Exogenous IL-9 Ameliorates Experimental Autoimmune Myasthenia Gravis Symptoms in Rats.

Interleukin-9 (IL-9) is a multifunctional cytokine involved in protective immunity or immunopathology depending on the microenvironment and specific disease settings. Our early study determined that IL-9 and Th9 cells participate in and promote the progression of experimental autoimmune myasthenia gravis (EAMG). The data from this study showed that exogenous recombinant rat IL-9 (rrIL-9) acted as an IL-9 receptor antagonist, reduced the incidence of EAMG in rats, alleviated the severity of the disease, and reduced the anti-acetylcholine receptor (AChR) IgG antibody levels by altering the Th-subset distribution. These data suggest that administration of rrIL-9 may provide a novel therapeutic strategy against MG or related autoimmune diseases. Abbreviations: 2-Mercaptoethanol (2-ME); antibodies (Abs); ?-bungarotoxin (?-BTX); acetylcholine receptor (AChR); airway hyper-reactivity (AHR); allophycocyanin-conjugated (APC); antigen presenting cells (APCs); complete Freund's adjuvant (CFA); Cyanine dye 3 (Cy3); dendritic cells (DCs); experimental autoimmune encephalomyelitis (EAE); experimental autoimmune myasthenia gravis (EAMG); flow cytometry (FACS); fetal bovine serum (FBS); fetal calf serum (FCS); Fluorescein isothiocyanate (FITC); gamma chain (?c); intraperitoneally (i.p.); Incomplete Freund's adjuvant (IFA); interferon (IFN); immunoglobulin (Ig); Interleukin (IL); Janus kinase (JAK); myasthenia gravis (MG); Mononuclear cells (MNC); neuromuscular junctions (NMJ); optical density (OD); ovalbumin (OVA); phosphate-buffered saline (PBS); phycoerythrin (PE); Peridinin chlorophyll protein complex (Percp); Rat AChR ? subunit (R-AChR97-116); Recombinant Rat (rr); room temperature (RT); signal transducer and activator of transcription (STAT); T helper cells (Th).

Specific removal of autoantibodies by extracorporeal immunoadsorption ameliorates experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) is caused by autoantibodies, the majority of which target the muscle acetylcholine receptor (AChR). Plasmapheresis and IgG-immunoadsorption are useful therapy options, but are highly non-specific. Antigen-specific immunoadsorption would remove only the pathogenic autoantibodies, reducing the possibility of side effects while maximizing the benefit. We have extensively characterized such adsorbents, but in vivo studies are missing. We used rats with experimental autoimmune MG to perform antigen-specific immunoadsorptions over three weeks, regularly monitoring symptoms and autoantibody titers. Immunoadsorption was effective, resulting in a marked autoantibody titer decrease while the immunoadsorbed, but not the mock-treated, animals showed a dramatic symptom improvement. Overall, the procedure was found to be efficient, suggesting the subsequent initiation of clinical trials.

Regulatory T cells in multiple sclerosis and myasthenia gravis.

Multiple sclerosis (MS) is a chronic debilitating disease of the central nervous system primarily mediated by T lymphocytes with specificity to neuronal antigens in genetically susceptible individuals. On the other hand, myasthenia gravis (MG) primarily involves destruction of the neuromuscular junction by antibodies specific to the acetylcholine receptor. Both autoimmune diseases are thought to result from loss of self-tolerance, which allows for the development and function of autoreactive lymphocytes. Although the mechanisms underlying compromised self-tolerance in these and other autoimmune diseases have not been fully elucidated, one possibility is numerical, functional, and/or migratory deficits in T regulatory cells (Tregs). Tregs are thought to play a critical role in the maintenance of peripheral immune tolerance. It is believed that Tregs function by suppressing the effector CD4+ T cell subsets that mediate autoimmune responses. Dysregulation of suppressive and migratory markers on Tregs have been linked to the pathogenesis of both MS and MG. For example, genetic abnormalities have been found in Treg suppressive markers CTLA-4 and CD25, while others have shown a decreased expression of FoxP3 and IL-10. Furthermore, elevated levels of pro-inflammatory cytokines such as IL-6, IL-17, and IFN-γ secreted by T effectors have been noted in MS and MG patients. This review provides several strategies of treatment which have been shown to be effective or are proposed as potential therapies to restore the function of various Treg subsets including Tr1, iTr35, nTregs, and iTregs. Strategies focusing on enhancing the Treg function find importance in cytokines TGF-ß, IDO, interleukins 10, 27, and 35, and ligands Jagged-1 and OX40L. Likewise, strategies which affect Treg migration involve chemokines CCL17 and CXCL11. In pre-clinical animal models of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune myasthenia gravis (EAMG), several strategies have been shown to ameliorate the disease and thus appear promising for treating patients with MS or MG.

Immature Exosomes Derived from MicroRNA-146a Overexpressing Dendritic Cells Act as Antigen-Specific Therapy for Myasthenia Gravis.

Myasthenia gravis (MG) is a neurological autoimmune disease characterized by fluctuating weakness of certain voluntary muscles. Current treatments for MG are largely directed at suppressing the whole immune system by using immunosuppressants or glucocorticoids and often cause several side effects. The ideal therapeutic methods for MG should suppress aberrant immunoactivation specifically, while retaining normal function of the immune system. In this study, we first produced exosomes from microRNA-146a overexpressing dendritic cells (DCs). Then, we observed suppressive effects of those exosomes in experimental autoimmune myasthenia gravis (EAMG) mice. Results showed that exosomes from microRNA-146a overexpressing DCs expressed decreased levels of CD80 and CD86. In experimental autoimmune MG, exosomes from microRNA-146a overexpressing DCs suppressed ongoing clinical MG in mice and altered T helper cell profiles from Th1/Th17 to Th2/Treg both in serum and spleen, and the therapeutic effects of those exosomes were antigen-specific and partly dose dependent. All the findings provide experimental basis for antigen-specific therapy of MG.

Preconditioned mesenchymal stem cells treat myasthenia gravis in a humanized preclinical model.

Myasthenia gravis (MG) with anti-acetylcholine receptor (AChR) Abs is an autoimmune disease characterized by severe defects in immune regulation and thymic inflammation. Because mesenchymal stem cells (MSCs) display immunomodulatory features, we investigated whether and how in vitro-preconditioned human MSCs (cMSCs) could treat MG disease. We developed a new humanized preclinical model by subcutaneously grafting thymic MG fragments into immunodeficient NSG mice (NSG-MG model). Ninety percent of the animals displayed human anti-AChR Abs in the serum, and 50% of the animals displayed MG-like symptoms that correlated with the loss of AChR at the muscle endplates. Interestingly, each mouse experiment recapitulated the MG features of each patient. We next demonstrated that cMSCs markedly improved MG, reducing the level of anti-AChR Abs in the serum and restoring AChR expression at the muscle endplate. Resting MSCs had a smaller effect. Finally, we showed that the underlying mechanisms involved (a) the inhibition of cell proliferation, (b) the inhibition of B cell-related and costimulatory molecules, and (c) the activation of the complement regulator DAF/CD55. In conclusion, this study shows that a preconditioning step promotes the therapeutic effects of MSCs via combined mechanisms, making cMSCs a promising strategy for treating MG and potentially other autoimmune diseases.

ROCK inhibitor abolishes the antibody response in experimental autoimmune myasthenia gravis.

The Rho/Rho kinase (ROCK) pathway serves as molecular switches in many biological processes including the immune response. ROCK inhibitors lead to amelioration of some autoimmune diseases. The present study was designed to define whether a selective ROCK inhibitor, fasudil, was effective in experimental autoimmune myasthenia gravis (EAMG) and investigate the underlying mechanisms. Here we found fasudil effectively attenuated the development of ongoing EAMG. Fasudil abolished the antibody production and function by decreasing follicular helper T cells and CD19(+) B cells, especially germinal center B cells. Moreover, fasudil reduced the expression of CD80 on lymph node mononuclear cells. These findings suggest the inhibition of ROCK might be a potential therapeutic strategy for antibody-mediated autoimmune diseases.

Silencing miR-146a influences B cells and ameliorates experimental autoimmune myasthenia gravis.

MicroRNAs have been shown to be important regulators of immune homeostasis as patients with aberrant microRNA expression appeared to be more susceptible to autoimmune diseases. We recently found that miR-146a was up-regulated in activated B cells in response to rat acetylcholine receptor (AChR) α-subunit 97-116 peptide, and this up-regulation was significantly attenuated by AntagomiR-146a. Our data also demonstrated that silencing miR-146a with its inhibitor AntagomiR-146a effectively ameliorated clinical myasthenic symptoms in mice with ongoing experimental autoimmune myasthenia gravis. Furthermore, multiple defects were observed after miR-146a was knocked down in B cells, including decreased anti-R97-116 antibody production and class switching, reduced numbers of plasma cells, memory B cells and B-1 cells, and weakened activation of B cells. Previously, miR-146a has been identified as a nuclear factor-κB-dependent gene and predicted to base pair with the tumour necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) genes to regulate the immune response. However, our study proved that miR-146a inhibition had no effect on the expression of TRAF6 and IRAK1 in B cells. This result suggests that the function of miR-146a in B cells does not involve these two target molecules. We conclude that silencing miR-146a exerts its therapeutic effects by influencing the B-cell functions that contribute to the autoimmune pathogenesis of myasthenia gravis.

A novel antibody against human factor B that blocks formation of the C3bB proconvertase and inhibits complement activation in disease models.

The alternative pathway (AP) is critical for the efficient activation of complement regardless of the trigger. It is also a major player in pathogenesis, as illustrated by the long list of diseases in which AP activation contributes to pathology. Its relevance to human disease is further emphasized by the high prevalence of pathogenic inherited defects and acquired autoantibodies disrupting components and regulators of the AP C3-convertase. Because pharmacological downmodulation of the AP emerges as a broad-spectrum treatment alternative, there is a powerful interest in developing new molecules to block formation and/or activity of the AP C3-convertase. In this paper, we describe the generation of a novel mAb targeting human factor B (FB). mAb FB48.4.2, recognizing with high affinity an evolutionary-conserved epitope in the Ba fragment of FB, very efficiently inhibited formation of the AP C3-proconvertase by blocking the interaction between FB and C3b. In vitro assays using rabbit and sheep erythrocytes demonstrated that FB28.4.2 was a potent AP inhibitor that blocked complement-mediated hemolysis in several species. Using ex vivo models of disease we demonstrated that FB28.4.2 protected paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and inhibited both C3 fragment and C5b-9 deposition on ADP-activated HMEC-1 cells, an experimental model for atypical hemolytic uremic syndrome. Moreover, i.v. injection of FB28.4.2 in rats blocked complement activation in rat serum and prevented the passive induction of experimental autoimmune Myasthenia gravis. As a whole, these data demonstrate the potential value of FB28.4.2 for the treatment of disorders associated with AP complement dysregulation in man and animal models.

Myeloid-derived suppressor cells as a potential therapy for experimental autoimmune myasthenia gravis.

We recently demonstrated that hepatic stellate cells induce the differentiation of myeloid-derived suppressor cells (MDSCs) from myeloid progenitors. In this study, we found that adoptive transfer of these MDSCs effectively reversed disease progression in experimental autoimmune myasthenia gravis (EAMG), a T cell-dependent and B cell-mediated model for myasthenia gravis. In addition to ameliorated disease severity, MDSC-treated EAMG mice showed suppressed acetylcholine receptor (AChR)-specific T cell responses, decreased levels of serum anti-AChR IgGs, and reduced complement activation at the neuromuscular junctions. Incubating MDSCs with B cells activated by anti-IgM or anti-CD40 Abs inhibited the proliferation of these in vitro-activated B cells. Administering MDSCs into mice immunized with a T cell-independent Ag inhibited the Ag-specific Ab production in vivo. MDSCs directly inhibit B cells through multiple mechanisms, including PGE2, inducible NO synthase, and arginase. Interestingly, MDSC treatment in EAMG mice does not appear to significantly inhibit their immune response to a nonrelevant Ag, OVA. These results demonstrated that hepatic stellate cell-induced MDSCs concurrently suppress both T and B cell autoimmunity, leading to effective treatment of established EAMG, and that the MDSCs inhibit AChR-specific immune responses at least partially in an Ag-specific manner. These data suggest that MDSCs could be further developed as a novel approach to treating myasthenia gravis and, even more broadly, other diseases in which T and B cells are involved in pathogenesis.

Recombinant IgG2a Fc (M045) multimers effectively suppress experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disorder caused by target-specific pathogenic antibodies directed toward postsynaptic neuromuscular junction (NMJ) proteins, most commonly the skeletal muscle nicotinic acetylcholine receptor (AChR). In MG, high-affinity anti-AChR Abs binding to the NMJ lead to loss of functional AChRs, culminating in neuromuscular transmission failure and myasthenic symptoms. Intravenous immune globulin (IVIg) has broad therapeutic application in the treatment of a range of autoimmune diseases, including MG, although its mechanism of action is not clear. Recently, the anti-inflammatory and anti-autoimmune activities of IVIg have been attributed to the IgG Fc domains. Soluble immune aggregates bearing intact Fc fragments have been shown to be effective treatment for a number of autoimmune disorders in mice, and fully recombinant multimeric Fc molecules have been shown to be effective in treating collagen-induced arthritis, murine immune thrombocytopenic purpura, and experimental inflammatory neuritis. In this study, a murine model of MG (EAMG) was used to study the effectiveness of this novel recombinant polyvalent IgG2a Fc (M045) in treating established myasthenia, with a direct comparison to treatment with IVIg. M045 treatment had profound effects on the clinical course of EAMG, accompanied by down-modulation of pathogenic antibody responses. These effects were associated with reduced B cell activation and T cell proliferative responses to AChR, an expansion in the population of FoxP3(+) regulatory T cells, and enhanced production of suppressive cytokines, such as IL-10. Treatment was at least as effective as IVIg in suppressing EAMG, even at doses 25-30 fold lower. Multimeric Fc molecules offer the advantages of being recombinant, homogenous, available in unlimited quantity, free of risk from infection and effective at significantly reduced protein loads, and may represent a viable therapeutic alternative to polyclonal IVIg.

RNA interference targeting Bcl-6 ameliorates experimental autoimmune myasthenia gravis in mice.

Follicular helper T (Tfh) cells are dedicated to providing help to B cells and are strongly associated with antibody-mediated autoimmune disease. B cell lymphoma 6 (Bcl-6) is a key transcription factor of Tfh cells, and IL-21 is known to be a critical cytokine produced by Tfh cells. We silenced Bcl-6 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of Bcl-6 short hairpin RNAs (shRNAs) in experimental autoimmune myasthenia gravis (EAMG). Our data demonstrate that CD4(+)CXCR5(+)PD-1(+) Tfh cells, Bcl-6 and IL-21 were significantly increased in EAMG mice, compared with controls. In addition, we found that frequencies of Tfh cells were positively correlated with the levels of serum anti-AChR Ab. In-vivo transduction of lenti-siRNA-Bcl6 ameliorates the severity of ongoing EAMG with decreased Tfh cells, Bcl-6 and IL-21 expression, and leads to decreased anti-AChR antibody levels. Furthermore, we found that siRNA knockdown of Bcl-6 expression increases the expression of Th1(IFN-γ, T-bet) and Th2 markers (IL-4 and GATA3), but failed to alter the expression of Th17-related markers (RORγt, IL-17) and Treg markers (FoxP3). Our study suggests that Tfh cells contribute to the antibody production and could be one of the most important T cell subsets responsible for development and progression of EAMG or MG. Bcl-6 provides a promising therapeutic target for immunotherapy not only for MG, but also for other antibody-mediated autoimmune diseases.

Atorvastatin-modified dendritic cells in vitro ameliorate experimental autoimmune myasthenia gravis by up-regulated Treg cells and shifted Th1/Th17 to Th2 cytokines.

Conventional therapies for autoimmune diseases produce nonspecific immune suppression, which are usually continued lifelong to maintain disease control, and associated with a variety of adverse effects. In this study, we found that spleen-derived dendritic cells (DCs) from the ongoing experimental autoimmune myasthenia gravis (EAMG) rats can be induced into tolerogenic DCs by atorvastatin in vitro. Administration of these tolerogenic DCs to EAMG rats on days 5 and 13 post immunization (p.i.) resulted in improved clinical symptoms, which were associated with increased numbers of CD4(+)CD25(+) T regulatory (Treg) cells and Foxp3 expression, decreased lymphocyte proliferation among lymph node mononuclear cells (MNC), shifted cytokine profile from Th1/Th17 to Th2 type cytokines, decreased level of anti-R97-116 peptide (region 97-116 of the rat acetylcholine receptor α subunit) IgG antibody in serum. These tolerogenic DCs can migrate to spleen, thymus, popliteal and inguinal lymph nodes after they were injected into the EAMG rats intraperitoneally. Furthermore, these tolerogenic DCs played their immunomodulatory effects in vivo mainly by decreased expression of CD86 and MHC class II on endogenous DCs. All these data provided us a new strategy to treat EAMG and even human myasthenia gravis (MG).

Complement C2 siRNA mediated therapy of myasthenia gravis in mice.

Activation of complement components is crucial in the progression and severity of myasthenia gravis and experimental autoimmune myasthenia gravis (EAMG). Mice deficient in complement component C4 or treated with monoclonal antibody to C1q are resistant to EAMG. In this study, we show that inhibition of complement cascade activation by suppressing the expression of a critical low-abundant protein, C2, in the classical complement pathway, significantly improved clinical and immunopathological manifestations of EAMG. Two weeks after a second booster immunization with acetylcholine receptor, when mice exhibit muscle weakness, i.p. injection of C2 siRNA significantly suppressed C2 mRNA in the blood cells and liver of EAMG mice. Treatment of EAMG mice with C2 siRNA, once a week for 5 weeks, significantly improved muscle strength, which was further evidenced by functional AChR preservation in muscle, reduction in number of C3 and membrane-attack complexes at neuro-muscular junctions in forelimb muscle sections, and a transient decrease in serum IgG2b levels. Our study shows for the first time that siRNA-mediated suppression of C2, a component of the classical complement system, following established disease, can effectively contribute to the remission of EAMG. Therefore, C2 siRNA mediated therapy can be applied in all complement mediated autoimmune diseases.

Specific binding of collagen Q to the neuromuscular junction is exploited to cure congenital myasthenia and to explore bases of myasthenia gravis.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.

Regulatory T cell-based immunotherapies in experimental autoimmune myasthenia gravis.

Establishment of tolerance in myasthenia gravis (MG) involves regulatory T (T(reg)) cells. Experimental autoimmune MG (EAMG) in rats is a suitable model for assessing the contribution of T(reg) cells to the immunopathology of the disease and for testing novel T(reg) cell-based treatment modalities. We have studied two immunotherapeutic approaches for targeting of T(reg) cells in myasthenia. By one approach we demonstrated that treatment of sick rats by ex vivo-generated exogenous T(reg) cells derived from healthy donors suppressed EAMG. By a different approach, we aimed at affecting the endogenous T(reg)/Th17 cell balance by targeting IL-6, which has a key role in controlling the equilibrium between pathogenic Th17 and suppressive T(reg) cells. We found that treatment of myasthenic rats by neutralizing anti-IL-6 antibodies shifted this equilibrium in favor of T(reg) cells and led to suppression of EAMG. Our results show that T(reg) cells could serve as potential targets in treating MG patients.

Specific immunotherapy of experimental myasthenia gravis in vitro and in vivo: the Guided Missile strategy.

Current immunotherapy of myasthenia gravis (MG) is often effective, but entails risks of infection and neoplasia. The "Guided Missile" strategy described here is designed to target and eliminate the individual's unique AChR-specific T cell repertoire, without otherwise interfering with the immune system. We genetically engineered dendritic cells to present AChR epitopes and simultaneously express Fas ligand in an ongoing EAMG model. In both in vitro and in vivo experiments, these engineered cells specifically killed AChR-responsive T cells without otherwise damaging the immune system. AChR antibodies were markedly reduced in the treated mice. Translation of this method to treat human MG is possible.

Acetylcholine receptor-induced experimental myasthenia gravis: what have we learned from animal models after three decades?

Myasthenia gravis (MG) is an autoimmune disease caused by an immunological response against the acetylcholine receptor (AChR) at the neuromuscular junction. Anti-AChR antibodies induce degradation of the receptor, activation of complement cascade and destruction of the post-synaptic membrane, resulting in a functional reduction of AChR availability. The pathophysiological role of autoantibodies (auto-Abs) and T helper lymphocytes has been studied in the experimental autoimmune MG (EAMG) models. EAMG models have been employed to investigate the factors involved in the development of MG and to suggest new therapies aimed to preventing or modulating the ongoing disease. EAMG can be induced in susceptible mouse and rat strains, which develop clinical symptoms such as muscular weakness and fatigability, mimicking the human disease. Two major types of EAMG can be induced, passive and active EAMG. Passive transfer MG models, involving the injection of auto-Abs, are helpful for studying the role of complement molecules and their regulatory proteins, which can prevent neuromuscular junction degradation. Active models, induced by immunization, are employed for the analysis of antigen-specific immune responses and their modulation in order to improve disease progression. In this review, we will concentrate on the main pathogenic mechanisms of MG, focusing on recent findings on EAMG experimental models.

[Umbilical cord mesenchymal stem cell transplantation for treatment of experimental autoimmune myasthenia gravis in rats].

Umbilical cord mesenchymal stem cell (UCMSC) transplantation has been widely used in the treatment of a variety of diseases due to their advantages such as abundant resources, low immunogenicity and large ex vivo expansion capacity. This study was aimed to investigate the effects of UCMSC on experimental autoimmune myasthenia gravis (EAMG) rats. The distribution of human-derived cells was observed by immunofluorescence method, the effect of MSC on B-cell in situ-secreted antibodies was assayed by ELISPOT, the secreted IFN-γ level was detected by using Transwell test. The results showed that UCMSC were able to migrate to inflammation region and lymph nudes, moreover human-derived cells could be detected in medulla zone of lymph nudes. In vitro in situ detection of AchR specific antibody secretion revealed that the full contact of MSC with lymphnode-derived lymphocytes could effectively inhibit production of AchR antibody. Transwell test indicated that the direct contact of UCMSC with CD4 T cells could effectively decrease production of IFN-γ, which modulated the unbalance between Th1/Th2 to a certain extent. It is concluded that UCMSC can regulate the immune system by direct cell-cell contact or/and release of cytokines, which bring a new insight into knowledge about MSC-based therapy for EAMG.

Intravenous administration of bone marrow mesenchymal stem cells benefits experimental autoimmune myasthenia gravis mice through an immunomodulatory action.

Mesenchymal stem cells (MSC) are potent in immunomodulation. It has been proven that MSC functioned to correct immune disorder in several immune diseases. Here, we tested the hypothesis that MSC from human bone marrow (hMSC) can provide a potential therapy for experimental autoimmune myasthenia gravis (EAMG). EAMG mice model was established by subcutaneous injection of synthetic analogue of acetylcholine receptor (AchR), then, hMSC were intravenously delivered into these mice repeatedly. The results showed that hMSC could specifically home to spleen tissue and hMSC treatment significantly improved the functional deficits of EAMG mice. In addition, AchR antibody level was dramatically decreased in cell-treated group when compared with untreated control on 10 days after the second cell injection. Moreover, both in vivo and in vitro mixed lymphocyte proliferation assays revealed that hMSC could definitely inhibit the proliferation of AchR-specific lymphocyte. In conclusion, our study demonstrated that hMSC treatment was therapeutically useful in autoimmune myasthenia gravis mice, and the underlying mechanism may relate with their immunomodulatory potential.

Specific immunotherapy of experimental myasthenia gravis by a novel mechanism.

OBJECTIVE: Myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), are antibody (Ab)-mediated autoimmune diseases, in which autoantibodies bind to and cause loss of muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. To develop a specific immunotherapy of MG, we treated rats with ongoing EAMG by intraperitoneal injection of bacterially-expressed human muscle AChR constructs. METHODS: Rats with ongoing EAMG received intraperitoneal treatment with the constructs weekly for 5 weeks beginning after the acute phase. Autoantibody concentration, subclassification, and specificity were analyzed to address the underlying therapeutic mechanism. RESULTS: EAMG was specifically suppressed by diverting autoantibody production away from pathologically relevant specificities directed at epitopes on the extracellular surface of muscle AChRs toward pathologically irrelevant epitopes on the cytoplasmic domain. A mixture of subunit cytoplasmic domains was more effective than a mixture containing both extracellular and cytoplasmic domains or than only the extracellular domain of alpha1 subunits. INTERPRETATION: Therapy using only cytoplasmic domains, which lack pathologically relevant epitopes, avoids the potential liability of boosting the pathological response. Use of a mixture of bacterially-expressed human muscle AChR cytoplasmic domains for antigen-specific immunosuppression of myasthenia gravis has the potential to be specific, robust, and safe.