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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.

Chronic inflammatory diseases such as insulin resistance, Type 2 diabetes, neurodegenerative diseases etc., are shown to be caused due to imbalanced activation states of macrophages. MicroRNAs which are transcriptional/post-transcriptional regulators of gene expression drive several pathophysiological processes including macrophage polarization. However the functional role of microRNAs in regulating inflammation induced insulin resistance is ill defined. In our current study we observed that the expression of miR-712 was reduced in macrophages exposed to LPS and IFN-γ. Ectopic expression of miR-712 in RAW 264.7 mouse macrophages impaired the expression of iNOS protein and secretion of pro-inflammatory cytokines such as TNF-α, IL-6 and IFN-β which in turn led to improved insulin stimulated glucose uptake in co-cultured L6 myoblasts. Mechanistically, we identified that miR-712 targets the 3'UTR of a potent inflammatory gene LRRK2 and dampens the phosphorylation of p38 and ERK1/2 kinases. Taken together, our data underscore the regulatory role of miR-712 in restoring insulin stimulated glucose uptake by myoblasts through down-regulating macrophage mediated inflammatory responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Idiopathic inflammatory myopathies (IIMs) are diseases with muscle weakness, morphologically characterized by inflammatory infiltration and increased expression of MHC class I molecule on myofibers. Immunoproteasome, as a proteolytic complex that shapes the repertoire of antigenic peptides, has been previously demonstrated to be over-expressed in IIMs at mRNA level. In this study, we investigated the expression and the function of the immunoproteasome in IIMs in more detail. As shown by immunofluorescence staining, expression of relevant players of the immunoproteasome was detectable in the inflamed skeletal muscle tissue from IIM patients. In fact, two subunits of the immunoproteasome, β1i or β5i were upregulated in sporadic inclusion body myositis, immune-mediated necrotizing myopathies and dermatomyositis muscle biopsies and co-localized with the MHC class I expressing myofibers. Double immunofluorescence revealed that both myofibers and muscle infiltrating cells, including CD8 + T-cells and CD68  +  macrophages in IIMs expressed β1i or β5i. In addition, we have also investigated the role of the immunoproteasome in myoblasts during in vitro inflammatory conditions. Using human primary myoblasts cultures we found that pro-inflammatory cytokines, TNF-α or IFN-γ upregulate β1i or β5i. Selective inhibition or depletion of β5i amplified the TNF-α or IFN-γ mediated expression of cytokines/chemokines (myokines) in myoblasts. Furthermore, we demonstrated that specific inhibitors of β1i or β5i reduced the cell surface expression of MHC class I in myoblasts induced by IFN-γ. Taken together, our data suggest that the immunoproteasome is involved in pathologic MHC class I expression and maintenance of myokine production in IIMs. Thus, induction of the immunoproteasome was identified as a pathomechanism underlying inflammation in IIMs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Emodin, a major component of Rheum palmatum, has been reported to significantly protect neural tissue against apoptosis and autophagy. However, the effects and underlying mechanisms of action of emodin in muscle atrophy are still poorly defined. In this study, we investigated the protective effects and the underlying mechanisms by which emodin acts on tumor necrosis factor alpha (TNF-α)-induced apoptosis and autophagy in mouse C2C12 myoblasts. Emodin, at various concentrations, decreased TNF-α-induced apoptosis in C2C12 myoblasts, which were analyzed by Hoechst 33342 staining and annexin V/PI analysis. Emodin also inhibited the collapse of the mitochondrial membrane potential and the generation of reactive oxygen species in TNF-α-stimulated C2C12 myoblasts. Consistent with these results, the expression of Bcl-2 was increased, whereas the expression of Bax, cleaved-caspase 3 and cleaved-PARP was decreased after emodin treatment. These data demonstrate that emodin attenuated apoptosis in TNF-α-stimulated C2C12 myoblasts through mitochondrial signaling pathways. In addition, emodin inhibited autophagy in TNF-α-stimulated C2C12 myoblasts by suppressing the expression of LC3-II, Beclin-1 and Atg7. Emodin also resulted in the upregulation of the phosphorylated forms of Akt. Taken together, these results suggest that emodin inhibited apoptosis and autophagy in TNF-α-induced C2C12 myoblasts, possibly through the activation of phosphorylated Akt. Our findings suggest that emodin could be a potential therapeutic agent in the treatment of muscle atrophy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Uncontrolled inflammation leads to several diseases such as insulin resistance, T2D and several types of cancers. The functional role of microRNAs in inflammation induced insulin resistance is poorly studied. MicroRNAs are post-transcriptional regulatory molecules which mediate diverse biological processes. We here show that miR-16 expression levels are down-regulated in different inflammatory conditions such as LPS/IFNγ or palmitate treated macrophages, palmitate exposed myoblasts and insulin responsive tissues of high sucrose diet induced insulin resistant rats. Importantly, forced expression of miR-16 in macrophages impaired the production of TNF-α, IL-6 and IFN-β leading to enhanced insulin stimulated glucose uptake in co-cultured skeletal myoblasts. Further, ectopic expression of miR-16 enhanced insulin stimulated glucose uptake in skeletal myoblasts via the up-regulation of GLUT4 and MEF2A, two key players involved in insulin stimulated glucose uptake. Collectively, our data highlight the important role of miR-16 in ameliorating inflammation induced insulin resistance., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

In addition to fever, rash, and arthralgia/arthritis, myalgia is another dominant symptom in Chikungunya virus (CHIKV) infection. How CHIKV induces myalgia is unclear. To better understand the viral factors involved in CHIKV-induced myalgia, CHIKVs were isolated from patients with and without myalgia designated myalgia-CHIKV and mild-CHIKV, respectively. The response of myoblasts to infection by the two groups of clinical isolates of CHIKV was investigated. Both groups of CHIKV replicated well in primary human myoblasts. However, the myalgia-CHIKVs replicated to a higher titer and caused the death of infected myoblast more rapidly than the mild-CHIKVs. CHIKV-infected myoblasts increased production of four out of five inflammatory cytokines examined (MCP-1, IP-10, MIP-1α, and IL-8) in comparison to mock-infected cells. Comparison between the myoblast inflammatory cytokine responses showed that myalgia-CHIKVs were stronger activators of cytokines than mild-CHIKVs. This means that recent epidemic strains of CHIKV exhibited different degrees of myoblast permissiveness as evidenced by differences in the ability to replicate and to stimulate inflammatory responses in myoblasts. This data suggest that the myopathic syndrome in recent epidemics is dependent upon the strain of CHIKV., (© 2015 Wiley Periodicals, Inc.)

Cachexia or muscle wasting is a common condition that occurs in many chronic diseases. The wasting conditions are characterized by increased levels of TNF-α which was also known as cachectin in the past. But how TNF-α exerts its cachetic effects remains controversial. To clarify this issue, we investigated the impact of TNF-α on C2C12 cell myogenic differentiation. Our results demonstrate that myotube formation was completely inhibited by TNF-α when added to differentiating C2C12 myoblasts. The inhibitory effect of TNF-α on differentiation was accompanied by activation of NF-κB and down regulation of myogenin and Akt. Importantly, TNF-α's effect on differentiation was abolished when IGF-1 was added to the culture. IGF-1 treatment also inhibited NF-κB reporter activity and restored Akt levels. Our data suggest that TNF-α inhibits myogenic differentiation through NF-κB activation and impairment of IGF-1 signaling pathway. The reversal of TNF-α induced inhibition of myogenesis by IGF-1 may have significant therapeutic potential., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To our knowledge, no study has evaluated the involvement of T helper (Th)1- and Th2-chemokines in extra-ocular muscle (EOM) myopathy in "patients with thyroid-associated ophthalmopathy" (TAO-p). We tested the effects of interferon (IFN)γ and tumor necrosis factor (TNF)α stimulation, and of increasing concentrations of peroxisome proliferator-activated receptor (PPAR)γ agonists (pioglitazone or rosiglitazone; 0.1 μM-20 μM), on Th1-chemokine [C-X-C motif ligand (CXCL)10] and Th2-chemokine [C-C motif ligand (CCL)2] secretion in primary EOM cultures from TAO-p vs. control myoblasts. Moreover, we evaluated serum CXCL10 and CCL2 in active TAO-p with prevalent EOM involvement (EOM-p) vs. those with prevalent orbital fat expansion (OF-p). Serum CXCL10 was higher in OF-p and EOM-p vs. controls, while serum CCL2 was not significantly different in controls, or in OF-p and EOM-p. We showed the expression of PPARγ in EOM cells. In primary EOM cultures from TAO-p: a) CXCL10 was undetectable in the supernatant, IFNγ dose-dependently induced it, whereas TNFα did not; b) EOM produced basally low amounts of CCL2, TNFα dose-dependently induced it, whereas IFNγ did not; c) the combination of TNFα and IFNγ had a significant synergistic effect on CXCL10 and CCL2 secretion; and d) PPARγ agonists have an inhibitory role on the modulation of CXCL10, while they stimulate CCL2 secretion. EOM participates in the self-perpetuation of inflammation by releasing both Th1 (CXCL10) and Th2 (CCL2) chemokines under the influence of cytokines, in TAO. PPARγ agonist activation plays an inhibitory role on CXCL10, but stimulates the release of CCL2., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Myostatin plays negative roles in muscle development. To block the inhibitory effects of myostatin on myogenesis, a 759 bp single chain variable fragment antibody (scFv) against myostatin was constructed and expressed in Escherichia coli. ELISA detection showed that the scFv could bind to myostatin, and change of the scFv N-terminal peptides decreased its binding affinity. MTT assay and cell morphology demonstrated that the cell number and viability of the C2C12 myoblast were enhanced by the scFv. Meanwhile, the scFv significantly inhibited the myostatin-induced expression of cyclin-dependent kinase inhibitor p21 and Smad binding element-luciferase activity. H2O2 increased the expression of Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx) in myoblasts as well as myostatin and MuRF1 in myotubes, and the scFv significantly decreased the H2O2-elevated expression of these genes. Conclusively, the scFv we developed could antagonize the inhibitory effects of myostatin on myogenesis through Smad pathway and regulation of p21, MuRF1 and MAFbx gene expression. The scFv may have application in the therapy of muscular dystrophy and improvement of animal meat production.

We investigated the effect of macrophage differentiation on the chemotactic activity to invade local damaged myoblasts using in vitro models of muscle injury. We found that: 1) the chemotactic activity of macrophages toward areas of damaged myoblasts might be induced more by live myoblasts than dead ones, 2) the chemotactic activity of macrophages is not due to velocity, but depends on the directionality toward damaged myoblast cells, and 3) macrophage differentiation strongly influence the chemotactic activity toward damaged myoblast cells through the expression of CCR2 and/or F-actin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiation of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We established a novel monoclonal antibody, Yaksa that is specific to a subpopulation of myogenic cells. The Yaksa antigen is not expressed on the surface of growing myoblasts but only on a subpopulation of myogenin-positive myocytes. When Yaksa antigen-positive mononucleated cells were freshly prepared from a murine myogenic cell by a cell sorter, they fused with each other and formed multinucleated myotubes shortly after replating while Yaksa antigen-negative cells scarcely generated myotubes. These results suggest that Yaksa could segregate fusion-competent, mononucleated cells from fusion-incompetent cells during muscle differentiation. The Yaksa antigen was also expressed in developing muscle and regenerating muscle in vivo and it was localized at sites of cell-cell contact between mono-nucleated muscle cells and between mono-nucleated muscle cells and myotubes. Thus, Yaksa that marks prefusion myocytes before myotube formation can be a useful tool to elucidate the cellular and molecular mechanisms of myogenic cell fusion.

Transforming Growth Factor-beta (TGF-β) is a pro-sclerotic cytokine widely associated with the development of fibrosis in diabetic nephropathy. Central to the underlying pathology of tubulointerstitial fibrosis is epithelial-to-mesenchymal transition (EMT), or the trans-differentiation of tubular epithelial cells into myofibroblasts. This process is accompanied by a number of key morphological and phenotypic changes culminating in detachment of cells from the tubular basement membrane and migration into the interstitium. Ultimately these cells reside as activated myofibroblasts and further exacerbate the state of fibrosis. A large body of evidence supports a role for TGF-β and downstream Smad signalling in the development and progression of renal fibrosis. Here we discuss a role for TGF-β as the principle effector in the development of renal fibrosis in diabetic nephropathy, focusing on the role of the TGF-β1 isoform and its downstream signalling intermediates, the Smad proteins. Specifically we review evidence for TGF-β1 induced EMT in both the proximal and distal regions of the nephron and describe potential therapeutic strategies that may target TGF-β1 activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In idiopathic inflammatory myopathies (IIMs), extracellular inflammatory stimulation is considered to induce secondary intracellular inflammatory changes including expression of major histocompatibility complex class-I (MHC-I) and to produce a self-sustaining loop of inflammation. We hypothesize that activation of calpain, a Ca(2+) -sensitive protease, bridges between these extracellular inflammatory stress and intracellular secondary inflammatory changes in muscle cells. In this study, we demonstrated that treatment of rat L6 myoblast cells with interferon-γ (IFN-γ) caused expression of MHC-I and inflammation-related transcription factors (phosphorylated-extracellular signal-regulated kinase 1/2 and nuclear factor-κB). We also demonstrated that treatment with tumor necrosis factor-α (TNF-α) induced apoptotic changes and activation of calpain and cyclooxygenase-2. Furthermore, we found that posttreatment with calpeptin attenuated the intracellular changes induced by IFN-γ or TNF-α. Our results indicate that calpain inhibition attenuates apoptosis and secondary inflammatory changes induced by extracellular inflammatory stimulation in the muscle cells. These results suggest calpain as a potential therapeutic target for treatment of IIMs., (Copyright © 2011 Wiley-Liss, Inc.)

Reports on muscle biology and regeneration often implicate immuno(cyto/histo)chemical protein characterization using rabbit polyclonal antibodies. In this study we demonstrate that newly formed myofibers are especially prone to false positive staining by rabbit antibodies and this unwanted staining is only recognized (1) by a negative muscle tissue control that does not harbor the protein to be examined (fx. from knockout mouse) or (2) by use of a nonsense rabbit antibody that has been prepared in the same way as the antibody of interest. However, many muscle immuno(cyto/histo)chemical studies only rely on controls that reveal non-specific binding by the secondary antibody and neglect that the primary rabbit antibody itself may cause false positive staining of the muscle. We suggest that reliable immuno-based protein detection in newly formed muscle fibers at least requires a nonsense rabbit antibody and optimally a negative muscle/cell control., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

Using a human primary muscle cell culture model the behaviour of myoblasts (satellite cells) cultured in human serum obtained from either young or elderly individuals was studied. Serum was obtained from a total of 13 young (7 males and 6 females aged, 23-36 years) and 9 elderly (4 males and 5 females aged 69-84 years) subjects and used in a number of experiments. Myoblasts were extracted from human muscle biopsy samples taken from the vastus lateralis. In the first experiment myoblasts were isolated immediately after extraction from the biopsy in media containing human sera to examine its effects on the onset and progression of Ki67 and desmin expression. No effect of the age of the serum was observed at 3, 5 or 7 days of culture. In addition, cells were studied that had been expanded initially in optimum myoblast growth medium (GM, containing foetal calf serum and additional growth factors) prior to culture in medium containing 15% human serum. The proportion of proliferating muscle cells coexpressing desmin and Ki67 antigens after 46 h was again similar in the young and old serum conditions. Culturing these myoblasts in media containing 2% human serum to study their fusion and differentiation also resulted in no difference between young and old serum conditions in terms of the percentage of nuclei inside myosin heavy chain positive myotubes. Despite the variability of different samples of myoblasts, the age of the serum donor has no effect on the expression of any measured index., (Copyright © 2010 Elsevier Inc. All rights reserved.)

FOXP3+ T regulatory cells (Tregs) are considered key players in the maintenance of immune homeostasis. Here we studied the presence and potential role of FOXP3+ Tregs in myositis. CD3 and FOXP3 expression in dermatomyositis, polymyositis and inclusion body myositis was assessed by immunohistochemistry and real-time PCR. FOXP3+ Tregs were found in close proximity to effector cells and their numbers correlated with the degree of inflammation. Despite divergent pathogenetic concepts, we observed no differences in the frequency of FOXP3 immunoreactive cells or FOXP3 mRNA expression between different myositis entities. Functional assays using human myoblasts as targets of CD8+ cells demonstrate that Tregs are capable to inhibit the lytic activity of cytotoxic cells. Our data suggest that FOXP3 Tregs serve to counterbalance muscle destruction by cytotoxic T cells in myositis., (Copyright 2010. Published by Elsevier B.V.)

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.

Here, we demonstrate that hepatic stellate cells (HSC) isolated from Lewis rats have in vitro antigen-presentation cell (APC) functionality and are able to process and present exogenous antigens. We show activation of a major histocompatibility complex II (RT1BI)-restricted T-cell hybridoma specific for guinea pig myelin basic protein (gpMBP) after coculture with HSC. During transdifferentiation of HSC into myofibroblasts (MFB) the APC function was markedly decreased but restorable by addition of interferon-gamma (IFN-gamma). Based on our findings we conclude that HSC play a key role in hepatic immune function and that IFN-gamma treatment might mediate its beneficial therapeutic effects via activation of APC function in MFB., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The association between malignancy and autoimmune myositis has been largely described and confirmed by numerous epidemiological studies. The temporal relationship between the two pathologic conditions can vary: malignancy may occur before, at the same time or following the diagnosis of myositis. Beside these observations, the molecular mechanisms underlying this association are still unknown, even though it has been demonstrated a possible antigenic similarity between regenerating myoblasts and some cancer cell populations. To better identify peculiar histopathologic features common to cancer and myositis, we screened muscle biopsies from patients affected with polymyositis, dermatomyositis, myositis in association to cancer, and from patients affected with newly diagnosed cancer, but without myositis. Similarly to the histopatologic features that were observed in the muscle from myositis patients, especially in those with cancer associated myositis, in patients affected with malignancy at the clinical onset of disease we observed early sign of myopathy, characterized by internally nucleated and regenerating myofibers, most of them expressing the neural cell adhesion molecule. The hypothesis that in a particular subset of individuals genetically predisposed to autoimmunity, an initial subclinical tumor-induced myopathy may result in an autoimmune myositis, represents a further intriguing link behind the association of these two conditions., (Copyright 2009 Elsevier B.V. All rights reserved.)

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.

Trogocytosis is a cell-contact dependent intercellular transfer of membrane fragments and associated molecules. We studied trogocytosis in the interaction of T cells with human skeletal muscle cells modeling muscle-immune cell interactions under pathophysiological conditions i.e. myositis. Human myoblasts donate membrane fragments to T cells. Acquisition of muscle-derived membrane molecules depended on T-cell activation, was independent of T-cell receptor engagement, sensitive to inhibition of actin polymerization and amplified by protein kinase C activation. Single-cell patch clamping was used to demonstrate the change in membrane capacitance upon incorporation of membrane fragments in T cells. Membrane uptake was fast and temporarily, but had clear functional consequences: T cells after intimate contact with myoblasts stimulated the proliferation of autologous T cells. Our observations raise the hypothesis that trogocytosis may modulate the outcome of T-T interactions within the micromilieu of skeletal muscle.

TWEAK, TNF-like weak inducer of apoptosis, is a relatively recently identified proinflammatory cytokine that functions through binding to Fn14 receptor in target cells. Although TWEAK has been shown to modulate several biological responses, the TWEAK-induced signaling pathways remain poorly understood. In this study, we tested the hypothesis that TAK1 (TGF-beta-activated kinase 1) is involved in TWEAK-induced activation of NF-kappaB and MAPK and expression of proinflammatory protein. TWEAK increased the phosphorylation and kinase activity of TAK1 in cultured myoblast and fibroblast cells. The activation of NF-kappaB was significantly inhibited in TAK1-deficient (TAK1(-/-)) mouse embryonic fibroblasts (MEF) compared with wild-type MEF. Deficiency of TAK1 also inhibited the TWEAK-induced activation of IkappaB kinase and the phosphorylation and degradation of IkappaBalpha protein. However, there was no difference in the levels of p100 protein in TWEAK-treated wild-type and TAK1(-/-) MEF. Furthermore, TWEAK-induced transcriptional activation of NF-kappaB was significantly reduced in TAK1(-/-) MEF and in C2C12 myoblasts transfected with a dominant-negative TAK1 or TAK1 short interfering RNA. TAK1 was also required for the activation of AP-1 in response to TWEAK. Activation of JNK1 and p38 MAPK, but not ERK1/2 or Akt kinase, was significantly inhibited in TAK1(-/-) MEF compared with wild-type MEF upon treatment with TWEAK. TWEAK-induced expression of proinflammatory genes such as MMP-9, CCL-2, and VCAM-1 was also reduced in TAK1(-/-) MEF compared with wild-type MEF. Furthermore, the activation of NF-kappaB and the expression of MMP-9 in response to TWEAK involved the upstream activation of Akt kinase. Collectively, our study demonstrates that TAK1 and Akt are the important components of TWEAK-induced proinflammatory signaling and gene expression.

Sclerosing angiomatoid nodular transformation (SANT) of the spleen is a rare inflammatory tumor-like lesion composed of vascular nodules and non-neoplastic stroma including spindle cells and inflammatory cells. The focus of our study was on the stromal proliferating process in SANT. Nine cases of SANT were examined. All cases showed alpha-smooth muscle actin (alpha-SMA) and vimentin on the spindle cells but not CD21, CD31, CD34, CD68, desmin, S100, human herpes virus-8, or anaplastic lymphoma kinase-1. In one case, 20-30% of the myofibroblasts in Epstein-Barr-virus (EBV)-positive spindle cells were detected using double-labeling immunohistochemistry for alpha-SMA and EBV-encoded small RNA in situ hybridization. A quantitative analysis of IgG and IgG4-positive plasma cells (pPCs) in SANT was performed. The median densities of IgG-pPCs and IgG4-pPCs in SANT were approximately four-fold and 13-fold higher than those in the normal spleens, respectively. In addition, there was a statistically significant increase of IgG4/IgG-pPCs ratio in SANT in comparison to the control specimens. In conclusion, the fibrogenesis in a subset of SANT may be associated with EBV-infected myofibroblasts in an overlapping immune reaction indicated by the presence of infiltrating IgG4-pPCs. Further investigation is needed to elucidate the association between SANT and IgG4-related sclerosing disease.

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.

Food poisoning due to staphylococcal enterotoxins A and B (SEA and SEB) affects hundreds of thousands of people annually. SEA and SEB induce massive intestinal cytokine production, which is believed to be the key factor in staphylococcal enterotoxin enteropathy. MHC class II molecules are the major receptors for staphylococcal enterotoxins. We recently demonstrated that normal human subepithelial intestinal myofibroblasts (IMFs) express MHC class II molecules. We hypothesized that IMFs are among the first cells to respond to staphylococcal enterotoxins and contribute to the cytokine production associated with staphylococcal enterotoxin pathogenesis. We demonstrated here that primary cultured IMFs bind staphylococcal enterotoxins in a MHC class II-dependent fashion in vitro. We also demonstrated that staphylococcal enterotoxins can cross a CaCo-2 epithelial monolayer in coculture with IMFs and bind to the MHC class II on IMFs. IMFs responded to SEA, but not SEB, exposure with 3- to 20-fold increases in the production of proinflammatory chemokines (MCP-1, IL-8), cytokines (IL-6), and growth factors (GM-CSF and G-CSF). The SEA induction of the proinflammatory mediators by IMFs resulted from the efficient cross-linking of MHC class II molecules because cross-linking of class II MHC by biotinylated anti-HLA-DR Abs induced similar cytokine patterns. The studies presented here show that MCP-1 is central to the production of other cytokines elicited by SEA in IMFs because its neutralization with specific Abs prevented the expression of IL-6 and IL-8 by IMFs. Thus, MCP-1 may play a leading role in initiation of inflammatory injury associated with staphylococcal enterotoxigenic disease.

Background: FOXP3+ regulatory T-cells (Treg) are important regulators of allo-reactivity and may therefore represent an important predictor for the risk of graft versus-host disease (GVHD) after allogeneic stem cell transplantation., Methods: To determine the clinical significance of Treg-content in stem cell grafts, we analyzed 58 human leukocyte antigen (HLA)-identical sibling donors (34 patients received myeloablative and 24 patients reduced intense conditioning regimens) and correlated the Treg frequency with clinical outcome after stem cell transplantation (SCT)., Results: A mean value of 9.1 x 10(6) CD4+ FOXP3+ Treg per kg body weight (bw) of the recipient was transplanted (ranging from 0.7 to 33.7 x 10(6) Treg/kg bw). Graft content of Treg correlated with mononuclear cells and CD3+ T-cells. Patients receiving low numbers of Treg (Treg(low)) after myeloablative conditioning for SCT had a significantly increased cumulative incidence of 76% for acute GVHD when compared with 23% for individuals receiving high numbers of Treg (Treg(high)). This observation, however, was not made in patients after reduced intense conditioning-SCT. Notably, relapse rate was not significantly different between Treg(low) and Treg(high) patients in either patient group and overall survival was even increased in Treg(high) patients after myeloablative SCT. Finally, low Treg graft levels represent an independent prognostic factor in multivariate analysis for the appearance of acute GHVD., Conclusion: Donor-derived Treg might be of particular significance for the development of acute GVHD after myeloablative SCT using HLA-identical sibling donors.

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.

Tumour growth is characterised by the formation of a fine vessel network or neovasculature which nourishes tumour cells. Two kinds of novel anti-angiogenic therapies are based on the prevention of vessels growth and on the destruction of those vessels already formed. We report here on the design and construction of a novel immunotoxin formed with the non-toxic type II ribosome-inactivating protein ebulin l and the mouse anti-human CD105 monoclonal antibody 44G4. The 44G4-ebulin immunotoxin was formed by covalent linking of both proteins with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and was purified by chromatography on Superdex 200 HiLoad. The analysis of the anti-ribosomal effects in a cell-free translation system indicated that conjugation does not affect the activity of ebulin l. The immunotoxin displays cytotoxicity with nanomolar IC50 values on human CD105+ cells like the mouse fibroblasts L929 cells transfected with the short form of human CD105 and the rat myoblasts L6E9 transfected with the long form of human CD105. In contrast, cells lacking human CD105 were 2-2.5 logs less sensitive to the immunotoxin. Free ebulin displays IC50 values in the range 10(-6) M. Since CD105 is being considered as a potential target for the anti-vascular therapy of tumours, the present immunotoxin could be a promising tool for the anticancer therapy, especially due to the very low in vivo toxicity of ebulin l as compared ricin and other toxins used for immunotoxins.

Myogenesis is regulated by the MyoD class of myogenic regulatory factors (MRFs). These basic helix-loop-helix transcription factors dimerize with E proteins to bind conserved E-box sequences in the promoter regions of muscle-specific genes. Perhaps due to their expression in a wide array of tissues, the specific interactions of E proteins with different MRFs have been largely ignored. Likewise, the expression of E proteins in muscle tissue remains mostly uncharacterized. We investigated the expression of the E proteins HEB, E12, and E47 in rat L6 myoblasts, which express only embryonic and fast (2X) myosin heavy chains (MyHCs) in vitro, C2C12 myosatellite cells, and a number of muscle tissues, to determine whether myosin heavy chain diversity is mirrored by diversity in E protein or MRF expression. Although L6 and C2C12 myotubes demonstrate strong expression of embryonic and 2X (fast) MyHCs, immunofluorescence demonstrated the additional expression of type 1 (slow), 2A, and 2B MyHCs in the C2C12 cell line. Immunofluorescence and western blot analyses show that HEB was expressed in differentiating L6 myoblasts, C2C12 cells, and neonatal rat primary myotubes. In contrast, E12 and E47 expression was not detected in either cell line or in any adult muscle tissue examined. These data strongly implicate HEB in the development of skeletal muscle. However, because HEB is expressed in L6 myoblasts, C2C12 myosatellite cells, and neonatal hindlimb muscles, it is unlikely to be involved in a fiber type-specific manner, and may have a more general role in differentiation of myotubes.

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.

Muscle is the site or the target of immunologic injury in several diseases. Whereas under physiologic conditions muscle fibers are negative for major histocompatibility complex (MHC) class I antigens, these are upregulated under pathologic conditions, thus rendering muscle a possible target for the recognition by cytotoxic CD8 T cells. Cultured muscle cells are capable of presenting antigens to CD4 and CD8 T cells, further indicating that muscle fibers in vivo are critically involved in the initiating or perpetuating steps of inflammatory responses. The finding that muscle fibers in autoimmune inflammatory myopathies in vivo and cultured muscle cells in vitro express the nonclassical major histocompatibility complex molecule HLA-G raises several hypothesis concerning its possible pathophysiologic role. We review present knowledge on the functional consequences of muscle-related HLA-G and provide concepts of its relevance under pathologic conditions. We further speculate on the potential therapeutic implications of HLA-G that relate to special approaches such as myoblast transplantation or strategies against inflammatory aggression in general.

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.

The immunological properties of muscle cells are of critical importance for both the pathogenesis of inflammatory muscle disorders as well as for understanding and controlling novel therapeutic strategies. Muscle cells can present antigens to both CD4 and CD8 cells. However, the cellular biochemistry of antigen processing and presentation by muscle cells is not clear. Cathepsins play a central role in the generation of antigenic peptide and control transport and maturation of MHC class II molecules. To further elucidate the molecular basis for the MHC class II-mediated antigen presentation by muscle cells, we here analyzed cultured human myoblasts and biopsies from inflammatory myopathies with respect to the expression and function of the constituents of the MHC class II antigen presentation machinery. We identified cathepsin S (CatS) as the dominant endocytic protease that is specifically upregulated under inflammatory conditions to significant mRNA levels, synchronously with HLA-DR, -DM and the class II invariant chain (Ii), both in muscle biopsies from affected individuals with inflammatory myopathies and in human myoblasts cultured in the presence of IFN-gamma. This led to translation of the mature CatS polypeptide that was enzymatically active in human myoblasts under inflammatory conditions. By contrast, expression of CatL and CatB was unaffected by IFN-gamma at both the expression and activity levels. CatS activity is required for efficient surface display of MHC class II in this cell type: functional inhibition of CatS using a CatS-selective inhibitor reduced the levels of surface class II alphabeta:peptide complexes on stimulated myoblasts by almost 50%. Surprisingly, and in contrast to B cells and dendritic cells, this was not due to inefficient processing of Ii in the absence of CatS, which was unaffected by the elimination of CatS activity. We therefore conclude that CatS is involved in the regulation of class II expression in human myoblasts independently from Ii processing.

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.