Antibody to acetylcholine receptor in canine and human myasthenia gravis: differential cross-reactivity with human and rabbit receptor

Anti-acetylcholine receptor (AChR) antibody (ab) was found in the serum of a dog with acute myasthenia gravis (MG) by the use of Cowan 1 strain Staphylococcus aureus to bind radiolabeled anti-AChR ab-AChR immune complexes. Fifteen months later, when the dog was in remission, there was only a very low level of the anti-AChR ab. These observations strengthen the contention that anti-AChR ab is important in the pathophysiology of myasthenia gravis. Higher titers of the canine ab were measured with rabbit than with human AChR, whereas 17 human MG sera, selected to represent a wide range of anti-AChR ab titers, were all more reactive with human AChR. The degree of cross-reactivity of human anti-AChR ab with rabbit AChR varied widely, indicating a heterogeneous population of anti-AChR ab molecules in human myasthenia gravis sera.     

Enzyme-linked immunosorbent assay for antibody against the nicotinic acetylcholine receptor in human myasthenia gravis

Antibody against acetylcholine receptor (AChR) of human skeletal muscle was measured using enzyme-linked immunosorbent assay and found in 23 (74%) of 31 Japanese patients with generalized myasthenia gravis. In 15 patients with generalized myasthenia gravis who had not undergone thymectomy and who were not receiving adrenocorticosteroids, the antibody was found in 13 (87%). Antibody was also found in 13 (54%) of 24 patients with myasthenia gravis against AChR fractions obtained from fetal calf thymus. Based on the subunit structures of the AChR protein, the double precipitation assay using iodine 125-alpha-bungarotoxin is also capable of detecting antibody against the toxin binding site, by cross reactivity. This is among the first reports of experiments in which enzyme-linked immunosorbent assay was used to measure the antibodies in human myasthenia gravis and provides evidence of anti-AChR antibody against antigens from fetal calf thymus.     

Anti-acetylcholine receptor antibody specificities in serum and in thymic cell culture supernatants from myasthenia gravis patients

We investigated the role of the thymus in myasthenia gravis by comparing the antigenic specificities of anti-acetylcholine receptor antibodies (anti-AChR), defined by competition with mouse monoclonal antibodies that bind to five different regions on human muscle AChR, in thymic culture supernatants and in serum pre- and post-thymectomy. Anti-AChR specificities present in the serum were broadly unchanged in 16 non-thymoma and six thymoma patients 7-30 months after thymectomy compared with an initial sample, although total anti-AChR frequently fell. The fine specificities of the anti-AChR synthesized in vitro by cultured lymphocytes from the thymus of ten patients (without thymoma) correlated significantly with that of the anti-AChR in the serum at the same time. We conclude that AChR-specific B cells in the thymus are representative of the total AChR-specific repertoire, and that thymectomy does not selectively deplete particular B cell clones.     

Predictive value of serum anti-C1q antibody levels in experimental autoimmune myasthenia gravis

Components of the complement cascade and circulating immune complexes play important roles in both experimental autoimmune myasthenia gravis and myasthenia gravis in humans. Thus far, no serological factor has been identified to predict the clinical severity of either myasthenia gravis. Upon immunization with acetylcholine receptor, levels of complement factors C1q, C3 and CIC increase with time in sera from C57BL/6 (B6) mice. Both these and plasma samples from myasthenia gravis patients also contain anti-C1q antibodies. The serum levels of anti-C1q antibodies but not C1q, C3 and CIC are significantly correlated with the clinical severity in the experimental myasthenia mice. However, this correlation is not observed in myasthenia gravis patients.     

Acetylcholine release in myasthenia gravis: Regulation at single end-plate level

In myasthenia gravis, loss of acetylcholine receptors at motor end-plates is induced by antireceptor autoantibodies. At end-plates in rats in which myasthenia gravis–like symptoms are induced by chronic treatment with α-bungarotoxin, acetylcholine release is increased. Within muscles from such rats there is a strong correlation between the increase of acetylcholine release at an end-plate and the loss of postsynaptic acetylcholine receptors, caused by the toxin. The question is whether upregulation of acetylcholine release is a clinically relevant compensatory mechanism in myasthenia gravis or only a feature of the animal model using α-bungarotoxin. We investigated electrophysiologically the in vitro acetylcholine release at end-plates of muscles from patients with myasthenia gravis and rats with experimental autoimmune myasthenia gravis where acetylcholine receptor reduction is caused by autoantibody attack. In both human and rat autoimmune myasthenic muscle, the mean quantal content was considerably increased compared with control levels. At each individual myasthenic end-plate, the increase in quantal content appeared to be correlated with the reduction of the amplitude of the miniature end-plate potential. This finding suggests the existence of an important compensatory mechanism in myasthenia gravis, in which retrograde acting factors (i.e., from muscle fiber to nerve terminal) upregulate acetylcholine release.     

Anti-acetylcholine receptor antibodies in myasthenia gravis: Part 1. Relation to clinical parameters in 250 patients

We examined the significance of the presence or absence of anti-acetylcholine receptor (anti-AChR) antibodies in 250 myasthenia gravis (MG) patients and the relation between clinical features and anti-AChR levels.We found high anti-AChR levels in 2 out of 11 thymoma patients without MG, while 37 out of 250 MG patients had no detectable anti-AChR. The absence of these antibodies was related to purely ocular disease and to steroid therapy and/or thymectomy.Differences in anti-AChR levels did not correspond significantly to differences in disease activity when single measurements in patients were analysed. However, the results were influenced by both the presence or absence of a thymoma, the age at onset of disease and by steroid therapy. The thymic pathology and age at onset seemed to act independently. Early onset of disease was associated with high anti-AChR levels and absence of antibodies to striated muscle (anti-SM), whereas late onset was associated with low anti-AChR and the presence of anti-SM. Thymomas both have high anti-AChR and high anti-SM. The effect of steroid therapy on antibody levels was seen in all patient groups but was strongest in thymoma patients with early onset of disease.     

Neuromuscular junction autoimmune disease: muscle specific kinase antibodies and treatments for myasthenia gravis

PURPOSE OF REVIEW: Some of the 20% of myasthenia gravis patients who do not have antibodies to acetylcholine receptors (AChRs) have antibodies to muscle specific kinase (MuSK), but a full understanding of their frequency, the associated clinical phenotype and the mechanisms of action of the antibodies has not yet been achieved. Moreover, some patients do not respond well to conventional corticosteroid therapy. Here we review recent clinical and experimental studies on MuSK antibody associated myasthenia gravis, and summarize the results of newer treatments for myasthenia gravis. RECENT FINDINGS: MuSK antibodies are found in a variable proportion of AChR antibody negative myasthenia gravis patients who are often, but not exclusively, young adult females, with bulbar, neck, or respiratory muscle weakness. The thymus histology is normal or only very mildly abnormal. Surprisingly, limb or intercostal muscle biopsies exhibit no reduction in AChR numbers or complement deposition. However, patients without AChR or MuSK antibodies appear to be similar to those with AChR antibodies and may have low-affinity AChR antibodies. A variety of treatments, often intended to enable corticosteroid doses to be reduced, have been used in all types of myasthenia gravis with some success, but they have not been subjected to randomized clinical trials. SUMMARY: MuSK antibodies define a form of myasthenia gravis that can be difficult to diagnose, can be life threatening and may require additional treatments. An improved AChR antibody assay may be helpful in patients without AChR or MuSK antibodies. Clinical trials of drugs in other neuroimmunological diseases may help to guide the treatment of myasthenia gravis.     

Anti-idiotypic antibodies to anti-acetylcholinereceptor antibody: Characterization by ELISA and immunoprecipitation assays

The idiotype network is important both as a means of autoregulation of immune mechanisms and a potential tool for manipulation of abnormal responses. In the autoimmune disease myasthenia gravis the acetylcholine receptor (AChR) is the target of an aberrant immune response. In this study we compare 2 widely used methods of antibody determination--immunoprecipitation radioimmunoassay (IPRA) and enzyme-linked immunoassay (ELISA)--for their ability to detect both anti-AChR antibodies (polyclonal and monoclonal) and anti-idiotypic antibodies raised against polyclonal anti-AChR antibodies. Although the IPRA is considerably more sensitive for the detection of monoclonal anti-AChR antibodies, the 2 methods produce similar results in the detection of anti-idiotypic antibodies to the anti-AChR immune response. The 2 techniques also demonstrated specificity of the reagents for idiotypes associated with the anti-AChR response and absence of effect on an idiotype associated with the control antigen, ovalbumin. The results demonstrate that the idiotypic repertoire of the polyclonal anti-AChR response in C57B1/6 mice is sufficiently restricted that antigen-specific blocking anti-idiotypic antibodies can be raised in rabbits by immunization with anti-AChR antibodies.     

Anticorps dans la myasthénie

In autoimmune myasthenia gravis, 75 to 80% of patients have antiacetylcholine receptor antibodies (anti-AChR abs) quantified by immunoprecipitation. Anti-AChR abs are polyclonal, directed against all AChR subunits, with a major fraction against the main immunogenic region, (alpha-subunit, aminoacids 67-76); they cause AChR loss by three mechanisms: blocking of acetylcholine binding; accelerated degradation or AChR due to bridging of two adjacent AChR molecules (antigenic modulation); lysis of postsynaptic membrane induced by complement. Neither anti-AChR ab level nor antigenic repertoire are correlated with disease severity. Studies performed on rat and human myotubes have shown that capacity of myasthenic patients's sera or immunoglobulins to induce AChR loss was correlated with anti-AChR ab titer but not with severity. Highest anti-AChR ab titers are found in young women with hyperplastic thymus, lowest in older patients and atrophic thymus. Ten to fifteen percent of babies born to myasthenic mothers suffer from a transitory neonatal myasthenic syndrome due to passive transfer of maternal anti-AChR abs. There is no correlation between clinical condition of the baby (presence and severity of neonatal myasthenia) and severity of maternal myasthenia. The risk of neonatal myasthenia is high when maternal ab titer is elevated (≥ 100 nM). Very rare and severe cases of foetal neonatal myasthenia gravis with arthrogryposis, hypomobility are due to presence in maternal serum of anti-AChR ab directed against foetal (gamma) AChR. In generalized myasthenia without anti-AChR ab, antibodies directed against MuSK, a postsynaptic molecule involved in AChR aggregation, are detected in around 40% of patients. Features of MuSK+ myasthenia are the following: strong female preponderance, severity (respiratory and bulbar) requiring immunosuppressants, facial and tongue atrophy, poor response to anticholinesterase inhibitors, atrophic thymus and poor response to thymectomy. Low-affinity anti-AChR abs have been recently reported in myasthenia gravis without anti-AChR and anti-MuSK ABS.     

乙酰胆碱受体诱导实验性自身免疫性重症肌无力模型的建立

目的采用纯化乙酰胆碱受体(acetylcholine receptors,AChR)免疫大鼠和小鼠以建立实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)动物模型。方法以亲和层析法从电鳐电器官提取和纯化AChR,并用其免疫接种Lewis大鼠和C57BL/6小鼠,观察接种动物的临床症状、电生理变化以及AChR抗体产生的情况。结果动物模型临床肌无力症状、翻转悬挂时间(小鼠,P<0.05)、重复神经电刺激(repetitive nerve stimulation,RNS)动作电位衰减率、抗体吸光度(P<0.05)及新斯的明试验均为阳性或有统计学意义。结论纯化电器官AChR作为免疫原,成功诱导产生EAMG动物模型。Lewis大鼠和C57BL/6小鼠均对AChR免疫易感而产生EAMG表现,7~8周龄的C57BL/6更易诱导出类似人类MG表现的EAMG。     

The role of antibodies in myasthenia gravis

Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed. Experimental autoimmune myasthenia gravis, an experimental model closely mimicking the disease, has provided answers to many questions about the role of antibodies, complement macrophages and AChR anchor proteins. Genetically modified anti-AChR antibodies may also be used in the future to treat myasthenia.     

C5a is not involved in experimental autoimmune myasthenia gravis pathogenesis

C5 deficient mice are highly resistant to experimental autoimmune myasthenia gravis (EAMG) despite intact immune response to acetylcholine receptor (AChR), validating the pivotal role played by membrane attack complex (MAC, C5b-9) in neuromuscular junction destruction. To distinguish the significance of C5a from that of C5b in EAMG pathogenesis, C5a receptor (C5aR) knockout (KO) and wild-type (WT) mice were immunized with AChR to induce pathogenic anti-AChR antibodies. In contrast with C5 deficient mice, C5aR KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and lymphocyte proliferation response to AChR implicating that C5a is not involved in EAMG development.     

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.     

Ryanodine receptor antibodies related to severity of thymoma associated myasthenia gravis.

Ryanodine receptor (RyR) antibodies are detected in about 50% of patients with myasthenia gravis who have a thymoma. The RyR is a calcium release channel involved in the mechanism of excitation-contraction coupling in striated muscle. In this study the severity of myasthenia gravis assessed by a five point disability score was compared between 12 patients with myasthenia gravis, a thymoma, and RyR antibodies and 10 patients with myasthenia gravis and a thymoma but without such antibodies. Symptoms of myasthenia gravis were significantly more severe in patients with RyR antibodies. The mean (SD) disability scores were 3.7(0.5) in patients with antibodies and 2.7 (0.9) in those without at peak of illness, (p = 0.01) and 3.4(1.4) v 1.6(0.7) at the end of an average observation period of five years (p = 0.002). The number of deaths due to myasthenia gravis was five of 12 RyR antibody positive patients, and none of 10 RyR antibody negative patients (p = 0.04). RyR antibody levels correlated positively with severity of myasthenia gravis. The presence of circulating RyR antibodies seems to be associated with a severe form of thymoma associated myasthenia gravis.     

Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients

BACKGROUND: Titin is the major autoantigen recognized by anti-striated muscle antibodies, which are characteristic of generalized myasthenia gravis (MG). OBJECTIVE: To seek a correlation between anti-titin antibodies and other features of MG patients, including histopathology, age at diagnosis, anti-acetylcholine receptor (anti-AChR), autoantibody titers, and clinical severity. METHODS: A novel, highly specific radioligand assay was performed on a large group of 398 patients with generalized MG. RESULTS: Among thymectomized patients, anti-titin antibodies were present in most patients with thymoma (56/70 [80%]), contrasting with only a minority of patients with thymus atrophy or hyperplasia (17/165 [10%]). They were also present in 64 (41%) of 155 nonthymectomized patients who had a radiologically normal thymus. In these patients and in those who had a histologically normal thymus, anti-titin antibodies were associated with a later age at onset of disease and with intermediate titers of anti-AChR antibodies. After controlling for these 2 variables, disease severity was not significantly influenced by anti-titin antibodies. CONCLUSIONS: Anti-titin antibodies are a sensitive marker of thymoma associated with MG in patients 60 years and younger, justifying the insistent search for a thymoma in MG patients of this age group who have these antibodies. In nonthymoma patients, anti-titin antibodies represent an interesting marker complementary to the anti-AChR antibody titer, identifying a restricted subset of patients. These clinical correlations should prompt further studies to examine the mechanisms leading to the production of anti-titin antibodies.     

A sensitive rosetting assay for detection of acetylcholine receptor antibodies using BC3H-1 cells: positive results in 'antibody-negative' myasthenia gravis

Antibodies to acetylcholine receptor (AChR) were measured in a group of patients with myasthenia gravis (MG), some of whom had previously been classified as 'antibody negative' using the standard anti-AChR radioimmunoassay (RIA). AChR antibodies were measured using the rosetting assay, a new detection method which utilizes protein A-coated red blood cells and live BC3H-1 cells, a murine cell line which expresses muscle nicotinic AChR. The results of the rosetting assay were compared with those obtained in the anti-AChR RIA. 76% of all myasthenic sera tested showed rosetting at titers higher than any of the control sera (from patients with non-myasthenic neurologic disease and normal individuals). Of the myasthenic patients previously classified as 'antibody negative' in the RIA using human AChR, 71% demonstrated positive rosetting. There was no correlation between the anti-AChR antibody titer obtained in the rosetting assay and that obtained in the RIA using either human or denervated rat AChR. The results suggest that the rosetting assay may measure a subpopulation of antibodies that differs from those detected in the RIA.     

Function of circulating antibody to acetylcholine receptor in myasthenia gravis: investigation by plasma exchange

Plasma exchange has been used to investigate the function fo anti-acetylcholine receptor (anti-AChR) antibody in seven patients with acquired myasthenia gravis (MG) who had elevated antibody titers and in one patient with congenital MG who had normal titers. There was an inverse association between clinical indices of muscle strength and anti-AChR titers, with a minimum time lag of 2 days for the clinical response. The inverse association of the clinical state with anti-AChR antibody titers was closer than that with total immunoglobulin G or other immunoglobulins, and is consistent with the view that the anti-AChR antibody is the principal factor interfering with neuromuscular transmission in acquired MG.     

Detection of erythrocyte immune function and circulatory immune complex in myasthenia gravis

The circulating immune complexes (CIC) and erythrocyte immune function in myasthenia gravis were studied. In order to examine CIC in the patients with myasthenia gravis the complement sensitized yeast cell agglutination (CSYCA) test and anti-C3-ELISA were used. The CIC positive rate was 92.3% in the patients tested. The CIC test was all negative in the normal control subjects. The levels of CIC were remarkable elevated in the patients with myasthenia gravis (P less than 0.01). It was found that the rosette rate of red blood cell C3b receptor was 11.27 +/- 3.27% in the CIC negative patients with myasthenia gravis and 17.60 +/- 5.10% in CIC negative patients with myasthenia gravis. The erythrocyte immune function decreased remarkably in the CIC positive patients with myasthenia gravis (P less than 0.05). The relationship between the decrease of the erythrocyte immune function and myasthenia gravis is discussed.     

Immunohistochemical study of utrophin and dystrophin at the motor end-plate in myasthenia gravis

We studied the densities of utrophin and dystrophin at the motor end-plates of patients with myasthenia gravis (MG) using immunohistochemical analysis. The densities were compared with those found in patients with amyotrophic lateral sclerosis, Lambert-Eaton myasthenic syndrome and normal controls. Utrophin was reduced at the motor end-plates of MG patients, in association with a reduction of α-bungarotoxin binding sites. In contrast, the density of dystrophin at the motor end-plate of MG patients was not significantly different from that found in the controls. We conclude that, at the motor end-plate, utrophin may be more closely associated than dystrophin with the acetylcholine receptor, and that it plays a different role. Received: 8 June 1995 / Revised: 13 October 1995 / Accepted: 16 January 1996     

Specific immunoadsorption of the autoantibodies from myasthenic patients using the extracellular domain of the human muscle acetylcholine receptor alpha-subunit. Development of an antigen-specific therapeutic strategy

Antibodies against the acetylcholine receptor (AChR) are the main pathogenic factor in myasthenia gravis (MG). Clinical improvement correlates well with a reduction in levels of circulating anti-AChR antibodies, and plasmapheresis is an efficient short-term MG treatment. The Sepharose-immobilized N-terminal extracellular domain of human muscle AChR alpha-subunit was used to immunoadsorb anti-AChR autoantibodies from 50 MG patients sera. The immunoadsorbents removed 60-94% of the anti-AChR antibodies in 10 sera and a mean of 35% from all samples combined. Immunoadsorption was fast, efficient, and the columns could be used repeatedly without any release or proteolysis of the polypeptide, suggesting the feasibility of antigen-specific MG immunoadsorption therapy.