney phenotypes. We find that genetic dystrophin loss provides a second hit for MR-mediated cardiomyopathy in Duchenne muscular dystrophy model mice, as aldosterone worsens fibrosis, mass and dysfunction phenotypes. Vamorolone successfully prevents MR-activated phenotypes, whereas prednisolone activates negative MR and GR effects. In conclusion, vamorolone targets dual nuclear receptors to treat inflammation and cardiomyopathy with improved safety.

PCB exposure and potential future cancer incidence in Slovak children: an assessment from molecular finger printing by Ingenuity Pathway Analysis (IPA®) derived from experimental and epidemiological investigations.

The risk of cancer due to PCB exposure in humans is highly debated. In eastern Slovakia, high exposure of the population to organochlorines (especially PCBs) was associated with various disease and disorder pathways, viz., endocrine disruption, metabolic disorder & diabetes, and cancer, thereby disturbing several cellular processes, including protein synthesis, stress response, and apoptosis. We have evaluated a Slovak cohort (45-month children, at lower and higher levels of PCB exposure from the environment) for disease and disorder development to develop early disease cancer biomarkers that could shed new light on possible mechanisms for the genesis of cancers under such chemical exposures, and identify potential avenues for prevention.Microarray studies of global gene expression were conducted from the 45-month-old children on the Affymetrix platform followed by Ingenuity Pathway Analysis (IPA®) to associate the affected genes with their mechanistic pathways. High-throughput qRT-PCR TaqMan low-density array (TLDA) was performed to further validate the selected genes on the whole blood cells of the most highly exposed children from the study cohort (n = 71). TP53, MYC, BCL2, and LRP12 differential gene expressions suggested strong relationships between potential future tumor promotion and PCB exposure in Slovak children. The IPA analysis further detected the most important signaling pathways, including molecular mechanism of cancers, prostate cancer signaling, ovarian cancer signaling, P53 signaling, oncostatin M signaling, and their respective functions (viz., prostate cancer, breast cancer, progression of tumor, growth of tumor, and non-Hodgkin's disease). The results suggest that PCB exposures, even at the early age of these children, may have lifelong consequences for the future development of chronic diseases.

Expression of macrophage genes within skeletal muscle correlates inversely with adiposity and insulin resistance in humans.

Local inflammation in obese adipose tissue has been shown to contribute to insulin resistance; however, the role of macrophage infiltration within skeletal muscle is still debatable. This study aimed to evaluate the association of skeletal muscle macrophage gene expression with adiposity levels and insulin sensitivity in obese patients. Twenty-two nondiabetic obese patients and 23 healthy lean controls were included. Obese patients underwent a 3-month weight loss intervention. Macrophage gene expression in skeletal muscle (quantitative real-time polymerase chain reaction), body composition (dual-energy X-ray absorptiometry), and insulin sensitivity (homeostatic model assessment (HOMA) and oral glucose tolerance test) were compared between groups and their associations were analyzed. To validate skeletal muscle findings, we repeated the analyses with macrophage gene expression in adipose tissue. Expression levels of macrophage genes (CD68, CD11b, CD206, CD16, CD40, and CD163) were lower in skeletal muscle tissue of obese versus lean participants. Macrophage gene expression was also found to be inversely associated with adiposity, fasting insulin, and HOMA (r = -0.4 ∼ -0.6, p < 0.05), as well as positively associated with insulin sensitivity (r = 0.4 ∼ 0.8, p < 0.05). On the other hand, adipose tissue macrophage gene expression showed higher levels in obese versus lean participants, presenting a positive association with adiposity levels. Macrophage gene expression, in both skeletal and adipose tissue samples, was only minimally affected by the weight loss intervention. In contrast with the established positive relationship between adiposity and macrophage gene expression, an unexpected inverse correlation between these 2 variables was observed in skeletal muscle tissue. Additionally, muscle macrophage gene expression was inversely correlated with insulin resistance.

Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle.

Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.Exon skipping is a strategy for the treatment of Duchenne muscular dystrophy, but has variable efficacy. Here, the authors show that dystrophin restoration occurs preferentially in areas of myofiber regeneration, where antisense oligonucleotides are stored in macrophages and delivered to myoblasts and newly formed myotubes.

Osteopontin is linked with AKT, FoxO1, and myostatin in skeletal muscle cells.

INTRODUCTION: Osteopontin (OPN) polymorphisms are associated with muscle size and modify disease progression in Duchenne muscular dystrophy (DMD). We hypothesized that OPN may share a molecular network with myostatin (MSTN). METHODS: Studies were conducted in the golden retriever (GRMD) and mdx mouse models of DMD. Follow-up in-vitro studies were employed in myogenic cells and the mdx mouse treated with recombinant mouse (rm) or human (Hu) OPN protein. RESULTS: OPN was increased and MSTN was decreased and levels correlated inversely in GRMD hypertrophied muscle. RM-OPN treatment led to induced AKT1 and FoxO1 phosphorylation, microRNA-486 modulation, and decreased MSTN. An AKT1 inhibitor blocked these effects, whereas an RGD-mutant OPN protein and an RGDS blocking peptide showed similar effects to the AKT inhibitor. RMOPN induced myotube hypertrophy and minimal Feret diameter in mdx muscle. DISCUSSION: OPN may interact with AKT1/MSTN/FoxO1 to modify normal and dystrophic muscle. Muscle Nerve 56: 1119-1127, 2017.

African-American esophageal squamous cell carcinoma expression profile reveals dysregulation of stress response and detox networks.

BACKGROUND: Esophageal carcinoma is the third most common gastrointestinal malignancy worldwide and is largely unresponsive to therapy. African-Americans have an increased risk for esophageal squamous cell carcinoma (ESCC), the subtype that shows marked variation in geographic frequency. The molecular architecture of African-American ESCC is still poorly understood. It is unclear why African-American ESCC is more aggressive and the survival rate in these patients is worse than those of other ethnic groups. METHODS: To begin to define genetic alterations that occur in African-American ESCC we conducted microarray expression profiling in pairs of esophageal squamous cell tumors and matched control tissues. RESULTS: We found significant dysregulation of genes encoding drug-metabolizing enzymes and stress response components of the NRF2- mediated oxidative damage pathway, potentially representing key genes in African-American esophageal squamous carcinogenesis. Loss of activity of drug metabolizing enzymes would confer increased sensitivity of esophageal cells to xenobiotics, such as alcohol and tobacco smoke, and may account for the high incidence and aggressiveness of ESCC in this ethnic group. To determine whether certain genes are uniquely altered in African-American ESCC we performed a meta-analysis of ESCC expression profiles in our African-American samples and those of several Asian samples. Down-regulation of TP53 pathway components represented the most common feature in ESCC of all ethnic groups. Importantly, this analysis revealed a potential distinctive molecular underpinning of African-American ESCC, that is, a widespread and prominent involvement of the NRF2 pathway. CONCLUSION: Taken together, these findings highlight the remarkable interplay of genetic and environmental factors in the pathogenesis of African-American ESCC.

Genetic characterization of physical activity behaviours in university students enrolled in kinesiology degree programs.

Studies of physical activity behaviours have increasingly shown the importance of heritable factors such as genetic variation. Nonsynonymous polymorphisms of alpha-actinin 3 (ACTN3) and the ß-adrenergic receptors 1 and 3 (ADRB1 and ADRB3) have been previously associated with exercise capacity and cardiometabolic health. We thus hypothesized that these polymorphisms are also related to physical activity behaviours in young adults. To test this hypothesis we examined relationships between ACTN3 (R577X), ARDB1 (Arg389Gly), ADRB3 (Trp64Arg), and physical activity behaviours in university students. We stratified for student enrollment in kinesiology degree programs compared with nonmajors as we previously found this to be a predictor of physical activity. We did not identify novel associations between physical activity and ACTN3. However, the minor alleles of ADRB1 and ADRB3 were significantly underrepresented in kinesiology students compared with nonmajors. Furthermore, carriers of the ADRB1 minor allele reported reduced participation in moderate physical activity and increased afternoon fatigue compared with ancestral allele homozygotes. Together, these findings suggest that the heritability of physical activity behaviours in young adults may be linked to nonsynonymous polymorphisms within ß-adrenergic receptors.

Association Study of Exon Variants in the NF-κB and TGFß Pathways Identifies CD40 as a Modifier of Duchenne Muscular Dystrophy.

The expressivity of Mendelian diseases can be influenced by factors independent from the pathogenic mutation: in Duchenne muscular dystrophy (DMD), for instance, age at loss of ambulation (LoA) varies between individuals whose DMD mutations all abolish dystrophin expression. This suggests the existence of trans-acting variants in modifier genes. Common single nucleotide polymorphisms (SNPs) in candidate genes (SPP1, encoding osteopontin, and LTBP4, encoding latent transforming growth factor ß [TGFß]-binding protein 4) have been established as DMD modifiers. We performed a genome-wide association study of age at LoA in a sub-cohort of European or European American ancestry (n = 109) from the Cooperative International Research Group Duchenne Natural History Study (CINRG-DNHS). We focused on protein-altering variants (Exome Chip) and included glucocorticoid treatment as a covariate. As expected, due to the small population size, no SNPs displayed an exome-wide significant p value (< 1.8 × 10-6). Subsequently, we prioritized 438 SNPs in the vicinities of 384 genes implicated in DMD-related pathways, i.e., the nuclear-factor-κB and TGFß pathways. The minor allele at rs1883832, in the 5'-untranslated region of CD40, was associated with earlier LoA (p = 3.5 × 10-5). This allele diminishes the expression of CD40, a co-stimulatory molecule for T cell polarization. We validated this association in multiple independent DMD cohorts (United Dystrophinopathy Project, Bio-NMD, and Padova, total n = 660), establishing this locus as a DMD modifier. This finding points to cell-mediated immunity as a relevant pathogenetic mechanism and potential therapeutic target in DMD.

Identification of Pathway-Specific Serum Biomarkers of Response to Glucocorticoid and Infliximab Treatment in Children with Inflammatory Bowel Disease.

OBJECTIVE: Serum biomarkers may serve to predict early response to therapy, identify relapse, and facilitate drug development in inflammatory bowel disease (IBD). Biomarkers are particularly important in children, in whom achieving early remission and minimizing procedures are especially beneficial. METHODS: We profiled protein and micro RNA (miRNA) in serum from patients pre- and post-therapy, to identify molecular markers of pharmacodynamic effect. Serum was obtained from children with IBD before and after treatment with either corticosteroids (prednisone; n=12) or anti-tumor necrosis factor-α biologic (infliximab; n=7). Over 1,100 serum proteins were assayed using aptamer-based SOMAscan proteomics, and 22 miRNAs analyzed by quantitative real time PCR. Concordance of longitudinal changes between the groups was used to identify markers responsive to treatment. Bioinformatic analysis was used to build insight into mechanisms of changes in response to treatment. RESULTS: We identified 18 proteins and three miRNAs responsive to both prednisone and infliximab. Eight markers that decreased are associated with inflammation and have gene promoters regulated by nuclear factor (NF)-κB. Several that increased are associated with resolving inflammation and tissue damage. We also identified six markers that appear to be steroid-specific, three of which have glucocorticoid receptor binding elements in their promoter region. CONCLUSIONS: Serum markers regulated by the inflammatory transcription factor NF-κB are potential candidates for pharmacodynamic biomarkers that, if correlated with later outcomes like endoscopic or histologic healing, could be used to monitor treatment, optimize dosing, and enhance drug development. The pharmacodynamic biomarkers identified here hold potential to improve both clinical care and drug development. Further studies are warranted to investigate these markers as early predictors of response, or possibly surrogate outcomes.

OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages.

NEW FINDINGS: What is the central question of this study? What is the functional relevance of OPN isoform expression in muscle pathology? What is the main finding and its importance? The full-length human OPN-a isoform is the most pro-inflammatory isoform in the muscle microenvironment, acting on macrophages and myoblasts in an RGD-integrin-dependent manner. OPN-a upregulates expression of tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Blocking TLR4 signalling inhibits the pro-inflammatory effects of OPN-a, suggesting that a potential mechanism of OPN action is by promoting TNC-TLR4 signalling. Although osteopontin (OPN) is an important mediator of muscle remodelling in health and disease, functional differences in human spliced OPN variants in the muscle microenvironment have not been characterized. We thus sought to define the pro-inflammatory activities of human OPN isoforms (OPN-a, OPN-b and OPN-c) on cells present in regenerating muscle. OPN transcripts were quantified in normal and dystrophic human and dog muscle. Human macrophages and myoblasts were stimulated with recombinant human OPN protein isoforms, and cytokine mRNA and protein induction was assayed. OPN isoforms were greatly increased in dystrophic human (OPN-a > OPN-b > OPN-c) and dog muscle (OPN-a = OPN-c). In healthy human muscle, mechanical loading also upregulated OPN-a expression (eightfold; P < 0.01), but did not significantly upregulate OPN-c expression (twofold; P > 0.05). In vitro, OPN-a displayed the most pronounced pro-inflammatory activity among isoforms, acting on both macrophages and myoblasts. In vitro and in vivo data revealed that OPN-a upregulated tenascin-C (TNC), a known Toll-like receptor 4 (TLR4) agonist. Inhibition of TLR4 signalling attenuated OPN-mediated macrophage cytokine production. In summary, OPN-a is the most abundant and functionally active human spliced isoform in the skeletal muscle microenvironment. Here, OPN-a promotes pro-inflammatory signalling in both macrophages and myoblasts, possibly through induction of TNC-TLR4 signalling. Together, our findings suggest that specific targeting of OPN-a and/or TNC signalling in the damaged muscle microenvironment may be of therapeutic relevance.