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Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that target proteins at the neuromuscular junction, primarily the acetylcholine receptor (AChR) and the muscle-specific kinase. Because downstream of kinase 7 (Dok-7) is essential for the full activation of muscle-specific kinase and consequently for dense clustering of AChRs, we hypothesized that reduced levels of Dok-7 increase the susceptibility to passive transfer MG. To test this hypothesis, Dok-7 expression was reduced by transfecting shRNA-coding plasmids into the tibialis anterior muscle of adult rats by in vivo electroporation. Subclinical MG was subsequently induced with a low dose of anti-AChR monoclonal antibody 35. Neuromuscular transmission was significantly impaired in Dok-7-siRNA–electroporated legs compared with the contralateral control legs, which correlated with a reduction of AChR protein levels at the neuromuscular junction (approximately 25%) in Dok-7-siRNA–electroporated muscles, compared with contralateral control muscles. These results suggest that a reduced expression of Dok-7 may play a role in the susceptibility to passive transfer MG, by rendering AChR clusters less resistant to the autoantibody attack.

Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR) is characterized by a chronic, fatigable weakness of voluntary muscles. The production of autoantibodies involves the dysregulation of T cells which provide the environment for the development of autoreactive B cells. The symptoms are caused by destruction of the postsynaptic membrane and degradation of the AChR by IgG autoantibodies, predominantly of the G1 and G3 subclasses. Active immunization of animals with AChR from mammalian muscles, AChR from Torpedo or Electrophorus electric organs, and recombinant or synthetic AChR fragments generates a chronic model of MG, termed experimental autoimmune myasthenia gravis (EAMG). This model covers cellular mechanisms involved in the immune response against the AChR, e.g. antigen presentation, T cell-help and regulation, B cell selection and differentiation into plasma cells. Our aim is to define standard operation procedures and recommendations for the rat EAMG model using purified AChR from the Torpedo californica electric organ, in order to facilitate more rapid translation of preclinical proof of concept or efficacy studies into clinical trials and, ultimately, clinical practice.

Myasthenia gravis is an autoimmune disease characterized by muscle weakness due to neuromuscular junction (NMJ) damage by anti-acetylcholine receptor (AChR) auto-antibodies and complement. In experimental autoimmune myasthenia gravis (EAMG), which is induced by immunization with Torpedo AChR in CFA, anti-AChR IgG2b and IgG1 are the predominant isotypes in the circulation. Complement activation by isotypes such as IgG2b plays a crucial role in EAMG pathogenesis; this suggested the possibility that IgG1, which does not activate complement through the classical pathway, may suppress EAMG. In this study, we show that AChR-immunized BALB/c mice genetically deficient for IgG1 produce higher levels of complement-activating isotypes of anti-AChR, especially IgG3 and IgG2a, and develop increased IgG3/IgG2a deposits at the NMJ, as compared to wild type (WT) BALB/c mice. Consistent with this, AChR-immunized IgG1(-/-) BALB/c mice lose muscle strength and muscle AChR to a greater extent than AChR-immunized WT mice. These observations demonstrate that IgG1 deficiency leads to increased severity of EAMG associated with an increase in complement activating IgG isotypes. Further studies are needed to dissect the specific role or mechanism of IgG1 in limiting EAMG and that of EAMG exacerbating role of complement activating IgG3 and IgG2a in IgG1 deficiency.

Background miR-15b is significantly and consistently downregulated in different clinical phenotypes of myasthenia gravis (MG). However, its role in pathogenesis of MG is still not clear. This study aimed to explore the function of miR-15b in MG. Material and methods Blood samples from early-onset MG, late-onset MG, thymoma patients, and healthy participants were collected. The expression pattern of IL-15 and miR-15b was identified by qRT-PCR and ELISA in patient serum and mouse tissue samples. The regulative role of miR-15b on IL-15 expression was verified in an experimental autoimmune myasthenia gravis (EAMG) mice model. Results qRT-PCR and ELISA showed that miR-15b expression was significantly lower and IL-15 expression was significantly higher in all EMG, LMG, and thymoma cases compared to healthy controls. Based on mouse model, we confirmed that miR-15b knockdown could increase IL-15 expression in healthy mice, while miR-15b overexpression could inhibit IL-15 expression in EAMG mice. Through searching in bioinformatics databases, we identified a highly conserved consequential pairing between IL-15 and miR-15b. Subsequent dual luciferase assay further verified this match. Conclusions This study is the first to report the miR-15b-IL-15 axis can directly regulate IL15 expression, which helps to further explain the abnormal IL-15 expression in MG patients and the pathogenesis of MG.

Neuromyelitis optica (NMO) and myasthenia gravis (MG) are autoimmune diseases mediated by autoantibodies against either aquaporin 4 (AQP4) or acetylcholine receptor (AChR), respectively. Recently, we and others have reported an increased prevalence of NMO in patients with MG. To verify whether coexisting autoimmune disease may exacerbate experimental autoimmune MG, we tested whether active immunization with AQP4 peptides or passive transfer of NMO-Ig can affect the severity of EAMG. Injection of either AQP4 peptide or NMO-Ig to EAMG or to naive mice caused increased fatigability and aggravation of EAMG symptoms as expressed by augmented muscle weakness (but not paralysis), decremental response to repetitive nerve stimulation, increased neuromuscular jitter, and aberration of immune responses. Thus, our study shows increased disease severity in EAMG mice following immunization with the NMO autoantigen AQP4 or by NMO-Ig, mediated by augmented inflammatory response. This can explain exacerbation or increased susceptibility of patients with one autoimmune disease to develop additional autoimmune syndrome.

International audience; Myasthenia gravis (MG) is a neuromuscular disease caused in most cases by anti-acetyl-choline receptor (AChR) autoantibodies that impair neuromuscular signal transmission and affect skeletal muscle homeostasis. Myogenesis is carried out by muscle stem cells called satellite cells (SCs). However, myogenesis in MG had never been explored. The aim of this study was to characterise the functional properties of myasthenic SCs as well as their abilities in muscle regeneration. SCs were isolated from muscle biopsies of MG patients and age-matched controls. We first showed that the number of Pax7+ SCs was increased in muscle sections from MG and its experimental autoimmune myasthenia gravis (EAMG) mouse model. Myoblasts isolated from MG muscles proliferate and differentiate more actively than myoblasts from control muscles. MyoD and MyoG were expressed at a higher level in MG myoblasts as well as in MG muscle biopsies compared to controls. We found that treatment of control myoblasts with MG sera or monoclonal anti-AChR antibodies increased the differentiation and MyoG mRNA expression compared to control sera. To investigate the functional ability of SCs from MG muscle to regenerate, we induced muscle regeneration using acute cardiotoxin injury in the EAMG mouse model. We observed a delay in maturation evidenced by a decrease in fibre size and MyoG mRNA expression as well as an increase in fibre number and embryonic myosin heavy-chain mRNA expression. These findings demonstrate for the first time the altered function of SCs from MG compared to control muscles. These alterations could be due to the anti-AChR antibodies via the modulation of myogenic markers resulting in muscle regeneration impairment. In conclusion, the autoimmune attack in MG appears to have unsuspected pathogenic effects on SCs and muscle regeneration, with potential consequences on myogenic signalling pathways, and subsequently on clinical outcome, especially in the case of muscle stress.

Because of the failure of many promising therapeutics identified in preclinical evaluation, funding sources have established guidelines for increased rigor in animal evaluations. The myasthenia gravis (MG) community of scientists has developed guidelines for preclinical assessment for potential MG treatments. Here, we provide a focused summary of these recommendations and the role of complement in disease development in experimental models of MG.

The neuromuscular junction (NMJ) forms the synaptic connection between a motor neuron and a skeletal muscle fiber. In order to achieve a sustained muscle contraction, this synapse has to reliably transmit motor neuronal action potentials onto the muscle fiber. To guarantee successful transmission even during intense activation of the NMJ, a safety factor of neuromuscular transmission exists. In the neuromuscular disorder myasthenia gravis (MG), autoantibodies are directed against acetylcholine receptors or, in the rarer variants, against other postsynaptic NMJ proteins. This causes loss of functional acetylcholine receptors, which compromises the safety factor of neuromuscular transmission, leading to the typical fatigable muscle weakness of MG. With intracellular microelectrode measurement of (miniature) endplate potentials at NMJs in ex vivo nerve-muscle preparations from MG animal models, these functional synaptic defects have been determined in much detail. Here, we describe the electrophysiological events at the normal NMJ and the pathoelectrophysiology at NMJs of animal models for MG.

Generalized myasthenia gravis (gMG) is a rare autoimmune disorder characterized by skeletal muscle weakness caused by disrupted neurotransmission at the neuromuscular junction (NMJ). Approximately 74-88% of patients with gMG have acetylcholine receptor (AChR) autoantibodies. Complement plays an important role in innate and antibody-mediated immunity, and activation and amplification of complement results in the formation of membrane attack complexes (MACs), lipophilic proteins that damage cell membranes. The role of complement in gMG has been demonstrated in animal models and patients. Studies in animals lacking specific complement proteins have confirmed that MAC formation is required to induce experimental autoimmune MG (EAMG) and NMJ damage. Complement inhibition in EAMG models can prevent disease induction and reverse its progression. Patients with anti-AChR

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.

Experimental autoimmune myasthenia gravis (EAMG) and myasthenia gravis (MG) are caused by autoantibodies to the extracellular domain of muscle nicotinic acetylcholine receptors (AChRs). Autoantibodies to the cytoplasmic domain of AChRs do not cause EAMG because they cannot bind AChRs in vivo. The ideal MG therapy would quickly and permanently suppress only the pathological autoimmune response to AChRs. We have developed a specific immunosuppressive therapy for EAMG that involves immunizing rats with bacterially expressed cytoplasmic domains of human muscle AChRs. Therapy prevents onset of chronic EAMG, rapidly suppresses ongoing EAMG, and is potent, robust, long lasting, and safe, because the therapeutic antigen cannot induce EAMG. The therapy was developed using incomplete Freund's adjuvant, but is likely to work equally well with alum adjuvants routinely used for human immunizations. Therapeutic mechanisms may involve a combination of antibody-mediated feedback suppression and regulatory T and/or B lymphocytes.

While the majority of myasthenia gravis patients express antibodies targeting the acetylcholine receptor, the second most common cohort instead displays autoantibodies against muscle-specific kinase (MuSK). MuSK is a transmembrane tyrosine kinase found in the postsynaptic membrane of the neuromuscular junction. During development, MuSK serves as a signaling hub, coordinating the alignment of the pre- and postsynaptic components of the synapse. Adult mice that received repeated daily injections of IgG from anti-MuSK

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Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness and fatigue in the presence of circulating antibodies against components of the neuromuscular junction. Most patients have a good prognosis, but some are refractory to standard-of-care immunosuppressive treatment and suffer from recurrent myasthenic crises. Functional sphingosine-1-phosphate (S1P) antagonists like fingolimod and siponimod (BAF312) are successfully used for the treatment of multiple sclerosis, and fingolimod was shown to prevent the development of myasthenic symptoms in experimental autoimmune myasthenia gravis (EAMG), the standard model of MG. Here, we investigated whether fingolimod or siponimod improves outcome in EAMG mice when administered after disease onset, modeling the clinical setting in human MG. Both S1P antagonists inhibited lymphocyte egress, resulting in peripheral lymphopenia. After stimulation, there were differences in T-cell responses, but no change in either antibody titers or total or antigen-specific plasma cell populations after treatment. Most importantly, disease incidence and severity were not influenced by fingolimod or siponimod therapy. Although fingolimod and siponimod did lead to subtle changes in T-cell responses, they had no significant effect on antibody titers and disease severity. In conclusion, our data show no evidence of a therapeutic potential for S1P receptor antagonists in MG treatment.

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2

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.

Accumulating evidence shows that the immunoproteasome participates in the immune response, beyond its initial role in the protein degradation. Here, we tested the effects of the selective immunoproteasome inhibitor, ONX-0914, on experimental autoimmune myasthenia gravis (EAMG). We found that ONX-0914 ameliorated the severity of ongoing EAMG by reducing the autoantibody affinity, accompanied with decreased Tfh cells and antigen presenting cells. Also it reduced the percentage of Th17 cells and inhibited the secretion of IL-17. Our data indicated ONX-0914 may bring benefit for MG therapy.

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies.Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein.B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups.LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN-γ and IL-17) responses to LRP4 and AChR stimulation, respectively.Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG.

Agrin is essential for the formation and maintenance of neuromuscular junctions (NMJs). NT-1654 is a C-terminal fragment of mouse neural agrin. In this study, we determined the effects of NT-1654 on the severity of experimental autoimmune myasthenia gravis (EAMG).EAMG was induced in female Lewis rats by immunization with the Torpedo acetylcholine receptor (tAChR) and complete Freund's adjuvant (CFA). NT-1654 was dissolved in phosphate-buffered saline (PBS) and injected daily subcutaneously into tAChR immunized rats during the first 10 days after immunization, and then every other day for the following 20 days.We showed that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats.We demonstrated that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. Muscle Nerve 57: 814-820, 2018.

Properly sustained impulse transmission at the neuromuscular junction (NMJ) is crucial for successful muscle contraction. To guarantee this, NMJs not only possess a considerable safety factor in transmission but also have the ability to adjust the presynaptic acetylcholine release level to cope with any changes in the postsynaptic neurotransmitter sensitivity. This review will provide overview on the discovery and characterization of this synaptic homeostatic mechanism, especially in the condition of the neuromuscular disorder myasthenia gravis (MG) where the postsynaptic transmitter sensitivity at the NMJ becomes severely reduced due to autoimmune attack of acetylcholine receptors. Because homeostatic signalling and adaptation is presumably maximally active in this condition, NMJs from MG animal models are important study objects. Although candidate post- and presynaptic factors as well as the retrograde signals have been proposed, the homeostatic mechanism at the MG NMJ is still incompletely understood. Further identification and functional characterization of key factors is important because these may form new therapeutic targets in MG.

International audience; 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.

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.

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.

Interleukin-9 (IL-9) was initially thought to be a type 2 T helper (Th2)-associated cytokine involved in the regulation of autoimmune responses by affecting multiple cell types. However, it was recently shown that IL-9-producing CD4+ T cells represent a discrete subset of Th cells, designated Th9 cells. Although Th9 cells have been shown to be important in many diseases, their roles in myasthenia gravis (MG) are unclear. The aim of this study was to determine whether IL-9 and Th9 cells promote the progression of experimental autoimmune myasthenia gravis (EAMG). The results showed that the percentage of Th9 cells changed during the progression of EAMG, accompanied by an up-regulation of IL-9. Blocking IL-9 activity with antibodies against IL-9 inhibited EAMG-associated pathology in rats and reduced serum anti-acetylcholine receptor IgG levels. Neutralization of IL-9 altered the Th subset distribution in EAMG, reducing the number of Th1 cells and increasing the number of regulatory T cells. Administration of an anti-IL-9 antibody may represent an effective therapeutic strategy for MG-associated pathologies or other T-cell- or B-cell-mediated autoimmune diseases.

Extracellular adenosine is an essential negative regulator of immune reactions that acts by signaling via 4 distinct adenosine receptors. We evaluated adenosine receptor expression in Lewis rats presenting with experimental autoimmune myasthenia gravis (EAMG) to determine whether the expression of adenosine receptors are changed in the development and progression of EAMG. Lymphocyte A1AR and A2AAR mRNA and protein levels from lymphocytes harvested from the lymph nodes, spleen, and peripheral blood mononuclear cells (PBMCs) of EAMG rats were decreased. A modest but not significant increase in A2BAR levels was observed in EAMG lymphocytes harvested from lymph nodes and PBMCs. No changes in A3AR expression were observed in lymphocytes harvested from lymph nodes, spleen, or PBMCs following EAMG induction. Results presented in this report showed that the expression levels and the distribution pattern of adenosine receptors were altered in EAMG lymphocytes.

Summary MicroRNA-155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155-deficient B cells generated reduced germinal centre responses and failed to produce high-affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up-regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up-regulated in torpedo acetylcholine receptor (T-AChR)-stimulated B cells. We used an inhibitor of miR155 conjugated to anti-CD20 single-chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell-activating factor (BAFF)-R-related signalling pathway and reduced the translocation of nuclear factor (NF)-κB into the nucleus. Additionally, AChR-specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.

Myasthenia gravis (MG) is a prototypic antibody-mediated autoimmune disease, which fulfills strict criteria that define autoimmunity.1,2 MG is caused primarily by antibodies directed against the skeletal muscle nicotinic acetylcholine receptor (AChR), which is concentrated on the postsynaptic surface of the neuromuscular junction. The antibodies produce a reduction in AChR number and damage the muscle endplate. This leads to failure of neuromuscular transmission and produces weakness that may be life-threatening. AChR antibodies of patients with MG are polyclonal and recognize a complex epitope repertoire that differs among individuals. The heterogeneity of AChR antibodies among patients is increased further by the variety of light-chain and heavy-chain use. The antibody synthesis is T-cell–dependent, and the epitope repertoire of AChR CD4+ cells in MG patients is complex and characteristic of the individual. Due to extreme diversity of the immune response, specific suppression of autoantibody production is an elusive goal.1,3 Therapies for MG fall into 2 broad categories4,5: those that focus on enhancing neuromuscular transmission by inhibition of cholinesterase, such as pyridostigmine; or agents that suppress or modulate the immune system, such as corticosteroids, azathioprine, tacrolimus, and mycophenolate mofetil. Acute exacerbations of weakness are usually treated with plasma exchange or intravenous immunoglobulins, which act primarily through removal of pathogenic antibody. All these treatments were developed after initial use in other disorders6 and produce systemic adverse effects. The immunotherapies are not aimed at production of the specific autoantibody in MG, but rather general moderation of all immune responses, whether this occurs through reduction of autoantibody levels directly or indirectly through suppression of B- and T-cell activity. The approach described here attempts inhibition of a final effector mechanism of pathogenic AChR antibody. The AChR-specific antibodies induce disease by 3 mechanisms: (1) blocking binding and activation of the AChR; (2) accelerating degradation of AChR molecules cross-linked by antibodies (“antigenic modulation”); and (3) mobilizing complement at the NMJ.7–9 Autoantibodies are the only effectors of myasthenic pathology, as patients with MG do not have evidence of AChR-specific cell-mediated mechanisms of NMJ injury.1,10 Therefore, treatments focused on inhibiting antibody effector mechanisms are likely to work rapidly and be highly effective. Compromise of AChR function has not been found to be a major contributor to limitation of neuromuscular transmission.1,2 Antibodies from most MG patients accelerate the degradation rate of the AChR in whole muscle tissue culture and cultured muscle cells.11,12 However, not all AChR antibodies are capable of antigenic modulation, because the epitope location on the AChR surface restricts antibody ability to cross-link a second AChR molecule. Therefore, complement-mediated injury is the primary mechanism for pathology of the NMJ in MG. The autoantibodies that bind to the AChR can activate the complement cascade and produce lysis of the NMJ through insertion of the membrane attack complex (MAC). The NMJs of skeletal muscle are protected from complement attack by specific cell-surface complement-regulatory proteins.13 Decay-accelerating factor (DAF or CD55), CD59, and membrane cofactor protein (complement receptor 1–related gene/protein y is the rodent analog) reduce the activation of complement through various pathways.14–18 The role of DAF as a complement regulator is to accelerate the decay of autologous C3 and C5 convertases. We assessed the ability to target DAF to the NMJ and thereby reduce the damage produced by complement activation. Concentrating DAF to the cell surface by a fusion protein including an antibody binding domain has been demonstrated to be effective in inhibiting complement-mediated lysis.19 Eculizumab, the only complement inhibitor available for human use, demonstrated signs of efficacy in a small, pilot study of patients with severe MG, and thus there is some support for complement inhibitor–based therapeutic approaches for MG.20 However, to date, all inhibitor strategies have led to systemic inhibition of complement. Here we describe an approach that couples the complement-inhibitory domains of the DAF molecule to a single-chain antibody that binds the AChR. We used the single-chain antibody as a control to assess whether blocking pathogenic antibody binding could contribute to a treatment effect. The expectation was that concentration of complement inhibition to the site of pathology could enhance efficacy without compromising neuromuscular transmission.

Tissue plasminogen activator (tPA), a component of the PA/plasmin system, is elevated in inflammatory areas and plays a role in inflammatory neurological disorders. In the present study we explored the involvement of tPA and the potential immunomodulatory activity of tPA in experimental autoimmune myasthenia gravis (EAMG). Mice deficient in tPA (tPA(-/-)) present with a markedly more severe disease than wild type EAMG mice. In an attempt to treat EAMG with an 18aa peptide derived from the PA system inhibitor (PAI-1), designed to tether out the endogenous inhibitor, a significant suppression of disease severity was demonstrated. The more severe disease in tPA(-/-) mice was accompanied by a higher level of anti-AChR antibodies and increased expression of B-cell markers. In view of the essential role of B-cell activating factor (BAFF) in B-cell maturation, the expression of BAFF family components was tested. An increase in BAFF and BAFF receptor was observed in EAMG tPA(-/-) mice, whereas BCMA expression was reduced, consistent with the increased level of pathogenic antibodies and the more severe disease. Given the importance of T regulatory cells (Tregs) in EAMG, they were evaluated and their number was reduced in tPA(-/-) mice, in which EAMG was aggravated, whereas following PAI-1dp treatment, Tregs were replenished and the disease was ameliorated. The results show the involvement of tPA in EAMG, implying a protective role for tPA in EAMG pathogenesis. The amelioration of EAMG by PAI-1dp treatment suggests that the PA system may be considered a potential site for therapeutic intervention in neuroimmune diseases.

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.

Metformin, the most widely used medicine for type 2 diabetes, displays anti-inflammatory functions via activating AMP-activated protein kinase (AMPK). Circulating autoantibodies and disequilibrium of helper T cells and regulatory T cells are pathological hallmarks of myasthenia gravis (MG). Rectify the imbalance of different T cell populations has become an important therapeutic strategy to treat MG. In this study, we assessed the effect of metformin on the development of autoimmunity using an experimental autoimmune myasthenia gravis (EAMG) rat model. We first provided evidence that oral administration of metformin attenuated the onset of EAMG. This effect was accompanied by a substantial decrease of circulating auto-antibody levels with no effect on blood glucose level. While metformin treatment in vitro showed little effect on inducible Treg, metformin strongly inhibited Th17 cell differentiation through the increase of reactive oxygen species and AMPK. Furthermore, an attenuation of antigen-induced IgG2b antibody production by two different doses of metformin was also observed in the AChR-specific recall response. In conclusion, the above results indicate that metformin may have therapeutic value for the clinical treatment of MG.

Autoantibodies against three different postsynaptic antigens and one presynaptic antigen at the neuromuscular junction are known to cause myasthenic syndromes. The mechanisms by which these antibodies cause muscle weakness vary from antigenic modulation and complement-mediated membrane damage to inhibition of endogenous ligand binding and blocking of essential protein-protein interactions. These mechanisms are related to the autoantibody titre, specific epitopes on the target proteins and IgG autoantibody subclass. We here review the role of specific autoantibody-binding epitopes in myasthenia gravis, their possible relevance to the pathophysiology of the disease and potential implications of epitope mapping knowledge for new therapeutic strategies.

Emerging evidence demonstrates that miRNAs, a new family of key mRNA regulatory molecules, have crucial roles in controlling and modulating immunity. Their contribution to myasthenia gravis (MG), a T cell-dependent, antibody-mediated nervous system autoimmune disease, has not been thoroughly investigated. In the present study, using a highly sensitive microarray-based approach, we identified 11 miRNAs with differential expression between Peripheral Blood Mononuclear Cells (PBMC) from experimental autoimmune MG (EAMG) rats and control rats. miR-145 is one of the most significantly down-regulated miRNAs in PBMC from EAMG rats. Down-regulation of miR-145 expression was confirmed in PBMC and CD4+CD25- T cells (T effector cells) from both EAMG rats and MG patients by real-time PCR. Bioinformatics target prediction identified two crucial immune-related molecules-CD28 and NFATc1, as putative targets of miR-145. Furthermore, miR-145 inhibited CD28 and NFATc1 expression by directly targeting their 3'-UTRs, which was abolished by mutation of the miR-145 and CD28/NFATc1 binding sites. In vitro up-regulation of miR-145 in CD4+ T cells can significantly reduce CD28 protein levels accompanied by decreased proliferative response. In a dendritic cell (DC)-T cell coculture system, overexpression of miR-145 in AChR-specific CD4+ T cells suppresses NFATc1 expression and T Helper 17 cells level. Finally, we observed that administration of lentiviral-miR-145 decreased the severity of ongoing, established EAMG with decreased IL-17 production, and also decreased serum anti-AChR IgG levels. Our studies provide an important new insight into the pathogenesis of EAMG and MG, which may open a new perspective for the development of effective gene therapy for EAMG/MG.

The complement system is profoundly involved in the pathogenesis of acetylcholine receptor (AChR) antibody (Ab) related myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). The most characteristic finding of muscle pathology in both MG and EAMG is the abundance of IgG and complement deposits at the nerve-muscle junction (NMJ), suggesting that AChR-Ab induces muscle weakness by complement pathway activation and consequent membrane attack complex (MAC) formation. This assumption has been supported with EAMG resistance of complement factor C3 knockout (KO), C4 KO and C5 deficient mice and amelioration of EAMG symptoms following treatment with complement inhibitors such as cobra venom factor, soluble complement receptor 1, anti-C1q, anti-C5 and anti-C6 Abs. Moreover, the complement inhibitor decay accelerating factor (DAF) KO mice exhibit increased susceptibility to EAMG. These findings have brought forward improvisation of novel therapy methods based on inhibition of classical and common complement pathways in MG treatment.

Myasthenia gravis (MG) is an antibody mediated, T cell dependent autoimmune disease characterized by muscle fatigability in which autoantibodies directed to the acetylcholine receptor (AChR) impair neuromuscular transmission. The identification of CD4⁺CD25⁺Foxp3⁺Treg cells as important regulators of tolerance opened a major area of investigation raising the possibility that a dysfunction in the Treg compartment is involved in the etiology and pathogenesis of autoimmune diseases, including MG. In this paper we summarize shortly Treg abnormalities that were reported in MG patients and report on our studies of Treg in experimental autoimmune MG (EAMG). Hopefully these studies would pave the way towards the development of novel Treg-based treatment modalities that will restore self-tolerance in MG and other autoimmune diseases. In our previous studies in EAMG we have shown that Treg cells transferred from healthy rat donors to myasthenic rats suppress EAMG. However, Treg cells from sick animals do not have the same in vivo suppressive activity as those from healthy donors. The objective of the present study was to further characterize quantitative and qualitative alterations in Treg cells of rats with EAMG. We found that the frequency of CD4⁺CD25⁺Foxp3⁺Treg cells within the spleen and PBL was decreased in EAMG rats as compared to naïve and CFA-immunized healthy controls. Treg cells from myasthenic rats were less effective than Treg cells from controls in suppressing the proliferation of CD4⁺T effector cells in response to ConA and of B cells in response to LPS. Moreover, CD4⁺CD25⁺ cells from EAMG rats exhibited an elevated extent of apoptosis and expressed upregulated levels of FAS and of Th17-associated cytokines. Since EAMG is an induced disease, these quantitative and qualitative alterations in Treg cells do not reflect predisposing impairments and seem to be associated with the specific autoimmune response resulting from AChR immunization.

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.

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.

Objective To investigate the effects of tetramethylpyrazine (TMP) on experimental autoimmune myasthenia gravis (EAMG) in rats and explore the possible immune regulation mechanism. Methods Lewis rats were randomly divided into 4 groups: control group, EAMG group, TMP low-dose group (TMP-L, 10 mg/kg) and TMP high-dose group (TMP-H, 20 mg/kg). Except the control group, the other 3 groups were subjected to EAMG modeling. The body mass was determined and the symptoms of muscular weakness in rats were scored by Lennon EAMG criteria. The expressions of acetylcholine receptor (AChR), IgG and C3 in neuromuscular junctions were examined by immunofluorescence. The levels of R97-116 antibody (IgG1, IgG2a, IgG2b), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ) and interleukin 10 (IL-10) were detected by ELISA. Results TMP decreased the scores in the evaluation of the symptoms of muscle weakness in EAMG rats. Moreover, TMP administration significantly inhibited AChR expression and increased IgG and C3 expression in neuromuscular junctions of EAMG rats. In addition, after TMP treament, the levels of IgG1, IgG2a and TNF-α were remarkably reduced, while IL-10 level was elevated. However, there was no significant change in serum levels of IgG2b and IFN-γ. Conclusion TMP can exert inhibitory effects on Th1, Th2 and B cells and modulate the levels of IgG1, IgG2a, TNF-α and IL-10. All these effects contribute to relieve the symptoms of muscle weakness in EAMG rats.

Myasthenia gravis (MG) is usually caused by antibodies against the muscle acetylcholine receptor (AChR). Experimental autoimmune MG (EAMG) is the animal model of MG, typically induced by immunization of rodents with AChR isolated from the electric organ of Torpedo californica. We have successfully induced EAMG in Lewis rats by immunization with the extracellular domains (ECDs) of the human AChR subunits (α, β, γ, δ and e) expressed in yeast. Analysis of the antibody titers revealed a robust antigenic response against all the peptides, but a marked difference in their pathogenicity; the α subunit ECD was the most pathogenic, resulting in the highest percentage of affected animals. Measurements of antibody titers, electromyographic tests and quantitation of the muscle AChR content, offered further support to these findings. The EAMG models presented herein, could be used for studying subunit-specific pathogenic mechanisms, and, more importantly, as tools for the evaluation of antigen-specific therapeutic approaches, which rely on the human AChR.

Astilbin, a major bioactive compound extracted from Rhizoma smilacis glabrae (RSG), has been reported to possess immunosuppressive properties. Our study first evaluated the effect of astilbin on experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. The results showed that astilbin could attenuate the severity of EAMG by decreasing antigen-specific autoantibodies with up-regulation of regulatory T cells and down-regulation of Th17 cells. In addition to, astilbin also reduced the efficiency of the antigen presenting cells on which the expression of MHC class II decreased. These results suggest that astilbin might be a candidate drug for immunoregulation of EAMG, and provide us new treatment ideas for human myasthenia gravis (MG).

Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1 +, IgG2 + and IgG3 + peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis.

International audience; Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression. The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG. Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.

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.

Antibodies against acetylcholine receptors (AChRs) cause pathogenicity in myasthenia gravis (MG) patients through complement pathway-mediated destruction of postsynaptic membranes at neuromuscular junctions (NMJs). However, antibodies against muscle-specific kinase (MuSK), which constitute a major subclass of antibodies found in MG patients, do not activate the complement pathway. To investigate the pathophysiology of MuSK-MG and establish an experimental autoimmune MG (EAMG) model, we injected MuSK protein into mice deficient in complement component five (C5). MuSK-injected mice simultaneously developed severe muscle weakness, accompanied by an electromyographic pattern such as is typically observed in MG patients. In addition, we observed morphological and functional defects in the NMJs of EAMG mice, demonstrating that complement activation is not necessary for the onset of MuSK-MG. Furthermore, MuSK-injected mice exhibited acetylcholinesterase (AChE) inhibitor-evoked cholinergic hypersensitivity, as is observed in MuSK-MG patients, and a decrease in both AChE and the AChE-anchoring protein collagen Q at postsynaptic membranes. These findings suggest that MuSK is indispensable for the maintenance of NMJ structure and function, and that disruption of MuSK activity by autoantibodies causes MG. This mouse model of EAMG could be used to develop appropriate medications for the treatment of MuSK-MG in humans.

In myasthenia gravis (MG) and experimental autoimmune MG (EAMG), many pathologically significant autoantibodies are directed at the main immunogenic region (MIR), a conformation-dependent region at the extracellular tip of α1 subunits of muscle nicotinic acetylcholine receptors (AChRs). Human muscle AChR α1 MIR sequences were integrated into Aplysia ACh-binding protein (AChBP). The chimera was potent in inducing both acute and chronic EAMG, though less potent than Torpedo electric organ AChR. Wild-type AChBP also induced EAMG but was less potent, and weakness developed slowly without an acute phase. AChBP is more closely related in sequence to neuronal α7 AChRs that are also homomeric; however, autoimmune responses were induced to muscle AChR, but not to neuronal AChR subtypes. The greater accessibility of muscle AChRs to antibodies, compared to neuronal AChRs, may allow muscle AChRs to induce self-sustaining autoimmune responses. The human α1 subunit MIR is a potent immunogen for producing pathologically significant autoantibodies. Additional epitopes in this region or other parts of the AChR extracellular domain contribute significantly to myasthenogenicity. We show that an AChR-related protein can induce EAMG. Thus, in principle, an AChR-related protein could induce MG. AChBP is a water-soluble protein resembling the extracellular domain of AChRs, yet rats that developed EAMG had autoantibodies to AChR cytoplasmic domains. We propose that an initial autoimmune response, directed at the MIR on the extracellular surface of muscle AChRs, leads to an autoimmune response sustained by muscle AChRs. Autoimmune stimulation sustained by endogenous muscle AChR may be a target for specific immunosuppression.

Acetylcholine receptors (AchRs) and Na(+) channels (NaChs) are concentrated on neuromuscular junction (NMJ) postsynaptic folds; both are depleted in myasthenia gravis (MG), reducing the safety factor (SF) for neuromuscular transmission, especially in extraocular muscles (EOM). Studies of human myasthenic nerve-muscle preparations indicate that loss of endplate AChRs accounts for 59%, and NaChs for 40%, of SF reduction. Rodent models of MG indicate that NaChs and AChRs losses are due to complement-mediated destruction of postsynaptic folding. Saccades in MG show stereotyped, conjugate initial components, similar to normal but different from early disconjugacy with ocular nerve palsies. Loss of AChRs, NaChs, and postsynaptic folding all contribute to SF reduction in MG. EOM seem more susceptible to MG because of poor postsynaptic folding, lower baseline SF, and lower levels of intrinsic complement inhibitors. Initial conjugacy of saccades in MG reflects selective sparing of neuromuscular transmission of fast, pale global fibers, which have better developed postsynaptic folding.

MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) patients present a distinct selective fatigue, and sometimes atrophy, of bulbar, facial and neck muscles. Here, we study the mechanism underlying the focal muscle involvement in mice with MuSK+ experimental autoimmune MG (EAMG). 8 week-old female wildtype C57BL6 mice and transgenic mice, which express yellow fluorescence protein (YFP) in their motor neurons, were immunized with the extracellular domain of rat MuSK and compared with control mice. The soleus, EDL, sternomastoid, omohyoid, thoracic paraspinal and masseter muscles were examined for pre- and postsynaptic changes with whole mount immunostaining and confocal microscopy. Neuromuscular junction derangement was quantified and compared between muscles and correlated with transcript levels of MuSK and other postsynaptic genes. Correlating with the EAMG disease grade, the postsynaptic acetylcholine receptor (AChR) clusters were severely fragmented with a subsequent reduction also of the presynaptic nerve terminal area. Among the muscles analyzed, the thoracic paraspinal, sternomastoid and masseter muscles were more affected than the leg muscles. The masseter muscle was the most affected, leading to denervation and atrophy and this severity correlated with the lowest levels of MuSK mRNA. On the contrary, the soleus with high MuSK mRNA levels had less postsynaptic perturbation and more terminal nerve sprouting. We propose that low muscle-intrinsic MuSK levels render some muscles, such as the masseter, more vulnerable to the postsynaptic perturbation of MuSK antibodies with subsequent denervation and atrophy. These findings augment our understanding of the sometimes severe, facio-bulbar phenotype of MuSK+ MG.

The role of immunoadsorption therapy is well established in the management of myasthenia gravis (MG), an autoimmune disorder characterized by muscle weakness and caused by circulating IgG antibodies with specificity against the acetylcholine receptor. Conventional immunoadsorbents that employ recombinant protein A as immobilized ligand suffer from the drawbacks of high cost and low stability. The objective of this work is to assess the safety and efficacy of a synthetic adsorbent for treating MG. Adsorption columns were prepared from a Sepharose-based adsorbent coupled to 4-mercaptoethylpyridine (MEP), which acted as immobilized ligands. Animal model of experimental autoimmune MG (EAMG) using Lewis rats was developed and treated by whole blood perfusion. The results showed that the treatments provided a significant amelioration of clinical weakness for EAMG rats, with clinic score decreasing from 2.08 ± 0.38 to 1.25 ± 0.27. After a treatment session of about 1.5 h, blood cell counts were not significantly changed. Serum levels of total IgG and acetylcholine receptor antibody were reduced by 37.1 ± 6.5% and 35.6 ± 8.6%, respectively. In addition, reduction in complement components C3 (47.1 ± 6.7%), C4 (34.3 ± 3.4%), inflammatory cytokines interleukin-17 (10.4 ± 2.7%), and tumor necrosis factor-α (8.2 ± 3.1%) were also observed. This study demonstrated that MEP-based adsorbent not only removed pathogenic autoantibodies directly from the blood as with protein A adsorbents but also modulated cellular immunity through removal of complement components and related proinflammatory cytokines, thereby providing a potentially superior strategy for the treatment of MG.

Suppressive regulatory T cells (Treg) and pathogenic T helper 17 (Th17) cells are two lymphocyte subsets with opposing activities in autoimmune diseases. The proinflammatory cytokine IL-6 is a potent factor in switching immune responses in vivo from the induction of Treg to pathogenic Th17 cells. We studied the Treg and Th17 cell compartments in experimental autoimmune myasthenia gravis (EAMG) and healthy control rats in order to assess whether the equilibrium between Treg and Th17 cells is perturbed in the disease. We found that Th17 cell-related genes are upregulated and Treg-related genes are downregulated in EAMG. The shift in favor of Th17 cells in EAMG could be reversed by antibodies to IL-6. Administration of anti-IL-6 antibodies to myasthenic rats suppressed EAMG when treatment started at the acute or at the chronic phase of disease. Suppression of EAMG by anti-IL-6 antibodies was accompanied by a decrease in the overall rat anti-AChR antibody titer and by a reduced number of B cells as compared with control treatment. Administration of anti-IL-6 antibodies led to down-regulation of several Th17 related genes including IL-17, IL-17R, IL-23R and IL-21 but did not affect the number of Treg cells in the lymph nodes. These data identify IL-6 as an important target for modulation of autoimmune responses.

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.