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Myasthenia gravis (MG) presents with symptoms that significantly affect patients' daily lives. Long-term MG therapies may lead to substantial side effects, predominantly due to prolonged immune suppression. Sirt6, which plays a vital role in maintaining cellular homeostasis and is recognised for its involvement in cytokine production in immune cells, has not yet been explored in relation to MG. PBMCs and CD4 + T cells were isolated from blood samples. RT-qPCR, western blot and ELISA were used to assess the expression of target genes and proteins. Flow cytometry was used to identify the subsets of T helper cells. Co-IP was conducted to investigate the interaction between USP9X and Sirt6. Finally, the experimental autoimmune myasthenia gravis (EAMG) model was established. In MG patients, Sirt6 levels were downregulated compared to healthy controls. Sirt6 overexpression led to a reduction in Th1 and Th17 cell populations while augmenting Treg cells in PBMCs. USP9X interacted with Sirt6, leading to its deubiquitination and stabilisation. Elevated Sirt6 levels subsequently mitigated symptoms in the EAMG model. The stabilisation of Sirt6, mediated by USP9X, has been found to relieve symptoms of EAMG by influencing the subtypes of T helper cells. This highlights the promising potential of Sirt6 as a viable therapeutic target in the treatment of MG., (© 2024 John Wiley & Sons Australia, Ltd.)

Myasthenia gravis (MG) is a prototypical autoimmune disease of the neuromuscular junction (NMJ). The study of the underlying pathophysiology has provided novel insights into the interplay of autoantibodies and complement-mediated tissue damage. Experimental autoimmune myasthenia gravis (EAMG) emerged as a valuable animal model, designed to gain further insight and to test novel therapeutic approaches for MG. However, the availability of native acetylcholine receptor (AChR) protein is limited favouring the use of recombinant proteins. To provide a simplified platform for the study of MG, we established a model of EAMG using a recombinant protein containing the immunogenic sequence of AChR in mice. This model recapitulates key features of EAMG, including fatigable muscle weakness, the presence of anti-AChR-antibodies, and engagement of the NMJ by complement and a reduced NMJ density. Further characterization of this model demonstrated a prominent B cell immunopathology supported by T follicular helper cells. Taken together, the herein-presented EAMG model may be a valuable tool for the study of MG pathophysiology and the pre-clinical testing of therapeutic applications.

Background: Myasthenia gravis (MG) and the experimental autoimmune MG (EAMG) animal model are characterized by T-cell-induced and B-cell-dominated autoimmune diseases that affect the neuromuscular junction. Several subtypes of CD4 + T cells, including T helper (Th) 17 cells, follicular Th cells, and regulatory T cells (Tregs), contribute to the pathogenesis of MG. However, increasing evidence suggests that CD8 + T cells also play a critical role in the pathogenesis and treatment of MG., Main Body: Herein, we review the literature on CD8 + T cells in MG, focusing on their potential effector and regulatory roles, as well as on relevant evidence (peripheral, in situ, cerebrospinal fluid, and under different treatments), T-cell receptor usage, cytokine and chemokine expression, cell marker expression, and Treg, Tc17, CD3 + CD8 + CD20 + T, and CXCR5 + CD8 + T cells., Conclusions: Further studies on CD8 + T cells in MG are necessary to determine, among others, the real pattern of the Vβ gene usage of autoantigen-specific CD8 + cells in patients with MG, real images of the physiology and function of autoantigen-specific CD8 + cells from MG/EAMG, and the subset of autoantigen-specific CD8 + cells (Tc1, Tc17, and IL-17 + IFN-γ + CD8 + T cells). There are many reports of CD20-expressing T (or CD20 + T) and CXCR5 + CD8 T cells on autoimmune diseases, especially on multiple sclerosis and rheumatoid arthritis. Unfortunately, up to now, there has been no report on these T cells on MG, which might be a good direction for future studies., (© 2024. The Author(s).)

Background and Objectives: Complement regulatory proteins at the neuromuscular junction (NMJ) could offer protection against complement-mediated damage in myasthenia gravis (MG). However, there is limited information on their expression at the human NMJ. Thus, this study aimed at investigating the expression of the cluster of differentiation 59 (CD59) at the NMJ of human muscle specimens and demonstrating the overexpression of CD59 mRNA and protein in the muscles of patients with MG., Methods: In this observational study, muscle specimens from 16 patients with MG (9 and 7 patients with and without thymoma, respectively) and 6 nonmyopathy control patients were examined. Immunohistochemical stains, Western blot analysis, and quantitative real-time reverse transcription PCR were used to evaluate the CD59 expression., Results: A strong localized expression of CD59 was observed at the NMJ in both patients with and without MG. Moreover, the CD59/glyceraldehyde-3-phosphate dehydrogenase protein ratio in patients with MG was significantly higher than that in the nonmyopathy controls (MG; n = 16, median 0.16, interquartile range (IQR) 0.08-0.26 and nonmyopathy controls; n = 6, median 0.03, IQR 0.02-0.11, p = 0.01). The proportion of CD59 mRNA expression relative to AChR mRNA expression (ΔCt CD59/AChR ) was associated with the quantitative MG score, MG activities of daily living score, and MG of Foundation of America Clinical Classification ( r = 0.663, p = 0.01; r = 0.638, p = 0.014; and r = 0.715, p = 0.003, respectively)., Discussion: CD59, which acts as a complement regulator, may protect the NMJ from complement attack. Our findings could provide a basis for further research that investigates the underlying pathogenesis in MG and the immunomodulating interactions of the muscle cells., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Diabetes mellitus (DM) is a common concomitant disease of late-onset myasthenia gravis (MG). However, the impacts of DM on the progression of late-onset MG were unclear., Methods: In this study, we examined the immune response in experimental autoimmune myasthenia gravis (EAMG) rats with DM or not. The phenotype and function of the spleen and lymph nodes were determined by flow cytometry. The serum antibodies, Tfh cells, and germinal center B cells were determined by ELISA and flow cytometry. The roles of advanced glycation end products (AGEs) in regulating Tfh cells were further explored in vitro by co-culture assays., Results: Our results indicated clinical scores of EAMG rats were worse in diabetes rats compared to control, which was due to the increased production of anti-R97-116 antibody and antibody-secreting cells. Furthermore, diabetes induced a significant upregulation of Tfh cells and the subtypes of Tfh1 and Tfh17 cells to provide assistance for antibody production. The total percentages of B cells were increased with an activated statue of improved expression of costimulatory molecules CD80 and CD86. We found CD4 + T-cell differentiation was shifted from Treg cells towards Th1/Th17 in the DM+EAMG group compared to the EAMG group. In addition, in innate immunity, diabetic EAMG rats displayed more CXCR5 expression on NK cells. However, the expression of CXCR5 on NKT cells was down-regulated with the increased percentages of NKT cells in the DM+EAMG group. Ex vivo studies further indicated that Tfh cells were upregulated by AGEs instead of hyperglycemia. The upregulation was mediated by the existence of B cells, the mechanism of which might be attributed the elevated molecule CD40 on B cells., Conclusions: Diabetes promoted both adaptive and innate immunity and exacerbated clinical symptoms in EAMG rats. Considering the effect of diabetes, therapy in reducing blood glucose levels in MG patients might improve clinical efficacy through suppressing the both innate and adaptive immune responses. Additional studies are needed to confirm the effect of glucose or AGEs reduction to seek treatment for MG., (© 2021. The Author(s).)

Myasthenia gravis (MG) is an autoimmune disease with autoantibodies against the mainly nicotinic acetylcholine receptor (AChR). High mobility group box1 (HMGB1) acts as a danger signal and drives the pathogenesis of autoimmune-mediated diseases. However, the role of HMGB1 in the pathogenesis of MG is not fully understood. Therefore, in this study, we analyzed serum levels of HMGB1 and immunohistochemical HMGB1 staining of muscle tissues in the passive transfer MG model to investigate the role of HMGB1 in MG. As a result, serum HMGB1 levels tended to be higher and the quantitative score of muscle pathology showed greater HMGB1 deposition (P = 0.02) along with sparser AChR staining and more severe inflammation in the passive transfer MG rats (n = 6) than those in control rats (n = 6). These findings indicate that HMGB1 is an important mediator and biomarker for inflammation in the pathogenesis of MG and can be a therapeutic target in MG., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Myasthenia gravis (MG) is a rare autoimmune disease mainly mediated by autoantibodies against the acetylcholine receptor (AChR) at the neuromuscular junction. The thymus is the effector organ, and its removal alleviates the symptoms of the disease. In the early-onset form of MG, the thymus displays functional and morphological abnormalities such as B cell infiltration leading to follicular hyperplasia, and the production of AChR antibodies. Type-I interferon (IFN-I), especially IFN-β, is the orchestrator of thymic changes observed in MG. As Dicer and miR-29 subtypes play a role in modulating the IFN-I signalization in mouse thymus, we investigated their expression in MG thymus., Methods: The expression of DICER and miR-29 subtypes were thoroughly investigated by RT-PCR in human control and MG thymuses, and in thymic epithelial cells (TECs). Using miR-29a/b-1-deficient mice, with lower miR-29a/b-1 expression, we investigated their susceptibility to experimental autoimmune MG (EAMG) as compared to wild-type mice., Results: DICER mRNA and all miR-29 subtypes were down-regulated in the thymus of MG patients and DICER expression was correlated with the lower expression of miR-29a-3p. A decreased expression of miR-29 subtypes was similarly observed in MG TECs; a decrease also induced in TECs upon IFN-β treatment. We demonstrated that miR-29a/b-1-deficient mice were more susceptible to EAMG without higher levels of anti-AChR IgG subtypes. In the thymus, if no B cell infiltration was observed, an increased expression of Ifn-β associated with Baff expression and the differentiation of Th17 cells associated with increased expression of Il-6, Il-17a and Il-21 and decreased Tgf-β1 mRNA were demonstrated in miR-29a/b-1-deficient EAMG mice., Conclusions: It is not clear if the decreased expression of miR-29 subtypes in human MG is a consequence or a causative factor of thymic inflammation. However, our results from the EAMG mouse model indicated that a reduction in miR-29a/b1 may contribute to the pathophysiological process involved in MG by favoring the increased expression of IFN-β and the emergence of pro-inflammatory Th17 cells.

MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) typically affects skeletal muscles of the bulbar area, including the omohyoid muscle, causing focal fatigue, weakness and atrophy. The profile of circulating extracellular microRNA (miRNA) is changed in MuSK + MG, but the intracellular miRNA profile in skeletal muscles of MuSK + MG and MuSK + experimental autoimmune MG (EAMG) remains unknown. This study elucidated the intracellular miRNA profile in the omohyoid muscle of mice with MuSK + EAMG. The levels of eleven mouse miRNAs were elevated and two mouse miRNAs were reduced in muscles of MuSK + EAMG mice. Transient expression of miR-1933-3p and miR-1930-5p in mouse muscle (C2C12) cells revealed several downregulated genes, out of which five had predicted binding sites for miR-1933-3p. The mRNA expression of mitochondrial ribosomal protein L27 (Mrpl27) and Inositol monophosphatase I (Impa1) was reduced in miR-1933-3p transfected C2C12 cells compared to control cells (p = 0.032 versus p = 0.020). Further, transient expression of miR-1933-3p reduced Impa1 protein accumulation in C2C12 cells. These findings provide novel insights of dysregulated miRNAs and their intracellular pathways in muscle tissue afflicted with MuSK + EAMG, providing a possible link to mitochondrial dysfunction and muscle atrophy observed in MuSK + MG., (Copyright © 2019 Elsevier B.V. and Japan Neuroscience Society. All rights reserved.)

Background: While extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific 'myocyte' cultures., Methods: From well-characterised MG patients we developed the 'myocyte' culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG)., Results: OP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p < 0.05). A correlation matrix of gene pair expression levels revealed that 15% of gene pairs were strongly correlated in OP-MG samples (r > 0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10 - 6 ) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway., Conclusion: Using a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies.

The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1 R63W ). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1 R63W ) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1 R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4 + T cells, as well as an increased frequency of antigen-specific CD4 + T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.

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.

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2  b ) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.

We have previously demonstrated that Cysteinyl aspartate-specific proteinase-1 (caspase-1) inhibitor ameliorates experimental autoimmune myasthenia gravis (EAMG) by inhibited cellular immune response, via suppressing DC IL-1 β, CD4 + T and γdT cells IL-17 pathways. In this study, we investigated the effect of caspase-1 inhibitor on humoral immune response of EAMG and further explore the underlying mechanisms. An animal model of MG was induced by region 97-116 of the rat AChR α subunit (R97-116 peptide) in Lewis rats. Rats were treated with caspase-1 inhibitor Ac-YVAD-cmk intraperitoneally (i.p.) every second day from day 13 after the first immunization. Flow cytometry, western blot, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the neuroprotective effect of caspase-1 inhibitor on humoral immune response of EAMG. The results showed that caspase-1 inhibitor reduced the relative affinity of anti-R97-116 IgG, suppressed germinal center response, decreased follicular helper T cells, and increased follicular regulatory T cells and regulatory B cells. In addition, we found that caspase-1 inhibitor inhibited humoral immunity response in EAMG rats via suppressing IL-6-STAT3-Bcl-6 pathways. These results suggest that caspase-1 inhibitor ameliorates EAMG by regulating humoral immune response, thus providing new insights into the development of myasthenia gravis and other autoimmune diseases therapies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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-β, 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.

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 ε) 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Curcumin is a traditional Asian medicine with diverse immunomodulatory properties used therapeutically in the treatment of many autoimmune diseases. However, the effects of curcumin on myasthenia gravis (MG) remain undefined. Here we investigated the effects and potential mechanisms of curcumin in experimental autoimmune myasthenia gravis (EAMG). Our results demonstrated that curcumin ameliorated the clinical scores of EAMG, suppressed the expression of T cell co-stimulatory molecules (CD80 and CD86) and MHC class II, down-regulated the levels of pro-inflammatory cytokines (IL-17, IFN-γ and TNF-α) and up-regulated the levels of the anti-inflammatory cytokine IL-10, shifted the balance from Th1/Th17 toward Th2/Treg, and increased the numbers of NKR-P1(+) cells (natural killer cell receptor protein 1 positive cells, including NK and NKT cells). Moreover, the administration of curcumin promoted the differentiation of B cells into a subset of B10 cells, increased the anti-R97-166 peptide IgG1 levels and decreased the relative affinity indexes of anti-R97-116 peptide IgG. In summary, curcumin effectively ameliorate EAMG, indicating that curcumin may be a potential candidate therapeutic agent for MG., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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.

Adoptive transfer of regulatory T (Treg) cells have been employed effectively for suppression of several animal models for autoimmune diseases. In order to employ Treg cell therapy in patients, it is necessary to generate Treg cells from the patient's own cells (autologous) that would be able to suppress effectively the disease in vivo, upon their reintroduction to the patient. Towards this objective, we report in the present study on a protocol for a successful immune-regulation of experimental autoimmune myasthenia gravis (EAMG) by ex vivo--generated autologous Treg cells. For this protocol bone marrow (BM) cells, are first cultured in the presence of GM-CSF, giving rise to a population of CD11c(+)MHCII(+)CD45RA(+)CD8(-) DCs (BMDCs). Splenic CD4(+) T cells are then co-cultured with the differentiated BM cells and expand to 90% of Foxp3(+) Treg cells. In vitro assay exhibits a similar dose dependent manner in the suppression of T effector cells proliferation between Treg cells obtained from either healthy or sick donors. In addition, both Treg cells inhibit similarly the secretion of IFN-γ from activated splenocytes. Administration of 1 × 10(6) ex-vivo generated Treg cells, I.V, to EAMG rats, modulates the disease following a single treatment, given 3 days or 3 weeks after disease induction. Similar disease inhibition was achieved when CD4 cells were taken from either healthy or sick donors. The disease suppression was accompanied by reduced levels of total AChR specific antibodies in the serum. Moreover, due to the polyclonality of the described Treg cell, we have examined whether this treatment approach could be also employed for the treatment of other autoimmune diseases involving Treg cells. Indeed, we demonstrated that the ex-vivo generated autologous Treg cells suppress Adjuvant Arthritis (AA) in rats. This study opens the way for the application of induced autologous Treg cell therapy for myasthenia gravis, as well as for other human autoimmune diseases involving Treg cells., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.

Terminal Schwann cells (TSCs) are key components of the mammalian neuromuscular junction (NMJ). How the TSCs sense the synaptic activity in physiological conditions remains unclear. We have taken advantage of the distinct localization of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at the NMJ to bring out the function of different ACh receptors (AChRs). AChE is clustered by the collagen Q in the synaptic cleft and prevents the repetitive activation of muscle nicotinic AChRs. We found that BChE is anchored at the TSC by a proline-rich membrane anchor, the small transmembrane protein anchor of brain AChE. When BChE was specifically inhibited, ACh release was significant depressed through the activation of α7 nAChRs localized on the TSC and activated by the spillover of ACh. When both AChE and BChE were inhibited, the spillover increased and induced a dramatic reduction of ACh release that compromised the muscle twitch triggered by the nerve stimulation. α7 nAChRs at the TSC may act as a sensor for spillover of ACh adjusted by BChE and may represent an extrasynaptic sensor for homeostasis at the NMJ. In myasthenic rats, selective inhibition of AChE is more effective in rescuing muscle function than the simultaneous inhibition of AChE and BChE because the concomitant inhibition of BChE counteracts the positive action of AChE inhibition. These results show that inhibition of BChE should be avoided during the treatment of myasthenia and the pharmacological reversal of residual curarization after anesthesia., (Copyright © 2014 the authors 0270-6474/14/3411870-14$15.00/0.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Myasthenia gravis (MG) is an autoimmune disease caused by circulating antibodies that block acetylcholine receptor (AchR) at the neuromuscular junction. There is the cognitive and memory impairment in patients with MG. However, the molecular mechanisms underlying the alteration of central nervous system in MG remain unknown. In the present study, we found that the level of malondialdehyde (MDA) was increased in the brain of experimental autoimmune myasthenia gravis (EAMG). Furthermore, the expression of thioredoxin-1 (Trx-1) and the activity of cAMP response element-binding protein (CREB) were significantly decreased in frontal lobe and hippocampus of mice with EAMG. We also found that the expression of pro-apoptotic C/EBP homologous protein (CHOP) was increased in the frontal lobe and hippocampus of mice. However, the expressions of glucose regulated protein 78 (GRP78/Bip) was not changed in same areas. Inversely, the expressions of pro-caspase-12, pro-caspase-3 and pro-caspase-9 were decreased. These data indicate that Trx-1 mediated endoplasmic reticulum and mitochondria pathways are involved in brain damage in MG. Trx-1 may be a pivotal target for brain protective treatment in MG., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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). We exploited the proprietary NMJ-targeting signals of ColQ to treat congenital myasthenia and to explore the mechanisms of autoimmune myasthenia gravis (MG). Mutations in COLQ cause congenital endplate AChE deficiency (CEAD). First, a single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq−/− mice normalized motor functions, synaptic transmission, and partly the NMJ ultrastructure. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. Second, MuSK antibody-positive MG accounts for 5-15 % of MG. In vitro overlay of AChE/ColQ to muscle sections of Colq−/− mice, as well as in vitro plate-binding of MuSK to ColQ, revealed thatMuSK-IgG blocks binding of ColQ to MuSK in a dose-dependent manner. Passive transfer of MuSK-IgG to wild-type mice markedly reduced the size and intensity of ColQ signals at NMJs. MuSK-IgG thus interferes with binding of ColQ to MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq−/− mice and also to reveal underlying mechanisms of anti-MuSK-MG.

Purpose: Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles (EOM), and we have hypothesized that this may be due to a unique immunological environment. To assess this hypothesis, we took an unbiased approach to analyze RNA expression profiles in EOM, diaphragm, and extensor digitorum longus (EDL) in rats with experimentally acquired myasthenia gravis (EAMG)., Methods: Experimentally acquired myasthenia gravis was induced in rats by intraperitoneal injection of antibody directed against the acetylcholine receptor (AChR), whereas control rats received antibody known to bind the AChR but not induce disease. After 48 hours, animals were killed and muscles analyzed by RNA expression profiling. Profiling results were validated using qPCR and immunohistochemical analysis., Results: Sixty-two genes common among all muscle groups were increased in expression. These fell into four major categories: 12.8% stress response, 10.5% immune response, 10.5% metabolism, and 9.0% transcription factors. EOM expressed 212 genes at higher levels, not shared by the other two muscles, and a preponderance of EOM gene changes fell into the immune response category. EOM had the most uniquely reduced genes (126) compared with diaphragm (26) and EDL (50). Only 18 downregulated genes were shared by the three muscles. Histological evaluation and disease load index (sum of fold changes for all genes) demonstrated that EOM had the greatest degree of pathology., Conclusions: Our studies demonstrated that consistent with human myasthenia gravis, EOM demonstrates a distinct RNA expression signature from EDL and diaphragm, which is based on differences in the degree of muscle injury and inflammatory response., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Myasthenia gravis (MG) patients with antibodies against muscle specific tyrosine kinase (MuSK+) typically present focal fatigue and atrophy of the facial and bulbar muscles, including the masseter muscle, whereas leg muscles often are clinically spared. This study addresses the regulation of the mTOR signaling pathway in the masseter muscle versus the leg muscle tibialis anterior (TA). We analyzed muscle morphology, protein levels of mTOR components as well as atrogenes and mitochondrial markers in these muscles of healthy control mice and mice with different clinical severity grades of MuSK+ experimental autoimmune MG (EAMG). Protein levels of mTOR components were reduced in the atrophic masseter muscle of MuSK+ EAMG mice, whereas enhanced accumulation of mTOR components was observed in the TA muscles. Two other muscles: omohyoid and soleus showed intermediate spectra. In conclusion, the anabolic mTOR signaling pathway is differentially regulated even in muscles with the same activity pattern in the same neuromuscular disease. This could in part explain the clinical phenotype in MuSK+ EAMG as well as in muscular dystrophies., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies, most commonly directed against the acetylcholine receptor (AChR), impair neuromuscular transmission and cause muscle weakness. In this study, we utilized two-dimensional difference in-gel electrophoresis (2D-DIGE) to analyze the muscle's proteomic profile at different stages of experimental autoimmune myasthenia gravis (EAMG). We identified twenty-two differentially expressed proteins, mainly related to metabolic and stress-response pathways. Interestingly, these identified proteins have also been associated with other contraction-impairing muscle pathologies (e.g. inclusion body myositis), suggesting a similar response of the muscle to such conditions., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Myasthenia gravis (MG) is a T cell-dependent antibody-mediated autoimmune neuromuscular disease. Antibodies to the nicotinic acetylcholine receptor (AChR) destroy the AChR, thus leading to defective neuromuscular transmission of electrical impulse and to muscle weakness. This unit is a practical guide to the induction and evaluation of experimental autoimmune myasthenia gravis (EAMG) in the mouse, the animal model for MG. Protocols are provided for the extraction and purification of AChR from the electric organs of Torpedo californica, or the electric ray. The purified receptor is used as an immunogen to induce autoimmunity to AChR, thus causing EAMG. The defect in neuromuscular transmission can also be measured quantitatively by electromyography. In addition, EAMG is frequently characterized by the presence of serum antibodies to AChR, which are measured by radioimmunoassay and by a marked antibody-mediated reduction in the number of muscle AChRs. AChR extracted from mouse muscle is used in measuring serum antibody levels and for quantifying muscle AChR content. Another hallmark of the disease is complement and IgG deposits located at the neuromuscular junction, which can be visualized by immunofluorescence techniques., (© 2013 by John Wiley & Sons, Inc.)

Complement plays an important role in the pathophysiology of experimental autoimmune myasthenia gravis (EAMG). The deposition of IgG at the neuromuscular junction, followed by the activation and observance of C3 at the site, and finally the insertion of the membrane attack complex results in the destruction of the plasma membrane at the neuromuscular junction. Animal models of complement-deficient components show the importance of the mediated lysis in EAMG. These events have regulators that allow for the limitation in the cascade and the ability of the cell to inhibit complement at many places along the pathway. The complement regulatory proteins have many roles in reducing the activation of the complement cascade and the inflammatory pathways. Mice deficient in complement regulatory proteins, decay accelerating factor, and CD59 demonstrate a significant increase in the destruction at the neuromuscular junction. Inhibition of complement-mediated lysis is an attractive therapeutic in MG., (© 2012 New York Academy of Sciences.)

Antimuscle-specific kinase (anti-MuSK) myasthenia (AMM) differs from antiacetylcholine receptor myasthenia gravis in exhibiting more focal muscle involvement (neck, shoulder, facial, and bulbar muscles) with wasting of the involved, primarily axial, muscles. AMM is not associated with thymic hyperplasia and responds poorly to anticholinesterase treatment. Animal models of AMM have been induced in rabbits, mice, and rats by immunization with purified xenogeneic MuSK ectodomain, and by passive transfer of large quantities of purified serum IgG from AMM patients into mice. The models have confirmed the pathogenic role of the MuSK antibodies in AMM and have demonstrated the involvement of both the presynaptic and postsynaptic components of the neuromuscular junction. The observations in this human disease and its animal models demonstrate the role of MuSK not only in the formation of this synapse but also in its maintenance., (© 2012 New York Academy of Sciences.)

Controlled regulation of synaptic nicotinic acetylcholine receptors (AChRs) and acetylcholinesterase (AChE), together with maintenance of a dynamic balance between them, is a requirement for proper function of cholinergic synapses. In the present study we assessed whether pathological changes in AChR perturb this balance, and whether such changes can be corrected. We studied the influence of AChR loss, caused by experimental autoimmune myasthenia gravis (EAMG), on muscle AChE, as well as the reciprocal effect of an antisense targeted towards AChE on both AChR and AChE at the neuromuscular synapse. The extensor digitorum longus (EDL) muscles of EAMG Lewis rats were isolated, and AChE levels and isoform compositions were examined. Although AChE levels in the muscles of healthy and EAMG rats were similar, marked changes were observed in isoform composition. Healthy EDL muscles contained globular (G(1,2) , G(4) ) and asymmetric (primarily A(12) ) isoforms. G(1,2) -AChE was significantly reduced in EAMG muscles, whereas both G(4) - and A(12) -AChE remained unchanged. Treatment of EAMG rats with the antisense EN101 resulted in decreased total muscle AChE, with recovery in G(1,2) and reduction in A(12) -AChE. AChE/AChR ratios were determined at the neuromuscular junctions (NMJ). The decrease in AChR levels that occurred as the disease progressed resulted in a dramatic increase in this ratio, and a significant recovery towards normal ratios occurred after EN101 treatment. This improvement was primarily due to increased synaptic AChR content. Our findings emphasise the tight connection between AChR and AChE at the myasthenic NMJ, and the importance of the AChE/AChR ratio in maintaining the required cholinergic balance., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Probiotics are live bacteria that confer health benefits to the host physiology. Although protective role of probiotics have been reported in diverse diseases, no information is available whether probiotics can modulate neuromuscular immune disorders. We have recently demonstrated that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse experimental disorders in mice model. In this study we further investigated whether IRT5 probiotics could modulate the progression of experimental autoimmune myasthenia gravis (EAMG). Myasthenia gravis (MG) is a T cell dependent antibody mediated autoimmune disorder in which acetylcholine receptor (AChR) at the neuromuscular junction is the major auto-antigen. Oral administration of IRT5 probiotics significantly reduced clinical symptoms of EAMG such as weight loss, body trembling and grip strength. Prophylactic effect of IRT5 probiotics on EMAG is mediated by down-regulation of effector function of AChR-reactive T cells and B cells. Administration of IRT5 probiotics decreased AChR-reactive lymphocyte proliferation, anti-AChR reactive IgG levels and inflammatory cytokine levels such as IFN-γ, TNF-α, IL-6 and IL-17. Down-regulation of inflammatory mediators in AChR-reactive lymphocytes by IRT5 probiotics is mediated by the generation of regulatory dendritic cells (rDCs) that express increased levels of IL-10, TGF-β, arginase 1 and aldh1a2. Furthermore, DCs isolated from IRT5 probiotics-fed group effectively converted CD4(+) T cells into CD4(+)Foxp3(+) regulatory T cells compared with control DCs. Our data suggest that IRT5 probiotics could be applicable to modulate antibody mediated autoimmune diseases including myasthenia gravis.

Objective: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%-15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK-immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK., Methods: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq-/- mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice., Results: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dose-dependent block of MuSK binding to ColQ by but not to LRP4. 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 size and density of AChR and MuSK., Conclusions: As lack of ColQ compromises agrin-mediated AChR clustering in Colq-/- mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox.

RAGE belongs to immunoglobulin superfamily and serves as a ligand for various immunoregulatory molecules including S100B that has been demonstrated important to T cell mediated autoimmune diseases. In this context, we hypothesized that RAGE could also impact B cell mediated, T cell-dependent autoimmune diseases. This was tested using myasthenia gravis (MG) animal model, EAMG. We show that expression of both RAGE and S100B are increased during EAMG and the interaction between RAGE and S100B affected the Th1/Th2/Th17/Treg cell equilibrium, up-regulate AChR-specific T cell proliferation. Furthermore, addition of S100B in vitro stimulated splenocyte activity linked to COX-2 up-regulation. NS-398, a selective COX-2 inhibitor, effectively diminished S100B mediated activity of AChR-specific antibody secreting splenocytes. These findings suggested that a reciprocal relationship between RAGE and S100B promoted the development of EAMG, highlighting the importance of understanding the mechanisms of EAMG disease as a means of developing new therapies for the treatment of MG., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: To observe the therapeutic effect of RelB-silenced dendritic cells (DCs) in experimental autoimmune myasthenia gravis (EAMG), and further to investigate the mechanism of this immune system therapeutic., Methods: (1) RelB-silenced DCs and control DCs were prepared and the supernatants were collected for IL-12p70, IL-6, and IL-23 measurement by ELISA. (2) RelB-silenced DCs and control DCs were co-cultured with AChR-specific T cells, and the supernatant was collected to observe the IL-17, IFN-gamma, IL-4 production. (3) EAMG mice with clinical scores of 1 to 2 were collected and enrolled randomly into the RelB-silenced DC group or the control DC group. RelB-silenced DCs or an equal amount of control DCs were injected intravenously on days 0, 7, and 14 after enrollment. Clinical scores were evaluated every other day. Twenty days after allotment, serum from individual mice was collected to detect serum concentrations of anti-mouse AChR IgG, IgG1, IgG2b, and IgG2c. The splenocytes were isolated for analysis of lymphocyte proliferative responses, cytokine (IL-17, IFN-gamma, IL-4) production, and protein levels of RORgammat, T-bet, GATA-3, and FoxP3 (the special transcription factors of Th17, Th1, Th2, and Treg, respectively)., Results: (1) RelB-silenced DCs produced significantly reduced amounts of IL-12p70, IL-6, and IL-23, as compared with control DCs. (2) RelB-silenced DCs spurred on the CD4(+) T cells from Th1/Th17 to the Th2 cell subset in the co-culture system. (3) Treatment with RelB-silenced DCs effectively reduced myasthenic symptoms and levels of serum anti-acetylcholine receptor autoantibody in mice with ongoing EAMG. Th17-related markers (RORgammat, IL-17) and Th1-related markers (T-bet, IFN-gamma) were downregulated, whereas Th2 markers (IL-4 and GATA3) and Treg marker (FoxP3) were upregulated., Conclusions: RelB-silenced DCs were able to create a particular cytokine environment that was absent of inflammatory cytokines. RelB-silenced DCs provide a practical means to normalize the differentiation of the four T-cell subsets (Th17, Th1, Th2, and Treg) in vivo, and thus possess therapeutic potential in Th1/Th17-dominant autoimmune disorders such as myasthenia gravis.

The study reported below describes increased expression of IL-4 receptor in cultured rat myocytes following exposure to an antibody reactive with the acetylcholine receptor (AChR). In addition, upon up-regulation of IL-4R, myocytes demonstrated an increased responsiveness to IL-4 by producing increased levels of IL-15. Moreover, following passive transfer of AChR antibody into Lewis rats, both the increased IL-4R expression and IL-15 production were also observed in intact skeletal muscle, co-localizing in particular individual muscle fibers; the same muscle fibers also produced the chemokine MCP-1 to which IL-4-producing T cells were attracted. A model is proposed in which these muscle activities participate in disease progression in experimental myasthenia gravis.

Previous studies have shown increased expression of acetylcholine receptor-alpha (AChR-alpha) subunit transcripts in myasthenia gravis (MG) and experimental MG (EAMG), but none examined the functional properties of this overexpression. In this study we examined the mRNA and protein expression of AChR-alpha as well as the pattern of alpha-bungarotoxin labeling in muscle tissue from EAMG mice with varying disease severity. AChR-alpha expression was increased considerably in endplates from mice with severe EAMG, but it was distinct and greatly in excess of alpha-bungarotoxin labeling. This "aberrant expression" occurred in mice with morphologic endplate damage, and the pattern of complement and immunoglobulin deposition in muscle from these mice appeared to mirror the pattern of AChR-alpha expression. The loss of functional AChR in severe MG increases transcription of AChR-alpha mRNA, but the expressed protein is "functionally inert," failing to compensate for loss of AChR. This enhanced expression of AChR may play a role in driving the ongoing autoimmune response. Muscle Nerve 40: 279-286, 2009.

In addition to their capacity to differentiate, BM stromal cells (BMSC) have immunosuppressive qualities that make them strong candidates for use in cell therapy against human autoimmune diseases. We studied the immunoregulatory activities of BMSC on experimental autoimmune myasthenia gravis (EAMG) in vitro and in vivo. Intravenous administration of syngenic BMSC to EAMG-model rats on the day of their second immunization was effective in ameliorating the pathological features of the disease. In vitro, the proliferative ability of T cells or B cells from EAMG rats was inhibited when they were cocultured with BMSC at proper ratios. This inhibitory effect was at least partially dependent on the secretion of IDO. We also determined that the development of EAMG is accompanied by an imbalance among the Th1, Th2, Th17, and Treg cell subsets, and that this can be corrected by the administration of BMSC, which leads to an increase of Th2 (IL-4) and Treg (Foxp3) cells, and a reduction of Th1 (IFN-gamma) and Th17 (IL-17) cells, through an IDO-dependent mechanism. These results provide further insights into the pathogenesis of MG, EAMG, and other immune-mediated diseases, and support a potential role for BMSC in their treatment.

The development and function of Th17 cells are influenced in part by the cytokines TGF-beta, IL-23 and IL-6, but the mechanisms that govern recruitment and activity of Th17 cells during initiation of autoimmunity remain poorly defined. We show here that the development of autoreactive Th17 cells in secondary lymphoid organs in experimental autoimmune myasthenia gravis--an animal model of human myasthenia gravis--is modulated by IL-6-producing CD11b(+) cells via the CC chemokine ligand 2 (CCL2). Notably, acetylcholine receptor (AChR)-reactive Th17 cells provide help for the B cells to produce anti-AChR antibodies, which are responsible for the impairment of the neuromuscular transmission that contributes to the clinical manifestations of autoimmunity, as indicated by a lack of disease induction in IL-17-deficient mice. Thus, Th17 cells can promote humoral autoimmunity via a novel mechanism that involves CCL2.

Mice deficient for classical complement pathway (CCP) factor C4 are resistant to antibody and complement mediated experimental autoimmune myasthenia gravis (EAMG). Anti-C1q antibody administration before or following acetylcholine receptor immunization suppresses EAMG development by reducing lymph node cell IL-6 production and neuromuscular junction IgG, C3 and C5b-C9 deposition. This effect is achieved by treating mice with 10 microg of anti-C1q antibody, twice weekly for 4 weeks. Treatment with a higher amount of anti-C1q antibody gives rise to increased serum anti-acetylcholine receptor antibody, immune complex and C3 levels, facilitates kidney C3 and IgG deposits and thus reduces the treatment efficacy. C4 KO and anti-C1q antibody treated mice display normal immune system functions and intact antibody production capacity. Furthermore, CCP inhibition preserves alternative complement pathway activation, which is required for host defense against microorganisms. Therefore, CCP inhibition might constitute a specific treatment approach for not only myasthenia gravis but also other complement mediated autoimmune diseases.

Objective: To study the modulation of Jianjining Recipe (JJNR), a traditional Chinese compound herbal medicine for invigorating spleen and kidney on differential protein expression in spleen of rats with experimental autoimmune myasthenia gravis (EAMG)., Methods: EAMG rats were randomly divided into four groups: untreated group, JJNR-treated group, Qiangji Jianli capsule (QJJLC, a traditional Chinese compound herbal medicine)-treated group and prednisolone acetate (PA)-treated group. After therapeutic intervention with the above drugs for four consecutive weeks, the level of differential protein expression was analyzed by two-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry., Results: Twelve differential proteins were identified by comparing EAMG rats and normal rats. The levels of allograft inflammatory factor-1, peroxiredoxin I and actin-related protein 2/3 complex subunit 5 were significantly regulated by JJNR (P<0.01). These proteins were closely associated with immune response and cell movement., Conclusion: The results suggest that there are differential protein expressions between EAMG rats and normal rats. Furthermore, as a Chinese medicine prescription with effect of invigorating spleen and kidney, JJNR can effectively regulate the levels of some EAMG-related protein expression.