Titin and ryanodine receptor epitopes are expressed in cortical thymoma along with costimulatory molecules

Cortical-type thymomas are associated with myasthenia gravis (MG) in 50% of the cases. MG is caused by antibodies against the acetylcholine receptors (AChR), but additional non-AChR muscle autoantibodies such as those against titin and ryanodine receptor (RyR) are found in up to 95% of MG patients with thymoma. To elucidate the induction of non-AChR autoantibodies in thymoma-associated MG, we studied cortical-type thymomas from seven thymoma MG patients, and sera from six of them. All six had titin antibodies, and four had RyR antibodies. Titin and RyR epitopes were co-expressed along with LFA3 and B7 (BB1) costimulatory molecules on thymoma antigen-presenting cells (APC) in all thymomas. In normal thymus, the staining by anti-titin, anti-RyR, anti-LFA3, and anti-BB1 antibodies was weak and occurred exclusively in the medulla and perivascularly. Our results indicate a primary autosensitization against titin and RyR antigens inside the thymoma. In MG-associated thymoma, the mechanisms involved in the initial autosensitization against titin and RyR are probably similar to those implicated in the autosensitization against AChR. In all cases, there is an overexpression of muscle-like epitopes and costimulatory molecules indicating that the T-cell autoimmunization is actively promoted by the pathogenic microenvironment inside the thymoma.     

重症肌无力患者血清中Ryanodine受体抗体检测及其临床意义

目的　探讨Ryanodine受体 (RyR)抗体在重症肌无力 (MG)诊断中的临床意义。方法　以差速离心法提取富含RyR的肌质网 (SR)提取物 ,建立ELISA RyR抗体检测系统 ,分析 6 6例伴胸腺瘤的MG(MGT)、98例非胸腺瘤MG(NTMG)和 5 0例非重症肌无力 (NMG)及 12 3名健康人 (NC)血清中RyR抗体水平。 结果　MGT组RyR抗体阳性检出率显著性高于NTMG组和NMG组 (P <0 0 1) ,敏感性和特异性分别达 81 8%和 94 5 %。不同胸腺组织学类型MGT之间RyR抗体阳性检出率差异无显著意义 (P >0 0 5 )。RyR抗体呈阳性MG组的年龄、临床评分和乙酰胆碱受体抗体水平均显著高于RyR抗体阴性MG组 (P <0 0 1)。RyR抗体水平同MG患者临床症状的严重程度呈正相关 ,尤其是MGT组 (r =0 6 2 6 ,P <0 0 1)。不同胸腺组织学类型MGT中以上皮细胞型相关性最高 (r =0 5 92 ,P <0 0 1)。结论　RyR抗体检测对诊断MGT具有较高的敏感性和特异性 ,并且与MG患者临床症状的严重程度呈正相关。     

Disease severity and outcome in thymoma myasthenia gravis: a long-term observation study

Thymomas occurring in myasthenia gravis (MG) are usually of the cortical subtype and are usually treated by thymectomy. However, the factors that influence MG outcome in thymoma MG patients are not known. In a long-term study, MG severity and treatment was observed in 24 thymoma and 24 non-thymoma MG patients for up to 30 years, and the occurrence of muscle autoantibodies was assayed. The rate of complete stable remission was low and did not differ between the two groups. There was no significant difference in MG severity between thymoma and non-thymoma MG patients at any time during the study. Titin and ryanodine receptor (RyR) antibody occurrence was significantly higher in thymoma MG patients. Four thymoma (all titin and RyR antibody positive) and two non-thymoma (both titin and one RyR antibody positive) MG patients died from MG-related respiratory insufficiency. Seventy percent of thymoma and 75% of non-thymoma MG patients were treated with immunosuppressive drugs. The number of patients who received plasmapheresis did not differ in those who were treated because of acute MG deterioration, irrespective of planned surgery.This study shows equal MG severity and outcome in thymoma and non-thymoma MG, but the presence of RyR antibodies in thymoma MG and titin/RyR in non-thymoma MG indicates a less favorable prognosis.     

Myasthenia gravis sera containing antiryanodine receptor antibodies inhibit binding of [3H]-ryanodine to sacroplasmic reticulum

Myasthenia gravis (MG) patients with thymoma often have antibodies against the calcium-release channel of the sarcoplasmic reticulum (SR) in striated muscle, the ryanodine receptor (RyR). RyR function can be tested in vitro by measuring the degree of [³H]-ryanodine binding to SR. In this study, sera from 9 out of 14 MG patients containing RyR antibodies inhibited [³H]-ryanodine binding to SR membranes from rat skeletal muscle. The 9 patients with antibodies inhibiting ryanodine binding had more severe MG than those with noninhibiting antibodies (P = 0.006). Sera from MG patients with acetylcholine receptor and titin muscle antibodies but no antibodies against RyR and blood-donor sera did not have an inhibiting effect in the [³H]-ryanodine binding assay. The results show that RyR antibodies in MG patients have high affinity for the RyR, and that the binding of antibodies probably affects calcium release from SR by locking the RyR ion channel in a closed position. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:329–335.     

Anti-neuroblastoma antibodies in myasthenia gravis: clinical and immunological correlations

Forty-four of 109 myasthenia gravis (MG) patients (40%) had serum antibodies against human neuroblastoma cells (NBL). Anti-NBL antibodies were most frequent in the sera of MG patients who had either a hyperplastic thymus or a thymoma, clinically mild to moderately severe generalized MG, and a long disease duration (greater than or equal to 11 years). No correlation between individual anti-NBL antibody and anti-acetylcholine receptor (AChR) antibody titers was observed. Seven of the 19 patients negative for anti-AChR antibodies (37%) had anti-NBL antibodies in their sera. These findings provide further evidence for immunological heterogeneity in MG. In addition to the typical autoantibodies to the AChR, autoimmunization against neural antigens can frequently be detected in these patients.     

Myasthenia gravis: the specificities of skeletal muscle and thymus antibodies

Antibodies against skeletal muscle antigens and against thymic myoid cells were examined in sera from 40 patients with myasthenia gravis (MG). Using an indirect immunofluorescence technique, antibodies against the surface of muscle cells were found in 23 sera, and antibodies against muscle cell cross-striations in 28 sera. Antibodies against thymic myoid cells were found in 27 sera, stained cells also occurring in fetal thymus from 14 weeks of gestation and in hyperplastic thymus from MG patients. Sera which stained myoid cells also stained muscle cell cross-striations. Sera from all the 20 patients with thymoma contained antibodies to a citric acid extract of skeletal muscle (CAE) as detected by indirect haemagglutination, whereas sera from 20 comparable patients without thymoma did not contain CAE antibodies. The myoid cell antibodies could be absorbed by homogenized muscle, but not by CAE-coated sheep erythrocytes. Different antibody patterns are seen among different MG patients; close associations were found between cross-striational and myoid cell antibodies, and between CAE-antibodies and a thymoma.     

Muscle autoantibodies in subgroups of myasthenia gravis patients

Myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR), but several other muscle autoantibodies have also been identified in patient sera. We studied muscle autoantibodies against AChR, striated muscle tissue sections (SH), titin, citric acid antigen (CA), and ryanodine receptor (RyR) in sera from 146 consecutive MG patients to evaluate whether a single test or several tests together can predict a thymoma. The MG patients were divided into five subgroups; ocular MG, early-onset MG (< 50 years), late-onset MG (≥ 50 years), MG with thymoma, and AChR antibody negative MG. AChR, SH, titin, CA, and RyR antibodies were detected in 85%, 34%, 34%, 25%, and 14% of the MG patients, respectively. For thymoma MG, AChR, SH, titin, CA, and RyR antibodies were detected in 100%, 75%, 95%, 70%, and 70% respectively. SH, titin, CA, RyR antibodies, and computed tomography of the anterior mediastinum have similar sensitivity for thymoma MG. The specificity of RyR, titin, CA, and SH antibodies for thymoma was 70%, 39%, 38%, and 31%, respectively, which is significantly higher for RyR antibodies than for the others. No single muscle antibody assay can predict a thymoma, and a combination of several antibody assays is preferred, although RyR antibody testing alone showed 70% sensitivity and specificity for thymoma MG. SH and CA antibodies provided only little additional information. Received: 23 September 1999, Received in revised form: 6 December 1999, Accepted: 19 January 2000     

Ryanodine receptor antibodies are associated with severe myasthenia gravis

Sera from 32 thymoma patients, 29 of them with myasthenia gravis (MG), were tested for the presence of circulating antibodies to the ryanodine receptor (RyR) in Western blot RyR is a channel protein essential for the excitation-contraction coupling in skeletal muscle. MG severity was scored according to the Osserman classification during 1–17 years of follow-up (mean 7 years). Fifteen patients (14 MG and 1 non-MG) were RyR-antibody positive. RyR-positive patients had a significantly higher frequency of invasive thymomas (p = 0.01), and also a more severe MG than RyR-antibody negative patients (p = 0.04). The use of immunosuppressive drugs at latest follow-up was more frequent in RyR-antibody positive than in RyR-antibody negative patients (p = 0.02). Thus the presence of RyR-antibodies in thymoma patients is associated with a more severe disease and can be used as a prognostic marker.     

Autoantibodies in thymoma-associated myasthenia gravis with myositis or neuromyotonia

BACKGROUND: About 50% of patients with thymoma have paraneoplastic myasthenia gravis (MG). Myositis and myocarditis or neuromyotonia (NMT) will also develop in some. Patients with thymoma-associated MG produce autoantibodies to a variety of neuromuscular antigens, particularly acetylcholine receptor (AChR), titin, skeletal muscle calcium release channel (ryanodine receptor [RyR]), and voltage-gated potassium channels (VGKC). OBJECTIVE: To examine whether neuromuscular autoantibodies in patients with thymoma correlate with specific clinical syndromes. METHODS: Serum and plasma samples from 19 patients with thymoma-associated MG, of whom 5 had myositis and 6 had NMT, underwent testing for antibodies to AChR, titin, RyR, and VGKC. RESULTS: Antibodies to AChR and titin were found in 19 and 17 patients, respectively. Antibodies to RyR correlated with the presence of myositis (P = .03); they were found in all 5 patients with myositis and in only 1 patient with NMT, but also in 4 of 8 patients with neither disease. Antibodies to VGKC were found in 4 patients with NMT, 1 of 3 patients undergoing testing for myositis, and 2 of 7 patients undergoing testing with neither comorbidity. Presence of RyR antibodies correlated with high levels of titin antibodies. CONCLUSIONS: The results appear to distinguish partially between 3 groups of patients with thymoma-associated MG: the first with RyR antibodies and myositis or myocarditis, the second with NMT without RyR antibodies, and the third without RyR antibodies, myositis, or NMT. Differences in the thymoma may underlie these pathologic associations.     

Spontaneous thymoma rat as a model for myasthenic weakness caused by anti-ryanodine receptor antibodies

The mechanism of muscle weakness in myasthenia gravis and its possible relation to antibodies that are directed against the ryanodine receptor (RyR) were studied by the use of the spontaneous thymoma rat (Buffalo/Mna strain). The present study focused on the motor dysfunction as complicated by impaired subcellular machineries and noted particularly in patients with thymus abnormalities. Rats began to develop skeletal muscle weakness soon after birth and worsened progressively. Rats aged 3 months showed a benign thymoma characterized by proliferative lymphocytes; epithelial cells were stained with anti-RyR peptide antibody. The rat serum contained anti-RyR antibodies, but no anti-acetylcholine receptor antibodies. The electrophysiological study in muscle showed a reduction of contractile force without abnormality in synaptic transmission and membrane properties, suggesting a defect in excitation–contraction coupling. Hypothetically, thymic epithelial cells and skeletal muscles share a common RyR antigen, so that anti-RyR antibodies that target the thymic tissue may react with a homologous target in the muscle. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1655–1660, 1998     

Myasthenia gravis patients with ryanodine receptor antibodies have distinctive clinical features

Myasthenia gravis (MG) is an autoimmune disease caused in 85% of the patients by acetylcholine receptor (AChR) antibodies. Non-AChR muscle antibodies, against titin and ryanodine receptor (RyR) are mainly found in sera of patients with thymoma or late-onset MG. The occurrence of RyR antibodies increases the risk for severe MG and should lead to active immunomodulating treatment already at MG onset. The aim in this study was to describe the association between symptoms at MG onset and antibody profile in 152 patients. Patients with RyR antibodies had the highest rate of bulbar, respiratory and neck involvement at MG onset. They also had the highest frequency of non-limb MG symptoms. Neck weakness occurred in 40%. Respiratory difficulties at MG onset occurred in patients with titin antibodies, with and without RyR antibodies. Patients with RyR antibodies have a distinctive non-limb MG symptom profile, with bulbar, ocular, neck, and respiratory symptoms. These features, identified as early as at the first examination by a neurologist, characterize the RyR antibody positive subgroup at MG onset.     

重症肌无力患者血清Ryanodine受体抗体检测及意义

目的探讨Ryanodine受体（RyR）抗体在重症肌无力（myasthenia gravis，MG）诊断中的临床意义。方法采用ELISA法检测89例MG患者、66例其他神经系统疾病患者和66名正常对照者血清RyR抗体水平。结果MG组血清RyR抗体阳性率显著高于其他神经系统疾病组和正常对照组（P〈0．05），其敏感性和特异性分别为55．0％和91．7％。晚发型MG患者血清RyR抗体阳性率（74．4％）明显高于早发型MG（37．0％，P〈0．05）。MG合并胸腺瘤（MGT）和未合并胸腺瘤（nMGT）患者血清RyR抗体阳性率差异无统计学意义（P〉0．05）。将MG患者根据Osserman评分进行分型，各型血清RyR抗体阳性率及其吸光度值大小与病情严重程度无相关性（P〉0．05）。结论RyR抗体多见于晚发型MG，对诊断MG具有较高的敏感性和特异性。     

Complement activation by titin and ryanodine receptor autoantibodies in myasthenia gravis. A study of IgG subclasses and clinical correlations

To elucidate the mechanism of immune damage caused by titin and ryanodine receptor (RyR) autoantibodies in myasthenia gravis (MG), we studied the complement-activating capacity and the IgG subclass distribution of these autoantibodies in sera from 49 MG patients. Complement activation occurred in 38 out of 49 titin antibody positive sera, and in 14 out of 21 RyR antibody positive sera. The titin antibodies occurred only in the IgG 1 and IgG 4 subclasses, whereas the RyR antibodies occurred in all four IgG subclasses but with IgG 1 predominance. Complement-activating RyR antibodies occurred with higher frequency in sera of thymoma MG than of late-onset MG. RyR IgG 1 antibodies occurred more often in severe MG than in mild and moderate disease groups. Mean total IgG and IgG 1 titin and RyR antibody titers fell during long-time patient observation together with an improvement of the MG symptoms.     

Editorial introduction: from the structure and functions of the neuromuscular junction to the diseases

The neuromuscular junction has been recognized as a site for autoimmune and genetic disorders. Myasthenia gravis (MG) is mainly caused by postsynaptic nicotinic acetylcholine receptor (AChR) IgG1 antibodies that are directed against α-subunit 67-76 and 125-147 and activate complement. Thymic abnormalities are present in the autoimmune background. A proportion of MG patients without conformation-dependent AChR antibodies assayed by the cell-based method have muscle-specific tyrosine kinase (MuSK) antibodies which are largely IgG4 and partially IgG1. MuSK is activated by Dok-7 and Lrp4 (agrin receptor) and contributes to AChR clustering at the postsynaptic membrane via various kinase cascades in collaboration with Wnt-MuSK/Frizzled-Dishevelled signaling. Rapsyn interacts with MuSK-linked chaperones to stabilize postsynaptic architecture and also contributes to AChR phosphorylation. MG-associated thymomas express antigens that trigger antibody responses which play a part in disease generation and modification. Among these, ryanodine receptor-1 (RyR1; acts on sarcoplasmic Ca2+ release) antibodies cause muscle contractile weakness. Transient receptor potential canonical-3 (TRPC3) antibodies are also detected in thymoma-associated MG patients; they may participate in muscle contractile weakness because TRPC3 acts on RyR1, and may also impair the refill of sarcoplasmic Ca2+ stores since TRPCs contribute to the receptor-operated Ca2+ influx via the phospholipase C (PLC)-diacylglycerol (DAG) pathway in cooperation with the store-operated, STIM1/Orai1-mediated Ca2+ influx and TRPCs-Homerl-IP3R interaction. Lambert-Eaton myasthenic syndrome (LEMS) is caused by reduced ACh quantal release that occurs mainly because of presynaptic P/Q-type voltage-gated Ca2+ channel (VGCC) antibodies. Physicians should be vigilant for LEMS because it may predict an underlying malignancy, particularly small-cell lung carcinoma; SOX-1 antibodies are usually present in these patients and are absent in those who do not have caucer. Some patients with LEMS have antibodies against synaptotagmin-1, which associates with SNARE complex and functions as Ca2+ sensor for exocytosis. The stimulation of the M1-type presynaptic muscarinic AChR (mAChR)(G-proterin-coupled receptor) can compensate for the deficiency of Ca2+-mediated ACh quantal release via the PLC/DAG-mediated mechanism; This acts in a manner similar to the BDNF/NT4-TrkB interaction. The detection of M1 mAChR antibodies in LEMS suggests an impaired compensatory mechanism and corresponds, at least in part, to autonomic symptoms. Congenital myasthenic syndromes are classified into presynaptic, synaptic basal lamina and postsynaptic defects.     

Myasthenic thymus and thymoma are selectively enriched in acetylcholine receptor-reactive T cells

We compared T-cell proliferative responses to acetylcholine receptor (AChR) and to purified protein derivative (PPD) (of tuberculin) of hyperplastic thymus, thymoma, and blood cells from patients with myasthenia gravis (MG). Hyperplastic MG thymus cells gave significantly higher and more consistent responses to AChR than parallel cultures of autologous blood cells, whereas responses to PPD showed an opposite trend. Thus there was a preferential localization of AChR-reactive T cells in the hyperplastic MG thymus. Furthermore, there was a strong correlation between blood and thymus cell responses to PPD (but not to AChR), arguing that the hyperplastic MG thymus contains a sample of sensitized peripheral T cells. By contrast, both AChR- and PPD-responsive T cells were almost undetectable in thymus from nonmyasthenic patients, which is evidently much less receptive to circulating T cells. Cells from MG thymomas showed the highest stimulations by AChR but did not consistently react to PPD. However, the uninvolved thymus adjacent to these thymomas behaved almost identically to the hyperplastic samples described above. Our interpretation is that AChR-specific T cells are initially sensitized in the MG thymoma but are selectively trapped in the hyperplastic thymus after being primed elsewhere.     

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-ryanodine receptor antibodies and FK506 in myasthenia gravis

Anti-ryanodine receptor (RyR) antibodies were measured in sera from 33 myasthenia gravis (MG) patients using three peptides from the human RyR1 sequence, two C-terminal peptides included in the functional calcium release channel, and an N-terminal peptide implicated in ion-conduction. Antibodies were more frequently positive against the two C-terminal peptides, particularly in thymoma-associated MG. In a preliminary open trial with FK506, immunosuppressant and enhancer of RyR-related sarcoplasmic calcium release, the authors observed the sustained benefits in anti-RyR-positive MG patients.     

Surface phenotypes of lymphoid cells altered in the human myasthenic thymus

We investigated surface phenotypes of peripheral blood lymphocytes and thymic lymphoid cells from patients with myasthenia gravis (MG) by fluorocytometry, using monoclonal antibodies to human lymphoid cells. There were no significant differences in peripheral blood lymphocyte subsets in myasthenic patients, with or without a thymus. In the MG hyperplastic thymuses, the percentage of OKIa1+ cells or CCB1+ cells was significantly increased compared with controls. Although there were no significant differences in the percentage of T-cell lineage (CD3+, CD4+, CD8+, or CD1+ cells) between MG hyperplastic thymuses and the controls, the surface densities of T-cell lineage antigens (CD3, CD4, and CD8) were significantly increased on lymphoid cells in MG hyperplastic thymuses, compared with those in control thymuses. There were no significant differences in the percentage or fluorocytograph of lymphoid cells between the MG thymomas and the controls.     

Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients

Rabbit antiserum to a citric acid extract of human skeletal muscle (CA) stained both the cell membrane and the cross-striational bands of skeletal muscle cells. The rabbit antiserum also stained the cell membrane of epithelial thymoma cells from myasthenia gravis (MG) patients. Normal and hyperplastic thymus tissue were not stained, apart from scattered myoid thymic cells. Absorption of the antiserum with CA abolished staining of the thymoma, indicating that human skeletal muscle and epithelial thymoma cells possess common antigens.