Benfotiamine improves dystrophic pathology and exercise capacity in mdx mice by reducing inflammation and fibrosis.

Duchenne Muscular Dystrophy (DMD) is a progressive and fatal neuromuscular disease. Cycles of myofibre degeneration and regeneration are hallmarks of the disease where immune cells infiltrate to repair damaged skeletal muscle. Benfotiamine is a lipid soluble precursor to thiamine, shown clinically to reduce inflammation in diabetic related complications. We assessed whether benfotiamine administration could reduce inflammation related dystrophic pathology. Benfotiamine (10 mg/kg/day) was fed to male mdx mice (n = 7) for 15 weeks from 4 weeks of age. Treated mice had an increased growth weight (5-7 weeks) and myofibre size at treatment completion. Markers of dystrophic pathology (area of damaged necrotic tissue, central nuclei) were reduced in benfotiamine mdx quadriceps. Grip strength was increased and improved exercise capacity was found in mdx treated with benfotiamine for 12 weeks, before being placed into individual cages and allowed access to an exercise wheel for 3 weeks. Global gene expression profiling (RNAseq) in the gastrocnemius revealed benfotiamine regulated signalling pathways relevant to dystrophic pathology (Inflammatory Response, Myogenesis) and fibrotic gene markers (Col1a1, Col1a2, Col4a5, Col5a2, Col6a2, Col6a2, Col6a3, Lum) towards wildtype levels. In addition, we observed a reduction in gene expression of inflammatory gene markers in the quadriceps (Emr1, Cd163, Cd4, Cd8, Ifng). Overall, these data suggest that benfotiamine reduces dystrophic pathology by acting on inflammatory and fibrotic gene markers and signalling pathways. Given benfotiamine's excellent safety profile and current clinical use, it could be used in combination with glucocorticoids to treat DMD patients.

A phase 2 open-label study of the safety and efficacy of weekly dosing of ATL1102 in patients with non-ambulatory Duchenne muscular dystrophy and pharmacology in mdx mice.

BACKGROUND: ATL1102 is a 2'MOE gapmer antisense oligonucleotide to the CD49d alpha subunit of VLA-4, inhibiting expression of CD49d on lymphocytes, reducing survival, activation and migration to sites of inflammation. Children with DMD have dystrophin deficient muscles susceptible to contraction induced injury, which triggers the immune system, exacerbating muscle damage. CD49d is a biomarker of disease severity in DMD, with increased numbers of high CD49d expressing T cells correlating with more severe and progressive weakess, despite corticosteroid treatment. METHODS: This Phase 2 open label study assessed the safety, efficacy and pharmacokinetic profile of ATL1102 administered as 25 mg weekly by subcutaneous injection for 24 weeks in 9 non-ambulatory boys with DMD aged 10-18 years. The main objective was to assess safety and tolerability of ATL1102. Secondary objectives included the effect of ATL1102 on lymphocyte numbers in the blood, functional changes in upper limb function as assessed by Performance of Upper Limb test (PUL 2.0) and upper limb strength using MyoGrip and MyoPinch compared to baseline. RESULTS: Eight out of nine participants were on a stable dose of corticosteroids. ATL1102 was generally safe and well tolerated. No serious adverse events were reported. There were no participant withdrawals from the study. The most commonly reported adverse events were injection site erythema and skin discoloration. There was no statistically significant change in lymphocyte count from baseline to week 8, 12 or 24 of dosing however, the CD3+CD49d+ T lymphocytes were statistically significantly higher at week 28 compared to week 24, four weeks past the last dose (mean change 0.40x109/L 95%CI 0.05, 0.74; p = 0.030). Functional muscle strength, as measured by the PUL2.0, EK2 and Myoset grip and pinch measures, and MRI fat fraction of the forearm muscles were stable throughout the trial period. CONCLUSION: ATL1102, a novel antisense drug being developed for the treatment of inflammation that exacerbates muscle fibre damage in DMD, appears to be safe and well tolerated in non-ambulant boys with DMD. The apparent stabilisation observed on multiple muscle disease progression parameters assessed over the study duration support the continued development of ATL1102 for the treatment of DMD. TRIAL REGISTRATION: Clinical Trial Registration. Australian New Zealand Clinical Trials Registry Number: ACTRN12618000970246.

Generation of a human ACTA1-tdTomato reporter iPSC line using CRISPR/Cas9 editing.

We used gene editing to introduce DNA sequences encoding the tdTomato fluorescent protein into the α -skeletal actin 1 (ACTA1) locus to develop an ACTA1-tdTomato induced pluripotent stem cell reporter line for monitoring differentiation of skeletal muscle. This cell line will be used to better understand skeletal muscle maturation and development in vitro as well as provide a useful tool for drug screening and the evaluation of novel therapeutics for the treatment of skeletal muscle disease.

A Spotlight on T Lymphocytes in Duchenne Muscular Dystrophy-Not Just a Muscle Defect.

The lack of dystrophin in Duchenne muscular dystrophy (DMD) results in membrane fragility resulting in contraction-induced muscle damage and subsequent inflammation. The impact of inflammation is profound, resulting in fibrosis of skeletal muscle, the diaphragm and heart, which contributes to muscle weakness, reduced quality of life and premature death. To date, the innate immune system has been the major focus in individuals with DMD, and our understanding of the adaptive immune system, specifically T cells, is limited. Targeting the immune system has been the focus of multiple clinical trials for DMD and is considered a vital step in the development of better treatments. However, we must first have a complete picture of the involvement of the immune systems in dystrophic muscle disease to better understand how inflammation influences disease progression and severity. This review focuses on the role of T cells in DMD, highlighting the importance of looking beyond skeletal muscle when considering how the loss of dystrophin impacts disease progression. Finally, we propose that targeting T cells is a potential novel therapeutic in the treatment of DMD.

Generating an iPSC line (with isogenic control) from the PBMCs of an ACTA1 (p.Gly148Asp) nemaline myopathy patient.

To produce an in vitro model of nemaline myopathy, we reprogrammed the peripheral blood mononuclear cells (PBMCs) of a patient with a heterozygous p.Gly148Asp mutation in exon 3 of the ACTA1 gene to iPSCs. Using CRISPR/Cas9 gene editing we corrected the mutation to generate an isogenic control line. Both the mutant and control show a normal karyotype, express pluripotency markers and could differentiae into the three cell states that represent embryonic germ layers (endoderm, mesoderm and neuroectoderm) and the dermomyotome (precursor of skeletal muscle). When differentiated these cell lines will be used to explore disease mechanisms and evaluate novel therapeutics.

Resveratrol Promotes Hypertrophy in Wildtype Skeletal Muscle and Reduces Muscle Necrosis and Gene Expression of Inflammatory Markers in Mdx Mice.

Duchenne muscular dystrophy (DMD) is a progressive fatal neuromuscular disorder with no cure. Therapies to restore dystrophin deficiency have been approved in some jurisdictions but long-term effectiveness is yet to be established. There is a need to develop alternative strategies to treat DMD. Resveratrol is a nutraceutical with anti-inflammatory properties. Previous studies have shown high doses (100-400 mg/kg bodyweight/day) benefit mdx mice. We treated 4-week-old mdx and wildtype mice with a lower dose of resveratrol (5 mg/kg bodyweight/day) for 15 weeks. Voluntary exercise was used to test if a lower dosage than previously tested could reduce exercise-induced damage where a greater inflammatory infiltrate is present. We found resveratrol promoted skeletal muscle hypertrophy in wildtype mice. In dystrophic muscle, resveratrol reduced exercise-induced muscle necrosis. Gene expression of immune cell markers, CD86 and CD163 were reduced; however, signalling targets associated with resveratrol's mechanism of action including Sirt1 and NF-κB were unchanged. In conclusion, a lower dose of resveratrol compared to the dosage used by other studies reduced necrosis and gene expression of inflammatory cell markers in dystrophic muscle suggesting it as a therapeutic candidate for treating DMD.

Immunohistochemical analysis of laryngeal muscle of horses clinically affected with recurrent laryngeal neuropathy.

BACKGROUND: As myosin heavy chain (MyHC) profile of muscle fibres is heavily influenced by neural input, changes in MyHC expression are expected in horses clinically affected with recurrent laryngeal neuropathy (RLN) yet, this has not been thoroughly investigated. OBJECTIVES: To describe the changes in MyHC and fibre diameter in left cricoarytenoideus dorsalis (L-CAD) muscle of horses with clinical signs of RLN. STUDY DESIGN: Observational cohort study. METHODS: Immunohistochemistry was used to assess the MyHC-based fibre-type proportion, size and grouping in the L-CAD of 10 Thoroughbred horses, five clinically affected with RLN and five unaffected controls based on resting endoscopic examination. The Mann-Whitney U test was used to compare the two groups. RESULTS: Compared to controls (of mean age 3.0 ± 1.7 years) which only expressed type I, IIA and IIX MyHC, the L-CAD of affected horses (of mean age 2.8 ± 0.8 years) had obvious fibre-type grouping, and despite apparent compensatory hypertrophy of a small number of fibres, a decrease in overall fibre diameter (median difference -35.2 µm, 95% CI -47.4 to -7.9, P = .02) and diameter of type IIA fibres (median difference -46.8 µm, 95% CI -52.1 to -5.0, P = .03) was observed. Anti-fast MyHC (MY32) cross-immunoreacted with embryonic-MyHC. Whereas MY32-positive fibres were identified as type IIX in controls, in affected horses these fibres were less than 50 µm diameter with internal nuclei and were MYH3-positive for embryonic myosin indicating depletion of type IIX fibres, yet active regeneration and fibre renewal. MAIN LIMITATIONS: Small sample size that did not include subclinical cases. Fibre size and appearance rather than staining colour were relied upon to differentiate embryonic from type IIX MyHC. CONCLUSIONS: Horses clinically affected with RLN have overall atrophy of fibres, loss of IIX fibres and expression of embryonic myosin indicating regenerative capacity. Despite hypertrophy of some remaining fibres, the overall decline in the bulk of fibres, including those most fatigue-resistant, may be the critical change that results in failure to maintain arytenoid abduction during exercise although direct comparison to subclinical cases is needed to confirm this.

Expression profiling in exercised mdx suggests a role for extracellular proteins in the dystrophic muscle immune response.

Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disorder caused by mutations in the DMD gene that leads to the absence or severe reduction of dystrophin protein in muscle. The mdx mouse, also dystrophin deficient, is the model most widely used to study the pathology and test potential therapies, but the phenotype is milder than human DMD. This limits the magnitude and range of histological damage parameters and molecular changes that can be measured in pre-clinical drug testing. We used 3 weeks of voluntary wheel running to exacerbate the mdx phenotype. In mdx mice, voluntary exercise increased the amount of damaged necrotic tissue and macrophage infiltration. Global gene expression profiling revealed that exercise induced additional and larger gene expression changes in mdx mice and the pathways most impacted by exercise were all related to immune function or cell-extracellular matrix (ECM) interactions. When we compared the matrisome and inflammation genes that were dysregulated in mdx with those commonly differentially expressed in DMD, we found the exercised mdx molecular signature more closely resembled that of DMD. These gene expression changes in the exercised mdx model thus provide more scope to assess the effects of pre-clinical treatments. Our gene profiling comparisons also highlighted upregulation of ECM proteins involved in innate immunity pathways, proteases that can release them, downstream receptors and signaling molecules in exercised mdx and DMD, suggesting that the ECM could be a major source of pro-inflammatory molecules that trigger and maintain the immune response in dystrophic muscle.

Knockdown of a disintegrin A metalloprotease 12 (ADAM12) during adipogenesis reduces cell numbers, delays differentiation, and increases lipid accumulation in 3T3-L1 cells.

Mouse models have shown that a disintegrin A metalloprotease 12 (ADAM12) is implicated during adipogenesis; the molecular pathways are not well understood. Stealth RNA interference was used to knock down ADAM12 in 3T3-L1 cells. Using gene profiling and metabolic enzymatic markers, we have identified signaling pathways ADAM12 impacts upon during proliferation, differentiation, and maturation of adipocytes. ADAM12 reduced cell numbers in proliferating preadipocytes, delayed differentiation of preadipocytes to adipocytes, and increased lipid accumulation in mature adipocytes. The pathway most affected by ADAM12 knockdown was regulation of insulin-like growth factor (IGF) activity by insulin-like growth factor binding proteins (IGFBPs); ADAM12 is known to cleave IGFBP3 and IGFBP5. The IGF/mTOR signaling pathway was down-regulated, supporting a role for ADAM12 in the IGFBP/IGF/mTOR-growth pathway. PPARγ signaling was also down-regulated by ADAM12 knockdown. Gene ontology (GO) analysis revealed that the extracellular matrix was the cellular compartment most impacted. Filtering for matrisome genes, connective tissue growth factor ( Ctgf) was up-regulated. CTGF and IGBP3 can interact with PPARγ to hinder its regulation. Increased expression of these molecules could have influenced PPARγ signaling reducing differentiation and an imbalance of lipids. We believe ADAM12 regulates cell proliferation of preadipocytes through IGFBP/IGF/mTOR signaling and delays differentiation through altered PPAR signaling to cause an imbalance of lipids within mature adipocytes.

Ablation of Grb10 Specifically in Muscle Impacts Muscle Size and Glucose Metabolism in Mice.

Grb10 is an adaptor-type signaling protein most highly expressed in tissues involved in insulin action and glucose metabolism, such as muscle, pancreas, and adipose. Germline deletion of Grb10 in mice creates a phenotype with larger muscles and improved glucose homeostasis. However, it has not been determined whether Grb10 ablation specifically in muscle is sufficient to induce hypermuscularity or affect whole body glucose metabolism. In this study we generated muscle-specific Grb10-deficient mice (Grb10-mKO) by crossing Grb10flox/flox mice with mice expressing Cre recombinase under control of the human α-skeletal actin promoter. One-year-old Grb10-mKO mice had enlarged muscles, with greater cross-sectional area of fibers compared with wild-type (WT) mice. This degree of hypermuscularity did not affect whole body glucose homeostasis under basal conditions. However, hyperinsulinemic/euglycemic clamp studies revealed that Grb10-mKO mice had greater glucose uptake into muscles compared with WT mice. Insulin signaling was increased at the level of phospho-Akt in muscle of Grb10-mKO mice compared with WT mice, consistent with a role of Grb10 as a modulator of proximal insulin receptor signaling. We conclude that ablation of Grb10 in muscle is sufficient to affect muscle size and metabolism, supporting an important role for this protein in growth and metabolic pathways.

Nutraceuticals and Their Potential to Treat Duchenne Muscular Dystrophy: Separating the Credible from the Conjecture.

In recent years, complementary and alternative medicine has become increasingly popular. This trend has not escaped the Duchenne Muscular Dystrophy community with one study showing that 80% of caregivers have provided their Duchenne patients with complementary and alternative medicine in conjunction with their traditional treatments. These statistics are concerning given that many supplements are taken based on purely "anecdotal" evidence. Many nutraceuticals are thought to have anti-inflammatory or anti-oxidant effects. Given that dystrophic pathology is exacerbated by inflammation and oxidative stress these nutraceuticals could have some therapeutic benefit for Duchenne Muscular Dystrophy (DMD). This review gathers and evaluates the peer-reviewed scientific studies that have used nutraceuticals in clinical or pre-clinical trials for DMD and thus separates the credible from the conjecture.

Proliferation rates of bovine primary muscle cells relate to liveweight and carcase weight in cattle.

Muscling in cattle is largely influenced by genetic background, ultimately affecting beef yield and is of major interest to the beef industry. This investigation aimed to determine whether primary skeletal muscle cells isolated from different breeds of cattle with a varying genetic potential for muscling differ in their myogenic proliferative capacity. Primary skeletal muscle cells were isolated and cultured from the Longissimus muscle (LM) of 6 month old Angus, Hereford and Wagyu X Angus cattle. Cells were assessed for rate of proliferation and gene expression of PAX7, MYOD, MYF5, and MYOG. Proliferation rates were found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5-20 hours in culture) in vitro (P < 0.05). The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight and carcase weight of cattle (P < 0.05). Gene expression of MYF5 was also found to be significantly down-regulated in WagyuX compared with Angus cattle (P < 0.05). These findings suggest that early events during myogenesis are important for determining liveweight and caracase weights in cattle.

Versican processing by a disintegrin-like and metalloproteinase domain with thrombospondin-1 repeats proteinases-5 and -15 facilitates myoblast fusion.

Skeletal muscle development and regeneration requires the fusion of myoblasts into multinucleated myotubes. Because the enzymatic proteolysis of a hyaluronan and versican-rich matrix by ADAMTS versicanases is required for developmental morphogenesis, we hypothesized that the clearance of versican may facilitate the fusion of myoblasts during myogenesis. Here, we used transgenic mice and an in vitro model of myoblast fusion, C2C12 cells, to determine a potential role for ADAMTS versicanases. Versican processing was observed during in vivo myogenesis at the time when myoblasts were fusing to form multinucleated myotubes. Relevant ADAMTS genes, chief among them Adamts5 and Adamts15, were expressed both in developing embryonic muscle and differentiating C2C12 cells. Reducing the levels of Adamts5 mRNA in vitro impaired myoblast fusion, which could be rescued with catalytically active but not the inactive forms of ADAMTS5 or ADAMTS15. The addition of inactive ADAMTS5, ADAMTS15, or full-length V1 versican effectively impaired myoblast fusion. Finally, the expansion of a hyaluronan and versican-rich matrix was observed upon reducing the levels of Adamts5 mRNA in myoblasts. These data indicate that these ADAMTS proteinases contribute to the formation of multinucleated myotubes such as is necessary for both skeletal muscle development and during regeneration, by remodeling a versican-rich pericellular matrix of myoblasts. Our study identifies a possible pathway to target for the improvement of myogenesis in a plethora of diseases including cancer cachexia, sarcopenia, and muscular dystrophy.

1H NMR spectroscopy of serum reveals unique metabolic fingerprints associated with subtypes of surgically induced osteoarthritis in sheep.

Osteoarthritis (OA) is a highly prevalent joint disease. Its slow progressive nature and the correlation between pathological changes and clinical symptoms mean that OA is often well advanced by the time of diagnosis. In the absence of any specific pharmacological treatments, there is a pressing need to develop robust biomarkers for OA. We have adopted a nuclear magnetic resonance (NMR)-based metabolomic strategy to identify molecular responses to surgically induced OA in an animal model. Sheep underwent one of three types of surgical procedure (sham (control), meniscal destabilization, MD or anterior cruciate ligament transaction, ACLT), and for every animal a serum sample was collected both pre- and postoperatively, thus, affording two types of "control" data for comparison. 1D 1H NMR spectra were acquired from each sample at 800 MHz and the digitized spectral data were analyzed using principal components analysis and partial least-squares regression discriminant analysis. Our approach, combined with the study design, allowed us to separate the metabolic responses to surgical intervention from those associated with OA. We were able to identify dimethyl sulfone (DMSO2) as being increased in MD after 4 weeks, while ACLT-induced OA exhibited increased 3-methylhistidine and decreased branched chain amino acids (BCAAs). The findings are discussed in the context of interpretation of metabolomic results in studies of human disease, and the selection of appropriate "control" data sets.

Alterations in the expression of leukemia inhibitory factor following exercise: comparisons between wild-type and mdx muscles.

BACKGROUND: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine, belonging to the interleukin-6 family of cytokines, that has been suggested to have positive effects on myogenesis following injury and to minimise dystrophic pathology in mdx mice. Previous reports have suggested that Lif mRNA is up-regulated in the limb and diaphragm muscles of mdx mice, in human cases of dystrophy and acutely following exercise. This study examined expression of Lif mRNA in the quadriceps muscles of mdx and wild-type mice that were either sedentary or allowed to exercise voluntarily for two weeks. RESULTS: Exercise caused a decrease in Lif mRNA expression in wild-type muscle, but this was not the case in mdx muscle. Lif mRNA levels in sedentary mdx mice were similar to those in exercised wild type muscles, and in mdx mice there was no further decrease in levels following exercise. Similar down-regulation of Lif mRNA was observed in the tibialis anterior and diaphragm muscles of mdx mice at three and six weeks of age respectively, compared with wild-type controls. Transcripts for the LIF receptor (Lifr) were also down-regulated in these mdx muscles, suggesting LIF activity may be minimised in dystrophic muscle. However fluorescent immunohistochemical labeling of LIF did not correlate with transcript expression data, as LIF immunoreactivity could not be detected in wild-type muscle, where mRNA expression was high, but was present in dystrophic muscle where mRNA expression was low. This study also described the translocation of membrane proteins, including LIFR, to the nuclei of syncytial muscle cells during differentiation and fusion. In addition this study demonstrates that survival of donor myoblasts injected into dystrophic muscle was enhanced by co-administration of recombinant LIF. CONCLUSIONS: This study provides new evidence to support a role for LIF in normal muscle biology in response to exercise. Although expression levels of Lif transcript in mdx muscles were not consistent with previous studies, the detection of LIF protein in mdx muscle but not wild-type muscle supports a role for LIF in dystrophy. This study also provides evidence of the differential localisation of the LIFR, and the potential for anti-inflammatory actions of LIF that promote survival of transplanted myoblasts in dystrophic muscle.*corresponding author: Jason White, Muscular Dystrophy Research Group, Murdoch Childrens Research Institute; email: jasondw@unimelb.edu.au.

Proposed practice guidelines for continuous contact lens wear.

The introduction of silicone-containing hydrogel contact lenses (SCHCLs) for continuous wear (CW) presents practitioners with a unique challenge. To facilitate the transition, we propose a set of guidelines for the conduct of CW practice. The general concepts for the guide are derived from a range of similar codes of practice that have been produced for a range of aspects of eye care. The more general competencies required of eye care practice are assumed and are not discussed in detail. The document is based on the precept that the prescription of SCHCLs is an appropriate practice for suitably screened patients. Practitioners should acquire general and specific knowledge of information applicable to CW with SCHCLs. Support staff should be educated to handle a range of requirements specific to CW. There are no consulting room facilities required beyond those necessary for general optometric practice but we highly recommend that the CW practitioner use grading scales for anterior eye complications and a guide to corneal infiltrative conditions. More stringent examination protocols are required than for general contact lens practice, particularly in the selection of the patient. Patient management should be conducted to a medical standard of care. Documentation is critical and will involve general information about the product, an agreement, disclosure of risk factors, instructions for wear and care, recording of patient questions and answers provided, an informed consent form and detailed, accurate record keeping. A guideline such as this document cannot be positioned as a legal standard of care, but may be used in the formulation of such a standard.