Experimental Autoimmune Myasthenia Gravis Model Research Articles

Recombinant Acetylcholine Receptor Immunization Induces a Robust Model of Experimental Autoimmune Myasthenia Gravis in Mice.

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.

Promising Antigen-specific Immunotherapy for the Treatment of Myasthenia Gravis

Myasthenia gravis (MG) is a T cell-dependent, antibody-mediated, autoimmune disorder with well-established antigenic targets at the neuromuscular junction. MG autoantibodies mainly target the nicotinic acetylcholine receptor (AChR) and especially epitopes located in the extracellular domain of the α1 subunit (α1-ECD). Today, most therapeutic regimens for MG are non-specific and not curative, requiring chronic treatments that are associated with significant side effects. We aim to develop an antigen-specific therapeutic approach, based on reestablishing tolerance towards the AChR, the dominant autoantigen in MG. To this end, we used a soluble mutated form of the human α1-ECD, which incorporates a major fraction of MG autoreactive T cell epitopes and examined the therapeutic efficiency of intravenous administration in a rat experimental autoimmune MG model. We found that repeated intravenous administration of α1-ECD for up to 12 days led to a robust amelioration of disease symptoms in a dose and time-dependent manner. The observed therapeutic effect of α1-ECD was significantly better than the effect of two current mainstay drugs for MG treatment. There were no signs of toxicity in α1-ECD-treated animals and further studies are underway to fully elucidate the immunological mechanism underlying the treatment effect. Taken together, our preclinical data strongly suggest that intravenous administration of α1-ECD may represent an efficacious and safe strategy to treat MG and thus α1-ECD represents a new drug candidate for clinical application in MG.

Astragaloside IV ameliorates experimental autoimmune myasthenia gravis by regulating CD4 + T cells and altering gut microbiota

BackgroundMyasthenia gravis (MG) is an antibody-mediated autoimmune disease and its pathogenesis is closely related to CD4 + T cells. In recent years, gut microbiota is considered to play an important role in the pathogenesis of MG. Astragaloside IV (AS-IV) is one of the main active components extracted from Astragalus membranaceus and has immunomodulatory effects. To study the immunomodulatory effect of AS-IV and the changes of gut microbiota on experimental autoimmune myasthenia gravis (EAMG) mice, we explore the possible mechanism of AS-IV in improving MG.MethodsIn this study, network pharmacology was utilized to screen the crucial targets of AS-IV in the treatment of MG. Subsequently, a Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to identify potential pathways through which AS-IV acts against MG. Furthermore, experimental investigations were conducted to validate the underlying mechanism of AS-IV in MG treatment. Before modeling, 5 mice were randomly selected as the control group (CFA group), and the other 10 were induced to EAMG model. These mice were randomly divided into EAMG group and EAMG + AS-IV group, n = 5/group. In EAMG + AS-IV group, AS-IV was administered by gavage. CFA and EAMG groups were given the same volume of PBS. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. At the last administration, the feces were collected for 16S RNA microbiota analysis. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected by flow cytometry. The levels of IFN-γ, IL-17 and TGF-β in serum were measured by ELISA. Furthermore, fecal microbial transplantation (FMT) experiments were performed for exploring the influence of changed intestinal flora on EAMG. After EAMG model was induced, the mice were treated with antibiotics daily for 4 weeks to germ-free. Then germ-free EAMG mice were randomly divided into two groups: FMT EAMG group, FMT AS-IV group, n = 3/group. Fecal extractions from EAMG and EAMG + AS-IV groups as gathered above were used to administered daily to the respective groups for 4 weeks. Body weight, grip strength and clinical symptoms were assessed and recorded weekly. The levels of Treg, Th1 and Th17 cells in spleen and Th1 and Th17 cells in thymus were detected at the last administration. The levels of IFN-γ, IL-17 and TGF-β in serum were measured by ELISA.ResultsThe network pharmacology and KEGG pathway analysis revealed that AS-IV regulates T cell pathways, including T cell receptor signaling pathway and Th17 cell differentiation, suggesting its potential in improving MG. Further experimental verification demonstrated that AS-IV administration improved muscle strength and body weight, reduced the level of Th1 and Th17 cells, enhanced the level of Treg cells, and resulted in alterations of the gut microbiota, including changes in beta diversity, the Firmicutes/Bacteroidetes (F/B) ratio, and the abundance of Clostridia in EAMG mice. We further conducted FMT tests and demonstrated that the EAMG Abx-treated mice which were transplanted the feces of mice treated with AS-IV significantly alleviated myasthenia symptoms, reduced Th1 and Th17 cells levels, and increased Treg cell levels.ConclusionThis study speculated that AS-IV ameliorates EAMG by regulating CD4 + T cells and altering the structure and species of gut microbiota of EAMG.

Study on the Potential Mechanism of Semen Strychni against Myasthenia Gravis Based on Network Pharmacology and Molecular Docking with Experimental Verification.

Background Semen Strychni (SS) is an effective Chinese medicine formula for treating myasthenia gravis (MG) in clinics. Nonetheless, its molecular mechanism is largely unknown. Objective Using network pharmacology, molecular docking, and experimental validation, we aim to identify the therapeutic effect of SS on MG and its underlying mechanism. Methods The main ingredients of SS and their targets and potential disease targets for MG were extracted from public databases. The protein-protein interaction (PPI) network was constructed using the STRING 11.0 database, and Cytoscape was used to identify the hub targets. In addition, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to identify molecular biological processes and signaling pathways. Then, AutoDock Via conducted molecular docking. The experimental autoimmune myasthenia gravis (EAMG) model in female Lewis rats, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to confirm the effect and mechanism of SS on MG. Results The following active compounds and hub targets were identified by screening and analyzing: isobrucine, vomicine, (S)-stylopine, strychnine, brucine-N-oxide, brucine and AKT1, MAPK1, MAPK14, CHRM1, ACHE, and CHRNA4. KEGG enrichment analyses indicated that the cholinergic synapse and neuroactive ligand-receptor interaction signaling pathway may be necessary. The results of molecular docking revealed that the main active ingredients bind well to the hub targets. In vivo experiments proved that SS could improve the weight loss and Lennon scores in the EAMG model. Experiments in molecular biology showed that SS could treat MG by affecting the cholinergic synapse through the respective antibody, receptor, and key enzymes in the cholinergic pathway. Conclusion This study provided a preliminary overview of the active constituents, primary targets, and potential pathways of SS against MG. SS ameliorated EAMG by regulating the cholinergic synaptic junction.

A Targeted Complement Inhibitor CRIg/FH Protects Against Experimental Autoimmune Myasthenia Gravis in Rats via Immune Modulation

Antibody-induced complement activation may cause injury of the neuromuscular junction (NMJ) and is thus considered as a primary pathogenic factor in human myasthenia gravis (MG) and animal models of experimental autoimmune myasthenia gravis (EAMG). In this study, we tested whether CRIg/FH, a targeted complement inhibitor, could attenuate NMJ injury in rat MG models. We first demonstrated that CRIg/FH could inhibit complement-dependent cytotoxicity on human rhabdomyosarcoma TE671 cells induced by MG patient-derived IgG in vitro. Furthermore, we investigated the therapeutic effect of CRIg/FH in a passive and an active EAMG rodent model. In both models, administration of CRIg/FH could significantly reduce the complement-mediated end-plate damage and suppress the development of EAMG. In the active EAMG model, we also found that CRIg/FH treatment remarkably reduced the serum concentration of autoantibodies and of the cytokines including IFN-γ, IL-2, IL-6, and IL-17, and upregulated the percentage of Treg cells in the spleen, which was further verified in vitro. Therefore, our findings indicate that CRIg/FH may hold the potential for the treatment of MG via immune modulation.

Osteopontin exacerbates the progression of experimental autoimmune myasthenia gravis by affecting the differentiation of T cell subsets

Osteopontin (OPN) is a multifunctional extracellular matrix phosphoprotein that has a specific and complicated structure, and contributes to numerous physiological and pathological activities. The mechanism of OPN in many diseases has been confirmed; however, the role of OPN in myasthenia gravis (MG) remains unclear. In this study, we recombined rat OPN protein in vitro, and assessed how OPN affects the development of autoimmunity using an experimental autoimmune myasthenia gravis (EAMG) rat model. The results showed that the concentration of OPN in serum was up-regulated. Both mRNA and protein levels in splenocytes increased in the EAMG model. OPN treatment in vitro strongly promoted the differentiation of Th1 cells, and inhibited the differentiation of Treg cells. Intraperitoneal injection of OPN revealed the early incidence of EAMG, and more serious disease. This effect was accompanied by an increased percentage of Th1 cells. In conclusion, OPN likely exacerbates the pathogenesis of EAMG by promoting the differentiation of Th1 cells and inhibiting the differentiation of Treg cells.

Effect of Grilled Nux Vomica on Differential RNA Expression Profile of Gastrocnemius Muscle and Toll‑Like Receptor 4 (TLR-4)/Nuclear Factor kappa B (NF-κB) Signaling in Experimental Autoimmune Myasthenia Gravis Rats.

BackgroundMyasthenia gravis (MG) is a progressive autoimmune disorder caused by the production of antibodies directed against acetylcholine receptors (AChRs), resulting in muscle weakness and fatigue. This study aimed to explore the effect and mechanism of grilled nux vomica (GNV) in experimental autoimmune myasthenia gravis (EAMG) rats.Material/MethodsRat 97–116 peptides were used to mediate disease in the EAMG model in SPF female Lewis rats. The treatment groups received grilled nux vomica (75 mg/kg, 150 mg/kg, and 225 mg/kg). The autoantibody and inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). RNA profiling was performed on high-dose and model group rats. Profiling results and TLR-4/NF-κB signaling were validated by q-PCR and Western blot analysis.ResultsThe results showed that GNV could attenuate the symptoms of EAMG rats. There was a decreased level of AChR-ab, IFN-γ, TNF-α, IL-2, IL-4, and IL-17 levels, and an increased level of TGF-β1. In total, 235 differentially expressed genes (DEGs), consisting of 175 upregulated DEGs and 60 downregulated DEGs, were identified. Functional annotation demonstrated that DEGs were largely associated with leukocyte cell–cell adhesion, NF-kappa B signaling pathway, muscle contraction, and cardiac muscle contraction pathway. Rac2, Itgb2, Lcp2, Myl3, and Tnni1 were considered as hub genes with a higher degree value in the protein–protein interaction (PPI) network. The q-PCR and Western blot results of hub genes were consistent with RNA profiles. GNV treatment also significantly reduced the TLR-4 and NF-κB p65 protein expression in EAMG rats.ConclusionsThese results indicate that grilled nux vomica ameliorates EAMG by depressing the TLR-4/NF-κB signaling pathway, and hub genes may serve as potential targets for MG treatment.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

Use of Toll-Like Receptor Agonists to Induce Ectopic Lymphoid Structures in Myasthenia Gravis Mouse Models.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies against the acetylcholine receptor (AChR) at the neuromuscular junction. MG symptoms are characterized by muscle weaknesses. The thymus of MG patients is very often abnormal and possesses all the characteristics of tertiary lymphoid organs such as neoangiogenesis processes, overexpression of inflammatory cytokines and chemokines, and infiltration of B lymphocytes leading to ectopic germinal center (GC) development. We previously demonstrated that injections of mice with polyinosinic–polycytidylic acid [Poly(I:C)], a synthetic double-stranded RNA mimicking viral infection, induce thymic changes and trigger MG symptoms. Upon Poly(I:C) injections, we observed increased thymic expressions of α-AChR, interferon-β and chemokines such as CXCL13 and CCL21 leading to B-cell recruitment. However, these changes were only transient. In order to develop an experimental MG model associated with thymic GCs, we used Poly(I:C) in the classical experimental autoimmune MG model induced by immunizations with purified AChR emulsified in complete Freund’s adjuvant. We observed that Poly(I:C) strongly favored the development of MG as almost all mice displayed MG symptoms. Nevertheless, we did not observe any ectopic GC development. We next challenged mice with Poly(I:C) together with other toll-like receptor (TLR) agonists known to be involved in GC development and that are overexpressed in MG thymuses. Imiquimod and CpG oligodeoxynucleotides that activate TLR7 and TLR9, respectively, did not induce thymic changes. In contrast, lipopolysaccharide that activates TLR4 potentiated Poly(I:C) effects and induced a significant expression of CXCL13 mRNA in the thymus associated with a higher recruitment of B cells that induced over time thymic B-lymphoid structures. Altogether, these data suggest that tertiary lymphoid genesis in MG thymus could result from a combined activation of TLR signaling pathways.

Immunization with Recombinantly Expressed LRP4 Induces Experimental Autoimmune Myasthenia Gravis in C57BL/6 Mice

ABSTRACTBackground: 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.Objective: 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.Methods: 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.Results: 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.Conclusion: 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.

Characterization of a reproducible rat EAMG model induced with various human acetylcholine receptor domains

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.

Review on Toll-Like Receptor Activation in Myasthenia Gravis: Application to the Development of New Experimental Models.

Abnormal toll-like receptor (TLR) activation and uncontrolled resolution of inflammation are suspected to play a key role in the development of autoimmune diseases. Acquired myasthenia gravis (MG) is an invalidating neuromuscular disease leading to muscle weaknesses. MG is mainly mediated by anti-acetylcholine receptor (AChR) autoantibodies, and thymic hyperplasia characterized by ectopic germinal centers is a common feature in MG. An abnormal expression of certain TLRs is observed in the thymus of MG patients associated with the overexpression of interferon (IFN)-β, the orchestrator of thymic changes in MG. Experimental models have been developed for numerous autoimmune diseases. These models are induced by animal immunization with a purified antigen solubilized in complete Freund's adjuvant (CFA) containing heat-inactivated mycobacterium tuberculosis (MTB). Sensitization against the antigen is mainly due to the activation of TLR signaling pathways by the pathogen motifs displayed by MTB, and attempts have been made to substitute the use of CFA by TLR agonists. AChR emulsified in CFA is used to induce the classical experimental autoimmune MG model (EAMG). However, the TLR4 activator lipopolysaccharide (LPS) has proved to be efficient to replace MTB and induce a sensitization against purified AChR. Poly(I:C), the well-known TLR3 agonist, is also able by itself to induce MG symptoms in mice associated with early thymic changes as observed in human MG. In this review, we discuss the abnormal expression of TLRs in MG patients and we describe the use of TLR agonists to induce EAMG in comparison with other autoimmune experimental models.

Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice

Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG.

Standardization of the experimental autoimmune myasthenia gravis (EAMG) model by immunization of rats with Torpedo californica acetylcholine receptors — Recommendations for methods and experimental designs

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.

Guidelines for standard preclinical experiments in the mouse model of myasthenia gravis induced by acetylcholine receptor immunization

Myasthenia gravis (MG) is an autoimmune disorder characterized by generalized muscle weakness due to neuromuscular junction (NMJ) dysfunction brought by acetylcholine receptor (AChR) antibodies in most cases. Although steroids and other immunosuppressants are effectively used for treatment of MG, these medications often cause severe side effects and a complete remission cannot be obtained in many cases. For pre-clinical evaluation of more effective and less toxic treatment methods for MG, the experimental autoimmune myasthenia gravis (EAMG) induced by Torpedo AChR immunization has become one of the standard animal models. Although numerous compounds have been recently proposed for MG mostly by using the active immunization EAMG model, only a few have been proven to be effective in MG patients. The variability in the experimental design, immunization methods and outcome measurements of pre-clinical EAMG studies make it difficult to interpret the published reports and assess the potential for application to MG patients. In an effort to standardize the active immunization EAMG model, we propose standard procedures for animal care conditions, sampling and randomization of mice, experimental design and outcome measures. Utilization of these standard procedures might improve the power of pre-clinical EAMG experiments and increase the chances for identifying promising novel treatment methods that can be effectively translated into clinical trials for MG.

A new method to induce myasthenia gravis models and the protective effect of soluble decay accelerating factors.

It is expensive to induce experimental autoimmune myasthenia gravis (EAMG) by active immunity, and difficult to obtain natural acetylcholine receptor (AChR). We sought a new method of inducing EAMG by immunizing rats with artificially synthesized AChR. The AChR mRNA in TE671 cells was extracted and reverse transcribed. The inclusion body was purified and protein concentration was determined, and the EAMG animal model was used for induction. The serum was extracted from rat blood. The antibody titer was determined using enzyme-linked immunosorbant assay (ELISA). The concentration of decay accelerating factor (DAF) in the rat serum was determined by ELISA, and the metabolism of serum rDAF was determined by western blot. We evaluated the inhibition of rDAF by determining the 50% complement hemolysis unit in the rat serum. The extracellular domain (ECD) nucleotide sequence clone produced by polymerase chain reaction was completely consistent with that in the human gene bank; it was induced by isopropyl β-D-1-thiogalactopyranoside to express the protein after insertion into vector pET16b. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the inclusion body protein was the exact target. The ECD protein was able to bind with mAb35 after dialysis and renaturation, which demonstrated protein activity. The soluble ECD protein was used to immunize rats and obtain the EAMG models. The inhibitory effect of the complement was unsatisfactory owing to high decay rate after rDAF injection into the EAMG models. It is easy to induce the EAMG model by obtaining the AChRTEα1 subunit ECD protein using the substitution method.

Investigation of the impact of intravenous injection of mesenchymal stem cells from mouse bone marrow on muscle weakness and immunological parameters in experimental autoimmune myasthenia gravis model

Myasthenia gravis (MG) is a T cell dependent and antibody mediated autoimmune disease. Around 85% of the patients show AChR antibodies, 5% have muscle specific tyrosine kinase (MuSK) antibodies, whereas the remaining patients are seronegative. Although experimental autoimmune myasthenia gravis (EAMG) model has provided important information on MG pathogenesis, treatment strategies are still somewhat limited. In recent years, mesenchymal stem cells from human umbilical cord or blood marrow have been successfully tried in AChR related EAMG model, reducing muscle weakness and AChR reactive lymphocytes.

Autoimmune Channelopathies and Related Neurological Disorders

Ion channels are crucial elements in neuronal signaling and synaptic transmission, and defects in their function are known to underlie rare genetic disorders, including some forms of epilepsy. A second class of channelopathies, characterized by autoantibodies against ligand- and voltage-gated ion channels, cause a variety of defects in peripheral neuromuscular and ganglionic transmission. There is also emerging evidence for autoantibody-mediated mechanisms in subgroups of patients with central nervous system disorders, particularly those involving defects in cognition or sleep and often associated with epilepsy. In all autoimmune channelopathies, the relationship between autoantibody specificity and clinical phenotype is complex. But with this new information, autoimmune channelopathies are detected and treated with increasing success, and future research promises new insights into the mechanisms of dysfunction at neuronal synapses and the determinants of clinical phenotype.

Age-related resistance to experimental autoimmune myasthenia gravis in rats.

Abstract The influence of age on the induction of experimental autoimmune myasthenia gravis (EAMG) was investigated. Immunization with acetylcholine receptor (AChR) or injection of varying amounts of anti-AChR mAb 35 into young adult (10-12 wk) BN rats induced severe signs of EAMG including weight loss and decrement of muscle action potential, whereas aged BN rats (120-130 wk) did not show any clinical signs of EAMG. Serum anti-AChr mAb titers were not significantly different in young and aged rats up to 24 h after administration of mAb. No significant AChR loss was demonstrated in aged rats, whereas similarly treated young rats showed extensive AChR loss. In contrast to young rats, no degradation of the postsynaptic membrane could be demonstrated by electron microscopy in aged rats. C component C3 and C5b-9 membrane attack complex could be demonstrated at the neuromuscular junction in both young and aged mAb-treated rats. However, infiltrating macrophages and necrotic muscle fibers were seen only in young rats. These results suggest that the postsynaptic membrane in aged rats is resistant to autoantibody attack. AChR degradation by antigenic modulation may be less efficient in aged rats as a result of altered AChR density and distribution or rigidity of the postsynaptic membrane. Age-related resistance in the EAMG model can provide more information about the factors that determine the severity of myasthenia gravis. Manipulation of AChR density or lipid composition of the postsynaptic membrane may be of therapeutic interest in myasthenia gravis.

Age related resistance to experimental autoimmune myasthenia gravis in rats

The influence of age on the induction of experimental autoimmune myasthenia gravis (EAMG) was investigated. Immunization with acetylcholine receptor (AChR) or injection of varying amounts of anti-AChR mAb 35 into young adult (10-12 wk) BN rats induced severe signs of EAMG including weight loss and decrement of muscle action potential, whereas aged BN rats (120-130 wk) did not show any clinical signs of EAMG. Serum anti-AChr mAb titers were not significantly different in young and aged rats up to 24 h after administration of mAb. No significant AChR loss was demonstrated in aged rats, whereas similarly treated young rats showed extensive AChR loss. In contrast to young rats, no degradation of the postsynaptic membrane could be demonstrated by electron microscopy in aged rats. C component C3 and C5b-9 membrane attack complex could be demonstrated at the neuromuscular junction in both young and aged mAb-treated rats. However, infiltrating macrophages and necrotic muscle fibers were seen only in young rats. These results suggest that the postsynaptic membrane in aged rats is resistant to autoantibody attack. AChR degradation by antigenic modulation may be less efficient in aged rats as a result of altered AChR density and distribution or rigidity of the postsynaptic membrane. Age-related resistance in the EAMG model can provide more information about the factors that determine the severity of myasthenia gravis. Manipulation of AChR density or lipid composition of the postsynaptic membrane may be of therapeutic interest in myasthenia gravis.