Differences between ocular and generalized myasthenia gravis: binding characteristics of anti-acetylcholine receptor antibody against bovine muscles

We studied the binding characteristics of anti-acetylcholine receptor antibody (AChR Ab) in sera of nine patients with myasthenia gravis (MG) using affinity-purified extraocular muscles (EOM) and foot muscles (FM) of bovine species. The titer of AChR Ab measured with EOM was the same as that measured with FM for both ocular and generalized MG. In patients with ocular MG, the affinity of AChR Ab, determined by Scatchard analysis, was higher for FM than for EOM (P less than 0.05), whereas it was the same for EOM and FM in those with generalized MG. On the other hand, the affinity of AChR Ab for FM was lower in generalized MG than in ocular MG (P less than 0.05), but that for EOM did not differ between the two types of MG. The affinity of AChR Ab did not change after passing the patients' sera through an EOM-affinity column. The EOM-affinity column treatment of the sera decreased the AChR Ab titer measured with EOM in all patients, but the AChR Ab titer measured with FM was decreased in only some of the patients. These data indicate that there are polyclonal or heterogeneous AChR Abs in the sera of MG patients, which does not help to explain the clinical difference between ocular and generalized MG.     

Anti-alkaline phosphatase antibody positive myasthenia gravis

Anti-alkaline phosphatase antibody (AP Ab) was specific in 9% of 249 anti-acetylcholine receptor (AChR) Ab-positive myasthenia gravis (MG) (SPMG) patients but not in patients with AChR Ab-negative MG (SNMG), other neurological and immunological diseases, or healthy volunteers. No cross-reactivity and no significant titer correlation were found between AP Ab and AChR Ab. We confirmed immunologically by radioimmunoassay and western blot analysis the presence of antibodies directed against AP. AP Ab-positive SPMG patients were characterized clinically as having female predominance and a more severe form of generalized MG than AP Ab-negative SPMG patients, and about half required artificial ventilation at maximum severity. AP Ab's pathogenic role in MG is yet unclarified, but our findings show AP to be a novel antigen among the various autoantigens present in MG patients and in whom AP Ab may modify clinical symptoms.     

AChR myasthenia gravis switching to MuSK or double antibody positive myasthenia gravis in two children and literature review

Muscle–specific tyrosine kinase antibody (MuSK–Ab) and acetylcholine receptor antibody (AChR–Ab) coexistence in myasthenia gravis (MG) is very rare. In this report, two children with AChR–Ab switching to double antibody positive MG (DP–MG) or MuSK–Ab positive MG (MuSK–MG) are described. Six similar cases were found in the literature via online database search. Therefore, this study describes eight patients in total, six female and two male. The average age of onset was 7.25 ± 5.95 years. Four AChR–MG patients switched to DP–MG with no known precipitating factor and four switched after thymectomy (two to MuSK–MG and two to DP–MG). After the serological switch, the patients transitioned to the phenotype of MuSK–MG and responded poorly to cholinesterase inhibitors and well to corticosteroids and plasma exchange.     

Antibodies against the main immunogenic region of the acetylcholine receptor correlate with disease severity in myasthenia gravis

Objective We developed an assay that detects autoantibodies against the main immunogenic region (MIR) located at the extracellular end of the nicotinic acetylcholine receptor (AChR) α subunit, and investigated its clinical relevance in myasthenia gravis (MG). Methods In this retrospective cohort study, we measured MIR antibody (Ab) titres in sera obtained before treatment and analysed their associations with clinical parameters in 102 MG patients from two neurological centres. MIR Ab titres were determined using a modified competition immunoprecipitation assay in the presence or absence of monoclonal antibody 35. Results 11 of 23 (47.8%) ocular type and 66 of 72 (91.7%) generalised type MG patients were positive for the presence of MIR Abs, defined as a titre >16.8% (3 SDs above the mean for 70 healthy controls). A significantly higher MIR Ab titre (p<0.001) was shown in generalised type (47.9±19.2%) rather than in ocular type MG patients (16.4±8.4%). Bivariate regression analysis using both titre levels of MIR Ab and routine AChR binding Ab as variables revealed MIR Abs to be an exclusive indicator positively associated with disease severity (Myasthenia Gravis Foundation of America classification, p<0.0001; Quantitative MG score, p=0.008), the presence of bulbar symptoms (p<0.0001) and thymoma (p=0.016), and negatively associated with ocular MG (p<0.0001). Conclusions MIR Ab titre levels show much better correlations with factors related to disease severity compared with AChR binding Ab titres. The MIR Ab assay may be useful for predicting MG symptom severity, especially for discriminating between ocular and generalised types of MG.     

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.     

Passive transfer of myasthenia gravis by immunoglobulins: lack of correlation between AChR with antibody bound, acetylcholine receptor loss and transmission defect

The effects of serum Ig from 7 myasthenia gravis patients on neuromuscular transmission was investigated by passive transfer to mice. A protocol of 60 mg/day for 3 days produced mouse serum levels of anti-mouse AChR that were similar to those in the MG patients, and resulted in corresponding levels (2-76%) of mouse muscle AChR with antibody bound in situ. However, AChR loss was only greater than 20% with one MG preparation. Nevertheless, there was a marked neuromuscular defect in mice injected with 3 preparations which did not necessarily correlate with the degree of AChR loss or the amount of AChRs with antibody bound in situ. We conclude that in some MG patients part of the defect in neuromuscular transmission may result from antibodies binding to other components of the neuromuscular junction.     

B‐cell–activating factor is elevated in serum of patients with myasthenia gravis

Introduction: Myasthenia gravis (MG) is a B‐cell–mediated autoimmune disease. B‐cell–activating factor (BAFF) is a major factor in B‐cell development and activation. In this study we investigated serum BAFF levels in MG patients. Methods: We compared the serum BAFF levels of 20 MG patients with gender‐matched healthy controls. We assayed serum concentrations of BAFF and anti‐acetylcholine receptor antibody (AChR) titers. Results: Serum BAFF levels of MG patients with AChR antibodies were significantly higher than those of healthy controls. A significant positive correlation was observed between serum BAFF levels and anti‐AChR antibody titers. BAFF values did not correlate with disease severity. Conclusions: BAFF may play a major role in the pathogenesis of MG, and it may provide a potential target for therapy in patients with MG. Muscle Nerve 54 : 1030–1033, 2016     

Involvement of HLA in clinical courses of myasthenia gravis

The relationship between the histocompatibility leukocyte antigen (HLA) phenotypes and the clinical course of myasthenia gravis (MG) was studied in 53 Japanese patients with MG. The frequency of HLA-DRw9 antigen was high in the MG patients who did not need immunosuppressive therapy but only anticholinesterase agents (RR = 4.52; CP less than 0.02), who achieved remission of the disease (RR = 2.98; CP less than 0.05) or who showed a decrease in AChR antibody (Ab) titer (RR = 6.32; CP less than 0.0002), whereas the frequency of HLA-DRw8 antigen was increased in MG patients who underwent immunosuppressive therapy (RR = 4.03; CP less than 0.01), who did not have remission (RR = 4.75; CP less than 0.1) or who showed an increase in AChR Ab titer (RR = 6.48; CP less than 0.01). These data suggest that immunogenetic heterogeneity in MG might be reflected in its clinical course.     

Soluble terminal complement components in human myasthenia gravis

The loss of membrane acetylcholine receptor (AChR) leading to muscle weakness and impaired neuromuscular junction (NMJ) transmission in human myasthenia gravis (MG) is in part due to complement mediated muscle membrane damage. This has been supported by the histologic finding of C9 at the NMJ in human MG. We evaluated for evidence of terminal complement components in plasma by using an ELISA for SC5b-9 in 42 separate plasma samples from 31 patients with MG and from healthy controls. Abnormal elevations of SC5b-9 was found in 18 of 31 patients (58%) at one or more time points when plotted on a standard positive dilution curve. Multiple samples were available from 8 patients over time. Clinical deterioration in some, but not all, was accompanied by an increase in SC5b-9 values. There was no clear distinction in the group as a whole between MG severity or AChR antibody levels and SC5b-9 values. This supports the potential role of complement-mediated muscle membrane damage in the pathogenesis of human MG, but also demonstrates that plasma levels as measured by ELISA do not always correlate with disease activity.     

Effect of thymectomy and immunosuppressive therapy on anti-neuroblastoma antibody levels in patients with myasthenia gravis

Antibodies reacting with human neuroblastoma cells (NBL) are distinct from the "classical" anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG). The influence of therapeutic interventions on serum anti-NBL antibody levels was followed in 42 MG patients. Thymectomy alone was performed in 28 patients while immunosuppressive medication was given to 14 patients out of whom 10 also had a thymectomy. In most patients serum anti-NBL antibody titers declined after thymectomy and/or during immunosuppressive treatment, though individual variations in the antibody response could be observed. Sequential examinations of individual patients revealed an association between the clinical severity of MG and anti-NBL antibody levels. No correlation between the treatment-induced changes of anti-NBL and anti-acetylcholine receptor (AChR) antibody titers could be observed during the follow-up period in MG patients positive for both types of antibodies. These findings further emphasize the immunological complexity of MG. Anti-NBL antibodies represent a pathogenic marker of the disease and display a regulation different from that of the anti-AChR antibodies.     

Myasthenia in SCID mice grafted with myasthenic patient lymphocytes: role of CD4+ and CD8+ cells

OBJECTIVES: Acetylcholine receptor (AChR)-specific CD4+ cells are present in MG patients, and synthesis of the high-affinity immunoglobulin G (IgG) autoantibodies (autoAb) against the muscle AChR that causes MG symptoms requires intervention of CD4+ cells. The role of CD4+ cells in MG pathogenesis has been postulated but never proven. MG patients do not have anti-AChR cytotoxic phenomena, and it has been assumed that CD8+ cells do not have a pathogenic role in MG. However, CD8+ cells may facilitate rodent experimental MG, raising the possibility that CD8+ cells might be necessary also in MG. In this study we examined whether CD4+ and CD8+ cells play a role in the pathogenesis of MG and whether CD4+ cells specific for AChR epitope sequences recognized by most MG patients ("universal" epitopes) drive the synthesis of pathogenic antibodies. METHODS: First we characterized a chimeric human-mouse model of MG in severe combined immunodeficiency (SCID) mice engrafted with blood lymphocytes (BL) from MG patients. We used that model to determine whether CD4+ and CD8+ cells are necessary for transfer of MG symptoms. We engrafted SCID mice intraperitoneum with BL from 19 MG patients and 5 healthy controls. We engrafted some mice with either BL, BL depleted in CD4+ or CD8+ cells from the same patient, or CD4+ depleted BL reconstituted with CD4+ T cells from the same patient, specific for "universal" AChR epitopes or for two unrelated antigens, tetanus and diphtheria toxoids. We tested the mice for myasthenic symptoms for 7 to 18 weeks. RESULTS: Mice transplanted with BL, or CD8+ depleted BL, or CD4+-depleted BL reconstituted with anti-AChR CD4+ cells from MG patients frequently developed myasthenic weakness. The mice had human anti-AChR Ab in the serum and bound to muscle AChR. Mice transplanted with BL from controls, or CD4+-depleted BL from MG patients, or CD4+-depleted BL from an MG patient reconstituted with CD4+ cells specific for tetanus or diphtheria toxoids did not develop myasthenic weakness or anti-AChR Ab. CONCLUSIONS: CD4+ cells are necessary for MG pathogenesis; CD8+ cells may not be. CD4+ cells specific for "universal" AChR epitopes help the synthesis of pathogenic Ab.     

Does change in acetylcholine receptor antibody level correlate with clinical change in myasthenia gravis?

Introduction: The objective of this study is to determine if change in acetylcholine receptor antibody (AChR‐ab) levels reflects change in clinical severity in patients with myasthenia gravis (MG). Methods: We reviewed results from a prospective trial in MG and from all 85 patients in an MG Clinic who had AChR‐ab determinations performed at least twice by the same commercial laboratory. Results: Change in AChR‐ab levels correlated only weakly with change in clinical severity. AChR‐ab levels fell in 92% of patients who improved and in 63% who did not. A fall in AChR‐ab level had a positive predictive value for clinical improvement of 83% and a negative predictive value of only 59%. Conclusions: AChR‐ab levels fell in almost all patients who improved, but also in most patients who did not. Thus, we do not recommend commercially available AChR‐ab levels as a biomarker of improvement in MG. However, antibody levels might be useful as a marker for inadequate immunotherapy. Muscle Nerve 49:483–486, 2014     

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.     

Repetitive nerve stimulation of facial muscles in MuSK antibody-positive myasthenia gravis

To better define electrophysiological abnormalities in myasthenia gravis (MG) patients with muscle-specific tyrosine kinase (MuSK) antibodies (Ab), we compared electrophysiological features of 14 MuSK Ab-positive, 73 acetylcholine receptor antibody (AChR Ab)-positive, and 22 MuSK and AChR Ab-negative (seronegative) patients with generalized disease. Repetitive nerve stimulation (RNS) abnormalities were observed in 86% of MuSK Ab-positive and 82% of AChR Ab-positive patients but in only 55% of seronegative patients. RNS decrements in the orbicularis oculi were more common and severe in the MuSK Ab-positive patients than the other two groups. Single-fiber electromyography (SFEMG) of the extensor digitorum communis was abnormal in 90% of MuSK Ab-positive patients. The high frequency of RNS abnormalities in facial muscles in the MuSK Ab-positive population reflects the propensity for facial muscle involvement in this form of MG and emphasizes the importance of including facial muscles in RNS protocols when evaluating these patients.     

Anti-MuSK antibody myasthenia gravis: clinical findings and response to treatment in two large cohorts

Introduction: Myasthenia gravis (MG) patients with autoantibodies to muscle‐specific tyrosine kinase (MuSK) represent a distinct subset of those with this disease. Treatment and outcomes data in these patients are limited and conflicting. Methods: We reviewed 110 MuSK‐MG patients from two large clinics in Italy and the USA. Results: Thirty‐nine to 49% of patients with generalized, acetylcholine receptor antibody (AChR‐Ab)–negative MG had MuSK‐MG. Eighty‐five percent were female, with disease onset typically in the fourth decade. Ocular and/or bulbar symptoms were present at onset in 79% of those studied. Eighty‐five percent were MGFA class III or greater, and crisis occurred in 28%. Plasma exchange (PLEX) produced improvement in 93%, whereas only 61% improved after intravenous immunoglobulin. Long‐term outcomes were comparable to those of patients with AChR‐Ab–positive MG. Conclusions: MuSK‐MG has a marked female predominance with frequent oculobulbar weakness and crises. Many patients deteriorate rapidly early in the disease, and PLEX is usually the preferred treatment. Long‐term outcomes are similar to those of patients with AChR‐Ab⁺ MG. Muscle Nerve 44: 36–40, 2011     

Multiple forms of anti-acetylcholine receptor antibody in myasthenia gravis

Sera of patients with myasthenia gravis (MG) contain anti-acetylcholine receptor (AChR) lgG antibodies (Ab) which have different antigenic specificities. Three Ab types were detected: (1) MG-I, which forms immune complexes with AChR; (2) MG-C, which decreases binding of AChR to concanavalin A; and MG-B, which blocks α-bungarotoxin binding to AChR. Sera from 152 MG patients were screened for the Ab types. Sixty-one percent contained MG-I, 26% contained MG-C, 10% contained MG-B, and 5% contained both MG-C and MG-B. The latter Ab types were associated with more severe forms of MG but showed no other clinical correlations. IgG antibodies of defined type were purified, and their interaction with unlabeled and toxin-prelabeled AChR from denervated rat muscle was studied in detail. Receptors are homogeneous with respect to determinants recognized by MG-I, but heterogeneous with respect to determinants recognized by MG-C (3 subpopulations, 22%, 28%, and 50% of AChR) and by MG-B (2 subpopulations, 30% and 70% of AChR). The stoichiometry of AChR interaction with the antibodies indicates that for each toxin-binding site, the receptor is divalent as an antigen for MG-I and MG-C but is tetravalent for MG-B. Denervated muscle AChR appears to be a mixture of at least 3 molecular forms of AChR, each of which has distinct immunological features as well as components common to all the receptor subpopulations.     

Mortality in myasthenia gravis: A nationwide population–based follow‐up study in Denmark

Introduction: In previous studies of myasthenia gravis (MG), increased mortality has been reported. The aim of this study was to estimate mortality in patients with acetylcholine receptor antibody–positive (AChR‐Ab–seropositive) MG in a nationwide population–based, long‐term follow‐up study. Methods: All AChR‐Ab–seropositive MG patients, diagnosed between 1985 and 2005, were identified. Defined by age at diagnosis (≤50 or >50 years), patients were classified as having early‐ or late‐onset MG. For comparison, 10 non‐MG individuals from the general population were matched with each patient. All patients and controls were followed until January 1, 2009. Mortality rates and estimated mortality rate ratios (MRRs) were calculated. Results: Of 702 AChR‐Ab–seropositive MG patients, 302 died during follow‐up. Overall mortality was higher for patients with MG (MRR = 1.41, range 1.24–1.60). In late‐onset women and men, the MRRs were 1.64 (1.36–1.99) and 1.22 (1.02–1.46), respectively. Total MRR was highest during the first 5 years after diagnosis. Conclusions: MG diagnosis is still associated with increased mortality. Muscle Nerve 53 : 73–77, 2016     

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.     

Immunogenetic heterogeneity and associated autoimmune disorders in myasthenia gravis: a population-based survey in the province of Ferrara, northern Italy

Introduction - The well-established relationship between myasthenia gravis (MG) and HLA antigens varies among different ethnic groups. In Caucasians B8 and/or DR3 alleles have been found associated with young MG women without thymoma and with high titres of acetylcholine-receptor antibody (AChR Ab). An increased frequency of haplotype HLA-A3, B7 and/or DR2 has been observed in older MG patients with low AChR Ab levels. So far, there is no convincing evidence for an association between a specific haplotype HLA and ocular MG or MG with thymoma. MG subjects often show other concurrent autoimmune disorders suggesting a more general inherited predisposition to autoimmunity. We performed a community-based study to verify the HLA-A, B, C, DR and DQ profile on ethnically homogeneous MG patients and with the aim to estimate the frequency of concurrent autoimmune diseases and to compare HLA phenotypes to autoimmune status in different MG patients groups. Methods - Forty-seven patients, living in the province of Ferrara, were followed-up in our neurologic department and typed for HLA Antigens. In addition a set of immunological laboratory tests was performed. Results - We found a trend towards an increased B8 and DR3 frequencies in total affected population; an association between B8 allele and early onset of generalized MG sustained by thymic hyperplasia. The DR3 allele is statistically associated with the presence of additional autoimmune disorders. Conclusions - Our data support the hypothesis of a genetically-based heterogeneity of the disease and show an increased prevalence of associate autoimmune conditions in MG patients.     

眼肌型重症肌无力临床分析

目的总结分析眼肌型重症肌无力患者的临床特征,以为诊断和治疗提供参考依据。方法回顾性分析113例眼肌型重症肌无力患者的临床资料。采用免疫荧光细胞染色方法检测血清乙酰胆碱受体(AChR)抗体和肌肉特异性受体酪氨酸激酶(MuSK)抗体表达水平,分析这两项免疫学指标对眼肌型重症肌无力向全身型转化的预测价值。结果成年发病的眼肌型重症肌无力好发于40岁以上男性,多以眼睑下垂(95例,84.07%)为首发症状,少数以复视(18例,1 5.93%)起病。疲劳试验和新斯的明试验阳性率分别为79.44%(85/107)和84.85%(84/99),低频重复神经电刺激和血清甲状腺抗体异常率分别为44.32%(39/88)和28%(14/50),胸腺增生和胸腺瘤阳性率分别为16.67%(17/102)和11.76%(12/102);血清AChR抗体阳性率为62.83%(71/113);但MuSK抗体均呈阴性。眼肌型重症肌无力向全身型转化率为12.39%(14/113),其中血清AChR抗体强阳性者(13例,28.26%)显著高于弱阳性者(1例,4%),二者差异有统计学意义(X~2=4.587,P=0.032)。结论成年发病的眼肌型重症肌无力好发于中年以上男性,主要表现为眼睑下垂和复视,大多数患者伴发胸腺和甲状腺异常。血清AChR抗体表达水平升高预示向全身型转化率升高,鲜有MuSK抗体阳性反应。