Search

Muscle-specific tyrosine kinase (MuSK) autoantibodies are the hallmark of a form of myasthenia gravis (MG) that can challenge the neurologist and the experimentalist. The clinical disease can be difficult to treat effectively. MuSK autoantibodies affect the neuromuscular junction in several ways. When added to muscle cells in culture, MuSK antibodies disperse acetylcholine receptor clusters. Experimental animals actively immunized with MuSK develop MuSK autoantibodies and muscle weakness. Weakness is associated with reduced postsynaptic acetylcholine receptor numbers, reduced amplitudes of miniature endplate potentials and endplate potentials, and failure of neuromuscular transmission. Similar impairments have been found in mice injected with IgG from MG patients positive for MuSK autoantibody (MuSK-MG). The active and passive models have begun to reveal the mechanisms by which MuSK antibodies disrupt synaptic function at the neuromuscular junction, and should be valuable in developing therapies for MuSK-MG. However, translation into new and improved treatments for patients requires procedures that are not too cumbersome but suitable for examining different aspects of MuSK function and the effects of potential therapies. Study design, conduct and analysis should be carefully considered and transparently reported. Here we review what has been learnt from animal and culture models of MuSK-MG, and offer guidelines for experimental design and conduct of studies, including sample size determination, randomization, outcome parameters and precautions for objective data analysis. These principles may also be relevant to the increasing number of other antibody-mediated diseases that are now recognized., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30μg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10μg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30μg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Activation of complement components is crucial in the progression and severity of myasthenia gravis and experimental autoimmune myasthenia gravis (EAMG). Mice deficient in complement component C4 or treated with monoclonal antibody to C1q are resistant to EAMG. In this study, we show that inhibition of complement cascade activation by suppressing the expression of a critical low-abundant protein, C2, in the classical complement pathway, significantly improved clinical and immunopathological manifestations of EAMG. Two weeks after a second booster immunization with acetylcholine receptor, when mice exhibit muscle weakness, i.p. injection of C2 siRNA significantly suppressed C2 mRNA in the blood cells and liver of EAMG mice. Treatment of EAMG mice with C2 siRNA, once a week for 5 weeks, significantly improved muscle strength, which was further evidenced by functional AChR preservation in muscle, reduction in number of C3 and membrane-attack complexes at neuro-muscular junctions in forelimb muscle sections, and a transient decrease in serum IgG2b levels. Our study shows for the first time that siRNA-mediated suppression of C2, a component of the classical complement system, following established disease, can effectively contribute to the remission of EAMG. Therefore, C2 siRNA mediated therapy can be applied in all complement mediated autoimmune diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Myasthenia gravis (MG) is a T cell-dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In a previous study we demonstrated that the human thyroglobulin (hTg) peptide p2340 (aa 2340-2359) can stimulate a T cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) mice. In the present study we examined whether p2340 can induce EAT in single HLA class II DR3 transgenic mice. This peptide was found to be immunogenic at the T cell level in DR3 mice, since it induced specific proliferative responses, as well as IL-2 and IFN-gamma secretion in secondary cultures of peptide-primed lymph node cells (LNC). Immunization of HLA-DR3 mice with p2340 in CFA elicited EAT (infiltration index of 1 to 2) in eight of nine mice. Peptide-primed LNC responded to intact hTg, whereas, hTg-primed LNC did not respond to p2340 in culture, suggesting that p2340 contains subdominant T cell epitope(s). P2340 was also found to be immunogenic at the B cell level, since strong p2340-specific IgG response was detected in all transgenic mice tested. Thus, we provide evidence for a pathogenic role of an hTg peptide in HLA-DR3 transgenic mice. Therefore, p2340 could be presented by DR3 molecule in patients with Hashimoto's thyroiditis and participate in the development of the disease.

Steroid-dependent myasthenia gravis patients improved following treatment with etanercept (recombinant human TNF receptor:Fc) in a prospective pilot trial. While the plasma anti-acetylcholine receptor antibody levels remained unaffected, etanercept treatment increased plasma levels of C3, circulating immune complexes, IL-10 and IFN-gamma. There was a direct correlation between plasma IL-6, TNF-alpha and IFN-gamma levels and the post-treatment clinical scores of patients. Moreover, patients with lower pre-treatment plasma IL-6 and IFN-gamma levels attained better clinical improvement following etanercept treatment.

To comprehend the reduced susceptibility of HLA-DQ6 transgenic mice in comparison with HLA-DQ8 mice, to experimental autoimmune myasthenia gravis (EAMG), we immunized them with acetylcholine receptor (AChR) and examined in vitro, the proliferative and cytokine responses to AChR. When immunized with AChR and examined for AChR-specific lymphocyte responses to AChR, EAMG-resistant DQ6 mice exhibited significantly reduced in vitro lymphoproliferative and cytokine responses to AChR, compared to DQ8 mice. The differences in susceptibility were not linked to a difference in peptide recognition by AChR-specific lymphocytes. AChR T cell epitope mapping showed that both DQ6 and DQ8 responded to the same epitopes, although to varying degrees. Resistance of DQ6 transgenic mice to EAMG was linked to a dramatic suppression of AChR-specific IFN-gamma, IL-2 and IL-10 productions by AChR-primed lymph node cells., (Copyright 2001 Academic Press.)

Myasthenia gravis (MG) is an antibody-mediated, autoimmune neuromuscular disease. Animal models of experimental autoimmune myasthenia gravis (EAMG) can be induced in vertebrates by immunization with Torpedo californica acetylcholine receptors (AChR) in complete Freund's adjuvant. The MHC class II genes influence the cellular and humoral immune response to AChR and are involved in the development of clinical EAMG in mice. A dominant epitope within the AChR alpha146-162 region activates MHC class II-restricted CD4 cells and is involved in the production of pathogenic anti-AChR antibodies by B cells. Neonatal or adult tolerance to this T-cell epitope could prevent EAMG. During an immune response to AChR in vivo, multiple TCR genes are used. The CD28-B7 and CD40L-CD40 interaction is required during the primary immune response to AChR. However, CTLA-4 blockade augmented T- and B-cell immune response to AChR and disease. Cytokines IFN-gamma and IL-12 upregulate, while IFN-alpha downregulates, EAMG pathogenesis. However, the Th2 cytokine IL-4 fails to play a significant role in the development of antibody-mediated EAMG. Systemic or mucosal tolerance to AChR or its dominant peptide(s) has prevented EAMG in an antigen-specific manner. Antigen-specific tolerance and downregulation of pathogenic cytokines could achieve effective therapy of EAMG and probably MG., (Copyright 2000 Academic Press.)

To study the involvement of B cells in the immune response to acetylcholine receptor (AChR), B-cell-deficient (mu mutant) and control wild-type C57BL/6 mice were immunized with AChR and assessed for clinical and immunopathological manifestations of experimental autoimmune myasthenia gravis (EAMG). The mu mutant mice failed to generate anti-AChR antibodies and were completely resistant to the induction of EAMG. However, mu mutant mice developed clinical EAMG when antibodies to the AChR main immunogenic region were passively transferred. Further, the in vivo expansion of lymph node cells after AChR immunization was greatly impaired in mu mutant mice. The mu mutant mice gave an effective in vitro T cell immune response to the immunodominant pathogenic AChR alpha chain peptide 146-162 (alpha 146-162) and to the whole AChR protein when tested on day 90 after immunization with AChR, whereas the response to both AChR and its alpha 146-162 peptide was reduced when tested on day 7 after immunization. The in vitro production of IFN-gamma and IL-2 by AChR-specific and alpha 146-162 peptide-specific lymphocytes was lower in mu mutant mice. The AChR immune mu mutant T cells proliferated and produced IFN-gamma when AChR or alpha 146-162 peptide was presented by wild-type irradiated AChR-primed antigen-presenting cells (APCs). This indicates that B cells are important in the processing and presentation of AChR dominant peptide in vitro during the initial immune response to AChR. However, APCs of non-B-cell lineage are sufficient to process AChR and prime the T cells to AChR dominant T cell epitope peptides.

Recombinant (r) human IL-2 was administered in vivo to improve homing and engraftment of rheumatoid arthritis (RA) patients' peripheral blood mononuclear cells (PBMC) into severe combined immunodeficient (SCID) mice. Human rIL-2 treatment resulted in augmented human Ig production and induced IgM rheumatoid factor (RF) of human origin in SCID-RA chimeras. The increment of human serum IgG in SCID-RA chimeras after IL-2 treatment ranged between 15 and 43% and for IgM between 50 and 98% during 2-8 weeks postengraftment. Human IgM-RF was detectable after 1 to 2 weeks after engraftment and persisted over a period of 10-13 weeks. No RF was produced in SCID mice engrafted with PBMC from healthy individuals with or without exogenous rIL-2 administration. Thus, human rIL-2 expanded autoreactive clones involved in the production of RF in the SCID-RA chimeras. The present study provides a novel approach to establish an in vivo SCID-RA model to study the cellular and molecular mechanisms involved in the production of RF and development of a RA-like lesion.

One of the dominant T cell epitopes in the acetylcholine receptor (AChR) alpha chain lies within the region 146-162 and has been implicated in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) in C57BL6 (B6) mice. To directly examine the pathogenic potential of alpha 146-162 in EAMG, B6 mice were primed with AChR in complete Freunds adjuvant (CFA) and subsequently boosted twice with either alpha 146-162 or a control peptide in CFA. Seventy percent of the mice boosted with alpha 146-162 developed muscle weakness characteristic of EAMG, while none of the mice boosted with the control peptide showed any clinical signs of the disease. Thus, the data provided evidence for epitope within AChR alpha 146-162 as one of the EAMG-inducing pathogenic epitopes in B6 mice.

A gene conversion event between Ebb and Abb in the B6.C-H-2bm12 (bm12) strain, which alters three amino acids in the C-terminal half of the first domain of Abb (Ile-67-->Phe; Arg-70-->Gln; Thr-71-->Lys) resulted in resistance to experimental autoimmune myasthenia gravis (EAMG) pathogenesis. To study the effect of bm12 mutation on the T-cell responses to epitopes of acetylcholine receptor (AChR)-alpha subunit, C57BL6 (B6) and bm12 mice were primed with Torpedo californica AChR, and the profiles of T-lymphocyte proliferation were determined with 18 synthetic overlapping peptides encompassing the entire extracellular portion of the AChR-alpha subunit. The proliferative responses of AChR-primed bm12 lymphocytes were markedly reduced to two (alpha 146-162 and alpha 182-198) of the three AChR peptides (alpha 111-126, alpha 146-162, and alpha 182-198) that are immunodominant in B6 mice. Thus, the Ab residues encompassing the region 67-71 determine the immunogenicity of two of the AChR-alpha subunit T-cell epitopes. To test the involvement of AChR-alpha chain epitopes within peptide alpha 146-162 in EAMG pathogenesis, B6 mice were neonatally tolerized with soluble peptide alpha 146-162, and subsequently immunized with AChR in complete Freund's adjuvant. Neonatal tolerance to AChR or to peptide alpha 146-162 reduced the incidence of clinical myasthenia gravis and suppressed serum anti-AChR antibodies. This indicates the involvement of T-cell epitopes within AChR-alpha subunit region alpha 146-162 in EAMG pathogenesis. Neonatal tolerance to peptide alpha 146-162 could have caused specific clonal deletion, and/or clonal anergy, and/or recruited suppressor cells to prevent clinical EAMG. Presumably, epitope(s) with AChR alpha 146-162, in the context of Ab encompassing region 67-71, stimulate specific T helper cells which interact with specific B cells to produce pathogenic antibodies, the primary culprit causing the end plate lesion in patients with myasthenia gravis.

The I-A beta gene has been implicated in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), with amino acids at positions 67, 70, and 71 of the I-A beta b chain that play a crucial role. In addition, the cell surface expression of the I-E molecule in C57BL10.E alpha k transgenic mice was associated with the partially suppressed serum anti-acetylcholine receptor antibody and clinical expression of EAMG. In this study, the contribution of the I-A alpha gene and the A beta b:A alpha k transpair in EAMG pathogenesis is assessed. The I-A alpha k transgene was introduced into the B10 (A beta b:A alpha b) strain, and the I-A alpha k chain was paired with I-A beta b; therefore, the A beta b:A alpha k complex was expressed on the cell surface. Expression of the A beta b:A alpha k transpair in C57BL10 transgenic mice suppressed the cellular and humoral autoimmune responses to acetylcholine receptors (AChR), reduced the amount of muscle AChR bound with antibody, and significantly reduced the incidence of muscle weakness and its associated abnormal electrophysiological response.

Myasthenia gravis (MG) is an autoimmune neuromuscular disease, characterized by muscle weakness and electrophysiological abnormality. No treatment which would reliably induce permanent clinical remission of MG is yet available. The therapeutic efficacy and toxic effect of daunomycin (Dm), an antibiotic of the rhodomycin group, was evaluated in murine experimental autoimmune MG. Low dosage Dm treatment effectively prevented the development of muscle weakness and its associated electrophysiological abnormality, without inducing detectable toxicity and global immunosuppression.