Evidence against acetylcholine receptor having a main immunogenic region as target for autoantibodies in myasthenia gravis

We investigated specificities of acetylcholine receptor (AChR) antibodies in 100 seropositive patients with myasthenia gravis (MG). Antibodies in 74 of these sera were inhibited by more than 50% from binding to human muscle AChR by a rat monoclonal antibody (mAb) of "main immunogenic region" (MIR) specificity. The mAb inhibition was not explainable by epitope competition because (1) the mAb was reactive with both Torpedo and human AChR, but antibodies in 85 of the MG sera did not bind to Torpedo AChR, and (2) the mAb blocked binding of rat anti-peptide antibodies to an alpha subunit region of the human AChR unrelated antigenically to the designated MIR region. Individual patients' sera had evidence of extensive antibody heterogeneity and revealed interspecies polymorphisms in AChR antigenicity, near and remote from the neurotransmitter-binding region. The data challenge the concept that a MIR of the AChR is the principal stimulus for antibody production in MG and emphasize a potential pitfall in assuming seronegativity in MG on the basis of a single assay system.     

Altered cellular distribution and subcellular sorting of gamma-tubulin in diffuse astrocytic gliomas and human glioblastoma cell lines

Centrosome amplification is a pivotal mechanism underlying tumorigenesis but its role in gliomas is underinvestigated. The present study specifically examines the expression and distribution of the centrosome-associated cytoskeletal protein gamma-tubulin in 56 primary diffuse astrocytic gliomas (grades II-IV) and in 4 human glioblastoma cell lines (U87MG, U118MG, U138MG, and T98G). Monoclonal anti-peptide antibodies recognizing epitopes in C-terminal or N-terminal domains of the gamma-tubulin molecule were used in immunohistochemical, immunofluorescence, and immunoblotting studies. In tumors in adults (n = 46), varying degrees of localization were detected in all tumor grades, but immunoreactivity was significantly increased in high-grade anaplastic astrocytomas and glioblastomas multiforme as compared to low-grade diffuse astrocytomas (p = 0.0001). A similar trend was noted in diffuse gliomas in children but the sample of cases was too small as to be statistically meaningful. Two overlapping patterns of ectopic cellular localization were identified in both primary tumors and glioblastoma cell lines: A punctate pattern, in which gamma-tubulin was partially co-distributed with pericentrin in the pericentriolar region, and a diffuse pattern, independent of pericentrin staining, denoting a soluble pool of gamma-tubulin. Cellular gamma-tubulin was detected in both soluble and insoluble (nocodazole-resistant) fractions of glioblastoma cells. Divergent localizations of gamma-tubulin and pericentrin suggest a differential distribution of these 2 centrosome-associated proteins in glioblastoma cell lines. Our results indicate that overexpression and ectopic cellular distribution of gamma-tubulin in astrocytic gliomas may be significant in the context of centrosome protein amplification and may be linked to tumor progression and anaplastic potential.     

Antibodies against neuronal nicotinic receptor subtypes in neurological disorders

Patients with myasthenia gravis (MG) have antibodies to the muscle nicotinic acetylcholine receptor (mAChR) which are responsible for their muscle weakness: but some patients with MG and other neuroimmunological disorders have autonomic symptoms. We characterised the neuronal forms of AChRs (nAChRs) into two neuroblastoma cell lines and developed immunoprecipitation assays to test for antibodies to the alpha7- and alpha3-containing nAChR subtypes, present in the autonomic ganglia. We then tested 70 sera samples from MG patients, 38 from subjects with other neurological diseases, and 30 from healthy individuals, for antibodies to these two forms of neuronal AChR subtypes. We used the alpha7 subtype extracted from the human neuroblastoma IMR32 cell line labeled with 125IalphaBungarotoxin (alphaBgtx), and the alpha3-containing subtype extracted from the human neuroblastoma SY5Y cell line labeled with 3H-Epibatidine (Epi). Nine subjects (five MG, one GBS, one CIPD and two LEMS) were positive for the alpha7 subtype; and four for the alpha3-containing subtype (two MG patients, one LEMS and the same GBS patient). None of the MG patients with undetectable levels of antibodies against muscle AChR were positive. The patients with serum antibodies to alpha7 or alpha3-containing neuronal AChRs showed a range of clinical features including autonomic symptoms and thymoma in two MG patients. These results indicate that patients with MG and other immune-mediated disorders can have antibodies to neuronal AChRs, and that these may contribute to the clinical characteristics of the diseases.     

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.     

重症肌无力患者血清Titin抗体和AchR抗体检测的意义

目的 探讨重症肌无力(MG)患者血清Titin抗体及乙酰胆碱受体(AchR)抗体的检测意义.方法 采用酶联免疫吸附试验(ELISA)对81例MG患者和80例对照组成员进行Titin和AchR抗体的检测.结果 Titin抗体对MG具有特异性,它在合并胸腺瘤的MG(MGT)、晚发型MG患者中有较高的阳性率(分别为80%、69.4%)和抗体水平,明显高于早发型MG患者的阳性率(25%)和抗体水平,差异均有统计学意义(P<0.05).而早发型MG患者中AchR抗体阳性率比晚发型MG患者明显增高.并且早发型MG患者的AchR抗体水平也明显高于晚发型MG和MGT患者,差异均有统计学意义(P<0.05).结论 两种抗体检测为MG诊断和病因学研究提供了重要证据,联合检测可以提高MG诊断的灵敏度.     

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.     

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     

Coexistence of primary sclerosing cholangitis in a patient with myasthenia gravis

Myasthenia gravis (MG) is an immune-mediated disease that compromises the postsynaptic membrane of the neuromuscular junction. Primary sclerosing cholangitis (PSC) is considered an immune-mediated cholestatic liver disease. Both MG and PSC include an autoimmune pathogenesis, so there is some evidence that patients with MG or PSC have a higher risk of developing autoantibodies and other immune disorders than normal controls, but the coexistence of these two disorders has never been documented. We report a 40-year-old woman who presented with MG when she was 20 years old and developed PSC 20 years after a thymectomy. Liver biochemistry revealed cholestasis. Magnetic resonance imaging showed multifocal strictures and beads involving the intrahepatic bile ducts. A liver biopsy confirmed sclerosing cholangitis. Serological analysis demonstrated positive autoantibodies (Anti-nuclear antibodies, anti-smooth muscle antibodies). Repetitive stimulation had a decremental response, and antibodies to acetylcholine receptors were detectable. To our knowledge, this is the first case of PSC in a patient with MG. The main characteristics of both MG and PSC combination are discussed.     

IgG from "seronegative" myasthenia gravis patients binds to a muscle cell line, TE671, but not to human acetylcholine receptor

Antibodies to acetylcholine receptor (AChR) are found in 85% of patients with myasthenia gravis (seropositive MG [SPMG]) and are thought to be pathogenic; but in 15% of MG patients, the standard immunoprecipitation test for anti-AChR is negative (seronegative MG [SNMG]). Here, we used a novel approach, fluorescence-activated cell sorting analysis, to measure binding of SPMG and SNMG IgG antibodies to rhabdomyosarcoma cell lines that express human adult (TE671-epsilon) or fetal (TE671-gamma) AChR, and to human embryonic kidney (HEK) fibroblasts that express adult human AChR (HEK-AChR). We found that whereas most SPMG antibodies bind to all three cell lines, IgG from 8 of 15 SNMG sera/plasmas bind to the surface of both TE671 cell lines but not to HEK-AChR cells. These results indicate that SNMG antibodies bind to a muscle surface antigen that is not the AChR, which strongly supports previous studies that suggest that SNMG should be considered a distinct subtype of MG.     

Myasthenia gravis and pregnancy: clinical course and management of delivery and the postpartum phase

OBJECTIVES: To study influences of pregnancy on the time-course of myasthenia gravis (MG) and of MG on pregnancy, delivery, postpartum and newborn. METHODS: We retrospectively collected data from 100 women affected with MG, hospitalized between 1994 and 2003 in departments of Neurology of Lille University Hospital. RESULTS: Eighteen patients had a total of 36 pregnancies, occurring 7.2 years on average after MG onset. MG exacerbation occurred in 7 patients (26 percent) during pregnancy and in 4 (14.8 percent) during postpartum. One patient died of acute respiratory failure during postpartum. Delay between the onset of MG and pregnancy was the only variable significantly associated with MG exacerbation: 5.8 years when exacerbation and 9.5 years when no exacerbation (p=0.03). Seven miscarriages, two therapeutic abortions and no death at birth were reported. Levels of anti-acetylcholine receptor antibodies were abnormal in 3 of 27 newborns (11 percent), but only one (3.7 percent) developed seronegative transient neonatal myasthenia gravis. DISCUSSION: During pregnancy, the clinical course of MG is variable but exacerbations were associated with a shorter delay between MG diagnosis and pregnancy. The risk of transient neonatal myasthenia gravis is relatively small but exists even when the parturient has stable MG without elevated levels of anti-acetylcholine receptor antibodies. CONCLUSION: Our study confirms pregnancy is more difficult to manage at the beginning of MG. Given the unpredictable course of MG during pregnancy, we recommend women affected with MG to begin a pregnancy when the disease is stable.     

Anti-presynaptic membrane receptor antibodies in myasthenia gravis

Myasthenia gravis (MG) is considered as an autoimmune disease of neuromuscular junction resulting from antibodies directed to acetylcholine receptors (AChR). We describe the use of beta-bungarotoxin (beta-BuTx) and alpha-bungarotoxin (alpha-BuTx) to capture their corresponding proteins from preparation of crude human muscle receptor. beta-BuTx binds to presynaptic membrane receptor (PsmR) of the whole receptor fraction, while alpha-BuTx binds to AchR. The captured proteins were used as antigen in ELISA to detect antibodies to PsmR and to AchR in sera from 82 Chinese patients with MG and in controls. In MG, antibodies to PsmR only were detected in 13%, to AchR only in 11% and both to PsmR and AchR in 54%. Only 3 of 50 patients with other neurological diseases and none of 50 healthy subjects had these antibodies. Specificity tests for antibodies showed that the detection systems which we used are specific and confident. No correlation was found between antibody levels and clinical status. The significance of the PsmR antibodies in the pathogenesis of MG is unknown. We suggest that myasthenia gravis is not only due to damage of the postsynaptic membrane, but of presynaptic structures as well.     

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.     

The features of myasthenia gravis with autoantibodies to MuSK

OBJECTIVES: To determine if myasthenia gravis (MG) with antibodies to MuSK is a distinct subgroup of seronegative MG. METHODS: We assayed antibodies to muscle specific tyrosine kinase (MuSK) in 55 MG patients who had no antibodies to acetylcholine receptors and looked for the specific phenotype, comparing clinical features of anti-MuSK positive and anti-MuSK negative MG patients. RESULTS: MG with anti-MuSK antibodies was characterised by a striking prevalence of female patients (15 women, two men). Age at onset ranged from 22 to 52 years, with 70.6% of patients presenting at < 40 years of age. The majority of patients (82.4%) had prevalent involvement of facial and bulbar muscles. One third of them did not respond well to anticholinesterase drugs. Steroid immunosuppression was effective in eight patients (44.4%). Nine patients underwent thymectomy; six of these had no thymus pathology, while three had a hyperplastic thymus. At the end of the observation period, six (35.3%) patients were in remission, five (29.4%) improved, four (23.6%) did not change, and two (11.7%) had died. CONCLUSIONS: MG patients with antibodies to MuSK have characteristic clinical features that are different from features of the remaining seronegative MG patients. This emphasises the predictive value of anti-MuSK antibody analysis in seronegative MG patients.     

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。     

The high frequency and clinical feature of seronegative myasthenia gravis in Southern China

Anti-acetylcholine receptor antibodies (anti-AChR-Ab) are responsible for the failure of neuromuscular junction in myasthenia gravis (MG). Some anti-AChR-Ab-seronegative MG patients have anti-muscle-specific tyrosine kinase antibodies (anti-MuSk-Ab). Here, the anti-AChR-Ab was tested in 250 MG outpatients from Southern China. While anti-MuSk-Ab was tested in 66 patients who had no anti-AChR-Ab in blood serum, but none of them was positive. The antibodies were measured by a radioimmunoprecipitation assay. The frequency of anti-AChR-Ab was 51.2 %. The percentage of anti-AChR-Ab in ocular type was lower than generalized type (44.9 vs. 66.2 %, P = 0.002). Seronegative MG was characterized by a lower percentage of thymoma than seropositive patients (P = 0.013). It seemed to be less severe in seronegative MG than seropositive MG in these 250 patients. In ocular type, seronegative MG mainly manifesting blepharoptosis but seldom diplopia or eyeball fixation related to ocular movement disability (P = 0.016). While in generalized type, seronegative MG was characterized by a lower percentage of bulbar muscle involvements than seropositive patients (P = 0.005). Logistic regression analysis revealed that bulbar weakness was affected by the existence of anti-AChR antibodies (OR = 3.524, P = 0.015). Besides, seronegative MG tended to be characterized by a lower percentage of neck extensor involvement, but this did not reach significance. The percentage of anti-AChR antibodies was much lower than other countries. Seronegative MG has characteristic clinical features that are different from features of the remaining seropositive MG. This emphasises the predictive value of anti-AChR antibodies analysis in MG patients.     

Acetylcholine receptors loss and postsynaptic damage in MuSK antibody-positive myasthenia gravis

Muscle-specific tyrosine kinase (MuSK) antibodies are found in some patients with "seronegative" myasthenia gravis (MG), but how they cause myasthenic symptoms is not clear. We visualized acetylcholine receptors (AChRs) and complement component 3 (C3) in muscle biopsies from 10 Japanese MG patients with MuSK antibodies, compared with 42 with AChR antibodies. The AChR density was not significantly decreased in MuSK antibody (Ab)-positive end-plates compared with AChR antibody-positive end-plates, and C3 was detected in only two of eight MuSK Ab-positive patients. MuSK antibodies do not appear to cause substantial AChR loss, complement deposition, or morphological damage. Effects on MuSK function need to be explored.     

Is the decremental pattern in Lambert–Eaton syndrome different from that in myasthenia gravis?

ObjectiveWe reviewed our experience to determine if the decremental pattern during low frequency repetitive nerve stimulation (LF-RNS) distinguishes between the Lambert–Eaton myasthenic syndrome (LEMS) and myasthenia gravis (MG).MethodsLF-RNS studies were reviewed from 34 LEMS and 44 MG patients, 4 of whom had antibodies to muscle specific kinase (MuSK). In each train we calculated the ratio between the early and the later decrement. Receiver-operator characteristic curves were calculated to determine the ratio that best distinguished between LEMS and MG.ResultsThe late decrement was more often greater in LEMS and the converse was true in MG, but with some overlap in values in individual patients. A late decrement more than 102% of the early decrement discriminated between LEMS and MG in 90% of studies. The decremental pattern in MG patients with MuSK antibodies resembled that in LEMS.ConclusionWhen the decrement becomes progressively greater during low frequency RNS, the patient is more likely to have LEMS than MG, and in MG, is more likely to have MuSK antibodies.SignificanceA progressive decrement in patients otherwise felt to have MG should prompt further clinical, serological and electrodiagnostic tests. Further studies are needed to assess the decremental pattern in MuSK MG.     

In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood mononuclear cells of patients with myasthenia gravis

We studied the in vitro synthesis of antibodies to acetylcholine receptor (anti-AChR) by peripheral blood mononuclear cells (PBM) of patients with myasthenia gravis (MG) and normal subjects (NS). PBM from three of eight patients with generalized MG (MG-G) synthesized anti-AChR in vitro in the absence of pokeweed mitogen (PWM), and seven of eight did so in the presence of PWM. In individual subjects with MG-G, the levels of anti-AChR secreted in vitro by PBM correlated with serum anti-AChR antibody levels (r = 0.77) but not with the amount of IgG secreted in vitro (r = 0.44). No anti-AChR secretion was seen in culture of PBM from a patient with ocular MG, a patient with thymoma without MG, or six NS.     

Immunochemical characterization of antibodies to the myelin proteolipid protein (PLP)

The immunochemical specificity of antibodies raised against the bovine myelin proteolipid protein (PLP) and a series of PLP synthetic peptides was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. Polyclonal rabbit anti-bovine PLP antibodies were cross-reactive with PLP isolated from rat, monkey, and human CNS white matter, as well as with PLP incorporated into rat myelin vesicles. Immunochemical analyses of 11 anti-peptide antisera revealed a more restrictive cross-reactivity pattern with only five of the 11 antisera, against peptides encompassing residues 48-59, 97-105, 183-193, 192-200 and 264-276, cross-reacting with bovine PLP by ELISA. Immunoblot analyses with the anti-peptide antisera demonstrated a similar recognition with four of the 11 antisera against peptides 48-59, 97-105, 192-200 and 264-276 recognizing the myelin PLP. These data suggest that, despite its strong phylogenetic conservation, multiple antigenic sites exist within the PLP molecule, and some of these determinants can be mimicked by synthetic peptides. However, the restrictive cross-reactivities of the anti-peptide antisera suggest that humoral recognition of the myelin PLP is conformationally dependent. The availability of anti-peptide antibodies of predetermined specificity, capable of recognizing the intact protein, should permit detailed examination of PLP topography in various experimental systems.     

Effect of sera from AChR-antibody negative myasthenia gravis patients on AChR and MuSK in cell cultures

A proportion of patients with myasthenia gravis (MG) do not have antibodies to the acetylcholine receptor (AChR). Some of these patients have antibodies to muscle specific kinase (MuSK), whereas others have neither antibody (seronegative MG, SNMG). Both MuSK antibody positive MG (MuSK-MG) and SNMG are antibody-mediated diseases but how they cause neuromuscular junction failure is not clear. One possibility is that they reduce the clustering and expression of AChRs. We looked at the effects of MuSK-MG and SNMG sera/IgG on surface AChR distribution and expression, and AChR subunit and MuSK mRNA by quantitative RT-PCR, in TE671 and C2C12 myotubes. In TE671 cells MuSK-MG sera reduced AChR expression by about 20%, but had no effect on AChR subunit or MuSK mRNA expression. In C2C12 myotubes, MuSK-MG sera caused a reduction in the number of agrin-induced clusters, but the clusters became larger and there was no significant effect on total surface AChR numbers or AChR subunit or MuSK mRNA. By contrast, SNMG sera not only reduced AChR numbers by about 20% in TE671 cells, but modestly upregulated AChR gamma subunit expression in TE671 cells and both AChR gamma subunit and MuSK expression in C2C12 myotubes. Thus, although these results have, disappointingly, demonstrated little effect of MuSK antibodies on AChR expression, they do imply that SNMG antibodies act on AChR-associated pathways.