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.     

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.     

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.     

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.     

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.     

The severity of myasthenia gravis correlates with the serum concentration of titin and ryanodine receptor antibodies

BACKGROUND: Myasthenia gravis (MG) is caused by autoantibodies to the acetylcholine receptor (AChR). Non-AChR muscle autoantibodies are present in many MG serum samples, mainly from patients with thymoma or late-onset MG. The exact relationship between MG severity and several non-AChR muscle antibodies is unknown. OBJECTIVE: To study the correlation between the severity of MG and the concentration of antibodies against striated muscle tissue sections, titin, citric acid antigen, ryanodine receptor, and AChR. SETTING: The severity of MG was graded in 146 consecutive patients with MG, and their serum samples were tested for the presence of autoantibodies. Ten patients who were titin antibody positive were observed in longitudinal follow-up. RESULTS: No significant difference was found in MG severity between late-onset and thymoma MG. Titin, citric acid antigen, and ryanodine receptor antibodies occurred significantly more often among patients with severe MG than among patients with less severe disease. Changes in MG severity correlated with changes in titin antibody titer in the individual patient. Titin antibodies showed a better longitudinal correlation with disease severity than the AChR antibodies. CONCLUSIONS: Non-AChR muscle autoantibodies occurred more frequently in severe MG regardless of MG subgroup. Thymoma per se does not generate a more severe MG. It may well be the presence of a humoral immune response to non-AChR muscle antigens such as titin, citric acid antigen, and ryanodine receptor that leads to a severe disease, not the presence of thymoma or a late age of onset. These antibodies can serve as important prognostic markers in MG regardless of the presence of thymoma.     

The significance of titin antibodies in myasthenia gravis

Myasthenia gravis (MG) is caused by autoantibodies to acetylcholine receptors (AChR). Non-AChR muscle autoantibodies, such as titin antibodies, are present in sera of many MG patients. To study the correlation between titin antibodies and the features of MG, the cDNA segment encoding MGT-30 was amplified and sequenced. The cloned MGT-30 cDNA was expressed in vector pET-30a, and then transfected into E.coli. BL21. We examined titin antibodies in sera of 265 normal subjects, 154 MG patients with different thymic pathology and 48 patients with other neurological diseases. Titin antibodies occurred more frequently in MGT, especially in MG with epithelial predominant-thymoma, and were correlated with the severity of disease. The levels of titin antibodies were reduced 6 months after thymectomy. The specificity of titin antibodies for the detection of thymoma was higher than that of CT examination in MG with thymoma. These results suggest that titin antibodies could be useful in both diagnosis and follow-up of MG patients with thymoma.     

Thymectomy and anti-muscle autoantibodies in late-onset myasthenia gravis

Thymectomy is still widely carried out in myasthenia gravis (MG) patients, but its role, especially in late-onset MG patients, is not established. These patients are immunologically heterogeneous, some with thymoma-like and others with early onset-like features. We evaluated whether any therapeutic effects of thymectomy correlate with the presence of non-acetylcholine receptor (AChR) muscle antibodies. The severity of MG, and titin and ryanodine receptor (RyR) antibodies, were assessed yearly starting from MG onset in 21 thymectomized and 22 non-thymectomized AChR antibody positive late-onset MG patients, who were followed for 2, 3 and 5 years. Clinical or pharmacological remission were seen in six of 11 titin antibody negative but none of the 10 titin antibody positive thymectomized patients, however, the non-thymectomized cases showed an opposite trend. The three MG-related deaths were all in patients with titin antibodies. There was no significant difference in MG severity between thymectomized and non-thymectomized patients; 2 years after MG onset, both groups were significantly improved. This study showed no dramatic benefit from thymectomy in late-onset MG in general. Any limited improvement appeared less likely in cases with titin and/or RyR antibodies.     

The role of complement in myasthenia gravis: serological evidence of complement consumption in vivo

BACKGROUND: Antibodies to the acetylcholine receptor (AChR) titin and the ryanodine receptor (RyR) occur in myasthenia gravis (MG). These antibodies are capable of complement activation in vitro. The involvement of the complement system should cause consumption of complement components such as C3 and C4 in vivo. MATERIALS AND METHODS: Complement components C3 and C4 were assayed in sera from 78 AChR antibody-positive MG patients and 52 healthy controls. Forty-eight of the patient sera contained titin antibodies as well, and 20 were also RyR antibody-positive. RESULTS: MG patients with AChR antibody concentrations above the median (11.2 nmol/l) had significantly lower mean C3 and C4 concentrations in serum compared to those with AChR antibody concentrations below the median. Titin antibody-positive MG patients, titin antibody-negative early-onset MG patients, titin antibody-negative late-onset MG patients, and controls had similar C3 and C4 concentrations. Nor did mean C3 and C4 concentrations differ in MG patients with RyR antibodies. Patients with severe MG (grades 4 and 5) had similar C3 and similar C4 levels compared to those with mild MG (grades 1 and 2). CONCLUSION: An increased in vivo complement consumption was detected in MG patients with high AChR antibody concentrations, unrelated to MG severity and non-AChR muscle antibodies.     

Thymectomy in nonthymoma early-onset myasthenia gravis in correlation with disease severity and muscle autoantibodies

OBJECTIVE: To study the clinical effect of thymectomy in a well-defined early-onset MG subgroup and to correlate it to MG severity, the presence of circulating muscle autoantibodies, and the need for pharmacological treatment in a long-term setting. METHODS: Fifty-two consecutive AChR antibody-positive early-onset MG patients (34 thymectomized and 18 nonthymectomized) were included. Severity was assessed and the pharmacological treatment monitored on a yearly basis, starting from the year of MG onset, for 5, 10, 15, and 20 consecutive years; AChR, titin, and RyR antibodies were assayed. RESULTS: In the four follow-up groups, MG severity was significantly higher in nonthymectomized compared to thymectomized MG patients. The postthymectomy MG improvement was significant and persistent. There were 21/34 remissions in thymectomized patients and only 4/18 in the nonthymectomized group. Patients with initially high or low AChR antibody concentration had a similar thymectomy outcome. Only 6 patients had titin antibodies, and none had RyR antibodies. CONCLUSION: The present study indicates a benefit of thymectomy in early-onset MG. The muscle autoantibody concentration does not influence the outcome of thymectomy in early-onset MG.     

Autoantibodies in thymoma-associated myasthenia gravis and their clinical significance

Myasthenia gravis (MG) is characterized by the development of antibodies that act against the acetylcholine receptor (AChR) present at the postsynaptic site of neuromuscular junctions. Some MG patients have antibodies that bind in a cross-striational pattern to skeletal and heart muscle tissue sections (striational antibodies). These antibodies react with the epitopes on the muscle protein titin, ryanodine receptor (RyR), and voltage-gated K channel α subunit, Kv1.4. Since these 3 molecules are expressed in the thymoma tissue of MG patients, striational antibodies are frequently detected in thymoma-associated MG. More severe MG symptoms in thymoma-associated MG may be due to the presence of striational autoantibodies. The anti-titin antibody, usually detected by enzyme-linked immunosorbent assay (ELISA), is found in 20-40% of all MG patients, and is more common in late-onset MG patients. The anti-RyR antibody, detected by Western blotting or ELISA, is found in 13-38% of all MG patients, and is known to inhibit Ca2+ release from sarcoplasmic reticulum and excitation-contraction coupling of the muscle. The anti-Kv1.4 antibody, detected by the immunoprecipitation assay with 35S-labeled extract from rhabdomyosarcoma cells, is found in 12-15% of all MG patients. Autoimmune myocarditis may develop in MG patients who have the anti-Kv1.4 antibody. In addition, the anti-Kv1.4 antibody is a useful marker to check the response to calcineurin inhibitors. In summary, the detection of striational antibodies provides more specific clinical information in MG patients.     

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.     

The occurrence of anti-titin antibodies and thymomas: a population survey of MG 1970-1999

OBJECTIVE: To estimate the incidence of elevated anti-titin antibodies titers and of thymomas in a population of patients with MG using various statistics and associations. METHODS: Extensive epidemiology, systematic measurement of anti-titin antibodies, and histologic assessment of thymomas according to the new World Health Organization classification. RESULTS: The mean annual incidence rate of MG per million population was 8.3. The analogous mean rate of thymomas was 2.0, out of which MG was encountered in about 20%. A thymoma was coexistent in 7% of the patients with MG. The finding of titin autoantibodies and the coexistence of thymomas were both associated with age at the appearance of MG. In patients with MG with a thymoma, the frequency of seropositivity was 68%, whereas acetylcholine receptor (AChR) autoantibodies were detected in all such sera. Titin autoantibody-positive sera were also anti-AChR antibodies positive. Further, all serum samples negative for anti-AChR antibodies were devoid of anti-titin antibodies. Titin autoantibodies were not detected in nonthymoma early-onset MG. CONCLUSION: Apart from MG with a thymoma, the finding of the titin autoantibodies was observed to be an exclusive feature of late-onset MG, the frequency being 55%. No data were found to suggest that patients with MG were more likely to present with thymic tumors than other patients exhibiting thymic neoplasia. In about 80%, such tumors in MG were composed of cortical cells. The concept of the anti-titin antibodies merely as a paraneoplastic marker in MG was not supported by these data.     

The diagnosis of thymoma in myasthenia gravis

Between 10 and 15 per cent of patients with myasthenia gravis (MG) have a thymoma. We have evaluated the possibility of detecting such tumours on the basis of clinical features, radiological findings and immunological tests in a group of 70 MG patients. The mean age at onset of myasthenic symptoms was 67.6 years in the thymoma group and 33.8 years in the non-thymoma group. Otherwise, the clinical picture was similar. Radiological examination revealed a mediastinal mass in 2 patients. Six of the 70 MG sera contained antibodies to a citric acid (CA) extract of striated muscle. At thymectomy, a thymoma was found in these 6 patients. None of the remaining 64 patients had evidence of a thymoma. Antibodies to AChR were detected in 52 of the 70 MG sera. There was no relation between titres of AChR and CA antibodies. Three out of 5 sera from non-MG patients with a thymoma contained CA antibodies. Apparently, this assay is a valuable technique for the identification of a thymoma at an early stage.     

Do titin and cytokine antibodies in MG patients predict thymoma or thymoma recurrence?

BACKGROUND: Patients with MG often have other autoantibodies in addition to those against the acetylcholine receptor (AChR). It has been suggested that antibodies to the muscle protein titin may be diagnostic of a thymoma, but they have also been found in patients with late-onset MG. Antibodies to certain cytokines have also been detected in patients with MG and thymoma, and it is not clear whether these antibodies could be more useful clinically. The authors measured antibodies against titin and the cytokines interferon alpha (IFNalpha) and interleukin 12 (IL12) in patients with MG and thymoma or thymoma recurrence, and in patients with MG but without thymoma presenting before (early-onset MG) or after (late-onset MG) 40 years of age. METHOD: Levels of titin, IFNalpha, and IL12 antibodies were determined by radioimmunoassay in 191 patients with MG and 82 controls. RESULTS: As previously reported, titin antibodies were uncommon in patients with early-onset MG. However, in patients with late-onset MG, titin antibodies had similar prevalence and levels to those in patients with MG and thymoma, although the antibodies were uncommon in patients between 40 and 60 years of age presenting without a tumor. By contrast, cytokine antibodies were more common in patients with thymoma than in patients without thymoma, and cytokine antibodies typically increased substantially if the thymoma recurred. CONCLUSIONS: Measurement of titin antibodies has limited use in predicting the presence of a tumor, unless the patient is less than 60 years of age, but measurement of IFNalpha and IL12 antibodies may be helpful in identifying patients with a thymoma recurrence, particularly when mediastinal imaging is equivocal.     

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.     

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。     

Incidence and reactivity patterns of skeletal and heart (SH) reactive autoantibodies in the sera of patients with myasthenia gravis

Serum from patients with myasthenia gravis (MG) contain antibodies to numerous skeletal muscle components in addition to the acetylcholine receptor (AChR). Certain non-AChR skeletal muscle autoantibodies have been shown by absorption to cross-react with cardiac muscle, leading to the designation of 'skeletal and heart' or SH antibodies. This study describes a new procedure for the extraction of human cardiac muscle which allows direct determination of SH antibody reactivity. Serologic evaluation of 17 patients with MG revealed 9/17 (53%) were seropositive for SH antibody to cardiac muscle. Absorption of selected MG serum samples with cardiac muscle extracts, reduced or eliminated reactivity to skeletal muscle in all cases, confirming the presence of cross-reactive antibodies. Immunoblot analysis of cardiac muscle extracts demonstrated several distinct antigenic components, which were unrelated to the acetylcholine receptor or to previously identified striational muscle proteins. Serum samples from individual MG patients displayed different immunoblot reactivity patterns ot the antigens in cardiac muscle extracts, providing the first evidence of multiple heart-reactive SH antibodies in MG.     

Characterization of ganglionic acetylcholine receptor autoantibodies

In myasthenia gravis (MG), autoantibodies bind to the alpha1 subunit and other subunits of the muscle nicotinic acetylcholine receptor (AChR). Autoimmune autonomic ganglionopathy (AAG) is an antibody-mediated neurological disorder caused by antibodies against neuronal AChRs in autonomic ganglia. Subunits of muscle and neuronal AChR are homologous. We examined the specificity of AChR antibodies in patients with MG and AAG. Ganglionic AChR autoantibodies found in AAG patients are specific for AChRs containing the alpha3 subunit. Muscle and ganglionic AChR antibody specificities are distinct. Antibody crossreactivity between AChRs with different alpha subunits is uncommon but can occur.     

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。