Suppression of experimental autoimmune myasthenia gravis by nasal administration of acetylcholine receptor

Experimental autoimmune myasthenia gravis (EAMG) is a well established animal model, which can be induced in various animal species and strains with acetylcholine receptor (AChR) and represents an experimental counterpart of human myasthenia gravis (MG). Current immunotherapies of both EAMG and MG are non-specific and limited by their toxicity. Tolerance to EAMG has been achieved by oral administration of milligram quantities of Torpedo AChR. In the present report we demonstrate that nasal administration of microgram doses of Torpedo AChR to female Lewis rats prior to immunization with Torpedo AChR and complete Freund's adjuvant resulted in the prevention of subsequently induced EAMG, the suppression of serum anti-AChR antibody levels, the decrease of delayed-type hypersensitivity responses to AChR, as well as the suppression of AChR-specific immunoglobulin G-secreting cells, AChR-reactive interferon-γ-secreting cells and T cell proliferation in peripheral lymphoid organs, particularly in popliteal and inguinal lymph nodes regional to immunization. We conclude that clinical signs of EAMG can be efficiently prevented by nasal administration of AChR in parallel with the downregulation of both B and T cell responses specific to AChR.     

Clonotypic analysis of the antibody response to the acetylcholine receptor in experimental autoimmune myasthenia gravis

Autoreactive B cells reactive with the acetylcholine receptor (AChR), and the antibodies produced by them, are proposed to play a primary role in the immunopathology of myasthenia gravis and its animal models. Therefore, the anti-AChR antibody response induced in rats was characterized for the clonotypic heterogeneity, isotype distribution, and affinity by isoelectric focusing (IEF) and affinity immunoblotting. It was determined that the rat anti-AChR serum antibody was relatively heterogeneous, reflecting the oligoclonality of the response. Furthermore, isotypic dominance by IgG2a was observed in that the majority of clonal products detected by IEF were of this isotype in both primary and secondary responses. Lastly, the clonotypic anti-AChR antibodies were of relatively low affinity (avidity) when compared to antibodies reactive with the highly immunogenic protein antigen, keyhole limpet hemocyanin; anti-AChR antibody avidity did not appear to increase when the antibodies in the secondary response were compared to antibodies in the primary response. These antibody characteristics are discussed in terms of their role in disease induction.     

Role of acetylcholine receptor antibody complexes in muscle in experimental autoimmune myasthenia gravis.

In experimental autoimmune myasthenia gravis anti-rat nicotinic acetylcholine receptor (AChR) antibody titers correlated significantly with the AChR-antibody complexes found in muscle. It was shown that at least a large part of the AChR-antibody complexes are formed in vitro, which can be prevented by washing of the muscle homogenate. Using a modified assay, no differences in AChR-antibody complexes could be detected between rats with and without symptoms of experimental autoimmune myasthenia gravis. Also no difference in AChR loss nor in inhibition of alpha-bungarotoxin binding to AChR was found between these groups of rats. However, a significant difference in the reduction of AChR function was found, using an assay measuring agonist-induced 22Na+ flux into the TE671 cell line.     

重组乙酰胆碱受体α-亚单位融合蛋白诱导实验性自身免疫性重症肌无力

目的　探讨重组乙酰胆碱受体 (Ach R)的α-亚单位融合蛋白诱导实验性自身免疫性重症肌无力动物模型的可行性 ,从而为重症肌无力的实验研究创造条件。方法　以重组 Ach R的α-亚单位融合蛋白主动免疫L ewis大鼠 ,末次免疫后观察临床表现 ,并进行低频重复电刺激、单纤维肌电图及血清乙酰胆碱受体抗体 (Ach Rab)滴度检测。结果　实验组 5只大鼠有临床症状者 4只 ,主要表现为摄食减少 ,活动减少 ,嘶咬无力且易疲劳 ,其中 1只有明显肌无力 ;实验组 5只大鼠有 3只低频重复电刺激衰减率 >10 % ,其中 1只衰减率 >2 0 % ;实验组大鼠 MCD值较正常组明显延长 (P<0 . 0 1) ,而 CFA对照组与正常组 MCD值无明显差异 (P>0 . 0 5) ;实验组 5只大鼠Ach Rab的 P/ N值 ,有 4只≥ 2 .1,1只介于 1.4和 2 .1之间。结论　以重组 Ach R的α-亚单位融合蛋白主动免疫L ewis大鼠成功地诱导了实验性自身免疫性重症肌无力动物模型     

重组人乙酰胆碱受体α亚基1-210诱导实验性自身免疫性重症肌无力模型

目的 克隆和表达人乙酰胆碱受体α亚基1-210(hAChRα1-210),并以该表达产物免疫Lewis鼠诱导实验性自身免疫性重症肌无力(EAMG)模型.方法 将经逆转录-PCR扩增出的目的 基因片段AChR α1-210在大肠杆菌中诱导表达,经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot鉴定表达产物为rhAChRα1-210.将凝胶上一定剂量rhAChRα1-210融合蛋白与完全氟氏佐剂(CFA)充分乳化后,经皮下多点注入实验组,对照组则注入等剂量与CFA充分混匀乳化的空菌总蛋白.观察免疫后鼠体重的变化,并给予Lennon临床评分;低频重复电刺激检查电衰减反应和ELISA法检测血清AChR-ab滴度作为评价造模是否成功的客观依据.结果 将一定量的表达产物rhAChR α1-210融合蛋白免疫Lewis鼠,并在4周后强化免疫一次.在强化免疫10 d后实验组(n=10)部分鼠出现体重下降和肌无力,至实验结束时实验组有7只鼠Lennon临床评分达1级以上,对照组未见体重下降和肌无力表现.低频重复电衰减检查显示实验组3 Hz和5 Hz衰减率分别为13.90%±4.20%、13.20%±2.62%,对照组则为5.60%±2.06%、7.70%±2.40%,两组比较差异有统计学意义.ELISA法检测血清AChR-ab滴度结果表明实验组阳性率为70%,对照组未出现阳性,两组比较差异有统计学意义.结论 采用rhAChRα1-210融合蛋白可成功诱导EAMG动物模型,与经典方法相比具有操作方法简单、成本较低、免疫原充足等优点.     

负载乙酰胆碱受体的未成熟树突状细胞对实验性自身免疫性重症肌无力大鼠细胞因子表达的影响

目的 探讨负载乙酰胆碱受体(AChR)的未成熟树突状细胞(iDC)对实验性自身免疫性重症肌无力(EAMG)大鼠细胞因子表达的影响.方法 30只雌性Lewis大鼠随机分为EAMG组、iDC组、成熟Dc(mDC)组、负载AChR的iDC(AChR+iDC)组、负载AChR的mDC(AChR+mDC)组和正常对照组.分别给...     

Breakage of tolerance to hidden cytoplasmic epitopes of the acetylcholine receptor in experimental autoimmune myasthenia gravis

The acetylcholine receptor (AChR) is the major autoantigen in the antibody-mediated disease myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). This study demonstrates that rats immunized with a recombinant fragment corresponding to the normally exposed extracellular region of the rat AChR -subunit first develop antibodies to the injected extracellular portion only, but later develop antibodies to intracellular cytoplasmic epitopes of AChR. The presence of autoantibodies to intracellular epitopes seems to be correlated with development of clinical signs of disease. We propose that a similar process of epitope spreading may take place in the natural course of myasthenia.     

Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model

Peroxidase-conjugated alpha-bungarotoxin (P-BGT) was used for the ultrastructural localization of the acetylcholine receptor in end-plates in external intercostal muscles of four patients with myasthenia gravis, in forelimb digit extensor muscles of rats with advanced chronic experimental autoimmune myasthenia gravis, and in suitable human and rat controls. In control end-plates, the previously reported localization of acetylcholine receptor on the terminal expansions of the postsynaptic folds and, in traces, on the presynaptic membrane was confirmed. By contrast, in myasthenia gravis some postsynaptic regions bound no P-BGT; in other regions, the folds displayed only faint traces of the reaction product, or only some segments of the postsynaptic membrane showed the reaction product; finally, in some regions there was no apparent decrease in reaction product. In general, those postsynaptic regions showing the greatest decrease in P-BGT binding were also the simplest or showed the most degenerative changes, and the presynaptic staining was decreased in proportion to the decrease in the adjacent postsynaptic P-BGT binding. In the experimental animals, the abnormalities in the amount and distribution of the acetylcholine receptor were essentially like those in the more severely affected patients. Morphometric estimates of the postsynaptic acetylcholine receptor surface correlated well with the patients' clinical status and linearly with the miniature end-plate potential amplitude.     

糖皮质激素受体阻断对大鼠实验性自身免疫性重症肌无力的影响

研究重症肌无力（ＭＧ）患者外周血白细胞糖皮质激素受体（ＧＲ）减少，而血浆皮质醇则在正常范围，探讨其与ＭＧ发病的关系。方法ＳＤ大鼠３２只，随机分成４组。实验组先以ＧＲ的竞争性拮抗剂米非司酮（ＲＵ３８４８６，ＲＵ４８６）阻断其ＧＲ，再以从人肌肉中粗提的乙酰胆碱受体（ｎＡＣｈＲ）进行免疫；实验对照组单用ｎＡＣｈＲ，试剂对照组只用ＲＵ４８６，而正常对照组仅用福氏佐剂。以临床症状、血清抗ｎＡＣｈＲ抗体（ｎＡＣｈＲ－ａｂ），重复刺激坐骨神经递减幅度为观察指标。结果实验组的临床症状和ｎＡＣｈＲ－ａｂ滴度升高及肌电图递减幅度均较明显，经ｔ检验分析，均与实验对照组有显著性差异（Ｐ＜０．０５），而试剂对照组和正常对照组均无ＭＧ的表现。结论ＧＲ被阻断后，对大鼠的实验性自身免疫性ＭＧ（ＥＡＭＧ）发病有易化作用。     

Neonatal experimental autoimmune myasthenia gravis

Neonatal rats born of and nursed by mothers immunized with Torpedo acetylcholine receptor protein developed a defect of neuromuscular transmission as indicated by reduced miniature endplate potential amplitudes. It is likely that antibodies to the Torpedo receptor protein were passively transferred to the neonates in the milk. With the exception of the route of transfer, this neonatal form of experimental autoimmune myasthenia gravis appears to be similar to its human counterpart, and thus can serve as an experimental model.     

A hierarchy of T cell receptor motifs determines responsiveness to the immunodominant epitope in experimental autoimmune myasthenia gravis

The predominant murine T lymphocyte population responding to Talpha146-162, the immunodominant epitope in EAMG, expresses the TCRBV 6 gene segment. However, cells expressing other TCRBV gene segments also react with this peptide. In order to more precisely characterize the Talpha146-162-specific TCR repertoire, we isolated CD4high cells from peptide-immunized mice. The majority of CD4high cells utilized an acidic TCR beta chain CDR3 motif regardless of TCRBV gene usage. Analysis of T cell clones demonstrated a fourfold higher avidity of Vbeta6+ than non-Vbeta6 cells for Talpha146-162 indicating that a hierarchy of TCR motifs determines T cell responsiveness in EAMG.     

Penicillamine-induced myasthenia gravis associated with antibodies to acetylcholine receptor

A woman with rheumatoid arthritis was treated with penicillamine and developed myasthenia gravis. This drug-induced disease was associated with characteristic autoantibodies to acetycholine receptor. After discontinuing the drug, her symptoms improved and the antibody titers fell. Penicillamine is now being used much more frequently in the treatment of rheumatoid arthritis and it is likely that this complication will become more prevalent.     

Antigenic modulation and receptor loss in experimental autoimmune myasthenia gravis

Immunization of groups of rats with 0.1- 100 microgram of acetylcholine receptor (AChR) purified from the electric organ of Torpedo californica resulted in dose-dependent (1) loss of acetylcholine receptor from the rats' muscles, (2) binding of antibodies to many of the receptors remaining in muscle and (3) production of antibodies in serum capable of cross-reacting with receptor solubilized from rat muscle. Addition of antibodies from rats immunized with electric organ acetylcholine receptors to muscle cells in culture caused loss of receptor by accelerating the rate of receptor degradation. Monovalent antibody fragments did not accelerate degradation unless antiantibody was added to cross-link the monovalent antibody fragments bound to receptors. This indicates that cross-linking of receptors by antibody molecules triggers accelerated receptor degradation, leading to receptor loss. The rate of increase in receptor destruction due to antigenic modulation observed in vitro appears sufficient to account for the extent of receptor loss observed in vivo. Endocytosis of antibody cross-linked receptors may be a rate-limiting step common to antigenic modulation in vitro and in vivo.     

Myasthenia gravis: Antibodies to acetylcholine receptor in ocular myasthenia gravis

Summary To improve the sensitivity of the radioimmunoassay method for anti-AChR-antibody, large amounts of sera from patients with myasthenia gravis, and higher concentrations of antigens and rabbit anti-human-IgG-antiserum, were used. These procedures enabled measurement of the titre value of over 0.04 pmol/ml serum and this value revealed a sensitivity about 10 times higher than that predicted using the previous model.Antibodies against AChR were found in 13 out of 17 ocular myasthenia patients (70%) and 35 of 37 with generalized myasthenia (90%). However, the average titre value of sera in those with ocular myasthenia was significantly lower than the value obtained in the generalized cases. Even in the patients with ptosis or ophthalmoparesis, in the early stage (less than one year) of ocular myasthenia, anti-AChR-antibodies were not detectable using this more sensitive assay method.

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Zusammenfassung Um die Empfindlichkeit der Radioimmunoassay-Methode für Anti-AChR-Antikörper zu verbessern, wurden eine große Menge des myasthenischen Serums, höhere Konzentration des Antigens und Kaninchen-Anti-Menschen-IgG-Antiserum gebraucht.Mit dieser Methode konnten wir den Wert über 0.04 pmol/ml Serum vermessen. Diese neue Methode zeigt etwa zehnfach höhere Empfindlichkeit als die vergangenen Methoden.Antikörper gegen AChR waren in 13 von 17 okulären Myastheniepatienten (70%) und in 35 von 37 generalisierten Myastheniepatienten (90%) gefunden worden. Aber der durchschnittliche Messungswert des Serums im Patienten der okulären Form war signifikant niedriger als der der generlisierten Form. Wenn der Patient auch Augenptose oder Augenparese hatte, waren die Anti-AChR-Antikörper mit unserer empfindlichen Methode im Frühstadium (innerhalb eines Jahres) nicht nachgewiesen worden.

     

Muscle acetylcholine receptor loss in murine experimental autoimmune myasthenia gravis: correlated with cellular, humoral and clinical responses

An extensive analysis of the relationship between immunological parameters and clinical responses and biochemical loss of muscle acetylcholine receptors (AChR) was performed in murine experimental autoimmune myasthenia gravis. The onset of clinical muscle weakness correlated strongly with the onset of significant muscle AChR loss. Mice with clinical muscle weakness had greater amount of muscle AChR loss. There was no correlation between the concentration of anti-AChR antibodies and the presence of clinical muscle weakness or amount of muscle AChR loss. However, the kinetics of autoantibody response correlated well with that of muscle AChR loss.     

Inhibitory IgG receptor FcgammaRIIB fails to inhibit experimental autoimmune myasthenia gravis pathogenesis

Deficiency of the inhibitory FcgammaRIIB renders mice susceptible to autoimmune disorders characterized with cellular infiltration of target tissue. To analyze the role of FcgammaRIIB in an antibody-mediated autoimmune disease, experimental autoimmune myasthenia gravis (EAMG), FcgammaRIIB knockout (KO) and wild-type mice were immunized with acetylcholine receptor (AChR). In contrast with previous reports, FcgammaRIIB KO mice were mildly resistant to EAMG despite preserved anti-AChR antibody production and neuromuscular junction complement deposition capacity. EAMG resistance was associated with reduced lymph node cell IL-6 and IL-10 production and increased CD4(+)CD25(+) cell ratios in lymph nodes. Our data suggest that FcgammaRIIB promotes antibody-mediated autoimmunity.     

Single-fiber electromyography in experimental autoimmune myasthenia gravis

The sensitivity of stimulated single-fiber electromyography in the detection of early abnormalities in neuromuscular transmission in experimental autoimmune myasthenia gravis (EAMG) was tested. Increased jitter and blocking were seen up to 3 weeks before clinical illness or decrement developed. Stimulation at 10 Hz appeared more sensitive in detection of abnormalities than stimulation at 3 or 5 Hz. Jitter values did not correlate with anti-Torpedo acetylcholine receptor (AChR), nor with anti-rat AChR antibody titer. No correlation was found between jitter and AChR loss or AChR-antibody complexes in muscle. It is concluded that, in addition to AChR loss and the presence of AChR-antibody complexes, other factors must determine the neuromuscular dysfunction in EAMG and possibly myasthenia gravis.     

Cellular response to human acetylcholine receptor in patients with myasthenia gravis

A preparation of human skeletal muscle acetylcholine receptor (AchR) was used in vitro as an antigen to stimulate lymphocytes from patients with myasthenia gravis (MG). Clinical data obtained from the patients included duration and severity of disease; history of steroid treatment or prior thymectomy; and the presence of thymoma. Lymphocytes from patients with MG showed a significantly higher response to human AchR antigen than did lymphocytes from control subjects. Previous studies of cellular response to AchR have used receptor prepared from eel or ray electric organs. By stimulating lymphocytes from MG patients with a preparation of human AchR, we have come one step closer to documenting a possible contribution of a cellular immune response to the pathogenesis of MG.