重症肌无力病人乙酰胆碱受体抗体的测定及临床意义

用ＥＬＩＳＡ（固相酶联免疫吸附）法测定１７２例ＭＧ病人血清乙酰胆碱受体抗体（ＡｃｈＲａｂ），结果显著高于健康献血员组和非ＭＧ病人组。不同性别、病程及临床类型与ＡｃｈＲａｂ无相关性，但４１～５０岁组的显著高于其他年龄组。６７例类固醇激素治疗组、２２例大剂量两种球蛋白治疗组、１２例胸腺切除术组及３例ＭＧ危象病人２４次血浆交换疗法（ＰＥ）组，治疗后伴随肌无力症状的好转，ＡｃｈＲａｂ均显著低于治疗前。结果表明：ＡｃｈＲａｂ测定为ＭＧ诊断提供了可靠的实验依据，为类固醇激素、大剂量丙种球蛋白、胸腺切除术和ＰＥ等治疗ＭＧ提供理论依据和疗效评定的实验指标，进一步证实了ＭＧ免疫学发病机理。     

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.     

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.     

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。     

重症肌无力血清乙酰胆碱受体抗体的阳性率及其影响因素

采用ABC-ELISA法检测162例重症肌无力患者的224份血清标本,100例正常人和78例其他疾病病人的血清中AchRab。重症肌无力病人的抗体阳性率为80.2％,其中单纯眼肌型63.1％,脊髓肌型90.0％,延髓肌型93.8％和全身肌型91.5％。合并胸腺瘤的阳性率为83.3％,与全组阳性率无显著性差异。激素和血浆交换治疗均未影响抗体阳性结果。随访观察表明,重症肌无力病人的AchRab阳性率与疾病严重程度不成线性相关。我们认为,AchRab滴度虽然与临床状况不相关,但在重症肌无力的诊断和自身免疫病因的研究中,仍是一项重要参数。     

Induction of the morphologic changes of both acute and chronic experimental myasthenia by monoclonal antibody directed against acetylcholine receptor

Summary To investigate pathogenic mechanisms in experimental autoimmune myasthenia gravis (EAMG) and myasthenia gravis (MG), we studied the acute and chronic effects in rats of injection of rat monoclonal antibodies (MCABs) directed against the acetylcholine receptor (AChR). Animals were severely weak 12 h after a single injection, at which time macrophages were found invading endplate regions of muscle and cholinesterase-stained regions were separted from the underlying muscle fibers. Ultrastructural studies showed findings identical to the acute phase of EAMG: degenerating postsynaptic membranes and invasion and phagocytosis of endplate regions by macrophages. Animals receiving sublethal doses of MCAB recovered clinically by 4–5 days after injection. Recovery was accompanied by a progressive decrease in the number of macrophages associated with endplates and reapposition to the myofibers of the cholinesterasestained regions. Animals injected once, or repeatedly over several months, remained clinically and electromyographically normal after recovery from the initial episode of weakness, but their endplate ultrastructure was highly simplified with blunted or absent synaptic folds and shallow or absent secondary synaptic clefts. These studies demonstrate that anti-AChR MCABs can induce the changes of both acute and chronic EAMG. There is good correlation between the inflammatory changes and the acute clinical disease but poor correlation between morphological and clinical parameters in the chronic syndrome. The latter observation suggests that severe ultrastructural changes, similar to those seen in chronic EAMG and MG, cannot account, at least in rats, for the clinical and electrophysiologic abnormalities of MG.Supported in part by grants from the Muscular Dystrophy Association and the National Institutes of Health (NS15462, A19268)     

重症肌无力患者血清IgG-乙酰胆硷受体抗体亚型研究

目的 探讨重症肌无力（ＭＧ）患者血清ＩｇＧ－乙酰胆硷受体抗体（ＡＣｈＲＡｂ）亚型的分布规律及其临床意义。方法 采用ＡＢＣ－ＥＬＩＳＡ法检测４３例ＭＧ组和２５例临床对照组、２０例正常对照组血清中ＩｇＧ－ＡＣｈＲＡｂ亚型ＩｇＧ１－４。结果 ＭＧ组与两对照组相比ＩｇＧ１和ＩｇＧ４亚型抗体无显著差别,ＩｇＧ２亚型抗体显著升高（Ｐ〈０．０５）,ＩｇＧ３亚型抗体显著降低（Ｐ〈０．０５）;ＭＧ组各临床类型间各亚型抗体无显著差别。结论 ＩｇＧ－ＡＣｈＲＡｂ亚型以ＩｇＧ２活性为主,但未显示与ＭＧ临床类型有关。     

VH gene family utilization of anti-acetylcholine receptor antibodies in experimental autoimmune myasthenia gravis

The immunoglobulin heavy chain (V_H) gene family usage in experimental autoimmune myasthenia gravis (EAMG) model was investigated by RNA slot blot hybridization using V_H gene family specific probes. Anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) isolated from susceptible C57BL/6 and resistant BALB/c mice were found to be encoded by V_H genes from at least six different families. The Vgam3.8 family was overrepresented in α-bungarotoxin blocking mAbs. Expression of cross-reactive idiotopes by anti-AChR mAbs was irrespective of the V_H gene family usage. Strain dependent differences in susceptibility for EAMG were not reflected in an aberrant V_H gene family usage of anti-AChR mAbs.     

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.     

激发型CD40单克隆抗体对实验性自身免疫性重症肌无力耐受的影响

目的 探讨激发型CD40单克隆抗体(CD40mAb)对未成熟树突状细胞(iDC)诱导实验性自身免疫性重症肌无力(EAMG)耐受的影响. 方法 20只Lewis大鼠按照随机数字表法分为正常组、EAMG对照组、耐受组和CD40mAb组,每组5只.耐受组和CD40mAb组每只大鼠于背部皮下分4点分别注射总量为1 mL的乙酰胆碱受体(AChR)+iDC,其中CD40mAb组每只大鼠在接种iDC的同时和次日分别给予CD40mAb腹腔注射,0.5mg/次.EAMG对照组注射1 mL无血清培养基.正常组不做任何处理.3周后用AChR和完全弗氏佐剂(CFA)免疫,观察免疫7周后重症肌无力(MG)相关指标的改变. 结果 耐受组大鼠和正常组大鼠一样,用AChR和CFA免疫后,MG相关指标无明显改变;而CD40mAb组大鼠和EAMG对照组一样,产生了明显的MG症状,重复电刺激出现明显衰减,血清AChRab滴度明显增高,神经肌肉接头呈现典型的MG样改变. 结论 激发型CD40mAb可阻止AChR负载的iDC诱导EAMG耐受,提示iDC功能异常可能与MG异常免疫反应的启动密切相关.

Abstract：

Objective To investigate the effect of agonist CD40 monoclonal antibody (CD40mAb) on the tolerance of experimental autoimmune myasthenia gravis (EAMG) induced by immature dendritic cells (iDCs). Methods Lewis rats were equally randomized into normal group,EAMG group, tolerance group and CD40mAb treatment group (n=5). Rats in the tolerance group and CD40mAb treatment group were vaccinated with AChR pulsed iDCs; and rats in the CD40mAb treatment group were intraperitoneally injected CD40mAb at a dosage of 0.5 mg once when performing the vaccination and on the 2rd d of vaccination. One mL serum-free medium was given to the rats in the EAMG group; normal group did not receive any treatment. Three weeks after that, rats in the above 4 groups were immunized with AChR and complete Freund's adjuvant (CFA). Seven weeks after the immunization, the corresponding indexes of MG were observed: behavioral assessment was performed and electromyogram was employed to detect the repetitive nerve stimulation on these rats; enzyme-linked immunosorbent assay (ELISA) was used to determine the level of AChRab; the pathological changes of neuromuscular junction were also detected. Results Just as the rats in the normal group, the rats in the tolerance group did not have significant changes in any of the corresponding indexes of MG after being immunized with AChR and CFA. In contrast, rats in both EAMG group and CD40mAb treatment group showed typical changes in the corresponding indexes of MG: their electromyogram wave amplitude obviously attenuated; the level of serum AChRab increased and neuromuscular junction appeared as a typical damage of MG. Conclusion Agonist CD40mAb could abrogate the tolerance of EAMG induced by AChR pulsed iDCs, suggesting that the dysfunction of DCs is related to the priming of abnormal immune of MG.     

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     

Antibody-secreting cells to acetylcholine receptor and to presynaptic membrane receptor in seronegative myasthenia gravis

Peripheral blood and bone marrow from seronegative and seropositive myasthenics were evaluated for antibody-secreting cells (ASC). Cells secreting antibody to acetylcholine receptor (AchR) and to presynaptic membrane receptor (prsmR) were counted using an immunospot assay. Immunoglobulin G (IgG) anti-AchR ASC were present in peripheral blood lymphocytes (PBL) from nine of 13 seronegative and nine of 12 seropositive myasthenics and in bone marrow lymphocytes (BML) from nine of 13 seronegative and eight of 12 seropositive myasthenics. The mean number of IgG anti-AchR ASC was lower for seronegative than for seropositive patients (P < 0.01 for PBL and P < 0.0001 for BML). In seropositive patients the mean number of IgG anti-AchR ASC was higher for BML than for PBL (P < 0.01); in seronegative patients it was not. IgG anti-prsmR ASC were detected in PBL from four of eight seronegative and six of eight seropositive myasthenics and in BML from three of eight seronegative and five of eight seropositive patients. The mean number of IgG-anti-prsmR ASC did not differ between seronegative and seropositive patients for PBL but for BML the value was higher for seropositive than for seronegative patients (P < 0.01). We conclude that seronegative myasthenia gravis is an autoimmune disease and that ASC to AchR and to prsmR are present both in the blood and the bone marrow in seronegative patients as in seropositive ones. A major difference between the groups lies in the significantly greater number of ASC found in the bone marrow in the seropositive cohort.     

重症肌无力患者血清白细胞介素—6水平测定

目的探讨重症肌无力(MG)与白细胞介素-6(IL-6)的关系.方法采用双抗体夹心ELISA法对30例MG患者用糖皮质激素(GC)治疗前、治疗2个月后和22例正常对照血清IL-6、乙酰胆碱受体抗体(AchRab)水平进行检测.结果MG患者组血清IL-6水平显著高于对照组(P＜0.01),MG患者组血清IL-6水平在用GC治疗2个月后显著降低(P＜0.01),其血清IL-6与血清AchRab水平呈正相关(r=0.693,P＜0.01).结论IL-6与MG发病密切相关,IL-6参加了MG的免疫病理过程;检测血清IL-6水平对MG临床有重要价值;GC可抑制IL-6合成及AchRab产生.     

Neonatal myasthenia gravis: Clinical and immunological study of seven mothers and their newborn infants

We studied 7 mothers with myasthenia gravis (MG) and their infants. We confirmed that the development of neonatal MG was not related to the serum titer of maternal anti-acetylcholine receptor antibody (anti-AChR ab). To investigate the possibility that specific immunization of the newborn infant had occurred, serial serum determinations of total and 'specific' anti-AChR IgG and IgM were performed. We found that: the decay in total IgG was within the normal range in all the babies; there was a shorter half-life of 'specific' IgG, compared to total IgG, in 3 of the cases, 2 of which did have neonatal MG; no difference was found between the decay of anti-AChR ab in the babies who had neonatal MG and those who did not; there was no anti-AChR IgM-associated activity. Our data suggest that neonatal MG is due to maternal anti-AChR abs and that affected infants do not produce specific antibodies.     

Modulation of acetylcholine receptor function in TE671 (rhabdomyosarcoma) cells by non-AChR ligands: possible relevance to seronegative myasthenia gravis

The acetylcholine receptor (AChR) is the main target antigen in myasthenia gravis (MG), but about 15% of patients with typical, immunologically mediated MG do not have detectable anti-AChR antibodies. Previous studies showed that plasma from these ‘seronegative’ patients (SNMG) reduced AChR function in the human AChR-expressing TE671 cell line, and it was proposed that SNMG plasmas may act indirectly via phosphorylation of AChR. We show here that substances such as the β₂-adrenergic agonist, salbutamol, calcitonin-gene-related-peptide (CGRP), and cholera toxin, that increase intracellular cAMP via binding to specific cell-surface receptors, reduced AChR function in TE671 cells. Moreover, non-specific activation of cell surface proteins by lectins achieved similar results. These observations lead us to hypothesise that SNMG immunoglobulins act in TE671 cells by cross-linking of specific cell surface antigen(s) resulting in generation of intracellular cAMP and/or other second messengers. The role of such antibodies at the neuromuscular junction in vivo could be reduction in AChR function by desensitization and/or damage to the postsynaptic membrane following complement activation.     

Prevention of passively transferred experimental autoimmune myasthenia gravis by Fab fragments of monoclonal antibodies directed against the main immunogenic region of the acetylcholine receptor

The muscle acetylcholine receptor loss, responsible for the clinical symptoms of myasthenia gravis, is due mainly to mechanisms dependent on the bivalent character of the anti-receptor antibodies. In cell culture, univalent Fab fragments of monoclonal antibodies (mAbs) directed against the main immunogenic region (MIR) of the acetylcholine receptor are able to protect the receptor against the action of the intact antibodies. To investigate the potential therapeutic use of this approach, we examined the ability of the Fab fragment of anti-MIR mAb195 (Fab195) to protect the receptor in vivo against two anti-MIR mAbs. Because of the rapid clearance of Fab fragments from the circulation, Lewis rats were treated repeatedly with Fab195. The Fab fragment significantly protected muscle receptors against antibody-mediated loss and was very efficient in providing protection against clinical symptoms when its administration was commenced before, simultaneously with, or 2 h after, mAb injection. Twenty-four hours after mAb injection, the protected rats only showed mild myasthenic symptoms, whereas those which only received intact antibodies were moribund or dead. These results suggest that, once modified to ensure their low immunogenicity and a long half-life, anti-MIR Fab fragments might be useful in the specific immunotherapy of myasthenia gravis.     

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.     

Serum acetylcholine receptor antibody measurement in myasthenia gravis: Some trials to background radioactivity in radioimmunoassay

Anti-acetylcholine receptor (AchR) antibodies in 19 non-myasthenic and 9 myasthenic sera were determined by radioimmunoassay, using [¹²⁵I]α-bungarotoxin-complexed receptors of human and denervated rat muscles (standard assay). Anti-AchR antibodies in the same sera blocked by antihuman IgG were assayed in parallel to measure background radioactivity (blank assay). The activity of blank assay did not differ significantly from that of standard assay in non-myasthenic sera. There was no remarkable difference in blank assay titers between non-myasthenic and myasthenic sera. The corrected titers, obtained by subtracting blank assay titers from standard assay titers, were in excess of those of non-myasthenic sera in 6 of 9 myasthenic sera with the denervated rat AchR and 8 of 9 myasthenic sera with human AchR.These findings suggest that this method is useful for the detection of the anti-AchR antibody in myasthenic sera.     

Widely varying TNF-α levels in patients with myasthenia gravis

Animal studies have indicated an important role of tumor necrosis factor-alpha (TNF-α) in the pathogenesis of myasthenia gravis (MG), and trials of monoclonal antibodies that block TNF-α have shown clinical improvement. However, before a TNF-α blocking agent is proposed for treatment of MG, whether serum TNF-α level correlates with the patient’s condition should be confirmed. Therefore, we evaluated the relationship between the serum TNF-α level and clinical factors, including the quantitative MG score and the anti-acetylcholine receptor antibody level, in 33 MG patients. TNF-α levels ranged from 0.44 to 3.63 pg/mL and did not correlate with clinical factors. Overall, we found that serum TNF-α levels varied greatly among MG patients.     

重症肌无力病人胸腺切除术后血清自身抗体水平变化和临床转归分析

６９例重症肌无力（ＭＧ）病人行胸腺切除术，术前血清乙酰胆碱受体抗体水平均高于正常，术后半年抗体水平明显下降，胸腺增生组（２０例）尤为显著．胸腺瘤组（４９例）术前胸腺瘤相关抗体水平增高，但术后半年内无明显变化．６９例ＭＧ患者胸腺切除术后４年，症状总缓解率为７６．８１％，其中胸腺增生组９５％，胸腺瘤组６９．３９％。作者认为，对于伴有胸腺增生或胸腺瘤的ＭＧ患者，胸腺切除术是有效的，应该列为治疗的第一选择。