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Objective: CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity., Results: We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPβ isoforms.

Objective: Although more than a dozen myositis-specific autoantibodies (MSAs) have been identified, most patients with myositis are positive for a single MSA. The specific overexpression of a given myositis autoantigen in myositis muscle has been proposed as initiating and/or propagating autoimmunity against that particular autoantigen. The present study was undertaken to test this hypothesis., Methods: In order to quantify autoantigen RNA expression, RNA sequencing was performed on muscle biopsy samples from control subjects, MSA-positive patients with myositis, regenerating mouse muscles, and cultured human muscle cells., Results: Muscle biopsy samples were available from 20 control subjects and 106 patients with autoantibodies recognizing hydroxymethylglutaryl-coenzyme A reductase (n = 40), signal recognition particles (n = 9), Jo-1 (n = 18), nuclear matrix protein 2 (n = 12), Mi-2 (n = 11), transcription intermediary factor 1γ (n = 11), or melanoma differentiation-associated protein 5 (n = 5). The increased expression of a given autoantigen in myositis muscle was not associated with autoantibodies recognizing that autoantigen (all q > 0.05). In biopsy specimens from both myositis muscle and regenerating mouse muscles, autoantigen expression correlated directly with the expression of muscle regeneration markers and correlated inversely with the expression of genes encoding mature muscle proteins. Myositis autoantigens were also expressed at high levels in cultured human muscle cells., Conclusion: Most myositis autoantigens are highly expressed during muscle regeneration, which may relate to the propagation of autoimmunity. However, factors other than overexpression of specific autoantigens are likely to govern the development of unique autoantibodies in individual patients with myositis., (© 2019 American College of Rheumatology. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

Background: IL-17A has effects on several cell types and is a therapeutic target in several inflammatory diseases. IL-17F shares 50% homology and biological activities with IL-17A. It is now of interest to target both cytokines. The objective was to compare the IL-17A and IL-17F effect on cytokine production by RA synoviocytes, and to extend to other cells. Methods: Cells (RA synoviocytes, psoriasis skin fibroblasts, endothelial cells, myoblasts, and hepatocytes) were cultured in the presence or not of: IL-17A, IL-17F, TNF, IL-1β alone or their combinations, IL-17A/TNF, IL-17A/IL-1β, IL-17A/TNF/IL-1β, IL-17F/TNF, IL-17F/IL-1β, and IL-17F/TNF/IL-1β. All experiments were performed in parallel to reduce variability. After 48 h, supernatants were recovered and IL-6 and IL-8 levels were measured by ELISA. Results: IL-17A and IL-17F alone increased significantly IL-6 and IL-8 productions by synoviocytes, with a stronger effect for IL-17A. For IL-6 production, TNF or IL-1β alone had the largest effect on myoblasts (5-fold increase), while for IL-8 production, it was on skin fibroblasts (5-fold increase). The IL-17A/TNF synergistic increase was observed on all cells for IL-6; and for IL-8, except for endothelial cells. For IL-17F/TNF, except with endothelial cells, a synergistic effect was also observed, but less powerful than with IL-17A/TNF. IL-17A/IL-1β or IL-17F/IL-1β effect was cell-type dependent, with an additive effect for synoviocytes (1.6 and 2-fold increase, respectively for IL-6, and 1.8 and 2-fold increase, respectively for IL-8) and a synergistic effect for hepatocytes (3.8 and 4.2-fold increase, respectively for IL-6, and 6 and 2-fold increase, respectively for IL-8). The three-cytokine combination induced an additive effect for synoviocytes and a synergistic effect for skin fibroblasts. Conclusion: IL-17A and IL-17F acted similarly by inducing pro-inflammatory cytokine secretion, with a stronger response intensity with IL-17A. Their activities were potentiated by the combination with TNF and IL-1β, with an effect dependent on the cell type.

Recent studies have reported that the immune system significantly mediates skeletal muscle repair and regeneration. Additionally, biological scaffolds have been shown to play a role in polarizing the immune microenvironment toward pro-myogenic outcomes. Moreover, myostatin inhibitors are known to promote muscle regeneration and ameliorate fibrosis in animal models of Duchenne muscular dystrophy (DMD), a human disease characterized by chronic muscle degeneration. Biological scaffolds and myostatin inhibition can potentially influence immune-mediated regeneration in the dystrophic environment, but have not been evaluated together. Toward this end, here we created an injectable biological scaffold composed of hyaluronic acid and processed skeletal muscle extracellular matrix. This material formed a cytocompatible hydrogel at physiological temperatures in vitro When injected subfascially above the tibialis anterior muscles of both WT and dystrophic mdx -5 Cv mice, a murine model of DMD, the hydrogel spreads across the entire muscle before completely degrading at 3 weeks in vivo We found that the hydrogel is associated with CD206 + pro-regenerative macrophage polarization and elevated anti-inflammatory cytokine expression in both WT and dystrophic mice. Co-injection of both hydrogel and myostatin inhibitor significantly increased FoxP3 + regulatory T cell modulation and Foxp3 gene expression in the scaffold immune microenvironment. Finally, delivery of myostatin inhibitor with the hydrogel increased its bioactivity in vivo , and transplantation of immortalized human myoblasts with the hydrogel promoted their survival in vivo This study identifies a key role for biological scaffolds and myostatin inhibitors in modulating a pro-regenerative immune microenvironment in dystrophic muscle.

Enterovirus 71 (EV71) has caused major outbreaks of hand, foot and mouth disease. EV71 infections increase the production of many host cytokines and pro-inflammatory factors, including interleukin (IL)-6, IL-10 and COX-2. Some of these molecules could stimulate the signal transducer and activator of transcription 3 (STAT3), which plays a key role in regulating host immune responses and several viral diseases. However, the role of STAT3 in EV71 infection remains unknown. This study found that the phosphorylation levels of STAT3 (p Y705 -STAT3) are closely related to EV71 infection. Further experiments revealed that STAT3 exerts an anti-EV71 activity. However, the antiviral activity of STAT3 is partially antagonized by EV71-induced miR-124, which directly targets STAT3 mRNA. Similarly, IL-6R, the α-subunit of the IL-6 receptor complex, exhibits anti-EV71 activity and is directly targeted by the virus-induced miR-124. These results indicate that EV71 can evade host IL-6R- and STAT3-mediated antiviral activities by EV71-induced miR-124. This suggests that controlling miR-124 and the downstream targets, IL-6R and STAT3, might benefit the antiviral treatment of EV71 infection.

Inflammatory myopathy is a rare autoimmune muscle disorder. Treatment typically focuses on skeletal muscle weakness or inflammation within muscle, as well as complications of respiratory failure secondary to respiratory muscle weakness. Impaired respiratory muscle function contributes to increased dyspnea and reduced exercise capacity in pulmonary hypertension (PH), a debilitating condition that has few treatment options. The initiation and progression of PH is associated with inflammation and inflammatory cell recruitment and it is established that hypoxia-induced mitogenic factor (HIMF, also known as resistin-like molecule α), activates macrophages in PH. However, the relationship between HIMF and inflammatory myoblasts remains unclear. This study investigated the signaling pathway involved in interleukin-18 (IL-18) expression and its relationship with HIMF in cultured myoblasts. We found that HIMF increased IL-18 production in myoblasts and that secreted IL-18 promoted tube formation of the endothelial progenitor cells. We used the mouse xenograft model and the chick chorioallantoic membrane assay to further explore the role of HIMF in inflammatory myoblasts and angiogenesis in vivo. Thus, our study focused on the mechanism by which HIMF mediates IL-18 expression in myoblasts through angiogenesis in vitro and in vivo. Our findings provide an insight into HIMF functioning in inflammatory myoblasts.

Background: Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or with various endogenous diseases that are associated with metabolic endotoxemia. Involuntary loss of skeletal muscle, termed muscle wasting, is commonly observed in these conditions, suggesting that circulating LPS might play an essential role in its development. Although impairment of muscle regeneration is an important determinant of skeletal muscle wasting, it is unclear whether LPS affects this process and, if so, by what mechanism. Here, we used the C2C12 myoblast cell line to investigate the effects of LPS on myogenesis., Methods: C2C12 myoblasts were grown to 80% confluence and induced to differentiate in the absence or presence of LPS (0.1 or 1 μg/mL); TAK-242 (1 μM), a specific inhibitor of Toll-like receptor 4 (TLR4) signaling; and a tumor necrosis factor (TNF)-α neutralizing antibody (5 μg/mL). Expression of a skeletal muscle differentiation marker (myosin heavy chain II), two essential myogenic regulatory factors (myogenin and MyoD), and a muscle negative regulatory factor (myostatin) was analyzed by western blotting. Nuclear factor-κB (NF-κB) DNA-binding activity was measured using an enzyme-linked immunosorbent assay., Results: LPS dose-dependently and significantly decreased the formation of multinucleated myotubes and the expression of myosin heavy chain II, myogenin, and MyoD, and increased NF-κB DNA-binding activity and myostatin expression. The inhibitory effect of LPS on myogenic differentiation was reversible, suggesting that it was not caused by nonspecific toxicity. Both TAK-242 and anti-TNF-α reduced the LPS-induced increase in NF-κB DNA-binding activity, downregulation of myogenic regulatory factors, and upregulation of myostatin, thereby partially rescuing the impairment of myogenesis., Conclusions: Our data suggest that LPS inhibits myogenic differentiation via a TLR4-NF-κB-dependent pathway and an autocrine/paracrine TNF-α-induced pathway. These pathways may be involved in the development of muscle wasting caused by sepsis or metabolic endotoxemia.

Stem cell therapy is a promising strategy for treatment of muscular dystrophies. In addition to muscle fiber formation, reconstitution of functional stem cell pool by donor cells is vital for long-term treatment. We show here that some CD133(+) cells within human muscle are located underneath the basal lamina of muscle fibers, in the position of the muscle satellite cell. Cultured hCD133(+) cells are heterogeneous and multipotent, capable of forming myotubes and reserve satellite cells in vitro. They contribute to extensive muscle regeneration and satellite cell formation following intramuscular transplantation into irradiated and cryodamaged tibialis anterior muscles of immunodeficient Rag2-/γ chain-/C5-mice. Some donor-derived satellite cells expressed the myogenic regulatory factor MyoD, indicating that they were activated. In addition, when transplanted host muscles were reinjured, there was significantly more newly-regenerated muscle fibers of donor origin in treated than in control, nonreinjured muscles, indicating that hCD133(+) cells had given rise to functional muscle stem cells, which were able to activate in response to injury and contribute to a further round of muscle regeneration. Our findings provide new evidence for the location and characterization of hCD133(+) cells, and highlight that these cells are highly suitable for future clinical application.

Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation.

Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers.

Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function.

Interleukin-27 (IL-27) is a new IL-12-related heterodimeric cytokine comprising a novel p28 molecule and the Epstein-Barr-virus-induced gene 3 (EBI3) molecules. It augments initiation of T helper type 1-mediated immunity by enhancing the proliferation and cytokine production of T cells. In this study, we examined whether a secreted form of IL-27 subunits would inhibit IL-27-mediated immunological responses. COS-7 cells transduced with the mouse (m) p28 gene secreted a monomeric mp28 protein; however, those transduced with the mEBI3 gene did not detect a mEBI3 protein in the culture supernatants. The secreted mp28 prevented the IL-27-mediated signal transduction and activator of transcription 1 phosphorylation and subsequently inhibited the IL-27-mediated intercellular adhesion molecule-1 induction and interferon-gamma production in CD4(+) T cells. We generated mp28-expressing murine carcinoma Colon 26 cells and inoculated a mixture of the mp28- and mIL-27-expressing Colon 26 cells into syngeneic BALB/c mice. Simultaneous production of mp28 and mIL-27 from Colon 26 cells suppressed IL-27-mediated anti-tumour effects in the mice. We examined the p28-mediated immune suppression by inoculating mp28-expressing myoblasts into allogeneic mice. Forced production of mp28 suppressed the allogeneic cytotoxic T-lymphocyte induction and subsequently retarded the graft rejection. Furthermore, production of both mp28 and mp40, which inhibits the functions of IL-12 and IL-23, prolonged the graft survival longer than the grafts expressing either mp28 or mp40. We propose that p28 can be a regulatory subunit for IL-27-mediated cellular immune responses and a possible therapeutic agent to suppress unfavourable immune responses.

Duchenne Muscular Dystrophy (DMD) is the most common and severe form of muscular dystrophy in humans. The goal of myogenic stem cell transplant therapy for DMD is to increase dystrophin expression in existing muscle fibers and to provide a source of stem cells for future muscle generation. Although syngeneic myogenic stem cell transplants have been successful in mice, allogeneic transplants of myogenic stem cells were ineffective in several human trials. To determine whether allogeneic muscle progenitor cells can be successfully transplanted in an immune-tolerant recipient, we induced immune tolerance in two DMD-affected (cxmd) dogs through hematopoietic cell transplantation (HCT). Injection of freshly isolated muscle-derived cells from the HCT donor into either fully or partially chimeric xmd recipients restored dystrophin expression up to 6.48% of wild-type levels, reduced the number of centrally located nuclei, and improved muscle structure. Dystrophin expression was maintained for at least 24 weeks. Taken together, these data indicate that immune tolerance to donor myoblasts provides an important platform from which to further improve myoblast transplantation, with the goal of restoring dystrophin expression to patients with DMD.

Tissue fibrosis evolving from impaired tissue remodeling after injury is characterized by myofibroblast accumulation. We propose that during the development of fibrosis myofibroblasts acquire an immune-privileged cell phenotype, allowing their uninterrupted accumulation. Using the murine model of bleomycin-induced lung fibrosis in mice, we show that myofibroblasts that accumulate in lungs with fibrosis, but not in normal lungs, kill Fas(+) lymphocytes, resist Fas-induced apoptosis, and survive longer when grafted into allogeneic mice. In contrast, bleomycin-treated FasLigand (FasL)-deficient (gld) chimeric mice did not accumulate myofibroblasts or collagen in their lungs, and their FasL(-) myofibroblasts did not survive after alloengraftment. This finding indicates that myofibroblasts possess Fas/FasL-pathway-dependent characteristics that allow them to escape from immune surveillance and resulting organ fibrosis.

Objective: We sought to investigate immune cell kinetics in relation to skeletal myoblast survival and heart function improvement after nonautologous skeletal myoblast transplantation in a rat model of myocardial infarction., Methods: One week after myocardial infarction, 208 Wistar rats were grouped into group 1 (n = 24, receiving 150 muL of medium only), group 2 (n = 24, receiving 150 muL of medium and cyclosporine [INN: ciclosporin]), group 3 (n = 40, human skeletal myoblast transplantation), group 4 (n = 40, human skeletal myoblast transplantation with cyclosporine treatment), group 5 (n = 40, rat skeletal myoblast transplantation), and group 6 (n = 40, rat skeletal myoblast transplantation with cyclosporine treatment). The hearts were harvested at 10 minutes and 1, 4, 7, and 28 days after cell transplantation. Skeletal myoblast survival was confirmed by means of immunohistochemical studies and quantified by using real-time polymerase chain reaction. Host immune responses were assessed by immunostaining for macrophages and CD4+ and CD8+ lymphocytes. Heart function was evaluated by means of echocardiographic analysis., Results: The majority of macrophages and lymphocytes infiltrated in the acute phase (from day 1 to day 7) and then subsided by day 28. The donor skeletal myoblasts survived and differentiated well in all skeletal myoblast transplantation groups. Allogeneic skeletal myoblasts showed a superior survival rate than xenogeneic skeletal myoblasts (P < .01). Cyclosporine inhibited the infiltration of the immunocytes, enhanced skeletal myoblast survival, and improved heart performance compared with that seen in the groups not receiving cyclosporine treatment (P < .05)., Conclusions: Allomyoblasts survive better than do xenomyoblasts after transplantation into infarcted myocardium. After inhibition of immunocyte infiltration by means of immunosuppressive treatment, skeletal myoblast survival is enhanced, with improved heart performance. These findings suggest the feasibility of nonautologous myoblast transplantation with immunosuppressive treatment.

The immune response to soluble antigens constitutes a current clinical problem impeding the development of protein therapeutics. We have developed an encapsulated-cell delivery system, which, transiently combined with an anti-CD154 antibody treatment, allows for the suppression of this immune response and the establishment of long-term secretion of a foreign antigen, human erythropoietin (huEPO). The chronic presence of antigen appears to be required to maintain this tolerance, as a 21-day gap in the exposure to huEPO is sufficient to restore the ability of mice to mount an antibody response. In contrast, chronic huEPO expression maintains tolerance even in the absence of further anti-CD154 treatment. These results suggest that a soluble antigenic protein can be continuously released, without inducing an antibody response, using encapsulated allogeneic cells. The temporary anti-CD154 treatment induces immune unresponsiveness to the delivered antigen, while the immunoprotected cell implant allows for chronic antigen release, favoring the establishment of tolerance.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease characterized by a typical regional distribution, featuring composed patterns of clinically affected and unaffected muscles. No treatment is available for this condition, in which the pathophysiological mechanism is still unknown. Autologous transfer of myoblasts from unaffected to affected territories could be considered as a potential strategy to delay or stop muscle degeneration. To evaluate the feasibility of this concept, we explored and compared the growth and differentiation characteristics of myoblasts prepared from phenotypically unaffected muscles of five FSHD patients and 10 control donors. According to a clinically approved procedure, 10(9) cells of a high degree of purity were obtained within 16-23 days. More than 80% of these cells were myoblasts, as demonstrated by labeling of the muscle markers CD56 and desmin. FSHD myoblasts presented a doubling time equivalent to that of control cells; they kept high proliferation ability and did not show early telomere shortening. In vitro, these cells were able to differentiate and to express muscle-specific antigens. In vivo, they participated to muscle structures when injected into immunodeficient mice. These data suggest that myoblasts expanded from unaffected FSHD muscles may be suitable tools in view of autologous cell transplantation clinical trials.

The treatment of several cancers with anti-idiotype antibodies has shown promising results in animals and clinical trials. A common drawback of many anti-idiotypic antibodies is their low immunogenicity. The aim of this work was to construct a new delivery form for the anti-idiotypic antibody ACA125hFc, with the goal of improving its immunogenicity as vaccine against ovarian carcinoma. Designed on bioencapsulation technologies, we generated an in vitro depot that acts as a continuous delivery system for the anti-idiotypic antibody ACA125hFc. C2C12 myoblasts were transfected with the anti-idiotypic chimeric antibody ACA125hFc, which mimics the CA125 tumor antigen and which consists of variable regions of the monoclonal murine antibody ACA125 (currently in clinical trial) and the human IgG Fc domain. Recombinant myoblasts were encapsulated in 1-cm semipermeable, polyethersulfone (PES), or polyvinylidene difluoride (PVDF) hollow fibers, which differ in their molecular weight cutoff (MWCO). Encapsulated cells were evaluated in vitro for viability and antibody secretion over a period of 3 months. PES hollow fibers with a higher MWCO showed a twofold higher secretion rate of chACA125hFc compared to PES devices with a lower MWCO. No remarkable level of ACA125hFc could be detected for PVDF devices. The expression levels of the anti-idiotypic antibody ACA125hFc from capsules with a lower MWCO could be improved substantially, in both PES as well as PVDF, by inserting an internal polyethyleneterephtalate (PET) yarn. We conclude that murine recombinant C2C12 myoblasts encapsulated in PES as well as PVDF hollow fibers containing an internal PET matrix can act as a long-term secretion system for anti-idiotypic antibodies.

Gene transfer with recombinant adenoviruses (rAds) is a powerful means of inducing an immune response against a transgene product. However, little is known about the mechanisms that underlie the induction of the immune response after intramuscular inoculation of adenovirus and, in particular, the relative role of the different cell types transduced. Several studies have suggested that CD8+ cytotoxic T lymphocyte responses elicited after inoculation of adenoviruses (Ads) are induced both by direct transduction of antigen presenting cells (APCs) and by cross-priming. In the present study, a library of fibre-chimeric rAds was screened in order to identify rAds with distinct capacities to express transgene product in murine cell types naturally found in muscle, i.e. myoblasts, endothelial cells (both representing non-APCs) and dendritic cells (representing APCs). Four selected pseudotypes, differing in their ability to infect muscular cells were used to immunize C57BL/6 mice. The relationship between the capacity to transduce non-APC or APC in vitro and the ability to induce humoral and cellular responses against the beta-galactosidase antigen after intramuscular inoculation were studied. Results indicate that CD8+ T cell responses against the beta-galactosidase antigen were similar after inoculation of the four viruses, thus revealing no direct relationship with their ability to transduce myoblasts, endothelial cells or dendritic cells in vitro.

There is a massive and rapid death of donor myoblasts (<20% surviving) within hours after intramuscular injection in myoblast transfer therapy (MTT), due to host immune cells, especially natural killer (NK) cells. To investigate the role of host immune cells in the dramatic death of donor myoblasts, MTT experiments were performed in irradiated host mice. Cultured normal C57BL/10ScSn male donor myoblasts were injected into muscles of female C57BL/10ScSn-Dmdmdx host mice after one of three treatments: whole body irradiation (WBI) to eliminate all circulating leukocytes, WBI and bone marrow reconstitution (BMR), or local irradiation (or protection) of one limb. Similar experiments were performed in host mice after antibody depletion of NK cells. Numbers of male donor myoblasts were quantified using a Y-chromosome-specific (male) probe following total DNA extraction of injected muscles. WBI prior to MTT resulted in dramatically enhanced survival (approximately 80%) of donor myoblasts at 1 hour after MTT, supporting a central role for host inflammatory cells in the initial death of donor myoblasts seen in untreated host mice. BMR restored the massive and rapid loss (approximately 25% surviving) of donor myoblasts at 1 hour after MTT. Local pre-irradiation also resulted in increased donor myoblast numbers (approximately 35-40%) compared with untreated controls (approximately 10%) at 3 weeks after MTT. Preirradiation of host muscle with 10 Gy did not significantly stimulate proliferation of the injected donor myoblasts. Serum protein levels of TNFalpha, IL-1beta, IL-6 and IL-12 fluctuated following irradiation treatments. These combined results strongly reinforce a major role for host immune cells in the rapid death of injected cultured donor myoblasts.

The observation that CD45(+) stem cells injected into the circulation participate in muscle regeneration raised the question of whether CD45(+) stem cells resident in muscle play a physiological role during regeneration. We found that CD45(+) cells cultured from uninjured muscle were uniformly nonmyogenic. However, CD45(+) cells purified from regenerating muscle readily gave rise to determined myoblasts. The number of CD45(+) cells in muscle rapidly expanded following injury, and a high proportion entered the cell cycle. Investigation of candidate pathways involved in embryonic myogenesis revealed that Wnt signaling was sufficient to induce the myogenic specification of muscle-derived CD45(+) stem cells. Moreover, injection of the Wnt antagonists sFRP2/3 into regenerating muscle markedly reduced CD45(+) stem cell proliferation and myogenic specification. Our data therefore suggest that mobilization of resident CD45(+) stem cells is an important factor in regeneration after injury and highlight the Wnt pathway as a potential therapeutic target for degenerative neuromuscular disease.

Determination and differentiation of cells in the skeletal muscle lineage is positively regulated by cell-cell contact. Cell-surface proteins proposed to mediate this effect include both classical cadherins and Ig superfamily members; potential interactions between the promyogenic activities of these classes of protein, however, are unknown. We show here that CDO and BOC, two promyogenic Ig superfamily members that bind to each other in a cis fashion, form complexes with N- and M-cadherin. These complexes contain beta-catenin and are enriched at sites of cell-cell contact between myoblasts. In transient expression assays, the ectodomains and intracellular regions of CDO, BOC, and N-cadherin each interact independently, suggesting that the interactions occur in a cis fashion; consistent with this conclusion, cadherin-mediated cell adhesion is not required for them to occur. Stable expression in myoblasts of a CDO deletion mutant deficient in its ability to associate with N-cadherin interferes with differentiation as assessed by biochemical, morphological, and reporter gene assays, suggesting that this interaction is functionally important in myogenesis. Thus, some of the cell-cell contact-mediated activities that are required for myogenesis seem to be based on interdependent activities of promyogenic classical cadherins and Ig superfamily members.

HLA-G is a non-classical MHC class I molecule with highly limited tissue distribution which has been attributed chiefly immune-regulatory functions. We previously have reported that HLA-G is expressed in inflamed muscle in vivo and by cultured myoblasts in vitro. Here, we used the in vitro models of human myoblasts or TE671 muscle rhabdomyosarcoma cells to characterize the functional role of HLA-G for muscle immune cell interactions. Gene transfer of the two major isoforms of HLA-G (transmembranous HLA-G1 and soluble HLA-G5) into TE671 rendered these cells resistant to alloreactive lysis by direct inhibition of natural killer (NK) cells, and CD4 and CD8 T cells. Further, HLA-G reduced alloproliferation, interfered with effective priming of antigen-specific cytotoxic T cells and reduced antigen-specific alloreactive lysis. HLA-G pre-induced on cultured myoblasts inhibited lysis by alloreactive peripheral blood mononuclear cells. This protection was reversed by a neutralizing HLA-G antibody. Interestingly, a few HLA-G-positive cells within a population of HLA-G-negative muscle target cells conveyed significant inhibitory effects on alloreactive lysis. Our results reveal further insights into the immunobiology of muscle and suggest that ectopic expression of HLA-G may promote the survival of transplanted myoblasts in the future treatment of hereditary muscle diseases. Further, HLA-G could represent a novel self-derived anti-inflammatory principle applicable in strategies against inflammatory aggression.