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Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

While skeletal muscle remodeling happens throughout life, diseases that result in its dysfunction are accountable for many deaths. Indeed, skeletal muscle is exceptionally capable to respond to stimuli modifying its homeostasis, such as in atrophy, hypertrophy, regeneration and repair. In particular conditions such as genetic diseases (muscular dystrophies), skeletal muscle's capacity to remodel is strongly affected and undergoes continuous cycles of chronic damage. This induces scarring, fatty infiltration, as well as loss of contractibility and of the ability to generate force. In this context, inflammation, primarily mediated by macrophages, plays a central pathogenic role. Macrophages contribute as the primary regulators of inflammation during skeletal muscle regeneration, affecting tissue-resident cells such as myogenic cells and endothelial cells, but also fibro-adipogenic progenitors, which are the main source of the fibro fatty scar. During skeletal muscle regeneration their function is tightly orchestrated, while in dystrophies their fate is strongly disturbed, resulting in chronic inflammation. In this review, we will discuss the latest findings on the role of macrophages in skeletal muscle diseases, and how they are regulated.

Muscular dystrophy is a well-known genetically heterogeneous group of rare muscle disorders. This progressive disease causes the breakdown of skeletal muscles over time and leads to grave weakness. This breakdown is caused by a diverse pattern of mutations in dystrophin and dystrophin associated protein complex. These mutations lead to the production of altered proteins in response to which, the body stimulates production of various cytokines and immune cells, particularly reactive oxygen species and NFκB. Immune cells display/exhibit a dual role by inducing muscle damage and muscle repair. Various anti-oxidants, anti-inflammatory and glucocorticoid drugs serve as potent therapeutics for muscular dystrophy. Along with the above mentioned therapeutics, induced pluripotent stem cells also serve as a novel approach paving a way for personalized treatment. These pluripotent stem cells allow regeneration of large numbers of regenerative myogenic progenitors that can be administered in muscular dystrophy patients which assist in the recovery of lost muscle fibers. In this review, we have summarized gene-editing, immunological and induced pluripotent stem cell based therapeutics for muscular dystrophy treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Muscular dystrophies and inflammatory myopathies are heterogeneous muscular disorders characterized by progressive muscle weakness and mass loss. Despite the high variability of etiology, inflammation and involvement of both innate and adaptive immune response are shared features. The best understood immune mechanisms involved in these pathologies include complement cascade activation, auto-antibodies directed against muscular proteins or de-novo expressed antigens in myofibers, MHC-I overexpression in myofibers, and lymphocytes-mediated cytotoxicity. Intravenous immunoglobulins (IVIGs) administration could represent a suitable immunomodulator with this respect. Here we focus on mechanisms of action of immunoglobulins in muscular dystrophies and inflammatory myopathies highlighting results of IVIGs from pre-clinical and case reports evidences., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Farini, Villa, Tripodi, Legato and Torrente.)

Background: Laminin-α2-related congenital muscular dystrophy (LAMA2-CMD) is a devastating genetic disease caused by mutations in the LAMA2 gene. These mutations result in progressive muscle wasting and inflammation leading to delayed milestones, and reduced lifespan in affected patients. There is currently no cure or treatment for LAMA2-CMD. Preclinical studies have demonstrated that mouse laminin-111 can serve as an effective protein replacement therapy in a mouse model of LAMA2-CMD., Methods: In this study, we generated a novel immunocompromised dy W mouse model of LAMA2-CMD to study the role the immune system plays in muscle disease progression. We used this immune-deficient dy W mouse model to test the therapeutic benefits of recombinant human laminin-111 and laminin-211 protein therapy on laminin-α2-deficient muscle disease progression., Results: We show that immunodeficient laminin-α2 null mice demonstrate subtle differences in muscle regeneration compared to immunocompetent animals during early disease stages but overall exhibit a comparable muscle disease progression. We found human laminin-111 and laminin-211 could serve as effective protein replacement strategies with mice showing improvements in muscle pathology and function. We observed that human laminin-111 and laminin-211 exhibit differences on satellite and myoblast cell populations and differentially affect muscle repair., Conclusions: This study describes the generation of a novel immunodeficient mouse model that allows investigation of the role the immune system plays in LAMA2-CMD. This model can be used to assess the therapeutic potential of heterologous therapies that would elicit an immune response. Using this model, we show that recombinant human laminin-111 can serve as effective protein replacement therapy for the treatment of LAMA2-CMD.

Muscular dystrophy describes generalized progressive muscular weakness due to the wasting of muscle fibers. The progression of the disease is affected by known immunological and mechanical factors, and possibly other unknown mechanisms. This article introduces a new mathematical model, the FRiND model, to further elucidate these known immunological actions. We will perform stability and sensitivity analyses on this model. The models time course results will be verified by biological studies in the literature. This model could be the foundation for further understanding of immunological muscle repair.

Mutations in the dystrophin and sarcoglycans genes result in muscular dystrophies causing severe disability and premature death and where no effective treatment is available. New therapeutic approaches targeting secondary disease mechanisms have a strong translational potential. Dystrophic muscle damage triggers release of ATP whilst loss of ecto-ATPase activity of sarcoglycan further elevates extracellular ATP (eATP) levels. Such a high eATP activates P2X7 purinoceptors on immune cells; these contribute to chronic inflammatory and immune responses that exacerbate the dystrophic pathology. Dystrophin mutations coincide with a significant P2X7 upregulation in Duchenne muscular dystrophy (DMD) muscle and alter receptor signalling in mouse dystrophic myoblasts and myofibers. P2X7 overexpression combined with the eATP-rich environment lead to cell dysfunction and death and ultimately to ineffective regeneration. P2X7 is therefore a therapeutic target for reducing damaging inflammation and supporting the repair of dystrophic muscles. Accordingly, genetic ablation and pharmacological inhibition of the eATP-P2X7 axis alleviated dystrophic phenotypes in mouse models of dystrophinopathy and sarcoglycanopathy. Thus, P2X7 inhibitors are good candidates for rapid re-purposing for the treatment of these highly debilitating diseases. Such a therapy is not constrained by causative mutations, so it would be suitable for all patients. Moreover, it appears effective in alleviating both muscle and non-muscle symptoms., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.

Muscular dystrophies represent a group of diseases which may develop in several forms, and severity of the disease is usually associated with gene mutations. In skeletal muscle regeneration and in muscular dystrophies, both innate and adaptive immune responses are involved. The regenerative potential of mesenchymal stem/stromal cells (MSCs) of bone marrow origin was confirmed by the ability to differentiate into diverse tissues and by their immunomodulatory and anti-inflammatory properties by secretion of a variety of growth factors and anti-inflammatory cytokines. Skeletal muscle comprises different types of stem/progenitor cells such as satellite cells and non-satellite stem cells including MSCs, interstitial stem cells positive for stress mediator PW1 expression and negative for PAX7 called PICs (PW1 + /PAX7 - interstitial cells), fibro/adipogenic progenitors/mesenchymal stem cells, muscle side population cells and muscle resident pericytes, and all of them actively participate in the muscle regeneration process. In this review, we present biological properties of MSCs of bone marrow origin and a heterogeneous population of muscle-resident stem/progenitor cells, their interaction with the inflammatory environment of dystrophic muscle and potential implications for cellular therapies for muscle regeneration. Subsequently, we propose-based on current research results, conclusions, and our own experience-hypothetical mechanisms for modulation of the complete muscle regeneration process to treat muscular dystrophies.

Background: Chronic increases in the levels of the inflammatory cytokine interleukin-6 (IL-6) in serum and skeletal muscle are thought to contribute to the progression of muscular dystrophy. Dystrophin/utrophin double-knockout (dKO) mice develop a more severe and progressive muscular dystrophy than the mdx mice, the most common murine model of Duchenne muscular dystrophy (DMD). In particular, dKO mice have smaller body sizes and muscle diameters, and develop progressive kyphosis and fibrosis in skeletal and cardiac muscles. As mdx mice and DMD patients, we found that IL-6 levels in the skeletal muscle were significantly increased in dKO mice. Thus, in this study, we aimed to analyze the effects of IL-6 receptor (IL-6R) blockade on the muscle pathology of dKO mice., Methods: Male dKO mice were administered an initial injection (200 mg/kg intraperitoneally (i.p.)) of either the anti-IL-6R antibody MR16-1 or an isotype-matched control rat IgG at the age of 14 days, and were then given weekly injections (25 mg/kg i.p.) until 90 days of age., Results: Treatment of dKO mice with the MR16-1 antibody successfully inhibited the IL-6 pathway in the skeletal muscle and resulted in a significant reduction in the expression levels of phosphorylated signal transducer and activator of transcription 3 in the skeletal muscle. Pathologically, a significant increase in the area of embryonic myosin heavy chain-positive myofibers and muscle diameter, and reduced fibrosis in the quadriceps muscle were observed. These results demonstrated the therapeutic effects of IL-6R blockade on promoting muscle regeneration. Consistently, serum creatine kinase levels were decreased. Despite these improvements observed in the limb muscles, degeneration of the diaphragm and cardiac muscles was not ameliorated by the treatment of mice with the MR16-1 antibody., Conclusion: As no adverse effects of treatment with the MR16-1 antibody were observed, our results indicate that the anti-IL-6R antibody is a potential therapy for muscular dystrophy particularly for promoting skeletal muscle regeneration.

Background: Collagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients., Methods: Many antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together., Results: Our analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains., Conclusions: This series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.

About 2-3 million years ago, Alu-mediated deletion of a critical exon in the CMAH gene became fixed in the hominin lineage ancestral to humans, possibly through a stepwise process of selection by pathogen targeting of the CMAH product (the sialic acid Neu5Gc), followed by reproductive isolation through female anti-Neu5Gc antibodies. Loss of CMAH has occurred independently in some other lineages, but is functionally intact in Old World primates, including our closest relatives, the chimpanzee. Although the biophysical and biochemical ramifications of losing tens of millions of Neu5Gc hydroxy groups at most cell surfaces remains poorly understood, we do know that there are multiscale effects functionally relevant to both sides of the host-pathogen interface. Hominin CMAH loss might also contribute to understanding human evolution, at the time when our ancestors were starting to use stone tools, increasing their consumption of meat, and possibly hunting. Comparisons with chimpanzees within ethical and practical limitations have revealed some consequences of human CMAH loss, but more has been learned by using a mouse model with a human-like Cmah inactivation. For example, such mice can develop antibodies against Neu5Gc that could affect inflammatory processes like cancer progression in the face of Neu5Gc metabolic incorporation from red meats, display a hyper-reactive immune system, a human-like tendency for delayed wound healing, late-onset hearing loss, insulin resistance, susceptibility to muscular dystrophy pathologies, and increased sensitivity to multiple human-adapted pathogens involving sialic acids. Further studies in such mice could provide a model for other human-specific processes and pathologies involving sialic acid biology that have yet to be explored., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Use of adeno-associated virus (AAV) to transduce genes into skeletal muscles can be associated with T-cell responses to viral capsid and/or to transgenic protein. Intramuscular mononuclear cell infiltrates primarily consisting of CD8+ T cells and also containing FOXP3+ regulatory T cells were present in rhesus macaque skeletal muscle treated with rAAVrh74.MCK.GALGT2 by vascular delivery. Administration of oral prednisone prior to AAV gene delivery and throughout the study reduced such infiltrates by 60% at 24 weeks post AAV delivery compared with AAV-treated animals not receiving prednisone, regardless of the presence of pre-existing AAV serum antibodies at the time of treatment. The majority of CD8+ T cells in AAV-treated muscles expressed activated caspase 3 and programmed cell death protein 1 (PD1), suggesting ongoing programmed cell death. AAV-transduced skeletal muscles also had elevated expression of programmed death ligand 2 (PDL2) on skeletal myofibers, and this increase in expression extended to muscles where transgene was not overexpressed. These data demonstrate that prednisone can reduce the extent of intramuscular T-cell infiltrates in AAV-treated muscles, which may aid in achieving long-term transgene expression, as may the induction of PDL2 expression on skeletal myofibers to promote PD1-mediated programmed T-cell death.

In skeletal muscles, levels and activity of Matrix MetalloProteinases (MMPs) and Tissue Inhibitors of MetalloProteinases (TIMPs) have been involved in myoblast migration, fusion and various physiological and pathological remodeling situations including neuromuscular diseases. This has opened perspectives for the use of MMPs' overexpression to improve the efficiency of cell therapy in muscular dystrophies and resolve fibrosis. Alternatively, inhibition of individual MMPs in animal models of muscular dystrophies has provided evidence of beneficial, dual or adverse effects on muscle morphology or function. We review here the role played by MMPs/TIMPs in skeletal muscle inflammation and fibrosis, two major hurdles that limit the success of cell and gene therapy. We report and analyze the consequences of genetic or pharmacological modulation of MMP levels on the inflammation of skeletal muscles and their repair in light of experimental findings. We further discuss how the interplay between MMPs/TIMPs levels, cytokines/chemokines, growth factors and permanent low-grade inflammation favor cellular and molecular modifications resulting in fibrosis.

Introduction: Microchimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues., Method: Fluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls., Results: Microchimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm(2) tissue) compared to controls (0.019 ± 0.007 cells/mm(2) tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm(2)). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm(2) versus 0 ± 0/mm(2) p = 0.003 and 0.043 ± 0.023/mm(2) versus 0 ± 0/mm(2) p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype., Conclusions: Microchimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.

Muscular dystrophies (MDs) are a heterogeneous group of diseases, caused by mutations in different components of sarcolemma, extracellular matrix, or enzymes. Inflammation and innate or adaptive immune response activation are prominent features of MDs. Various therapies under development are directed toward rescuing the dystrophic muscle damage using gene transfer or cell therapy. Here we discussed current knowledge about involvement of immune system responses to experimental therapies in MDs.

Background: In inflammatory myopathies muscle biopsy is a crucial diagnostic test. Misinterpretation between inflammatory myopathies and muscular dystrophies with inflammation is known., Materials and Methods: Thirty-one patients clinically and pathologically diagnosed to have polymyositis and dermatomyositis and 16 patients of muscular dystrophy with inflammation were studied for MHC-I, MHC-II, CD4 and CD8 expression in skeletal muscle tissue., Results: MHC-I upregulation was noted in all samples of PM and DM. Interstitial and perivascular inflammation in PM were predominantly CD8+ cells, in dermatomyositis, interstitial and perimysial perivascular inflammatory cells were CD4+ T cells and CD8+ T cells were seen around endomysial vessels. Interestingly MHC-I upregulation was seen in all 16 cases of muscular dystrophy with presence of inflammation., Conclusion: The pattern of MHC-I and II expression appeared to be similar in both inflammatory myopathies as well as in muscular dystrophies with inflammation and hence differentiating them on MHC - I expression was difficult.

Purpose of Review: Necrotizing myopathy exhibits a specific histological pattern, characterized by significant necrosis, with simultaneous muscle fiber regeneration, but without or with little inflammation. This histological pattern may be observed in acquired myopathies but also in muscular dystrophy. Acquired necrotizing myopathy can be secondary to drugs or toxics agents, and if not, autoimmune mechanisms have to be suspected. Necrotizing autoimmune myopathy (NAM) is now recognized as a subgroup of idiopathic inflammatory myopathies. This review aims to further describe the different acquired myopathies, with special focus on NAM since specific autoantibodies have been discovered., Recent Findings: Usually, acquired myopathy has an acute onset, but a slower progression may sometimes be observed that could lead to erroneous diagnosis of muscular dystrophy. NAM may be associated with specific autoantibodies against signal recognition particle, or, as more recently described, against 3-hydroxy-3-methylglutaryl-coenzyme A reductase, especially in statin-exposed patients. Their presence permits affirmation that muscular necrosis is immune-mediated. The antibody titer is correlated to the disease activity., Summary: NAMs are now considered as a new entity, treatable by immunosuppressants, and should not be misdiagnosed as a muscular dystrophy.

Inflammation plays a crucial role in muscle remodeling and repair after acute and chronic damage, in particular in muscular dystrophies, a heterogeneous group of genetic diseases leading to muscular degeneration. Defect of nitric oxide (NO) generation is a key pathogenic event in muscular dystrophies, thus NO donors have been explored as new therapeutics for this disease. We have investigated the immune-modulating effect of one of such drugs, molsidomine, able to slow the progression of muscular dystrophy in the α-Sarcoglican-null mice, a model for the limb girdle muscular dystrophy 2D, sharing several hallmarks of muscle degeneration with other muscular dystrophies. α-Sarcoglican-null mice were treated with molsidomine and drug effects on the inflammatory infiltrates and on muscle repair were assessed at selected time points. We found that molsidomine treatment modulates effectively the characteristics of the inflammatory infiltrate within dystrophic muscles, enhancing its healing function. Initially molsidomine amplified macrophage recruitment, promoting a more efficient clearance of cell debris and effective tissue regeneration. At a later stage molsidomine decreased significantly the extent of the inflammatory infiltrate, whose persistence exacerbates muscle damage: most of the remaining macrophages displayed characteristics of the transitional population, associated with reduced fibrosis and increased preservation of the muscle tissue. The dual action of molsidomine, the already known NO donation and the immunomodulatory function we now identified, suggests that it has a unique potential in tissue healing during chronic muscle damage. This, alongside its already approved use in human, makes molsidomine a drug with a significant therapeutic potential in muscular dystrophies., (© 2013 Elsevier B.V. All rights reserved.)

Cells encounter many physical, chemical and biological stresses that perturb plasma membrane integrity, warranting an immediate membrane repair response to regain cell homeostasis. Failure to respond properly to such perturbation leads to individual cell death, which may also produce systemic influence by triggering sterile immunological responses. In this review, we discuss recent progress on understanding the mechanisms underlying muscle cell membrane repair and the potential mediators of innate immune activation when the membrane repair system is defective, specifically focusing on pathology associated with dysferlin deficiency., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Genetic defects in like-glycosyltransferase (LARGE) cause congenital muscular dystrophy with central nervous system manifestations. The underlying molecular pathomechanism is the hypoglycosylation of α-dystroglycan (α-DG), which is evidenced by diminished immunoreactivity to IIH6C4 and VIA4-1, antibodies that recognize carbohydrate epitopes. Previous studies indicate that LARGE participates in the formation of a phosphoryl glycan branch on O-linked mannose or it modifies complex N- and mucin O-glycans. In this study, we overexpressed LARGE in neural stem cells deficient in protein O-mannosyltransferase 2 (POMT2), an enzyme required for O-mannosyl glycosylation. The results showed that overexpressing LARGE did not lead to hyperglycosylation of α-DG in POMT2 knockout (KO) cells but did generate IIH6C4 and VIA4-1 immunoreactivity and laminin-binding activity. Additionally, overexpressing LARGE in cells deficient in both POMT2 and α-DG generated laminin-binding IIH6C4 immunoreactivity. These results indicate that LARGE expression resulted in the glycosylation of proteins other than α-DG in the absence of O-mannosyl glycosylation. The IIH6C4 immunoreactivity generated in double-KO cells was largely removed by treatment either with peptide N-glycosidase F or with cold aqueous hydrofluoric acid, suggesting that LARGE expression caused phosphoryl glycosylation of N-glycans. However, the glycosylation of α-DG by LARGE is dependent on POMT2, indicating that LARGE expression only modifies O-linked mannosyl glycans of α-DG. Thus, LARGE expression mediates the phosphoryl glycosylation of not only O-mannosyl glycans including those on α-DG but also N-glycans on proteins other than α-DG.

To better understand the role of dendritic cells (DCs) in skeletal muscle, we investigated the migration of DCs from murine skeletal muscle and compared that to previously studied footpad (FP) DC trafficking. We adoptively transferred carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled mature DCs to syngeneic mice and followed them in various lymphatic tissues at different time points. Injection of DCs into the tibialis anterior muscle resulted in the peak number of CFSE(+) DCs recovered in spleen at 12h, not at 24h, when the largest number of these cells appeared in the draining lymph nodes. Interestingly, this result for adoptive transfer of DCs to skeletal muscle differs with what is previously reported for adoptive transfer to the FP, a result that we also confirmed in parallel studies. These findings could have a significant impact on (1) understanding muscle diseases with immunological complications such as muscular dystrophies and (2) the immunologic effects of treatments for muscle diseases., (Published by Elsevier GmbH.)

Recent discoveries reveal complex interactions between skeletal muscle and the immune system that regulate muscle regeneration. In this review, we evaluate evidence that indicates that the response of myeloid cells to muscle injury promotes muscle regeneration and growth. Acute perturbations of muscle activate a sequence of interactions between muscle and inflammatory cells. The initial inflammatory response is a characteristic Th1 inflammatory response, first dominated by neutrophils and subsequently by CD68(+) M1 macrophages. M1 macrophages can propagate the Th1 response by releasing proinflammatory cytokines and cause further tissue damage through the release of nitric oxide. Myeloid cells in the early Th1 response stimulate the proliferative phase of myogenesis through mechanisms mediated by TNF-alpha and IL-6; experimental prolongation of their presence is associated with delayed transition to the early differentiation stage of myogenesis. Subsequent invasion by CD163(+)/CD206(+) M2 macrophages attenuates M1 populations through the release of anti-inflammatory cytokines, including IL-10. M2 macrophages play a major role in promoting growth and regeneration; their absence greatly slows muscle growth following injury or modified use and inhibits muscle differentiation and regeneration. Chronic muscle injury leads to profiles of macrophage invasion and function that differ from acute injuries. For example, mdx muscular dystrophy yields invasion of muscle by M1 macrophages, but their early invasion is accompanied by a subpopulation of M2a macrophages. M2a macrophages are IL-4 receptor(+)/CD206(+) cells that reduce cytotoxicity of M1 macrophages. Subsequent invasion of dystrophic muscle by M2c macrophages is associated with progression of the regenerative phase in pathophysiology. Together, these findings show that transitions in macrophage phenotype are an essential component of muscle regeneration in vivo following acute or chronic muscle damage.

Aims: There has been much debate about the existence of juvenile polymyositis (JPM) as an entity distinct from muscular dystrophy (MD). The aim of this study was to retrospectively analyse muscle biopsies and clinical features of 13 Australian children given an initial diagnosis of JPM, to determine their clinicopathological, immunohistochemical and molecular characteristics., Methods and Results: The muscle biopsies on 13 cases were reviewed using detailed morphological and immunoperoxidase studies, with additional protein and molecular analyses, in conjunction with clinical review. Only one case had a true connective tissue disease inflammatory myopathy. Twelve (92.3%) cases with an initial diagnosis of JPM were found on clinical, pathological and molecular review to be MD., Conclusions: Inflammatory changes in apparently sporadic juvenile myopathies should prompt consideration of an early presentation of MD. Detailed analysis of muscle histopathology, specifically the detection of subsarcolemmal blebbing, isolated fibre degeneration occurring independent of inflammatory infiltrates, patchy clustered major histocompatibility complex-I expression and a CD68+/CD3+ perimysial infiltrate, assists in the diagnosis of early MD. Specific protein and gene analysis adds support to the pathological diagnosis of dystrophy. This series adds weight to suggestions that JPM may not represent a discrete clinical or pathological entity.

Skeletal muscle requires an efficient and active membrane repair system to overcome the rigours of frequent contraction. Dysferlin is a component of that system and absence of dysferlin causes muscular dystrophy (dysferlinopathy) characterized by adult onset muscle weakness, high serum creatine kinase levels and a prominent inflammatory infiltrate. We have observed that dysferlinopathy patient biopsies show an excess of immature fibres and therefore investigated the role of dysferlin in muscle regeneration. Using notexin-induced muscle damage, we have shown that regeneration is attenuated in a mouse model of dysferlinopathy, with delayed removal of necrotic fibres, an extended inflammatory phase and delayed functional recovery. Satellite cell activation and myoblast fusion appear normal, but there is a reduction in early neutrophil recruitment in regenerating and also needle wounded muscle in dysferlin-deficient mice. Primary mouse dysferlinopathy myoblast cultures show reduced cytokine release upon stimulation, indicating that the secretion of chemotactic molecules is impaired. We suggest an extension to the muscle membrane repair model, where in addition to fusing patch repair vesicles with the sarcolemma dysferlin is also involved in the release of chemotactic agents. Reduced neutrophil recruitment results in incomplete cycles of regeneration in dysferlinopathy which combines with the membrane repair deficit to ultimately trigger dystrophic pathology. This study reveals a novel pathomechanism affecting muscle regeneration and maintenance in dysferlinopathy and highlights enhancement of the neutrophil response as a potential therapeutic avenue in these disorders.

Skeletal muscle injury, despite the initial trigger, leads to a stereotypical cascade of events mediated by cells of the immune system. Acute damage recruits cells of the innate immune system (polymorphonuclear leukocytes and monocytes/macrophages) that initially release noxious molecules and clear the cellular debris. Macrophages in particular display two distinct differentiation patterns. At early times after acute damage inflammatory macrophages are predominant, and play a non-redundant role in the clearance of cellular debris. At later time points, when fibre regeneration occurs, macrophages acquire a de-activated phenotype, which has been associated to tissue remodelling. A role for cells of the acquired immune system, in particular antigen-specific T and B cells, in muscle regeneration has been envisaged, but still needs to be elucidated. Similar events possibly play a role during persistent muscle damage in which fibres never completely heal. As a consequence infiltrating leukocytes stay alive and are continuously activated. Their effector function in situ contributes to perpetuate the damage and results in the deposition of collagen with interstitial fibrosis and fat accumulation. In this review we will discuss the events characterising acute and persistent damage in stretch-induced injury, autoimmune polymyositis, inclusion bodies myositis and muscular dystrophies. We will focus on the molecular interactions involved in the positive and negative regulation of the inflammatory damage, with specific attention to their exploitation in the context of strategies to limit muscle wasting and supporting fibre regeneration.

Background: Miyoshi myopathy (MM) and limb girdle muscular dystrophy (LGMD2B) are distinct clinical entities because different muscle groups are involved at the onset. We describe the clinical features in 28 patients with dysferlin deficiency confirmed by muscle immunohistochemistry (IHC)., Settings and Design: A case series from a tertiary national referral center for neurological disorders., Materials and Methods: Patients with classical phenotype of MM and LGMD2B underwent a thorough phenotypic characterization followed by muscle histopathological study including IHC for dysferlin deficiency., Results: There were 28 patients (20 men and eight women) presenting with manifestations of distal myopathy or LGMD2B and had absence of dysferlin staining on IHC. Patients presented predominantly with distal myopathy of Miyoshi type (MM) or proximal LGMD type and were diagnosed to have dysferlinopathy on IHC. Two patients had the proximodistal form and two had onset as tibial muscular dystrophy. The main clinical features in these patients were onset in late adolescence or early adulthood (mean age of onset for MM was 22.0 +/- 6.7 years and for LGMD2B 19.4 +/- 5.1 years). There was early and predominant involvement of the posterior compartment muscles of the leg or proximal pelvic girdle muscles, dystrophic features with necrotic regeneration pattern without vacuoles on muscle biopsy and markedly elevated serum creatine kinase values with mean of 10033.8 +/- 9283 IU/l (range 402-27460). Consanguinity was reported in 46.4%. The mean duration of illness was 6.4 +/- 4.2 years. Dysferlinopathies formed nearly one-fourth of our patients with LGMD., Conclusion: In our experience dysferlinopathies was not an uncommon form of LGMD.

Signaling through nuclear factor-kappa B (NF-kappaB) is emerging as an important regulator of muscle development, maintenance, and regeneration. Classic signaling modulates early muscle development by enhancing proliferation and inhibiting differentiation, and alternative signaling promotes myofiber maintenance and metabolism. Likewise, NF-kappaB signaling is critical for the development of immunity. Although these processes occur normally, dysregulation of NF-kappaB signaling has prohibitive effects on muscle growth and regeneration and can perpetuate inflammation in muscle diseases. Aberrant NF-kappaB signaling from immune and muscle cells has been detected and implicated in the pathologic progression of numerous dystrophies and myopathies, indicating that targeted NF-kappaB inhibitors may prove clinically beneficial.

Several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis. Our previous work suggested the existence of molecular mimicry between antibodies raised against hydroxy-2-nonenal (HNE)-modified protein and anti-DNA autoantibodies, a serologic hallmark of systemic lupus erythematosus (SLE). In the present study, we investigated the possible involvement of HNE-modified proteins as the endogenous source of the anti-DNA antibodies. Accumulation of the antigen recognized by the antibody against the HNE-modified protein was observed in the nucleus of almost all of the epidermal cells from patients with autoimmune diseases, including SLE. The SLE patients also showed significantly higher serum levels of the anti-HNE titer than healthy individuals. To determine if a specific anti-DNA response could be initiated by the HNE-derived epitopes, we immunized BALB/c mice with the HNE-modified protein and observed a progressive increase in the anti-DNA response. Moreover, we generated the monoclonal antibodies, showing recognition specificity toward DNA, and found that they can bind to two structurally distinct antigens (i.e. the native DNA and protein-bound 4-oxo-2-nonenal). The findings in this study provide evidence to suspect an etiologic role for lipid peroxidation in autoimmune diseases.

Various clinical trials have documented the therapeutic benefit of glucocorticoids (GCs) in enhancing muscle strength and slowing disease progression of Duchenne and Becker muscular dystrophies (DMD/BMD). We hypothesized that GCs may have relevance to the differential anti-inflammatory effect on mononuclear inflammatory cells (MICs) and Dendritic cells (DCs) infiltrating the dystrophic muscles. In this prospective study, two muscle biopsies were obtained (before and after 6-month prednisone therapy) from 30 patients with dystrophies (DMD = 18; BMD = 6; and limb girdle muscular dystrophies (LGMD) = 6). MICs and DCs infiltrating the muscles were examined using mouse monoclonal antibodies and immunoperoxidase staining methods. Muscle strength was evaluated monthly by manual testing, motor ability and timed tests. Prednisone therapy was associated with: (i) functional improvement of overall motor disability, in upper limbs of DMD (P < 0.001) and BMD (P < 0.01) and lower limbs of DMD (P < 0.001) and BMD (P < 0.05); (ii) histological improvement such as fibre size variation (DMD, P < 0.01; BMD, P < 0.05), internalization of nuclei (DMD, P < 0.05), degeneration and necrosis (DMD and BMD, P < 0.01), regeneration (DMD, P < 0.001; BMD, P < 0.01) and endomysial connective tissue proliferation (DMD, P < 0.01; BMD, P < 0.05) and (iii) reduction of total MICs (P < 0.01) and DCs (P < 0.01). There was a positive correlation between the degree of improvement in overall motor disability and reduction of DCs numbers (In upper limbs; r = 0.638, P < 0.01 for DMD and r = 0.725, P < 0.01 for BMD, in Lower limbs; r = 0.547, P < 0.05 for DMD and r = 0.576, P < 0.05 for BMD). Such improvements and changes of MICs/DCs were absent in LGMD. In DMD/BMD, prednisone therapeutic effect was associated with reduced MICs and DCs numbers. Whether this therapeutic effect reflects targeting of the deleterious immune response produced by these cells mandates further investigations.

Various autoantibodies had been detected in patients with dilated cardiomyopathy (DCM). Among them, anti-beta 1 adrenoreceptor antibody (ARAb) had been proven to act as agonist on beta 1 adrenoreceptor and cause DCM. Cardiomyopathy is also serious problem in progressive muscular dystrophy (PMD). Because cardiac dysfunction is quite variable even in siblings sharing identical mutations, it is highly possible that there are some modifier factors. Thus, we measured ARAb in 93 patients with PMD and 11 patients with DCM to clarify immune function for cardiac impairment of PMD. The titer was abnormally elevated in 30.1% of PMD, 72.7% of DCM and 75.0% (9/12) of PMD patients with symptomatic cardiac failure. ARAb was weakly correlated to fractional shortening, brain natriuretic peptide, noradrenalin and severity of premature ventricular contractions (Lown grade). During the study period, four patients developed cardiac failure and ARAb was increased in all these patients. In DMD, although the patients receiving both beta blocker and angiotensin converting enzyme inhibitor showed worst cardiac function, the titers were rather low compared to patients with angiotensin converting enzyme inhibitor alone. Four of five patients initiating beta blocker showed decrease of ARAb. Autoantibodies for myocardium actually exist in PMD in certain ratio as is the case with DCM. It is highly possible that immune system plays some role in cardiac impairment even in PMD. We should pay enough attention to immune system to elucidate the mechanism of cardiac dysfunction and refine strategy of cardiac treatments.

Purpose of Review: This review provides an updated evaluation of the role of inflammation in muscular dystrophy, and presents findings which suggest that non-immunological factors promote idiopathic inflammatory myopathies. Recent findings are summarized which indicate that immune-targeted interventions may provide useful approaches to treat muscular dystrophy., Recent Findings: Elevated expression of the cytotoxic T-lymphocyte derived cytolytic molecule, perforin, and the inducible costimulatory molecule have been identified in muscles of Duchenne muscular dystrophy patients, which strengthens evidence that a cellular immune response contributes to dystrophinopathy. Conversely, new findings implicate non-immune factors in inflammatory myopathy pathogenesis. Muscles from healthy individuals expressed autoantigens typically present in inflammatory myopathies, and autoantigen expression increased along with elevated major histocompatibility complex class I expression at sites of muscle regeneration in inflammatory myopathies. Those observations suggest that regeneration could render conditions sufficient for an autoimmune response in inflammatory myopathies. Further studies of corticosteroids or tumor necrosis factor blockade in treating dystrophinopathy indicate that immunological interventions may yield improved therapies for muscular dystrophy. In addition, advancements in understanding the involvement of chemokines in muscular dystrophy and inflammatory myopathies suggest that targeting specific chemokines has potential therapeutic value., Summary: Our developing understanding of the pathogenesis of muscular dystrophies and inflammatory myopathies shows complex interactions between immunological and non-immunological features of these diseases that can affect disease onset and course. Among the muscular dystrophies, the best evidence for an immunological component to disease pathogenesis exists for dystrophinopathies. Conversely, muscle damage leading to regeneration may promote some inflammatory myopathies, although much remains to be learned concerning the identity and pathological significance of non-immunological features of inflammatory myopathies.

Dysferlin is expressed in skeletal and cardiac muscles. However, dysferlin deficiency results in skeletal muscle weakness, but spares the heart. We compared intraindividual mRNA expression profiles of cardiac and skeletal muscle in dysferlin-deficient SJL/J mice and found down-regulation of the complement inhibitor, decay-accelerating factor/CD55, in skeletal muscle only. This finding was confirmed on mRNA and protein levels in two additional dysferlin-deficient mouse strains, A/J mice and Dysf-/- mice, as well as in patients with dysferlin-deficient muscular dystrophy. In vitro, the absence of CD55 led to an increased susceptibility of human myotubes to complement attack. Evidence is provided that decay-accelerating factor/CD55 is regulated via the myostatin-SMAD pathway. In conclusion, a novel mechanism of muscle fiber injury in dysferlin-deficient muscular dystrophy is demonstrated, possibly opening therapeutic avenues in this to date untreatable disorder.

The basis of human prion diseases affecting the nervous system is accumulation of a disease-associated conformer (PrPSc) of the normal cellular prion protein (PrPC). Earlier studies demonstrated increased expression of PrPC in inclusion body myositis (IBM), dermato-, and polymyositis, as well as neurogenic muscle atrophy. To define the spectrum and reliability of PrPC immunoreactivity, its expression was examined systematically in a series of pathologically characterized muscular disorders by means of immunohistochemistry, confocal laser microscopy, and immunogold electron microscopy. Anti-PrPC immunolabelling of rimmed vacuoles was observed in IBM, inclusions of myofibrillary myopathy, targets, regenerating, and atrophic fibres, mononuclear cells, in addition to ragged red fibres in mitochondrial myopathies, and focal sarcolemmal immunostaining in non-diseased controls. Quantitative analysis demonstrated that, in neurogenic muscle lesions, anti-PrPC staining detects a significantly broader spectrum of fibres than anti-vimentin or anti-NCAM. In dystrophic muscle, PrPC expression was mainly restricted to regenerating fibres. In IBM, PrPC expression was not confined to rimmed vacuoles or vacuolated fibres and only a small percentage (7.1%) of rimmed vacuoles were PrPC positive. Ultrastructurally, PrPC was observed in the cytoplasm of lymphocytes, in the myofibrillar network of targets, and in rimmed vacuoles. Knowledge of disease circumstances with altered expression of PrPC is important in the setting of a potentially increased chance for extraneural PrPC-PrPSc conversion. In addition, our observations suggest that PrPC may have a general stress-response effect in various neuromuscular disorders., (Copyright (c) 2004 Pathological Society of Great Britain and Ireland.)

Research has shown that the use of a muscle-specific promoter can reduce immune response and improve gene transfer to muscle fibers. We investigated the efficiency of direct and ex vivo gene transfer to the skeletal muscles of 6- to 8-week-old mdx mice by using two adenoviral vectors: adenovirus (AD) encoding the luciferase gene under the cytomegalovirus (CMV) promoter (ADCMV) and AD encoding the same gene under the muscle creatine kinase (MCK) promoter (ADMCK). Direct intramuscular injection of ADMCK triggered a lower immune response that enabled more efficient delivery and more persistent expression of the transgene than did ADCMV injection. Similarly, ex vivo gene transfer using ADCMV-transduced muscle-derived stem cells (MDSCs) induced a stronger immune response and led to shorter transgene expression than did ex vivo gene transfer using ADMCK-transduced MDSCs. This immune response was due to the release of the antigen after MDSC death or to the ADCMV-transduced MDSCs acting as antigen-presenting cells (APCs) by expressing the transgene and rapidly initiating an immune response against subsequent viral inoculation. The use of a muscle-specific promoter that restricts transgene expression to differentiated muscle cells could prevent MDSCs from becoming APCs, and thereby could improve the efficiency of ex vivo gene transfer to skeletal muscle.

Skeletal muscle is a unique tissue whose function is dependent in great part on its ultrastructure. Repeated intense muscular contractions, especially those resulting in muscle lengthening, can lead to alterations in muscle structure (i.e., muscle damage) and subsequent decline in contractile force. The damage-induced decline in contractile force can have a significant impact on exercise performance during an athletic performance. In some disease conditions such as Duchenne muscular dystrophy (DMD), the muscles are more vulnerable to contraction-induced damage than normal muscle. In the case of the respiratory muscles, for example, the diaphragm, the consequences of muscle weakness secondary to damage are profound in that respiratory failure leading to premature death often ensues. In normal skeletal muscle, damage is followed by an inflammatory response involving multiple cell types that subsides after several days. This transient inflammatory response is a normal homeostatic reaction to muscle damage. In contrast, a persistent inflammatory response is observed in dystrophic skeletal muscle that leads to an altered extracellular environment, including an increased presence of inflammatory cells (e.g., macrophages) and elevated levels of various inflammatory cytokines (e.g., TNF-alpha, TGF-beta). The signals that lead to successful muscle repair in healthy muscle may promote muscle wasting and fibrosis in dystrophic muscle. Preliminary data indicate that immunosuppression in dystrophic (mdx) mice has beneficial effects on some indices of muscle dysfunction, thereby indicating that targeted immunosuppression may offer some promise in delaying the pathological progression of this insidious muscular disease.

Muscle inflammation is characteristic of inflammatory myopathies but also occurs in muscular dystrophy with lack of the sarcolemmal protein dysferlin. We quantified inflammatory cells and major histocompatibility complex (MHC) expression in muscle from 10 patients with dysferlinopathy. Infiltrating cells were always present although numbers varied considerably; macrophages were more common than T cells, T cytotoxicity was absent, and MHC class I was overexpressed on muscle fibers. These findings differ from polymyositis (PM) but are closely similar to those in SJL/J mice (which lack dysferlin) and emphasize the relationship between absence of dysferlin and immune system abnormalities in muscle.

Targeted correction of mutations in muscle can be delivered by direct i.m. injection of corrective DNA to the dystrophic muscle or by autologous injection of cells that have been genetically corrected after isolation from the individual with the dystrophic muscle. The successful application of chimeraplasty and short fragment homologous replacement to correct the exon 23 nonsense mdx transition at the mouse dys locus has opened up the possibility that with further development, targeted gene correction may have some future application for the treatment of muscular dystrophies. In vitro, application of targeted gene correction at the mdx dys locus results in better correction efficiencies than when applied directly to dystrophic muscle. This suggests that at least for the time being, a strategy involving ex vivo correction may be advantageous over a direct approach for delivery of gene correction to dystrophic muscle. This, particularly in view of recent developments indicating that bone-marrow-derived cells are able to systemically remodel dystrophic muscle, whilst penetration of DNA introduced to muscle is limited to individually injected muscles. Application of targeted gene correction to Duchenne dystrophy needs to account for the fact that about 65% of Duchenne muscular dystrophy cases involve large frame-shift deletion of gene sequence at the dys locus. Traditionally, whilst targeted gene correction is able to restore point mutations entirely, it remains to be seen as to whether a strategy for the 'correction' of frame shift deletions may be engineered successfully. This communication discusses the possibility of applying targeted gene correction to dystrophic muscle in Duchenne dystrophy.

Development of gene therapy for the muscular dystrophies represents a daunting challenge requiring significant advances in our knowledge of the defective genes, muscle promoters, viral vectors, immune system surveillance and methods for systemic delivery of vectors. However, tremendous progress has been made in developing improved viral vectors and avoiding immune reactions against gene transfer. This review summarizes recent progress and highlights problems that must be solved before an effective treatment is available.

Objective: To assess the presence of CD59 and the deposition of membrane attack complex (MAC) of complement system in skeletal muscle from patients with juvenile dermatomyositis (JDM), in comparison to patients with muscular dystrophies (MD) and children with normal muscle biopsies., Methods: Muscle specimens obtained for diagnostic purposes from 10 patients with JDM, 6 with MD, and 7 children whose biopsies showed normal histology were analyzed. Immunohistological staining was performed using Mab against CD59 (YTH 53.1) and MAC (WU 7.2)., Results: Immunohistochemical staining for CD59 was weak and irregularly distributed on muscle fibers of all patients with JDM. Two of the 9 biopsies that allowed analysis of vessels showed negative CD59 staining in all vessels; in the remaining 7 patients, there was weak staining in a proportion of the vessels. In contrast, uniform and strong or moderate immunoreactivity was detected on the sarcolemma and in intramuscular endothelium in all normal and MD samples. Immunostaining for MAC was strong in JDM muscle vessels, and weak in normal or MD muscle. An inverse relation was found between MAC deposition and presence of CD59 in vessels in 6/9 JDM biopsies and in all normal and MD samples., Conclusion: Decreased CD59 expression in JDM muscle fibers and vessels may be associated with muscle lesions mediated by deposition of MAC of complement in JDM.

Based on originally designed technique of myoblast cultivation and in accordance with the approved by the Russian Ministry of Health "one muscle treatment" protocol of myoblast transplantation to the Duchenne muscular dystrophy patients, the first in Russia clinical trial of this gene correction method was carried out. Immonologically related myoblast cultures (30 to 90 million cells per patient) were injected after all preliminary procedures into tibialis anterior muscles of four boys selected from a group of volunteer recipients (Duchenne muscular dystrophy patients) based on the analysis of a number of surface antigens in donor-recipient pairs. The condition of the patients remained satisfactory during the whole period of post-transplantation follow-up (from 6 months to 1.5 years). Six months after myoblast transplantation the presence of donor DNA or dystrophin synthesis was demonstrated in muscle biopsies of three out of four patients. This result confirms efficacy and safety of the procedure used.

Aim: At present, the diagnosis of muscular dystrophy is made by means of immunohistochemistry on frozen sections. The aim of this study was to develop a sensitive and reproducible immunohistochemical method for use on formalin fixed, paraffin wax embedded sections for the demonstration of dystrophin associated proteins and other muscle associated antigens., Methods: All the cases studied were from the files of the department of histopathology, Great Ormond Street Hospital for Children NHS Trust. Immunohistochemistry was performed on paraffin wax embedded sections with heat mediated antigen retrieval and overnight incubation with the antibodies at room temperature. Four different pretreatment buffers were tested in the attempt to optimise the immunostaining. Frozen sections were run in parallel for direct comparison., Results: All the antibodies except delta sarcoglycan gave strong, consistent immunostaining in paraffin wax embedded sections, comparable with the frozen sections. The most consistent results were obtained using citrate/EDTA as the pretreatment buffer., Conclusion: A reliable and reproducible technique has been established, using a heat mediated citrate/EDTA buffer antigen retrieval method, which works well for most of the antibodies needed to make the diagnosis of muscular dystrophy in formalin fixed, paraffin wax embedded sections. This technique overcomes some of the inherent problems encountered using frozen muscle tissue and it could become a valuable tool for the diagnosis of muscular dystrophy.

Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.

In previous in vitro studies, we proposed a role for the extracellular matrix component, laminin-2, and its integrin receptor, VLA-6, in thymocyte development. The characterization of two dystrophic mouse strains with different defects in laminin-2 allowed us to examine this proposal in vivo. Mice deficient in laminin-2, dy/dy, show a significant reduction in thymus size and number of thymocytes compared to normal littermates. These mice also exhibited apparent alterations of thymic architecture. Examination of the CD4/CD8 populations in dy/dy thymi showed large relative increases in the DN (CD4- CD8-) and SP (CD4+ CD8-, CD4- CD8+) populations and a significant decrease in the DP (CD4+ CD8+) population. Further examination of the DN population for CD44 and CD25 expression showed a remarkable decrease in the more mature pre-T cell populations. Analysis of apoptosis in situ, and by flow cytometry, in dy/dy thymi revealed a significant increase in apoptotic DN thymocytes in the capsule and subcapsular regions. Interestingly, thymocyte development appeared to proceed normally in dystrophic mice expressing a mutant form of laminin-2, dy2J, as well as, in fetal and neonatal dy/dy mice. We propose that laminin-2 plays an active role in thymocyte development by delivering cell survival and differentiation signals at specific stages of development in young adult mice.

A patient with Becker muscular dystrophy had transient arthritis. During the active illness his serum creatine kinase (CK) level normalized and serum levels of soluble interleukin 2 (IL-2) receptor, IL-6, IL-1 receptor antagonist, and soluble tumor necrosis factor receptor 2 were elevated. CK increased to his usual levels after arthritis remission whereas the levels of inflammatory cytokines and their inhibitors decreased.