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Background: The Thymectomy Trial in Non-Thymomatous Myasthenia Gravis Patients Receiving Prednisone (MGTX) showed that thymectomy combined with prednisone was superior to prednisone alone in improving clinical status as measured by the Quantitative Myasthenia Gravis (QMG) score in patients with generalised non-thymomatous myasthenia gravis at 3 years. We investigated the long-term effects of thymectomy up to 5 years on clinical status, medication requirements, and adverse events., Methods: We did a rater-blinded 2-year extension study at 36 centres in 15 countries for all patients who completed the randomised controlled MGTX and were willing to participate. MGTX patients were aged 18 to 65 years at enrolment, had generalised non-thymomatous myasthenia gravis of less than 5 years' duration, had acetylcholine receptor antibody titres of 1·00 nmol/L or higher (or concentrations of 0·50-0·99 nmol/L if diagnosis was confirmed by positive edrophonium or abnormal repetitive nerve stimulation, or abnormal single fibre electromyography), had Myasthenia Gravis Foundation of America Clinical Classification Class II-IV disease, and were on optimal anticholinesterase therapy with or without oral corticosteroids. In MGTX, patients were randomly assigned (1:1) to either thymectomy plus prednisone or prednisone alone. All patients in both groups received oral prednisone at doses titrated up to 100 mg on alternate days until they achieved minimal manifestation status. The primary endpoints of the extension phase were the time-weighted means of the QMG score and alternate-day prednisone dose from month 0 to month 60. Analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00294658. It is closed to new participants, with follow-up completed., Findings: Of the 111 patients who completed the 3-year MGTX, 68 (61%) entered the extension study between Sept 1, 2009, and Aug 26, 2015 (33 in the prednisone alone group and 35 in the prednisone plus thymectomy group). 50 (74%) patients completed the 60-month assessment, 24 in the prednisone alone group and 26 in the prednisone plus thymectomy group. At 5 years, patients in the thymectomy plus prednisone group had significantly lower time-weighted mean QMG scores (5·47 [SD 3·87] vs 9·34 [5·08]; p=0·0007) and mean alternate-day prednisone doses (24 mg [SD 21] vs 48 mg [29]; p=0·0002) than did those in the prednisone alone group. 14 (42%) of 33 patients in the prednisone group, and 12 (34%) of 35 in the thymectomy plus prednisone group, had at least one adverse event by month 60. No treatment-related deaths were reported during the extension phase., Interpretation: At 5 years, thymectomy plus prednisone continues to confer benefits in patients with generalised non-thymomatous myasthenia gravis compared with prednisone alone. Although caution is appropriate when generalising our findings because of the small sample size of our study, they nevertheless provide further support for the benefits of thymectomy in patients with generalised non-thymomatous myasthenia gravis., Funding: National Institutes of Health, National Institute of Neurological Disorders and Stroke., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Thymectomy has been a mainstay in the treatment of myasthenia gravis, but there is no conclusive evidence of its benefit. We conducted a multicenter, randomized trial comparing thymectomy plus prednisone with prednisone alone., Methods: We compared extended transsternal thymectomy plus alternate-day prednisone with alternate-day prednisone alone. Patients 18 to 65 years of age who had generalized nonthymomatous myasthenia gravis with a disease duration of less than 5 years were included if they had Myasthenia Gravis Foundation of America clinical class II to IV disease (on a scale from I to V, with higher classes indicating more severe disease) and elevated circulating concentrations of acetylcholine-receptor antibody. The primary outcomes were the time-weighted average Quantitative Myasthenia Gravis score (on a scale from 0 to 39, with higher scores indicating more severe disease) over a 3-year period, as assessed by means of blinded rating, and the time-weighted average required dose of prednisone over a 3-year period., Results: A total of 126 patients underwent randomization between 2006 and 2012 at 36 sites. Patients who underwent thymectomy had a lower time-weighted average Quantitative Myasthenia Gravis score over a 3-year period than those who received prednisone alone (6.15 vs. 8.99, P<0.001); patients in the thymectomy group also had a lower average requirement for alternate-day prednisone (44 mg vs. 60 mg, P<0.001). Fewer patients in the thymectomy group than in the prednisone-only group required immunosuppression with azathioprine (17% vs. 48%, P<0.001) or were hospitalized for exacerbations (9% vs. 37%, P<0.001). The number of patients with treatment-associated complications did not differ significantly between groups (P=0.73), but patients in the thymectomy group had fewer treatment-associated symptoms related to immunosuppressive medications (P<0.001) and lower distress levels related to symptoms (P=0.003)., Conclusions: Thymectomy improved clinical outcomes over a 3-year period in patients with nonthymomatous myasthenia gravis. (Funded by the National Institute of Neurological Disorders and Stroke and others; MGTX ClinicalTrials.gov number, NCT00294658.).

In early-onset myasthenia gravis (EOMG), the thymus is colonized by lymph node-like infiltrates including T cell areas and germinal centers. Our Group(1) showed (1978) spontaneous anti-acetylcholine receptor (AChR) autoantibody production by EOMG thymic cells. Especially after enzymic dispersal, these are enriched in plasma cells that are evidently autonomous, long-lived, terminally differentiated and radio-resistant. Radiolabeled AChR is highly sensitive both for localizing them in situ and detecting their ongoing antibody production in culture at limiting cell numbers. Thus EOMG thymi are a readily available source of specific autoimmune human plasma cells suitable for studying their biology and testing new therapies.

The role of antigen expression by thymomas in myasthenia gravis (MG) is not clear. Previous reports of acetylcholine receptor (AChR) mRNA expression by the highly sensitive reverse transcription-polymerase chain reactions (RT-PCR) produced varying results. To try to clarify this issue, we first used RT-PCR but then turned to the more accurate and quantitative RNase protection assays (RPA) to assess AChR subunit mRNA expression in thymomas from 25 patients (22 with MG). By RT-PCR, all five AChR subunits could be detected in many thymomas. However, by RPA, the mRNA for the adult-specific AChR epsilon-subunit was found in 13/25 (52%) thymomas, but not mRNA for the other subunits. AChR epsilon-subunit was more frequently detected in thymomas of A or AB histology (WHO classification) than those with B1-B3 histology. Overall, 6/6 with thymomas of A or AB histology were positive compared with only 8/19 with B histology (p=0.02). Autoantibodies in the two patients with the highest levels of epsilon-subunit mRNA bound better to adult (alpha(2)betadeltaepsilon) AChR than to fetal (alpha(2)betadeltagamma) AChR, whereas the other sera bound better to fetal AChR. The greater abundance of mRNA for AChR epsilon-subunit than for other subunits suggests that the AChR epsilon-subunit may play a distinctive role in autosensitization in MG-associated thymomas, particularly those of type A or AB.

The thymus is believed to play an important role in the pathogenesis of myasthenia gravis (MG). The 80% of MG patients with anti-acetylcholine receptor autoantibodies fall into three clinical subgroups: 1) thymoma; 2) early-onset MG (

We present our experience planning and launching a multinational, NIH/NINDS funded study of thymectomy in myasthenia gravis. We highlight the additional steps required for international sites and analyze and contrast the time investment required to bring U.S. and non-U.S. sites into full regulatory compliance. Results show the mean time for non-U.S. centers to achieve regulatory approval was significantly longer (mean 13.4+/0.96 [corrected] months) than for U.S. sites (9.67+/0.74 [corrected] months; p=0.003, [corrected] t-test). The delay for non-U.S. sites was mainly attributable to Federalwide Assurance certification and State Department clearance.

Antibodies to muscle-specific kinase (MuSK) are found in a variable proportion of patients with myasthenia without typical acetylcholine receptor (AChR) antibodies, but their characteristics and pathogenic mechanisms are not fully understood. We discuss the incidence and pathogenicity of MuSK antibodies and how clinical studies, animal models, and cultured cell lines can be used to elucidate their pathogenic mechanisms. Patients without either AChR or MuSK antibodies (seronegative myasthenia) appear to present another disease subtype that is highly similar to that of typical myasthenia gravis. We demonstrate a new method that detects AChR antibodies in these patients and show that these low-affinity AChR antibodies are predominantly IgG1 and can activate complement C3b deposition. Similarly MuSK antibodies, although mainly IgG4, are partially IgG1 and can activate C3b deposition. Overall, these results suggest that complement-activation may be an important pathogenic mechanism even in patients without conventional AChR antibodies.

The primary study [MGTX] aims to answer three questions: does extended transsternal thymectomy combined with the prednisone protocol, when compared with the prednisone protocol alone: (1) result in a greater improvement in myasthenic weakness, (2) result in a lower total dose of prednisone, thus decreasing the likelihood of concurrent and long-term toxic effects, (3) enhance the quality of life by reducing adverse events and symptoms associated with the therapies? Inclusion criteria are MGFA Class 2, 3, or 4; acetylcholine receptor antibody positive; age at least 18.0 years and <60.0 years; MG history of <3 years. Patients can be prednisone naïve or not. The National Institute for Neurological Disorders and Stroke awarded funding for MGTX in September 2005, and NIH awarded funding for the ancillary Biomarkers study (BioMG) in February 2006. Diverse regulatory obstacles have been encountered in this international study, but we now have a total of over 70 centers in 22 countries (North America, South America, Europe, Australasia, South Africa) either actively recruiting or at various levels of readiness.

Research advances over the last 30 years have shown that key transmembrane proteins at the neuromuscular junction are vulnerable to antibody-mediated autoimmune attack These targets are acetylcholine receptors (AChRs) and muscle specific kinase (MuSK) in myasthenia gravis, voltage-gated calcium channels (VGCCs) in the Lambert-Eaton myasthenic syndrome (LEMS), and voltage-gated potassium channels (VGKCs) in neuromyotonia. In parallel with these immunological advances, mutations identified in genes encoding pre-synaptic, synaptic and postsynaptic proteins that are crucial to neuromuscular transmission have revealed a similar diversity of congenital myasthenic syndromes (CMS). These discoveries have had a major impact on diagnosis and management.

Objective: Our aim was to study the pathophysiological process leading to facial muscle atrophy in 13 patients with MuSK antibody positive myasthenia gravis (MuSK-MG), and to compare with findings from 12 acetylcholine receptor antibody positive myasthenia patients (AChR-MG), selected because they suffered from the same degree of disease severity and required similar treatment., Methods: Motor unit action potential (MUAP) and interference pattern analysis from orbicularis oculi (O oculi) and orbicularis oris (O oris) muscles were studied using a concentric needle electrode, and compared with findings in 20 normal subjects, 6 patients receiving botulinum toxin injections (representing a neurogenic model) and 6 patients with a muscle dystrophy (representing a myopathic model). The techniques and control data have been reported previously., Results: The mean MUAP durations for O oculi and O oris were significantly reduced (p<0.001) in both MG cohorts when compared with healthy subjects, and were similar to those in the myopathic control group. They were significantly different from those obtained from the neurogenic control group (p<0.001 for both O oculi and O oris). The MUAP findings in O oculi occurred independently from neuromuscular blocking on single fibre EMG (SFEMG) in the same muscle. On turns amplitude analysis (TAA), 50% of MuSK-MG patients and 42% of AChR-MG patients had a pattern in O oculi which was similar to that in the myopathic control group, and 62% of MuSK-MG patients and 50% of AChR-MG patients had a pattern in O oris that was also similar to that in the myopathic control group. The TAA findings for O oculi and O oris in both MG cohorts were different from those obtained from the neurogenic control group., Conclusions: Facial muscle atrophy in MuSK-MG patients is not neurogenic and the pathophysiological changes are akin to a myopathic process. The selected AChR-MG patients also show evidence of a similar pathophysiological process in the facial muscles albeit to a lesser degree., Significance: We propose that muscle atrophy in MuSK-MG is a myopathic process consisting of either muscle fibre shrinkage or loss of muscle fibres from motor units. The duration of disease and long-term steroid treatment may be further contributory factors.

A proportion of patients with myasthenia gravis (MG) without acetylcholine receptor (AChR) antibodies have antibodies to muscle-specific kinase (MuSK). MG with MuSK antibodies (MuSK-MG) is often associated with persistent bulbar involvement, including marked facial weakness and tongue muscle wasting. The extent of muscle wasting in MuSK-MG, and whether it is also found in the few acetylcholine receptor (AChR-MG) patients who have persistent bulbar involvement, is not clear. We studied 12 MuSK-MG patients and recruited 14 AChR-MG patients matched broadly for age, sex ratio, duration of disease and degree of ocular, bulbar and facial weakness. We used coronal and sagittal T1-weighted (T1W) and T2-weighted (T2W) magnetic resonance imaging (MRI) to assess muscle wasting in facial and tongue muscles. Hyperintense signal on T1W MRI and comparison of axial T1W sequences with cUTE sequences were used to assess fibrous/fatty tissue in the tongue. We compared the results with those of four patients with myotonic dystrophy and 12 healthy individuals. We correlated the changes with clinical and treatment histories, and established a new ocular-bulbar-facial-respiratory (OBFR) score. At the time of study, none of the clinical measures, including the OBFR score, differed between the two MG groups. MRI demonstrated thinning of the buccinator, orbicularis oris (O.oris) and orbicularis oculi (O.oculi) muscles in MuSK-MG patients compared with healthy controls, whereas thinning of these muscles was not significant in AChR-MG. Tongue areas with T1W high signal were increased in MuSK-MG patients and the intensity of the signal on axial T1W sequences was greater in MuSK-MG than in controls. To look for possible correlations between imaging and clinical findings, we pooled results from all MG patients. The duration of treatment with prednisolone at >40 mg on alternate days (AD) correlated positively with the percentage of tongue area with high signal (P = 0.006) and negatively with MRI measurements of individual muscles and with the mean muscle dimensions (P = 0.001). The new OBFR score correlated positively with current Myasthenia Gravis Foundation of America grades and with the percentage of high signal (P = 0.004) and negatively with the mean muscle dimensions (P < 0.001). The results show that bulbar and facial muscle weakness and wasting are associated with significant muscle atrophy and fatty replacement in MuSK-MG, which was not found in the AChR-MG patients. MuSK antibodies per se may predispose to muscle thinning, but the difficulties in obtaining clinical remission under steroid therapy in some patients, resulting in long duration of treatment with higher doses (>40 mg AD), may be an additional factor.

We examined the findings from single-fiber electromyography in extensor digitorum communis (EDC) and orbicularis oculi (OOc) in 13 myasthenia gravis (MG) patients with muscle-specific kinase antibodies (MuSK-MG) and 12 MG patients with acetylcholine receptor antibodies (AChR-MG) with similar clinical scores. More than 70% of AChR-MG patients had abnormal jitter in both EDC and OOc, but the majority of MuSK-MG patients had normal jitter in EDC despite abnormal jitter in OOc. These findings demonstrate clear differences between the neurophysiology of MuSK-MG and AChR-MG.

The authors studied acetylcholine receptor antibody (AChR Ab) in twenty-six Thai patients diagnosed as having generalized myasthenia gravis and fifteen control cases. AChR Ab assay was done by radio-immunoassay technique and reported by titer in nmole/L. The positive result was defined by titer more than 0.5 nmole/L. In the myasthenia gravis group, age ranged from 18 to 64 years old with mean of 34 years old. The female: male ratio was 4.2:1. Duration of disease before taking blood sample ranged from 1 month to 14 years with a mean of 3.9 year The AChR Ab could be detected in 21 out of 26 patients (80.7%). In the control group, tests were all negative. The results of the test made the sensitivity of 80.7% and specificity of 100%. The positive predictive value was 100%, the negative predictive value was 75%, and the prevalence was 60.3%. There was no correlation between AChR Ab titer and clinical features. This test is a very valuable test in case of uncertainly in the diagnosis of myasthenia gravis.

In generalized myasthenia gravis (MG) patients without detectable acetylcholine receptor (AChR) antibodies (SNMG), the thymus is often reported as "normally involuted." We analyzed thymic compartments in 67 patients with generalized MG, with AChR antibodies (AChR+, n = 23), with muscle-specific kinase (MuSK) antibodies (MuSK+, n = 14) or with neither (MuSK-, n = 30), and in 11 non-MG controls. Four of 14 MuSK+ thymi had rare small germinal centers, but overall they were not different from age-matched controls. However, approximately 75% MuSK- samples showed lymph node-type infiltrates similar to those in AChR+ patients, but with fewer germinal centers. These variations may explain some apparent differences in responses to thymectomy in SNMG.

Antibodies to rat muscle specific kinase, MuSK, have recently been identified in some generalized "seronegative" myasthenia gravis (SNMG) patients, who are often females with marked bulbar symptoms. Using immunoprecipitation of (125)I-labelled-human MuSK, 27 of 66 (41%) seronegative patients were positive, but 18 ocular SNMG patients, 105 AChR antibody positive MG patients, and 108 controls were negative. The antibodies are of high affinity (Kds around 100 pM) with titers between 1 and 200 nM. They bind to the extracellular Ig-like domains of soluble or native MuSK. Surprisingly they are predominantly in the IgG4 subclass. MuSK-antibody associated MG may be different in etiological and pathological mechanisms.

Some myasthenia gravis (MG) patients do not have detectable acetylcholine receptor (AChR) antibodies and have been termed "seronegative" (SNMG) in many previous studies. A high proportion of patients with purely ocular symptoms, ocular MG, are seronegative; this may be because the sensitivity of the assay is insufficient to detect low levels of circulating AChR antibodies and because of intrinsic differences in the ocular muscles that make them more susceptible to circulating factors. Seronegative generalized myasthenia is proving to be heterogeneous both clinically and immunologically. Plasma from SNMG patients often contains a factor, probably an immunoglobulin M antibody, that alters AChR function in in vitro assays, but its target is not yet clear. A variable proportion of SNMG patients have antibodies to the muscle-specific tyrosine kinase (MuSK). These antibodies are directed against the extracellular domain of MuSK and inhibit agrin-induced AChR clustering in muscle myotubes. Although the role of these antibodies in causing myasthenic symptoms in vivo has not been elucidated, MuSK antibodies appear to define a group of patients who are often female with bulbar weakness, contrasting with MuSK antibody-negative SNMG patients who are more likely to have generalized weakness. MuSK antibody-positive patients may also differ in the distribution of their electrophysiological abnormalities and their responses to treatments.

Rapsyn mutations in 16 unrelated patients with a congenital/hereditary myasthenic syndrome were identified, and a mutation (N88K) common to each of them was found. Two distinct phenotypes were noted: early and late onset. The former is frequently associated with arthrogryposis multiplex congenita and life-threatening crises. The late-onset phenotype developed in adolescence or adulthood and was initially mistaken for seronegative myasthenia gravis. Recognition of this late-onset phenotype should prevent inappropriate immunotherapy.

Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

Thymectomy has been regularly used in the management of nonthymomatous autoimmune myasthenia gravis (MG), but its benefits have not been established in a randomized, controlled trial. The widespread use of thymectomy in MG patients without thymoma is largely based on retrospective, nonrandomized case series that have produced a consensus that the procedure is sometimes beneficial. Still, the benefits and utilization of thymectomy are actively debated among MG experts. In this paper, we describe the development of a multicenter, international trial to determine whether extended transsternal thymectomy reduces corticosteroid requirements for patients with generalized AChR antibody-positive nonthymomatous MG.

In humans, interleukin-12 (IL-12) and interferon-alpha (IFN-alpha) normally favor IFN-gamma-producing "Th1" T cell responses. Myasthenia gravis (MG) patients with thymomas frequently have high-titer neutralizing autoantibodies against these cytokines, but not against IFN-gamma. Because they occasionally develop intractable (even fatal) infections, we have tested effects of their sera on the generation of IFN-gamma responses by healthy adult T cells to autologous lipopolysaccharide (LPS)-treated dendritic cells (DC). Anti-IL-12(+) sera consistently reduced IFN-gamma responses substantially, whether assessed by intracellular staining or ELISA. Therefore, thymoma patients with intractable infections might benefit from cautious IFN-gamma therapy. We discuss wider implications of the surprising rarity of clear clinical hazards-or benefits-of these autoantibodies.

Myasthenia gravis (MG) is a heterogeneous disorder, a fact that needs to be kept in mind when considering treatment. Most patients benefit from pyridostigmine. In nonthymomatous ocular MG, prednisolone is often effective. Thymectomy is indicated for thymoma and is an option for acetylcholine receptor antibody-positive patients with generalized weakness developing under the age of 45 years. In older patients and in those failing to respond to thymectomy, prednisone alone or combined with azathioprine is the treatment of choice. Mycophenolate mofetil is an option in those intolerant of azathioprine. Lambert-Eaton myasthenic syndrome (LEMS) can exist in paraneoplastic (P-) and nonparaneoplastic (NP-) forms. Most patients benefit from 3,4-diaminopyridine. In P-LEMS, treatment of the tumor often results in neurological improvement. In both forms, prednisone alone is an option or combined with azathioprine in NP-LEMS. In both MG and LEMS, where weakness is severe, plasma exchange or intravenous immunoglobulin treatment may provide short-term benefit.

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

Myasthenia gravis (MG) is a well-recognised disorder of neuromuscular transmission that can be diagnosed by the presence of antibodies to the acetylcholine receptor (AChR). However, some patients (about 15%) with generalised MG do not have detectable AChR antibodies. There is some evidence, however, that this "seronegative" MG is an antibody-mediated disorder. Plasma from patients with the disorder seems to contain various distinct humoral factors: IgG antibodies that reversibly inhibit AChR function; a non-IgG (possibly IgM) factor that indirectly inhibits AChR function; and an IgG antibody against the muscle-specific kinase (MuSK). The presence of antibodies against MuSK appears to define a subgroup of patients with seronegative MG who have predominantly localised, in many cases bulbar, muscle weaknesses (face, tongue, pharynx, etc) and reduced response to conventional immunosuppressive treatments. Moreover, muscle wasting may be present, which prevents complete response to these therapies.

Background: Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR subunit are located in various functional domains, whereas fully described mutations in AChR non- subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two -subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern., Methods: These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells., Results: The authors identified two SCCMS mutations in the AChR subunit, L78P and L221F. Both mutations prolonged ACh-induced ion channel activations. L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and L221F shows variable penetrance in one of the two families that were identified harboring this mutation., Conclusion: SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

In early-onset myasthenia gravis (EOMG), the thymus usually shows medullary lymph node-type infiltrates, rearranged bands of hyperplastic epithelium and focal fenestrations in the intervening laminin borders. This resemblance to autoimmune target organs may reflect autoantigen expression by rare thymic myoid cells, long ago implicated circumstantially as agents provocateurs. In this quantitative study, they were frequently seen at the laminin fenestrations; if so, germinal centers (GC) were significantly commoner nearby, our most EOMG-specific finding (not seen in a distinct MG patient subset). As autoantibodies became detectable, myoid cell involvement apparently progressed. Our unifying hypothesis--that an early autoantibody attack on myoid cells provokes local GC formation--helps to resolve many puzzles.

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.

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific epsilon subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR delta subunit gene, delta 756ins2 and delta E59K. Expression studies demonstrate that delta 756ins2 is a null mutation. By contrast, both fetal and adult AChR containing delta E59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, delta E59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Antibodies to muscle acetylcholine receptors, to other muscle antigens, and to some cytokines are found in the majority of patients with thymic tumors (thymomas) and myasthenia gravis (MG). The role of the tumor in initiating autoimmunity, however, is unclear; in particular, it is not known whether the thymoma exports mature and long-lived T cells, which could provide help for antibody production in the periphery. Here, we quantified recently exported thymic T cells using the approach of measuring episomal DNA fragments [T-cell receptor excision circles (TRECs)], generated by T-cell receptor gene rearrangement. Compared to values in healthy individuals (n = 10) or in patients with late-onset MG (n = 8), TREC levels were significantly raised in both the CD4+ and CD8+ peripheral blood compartments of patients with thymoma and MG (n = 14, p = 0.002 and p = 0.0004 compared to healthy controls) but only in the CD8+ compartment of the three patients with thymoma without MG (p = 0.4 and p = 0.01 for CD4+ and CD8+). TREC levels decreased following thymectomy to values similar to controls but were substantially raised in patients who had developed tumor recurrence (n = 6, p = 0.04 and p = 0.02 for CD4+ and CD8+); this was associated with increased antibodies to interferon-alpha and interleukin-12 in the one case studied serially. Collectively, these results support the hypothesis that the neoplastic thymoma tissue itself can generate and export mature, long-lived T cells and that these T cells reflect the thymic pathology and are likely to be related to the associated autoimmune diseases. The results also provide a new approach for early diagnosis of thymoma recurrence.

Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction. In approximately 80% of patients, auto-antibodies to the muscle nicotinic acetylcholine receptor (AChR) are present. These antibodies cause loss of AChR numbers and function, and lead to failure of neuromuscular transmission with muscle weakness. The pathogenic mechanisms acting in the 20% of patients with generalized MG who are seronegative for AChR-antibodies (AChR-Ab) have not been elucidated, but there is evidence that they also have an antibody-mediated disorder, with the antibodies directed towards another, previously unidentified muscle-surface-membrane target. Here we show that 70% of AChR-Ab-seronegative MG patients, but not AChR-Ab-seropositive MG patients, have serum auto-antibodies against the muscle-specific receptor tyrosine kinase, MuSK. MuSK mediates the agrin-induced clustering of AChRs during synapse formation, and is also expressed at the mature neuromuscular junction. The MuSK antibodies were specific for the extracellular domains of MuSK expressed in transfected COS7 cells and strongly inhibited MuSK function in cultured myotubes. Our results indicate the involvement of MuSK antibodies in the pathogenesis of AChR-Ab-seronegative MG, thus defining two immunologically distinct forms of the disease. Measurement of MuSK antibodies will substantially aid diagnosis and clinical management.

We report six sibs with arthrogryposis multiplex congenita and a Pena-Shokeir phenotype, born to a healthy woman who was discovered to have asymptomatic myasthenia gravis (MG). This is the first report of anti-acetylcholine receptor (AChR) antibodies causing fetal akinesia/hypokinesia sequence in the offspring of an asymptomatic mother., (Copyright 2000 Wiley-Liss, Inc.)

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.

Acetylcholine receptor (AChR) deficiency is a recessively inherited congenital myasthenic syndrome in which fatigable muscle weakness results from impaired neuromuscular transmission caused by reduced AChR numbers. In mature muscle, AChRs consist of alpha2 betadelta together with the adult-specific epsilon subunit. We have identified a deletion of the first nucleotide in exon 12 of the AChR epsilon-subunit gene (epsilon1267delG) and demonstrate its recessive inheritance segregates with disease in 6 unrelated cases of AChR deficiency. In addition, we found that both healthy and AChR-deficient muscle contain a population of AChR epsilon-subunit mRNA transcripts that retain intron 11. We investigated the possible consequences of combining this mutation with the alternative mRNA species through AChR expression studies in human embryonic kidney cells and Xenopus oocytes. Epsilon1267delG generates a polypeptide that lacks M4 and is not detected in surface AChR, whereas retention of intron 11 in the RNA transcript restores the reading frame, conserves M4, and generates a polypeptide that is incorporated into functional surface AChR, although at a reduced level, consistent with the disease phenotype. Our results indicate that for some AChR deficiency mutations located between M3 and M4, the retention of intron 11 in the epsilon-subunit mRNA transcripts may rescue adult AChR function.

We have analyzed a series of HLA region markers in 207 UK Caucasoids with early-onset myasthenia gravis (EOMG, onset before age 40), where there is a strong female bias. The well known associations with HLA-DR3 and -B8 have now proved to be significantly stronger in the 165 females than in the 42 males. In patients (of either sex) lacking -DR3, there was also a significant increase in HLA-DR2. Although the muscle weakness in EOMG is clearly mediated by autoantibodies, the associations are consistently stronger with HLA-B8 (in class I) than with HLADR3 (in class II), as confirmed here. We therefore typed 87-137 cases for polymorphisms at four loci in the intervening class III region, and also at three in the adjacent stretch of class I. At each locus, one allele tended to co-occur with HLA-B8 and showed strong and highly significant associations in the patients. There appeared to be a region of maximal susceptibility extending from HSP70 (in class III) past HLA-B and HLA-C at least 600 kb telomerically into the class I region, which is now being mapped in detail. Any candidate genes here that act shortly after puberty may allow more precise localization of susceptibility.

The anti-rheumatoid drug D-penicillamine (D-pen) has a reactive sulfhydryl group capable of modifying self antigens, and can provoke typical autoantibody-mediated myasthenia gravis (MG), especially in DR1+ individuals. We have selected T cell clones from one such patient that were highly specific for D-pen but not its L-isomer or D-cysteine. Moreover, they were restricted to HLA-DR1, had a Th1 phenotype and used TCR V alpha4.1, V beta6.1. They responded well to blood mononuclear cells prepulsed with D-pen either in the absence of serum or after chloroquine treatment, but not to autologous D-pen-pulsed B cell lines. Thus, D-pen may directly couple to distinctive peptides resident in surface DR1 molecules on circulating macrophages or dendritic cells.

Low density cells can readily be enriched from thymus tissue both of children undergoing cardiac surgery and of older patients with myasthenia gravis, and can be cryostored in bulk. When fresh or thawed cells are cultured with granulocyte-macrophage colony-stimulating factor and stem cell factor with or without tumour necrosis factor-alpha (TNF-alpha), they generate numerous cells with the characteristic ultrastructural, phenotypic and functional properties of dendritic cells. These proved to be very potent, both as stimulators of primary mixed leucocyte responses and as costimulators in oxidative mitogenesis. Especially after exposure to TNF-alpha, these dendritic cells also processed a natural epitope from a 437-residue polypeptide and presented it efficiently to an autoimmune T-cell clone (of T helper type 0 phenotype). Thus, immunostimulatory dendritic cells can be cultured in relative abundance from progenitors in infant and adult human thymus. Both are convenient sources of potent antigen-presenting cells of identifiable origins, e.g. for use in selecting human T-cell lines.

Congenital myasthenic syndrome comprises a heterogeneous group of inherited disorders of neuromuscular transmission. Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome and in most cases results from mutations within the coding region of the AChR epsilon subunit. However, studies in mice have established that synapse-specific expression of AChR is dependent on a sequence contained within the AChR-subunit promoter regions, termed an N-box. We describe a consanguineous family in which 2 of 7 siblings had clinical and electromyographic features consistent with AChR deficiency. Muscle biopsy demonstrated low AChR numbers, establishing the disorder as postsynaptic. Single-strand conformational polymorphism analysis identified an abnormal conformer in the AChR epsilon-subunit gene promoter of the patients. DNA sequence and restriction endonuclease analysis shows that the disorder cosegregates with recessive inheritance of a single point mutation, a transition (C-->T) in the N-box of the epsilon-subunit promoter. Analysis of an intercostal biopsy from 1 of the patients showed a dramatic reduction in epsilon-subunit mRNA levels compared with disease and normal controls. This is the first evidence in humans that an N-box mutation can lead to disruption of epsilon-subunit transcription, resulting in the loss of adult AChR synthesis and the clinical phenotype of AChR-deficiency congenital myasthenic syndrome.

No immunodominant T-cell epitopes have yet been reported in the human acetylcholine receptor (AChR), the target of the pathogenic autoantibodies in myasthenia gravis (MG). We have selected and characterized T cells from MG patients by restimulation in culture with recombinant human AChR to alpha, gamma and epsilon subunits; the gamma and epsilon distinguish the fetal and adult AChR isoforms, respectively. We obtained clones specific for the epsilon, rather than the alpha or gamma, subunit in 3 of the first 4 early-onset MG cases tested. They all responded to peptide epsilon201-219 and to low concentrations of adult but not fetal AChR. Moreover, although using different T-cell receptor genes, they were all restricted to HLA-DR52a (DRB3*0101), a member of the strongly predisposing HLA-A1-B8-DR3 haplotype. This apparently immunodominant epsilon201-219 epitope (plus DR52a) was also recognized by clones from an elderly patient whose MG had recently been provoked by the drug D-penicillamine. In all 4 cases, however, the serum antibodies reacted better with fetal than adult AChR and may thus be end products of determinant spreading initiated by adult AChR-specific T cell responses. Furthermore, as these T cells had a pathogenic Th1 phenotype, with the potential to induce complement-activating antibodies, they should be important targets for selective immunotherapy.

We compared prednisolone (PRED) and azathioprine (AZA) versus prednisolone alone in the treatment of MG. Prednisolone alone or combined with azathioprine is widely used in the treatment of MG, but no randomized placebo-controlled comparative trial data are available. The prednisolone dose and clinical outcome were compared in a multicenter randomized double-blind study of 34 MG patients who were followed up for 3 years. One group (PRED + AZA) received prednisolone (on alternate days) plus azathioprine (2.5 mg/kg); the other group received prednisolone on alternate days plus placebo (PRED + PLAC). Initial high-dose prednisolone (1.5 mg/kg on alternate days) was tapered at remission to the minimal dose required to maintain remission. The prednisolone dose did not differ significantly between the two groups at 1 year (median values: PRED + AZA, 37.5 mg on alternate days; PRED + PLAC, 45 mg on alternate days) but was reduced at 2 and 3 years in the PRED + AZA group (median value at 3 years: PRED + AZA, 0 mg on alternate days; PRED + PLAC, 40 mg on alternate days; p=0.02). Relapses and failures to remit over the 3 years were more frequent in the PRED + PLAC group. There was a sharp rise in the anti-acetylcholine receptor (AChR) titers in the PRED + PLAC group at 2 years. Incidence of side effects was slightly less in the PRED + AZA group. Azathioprine as an adjunct to alternate day prednisolone in the treatment of antibody-positive generalized MG reduces the maintenance dose of prednisolone and is associated with fewer treatment failures, longer remissions, and fewer side effects.

Myasthenia gravis (MG) is caused by helper T cell-dependent autoantibodies against the muscle acetylcholine receptor (AChR). Thymic epithelial tumors (thymomas) occur in 10% of MG patients, but their autoimmunizing potential is unclear. They express mRNAs encoding AChR alpha and epsilon subunits, and might aberrantly select or sensitize developing thymocytes or recirculating peripheral T cells against AChR epitopes. Alternatively, there could be defective self-tolerance induction in the abundant maturing thymocytes that they usually generate. For the first time, we have isolated and characterized AChR-specific T cell clones from two MG thymomas. They recognize extracellular epitopes (alpha75-90 and alpha149-158) which are processed very efficiently from muscle AChR. Both clones express CD4 and CD8alpha, and have a Th-0 cytokine profile, producing IL-4 as well as IFN-gamma. They are restricted to HLA-DP14 and DR52a; expression of these minority isotypes was strong on professional antigen-presenting cells in the donors' tumors, although it is generally weak in the periphery. The two clones' T cell receptor beta chains are different, but their alpha chain sequences are very similar. These resemblances, and the striking contrasts with T cells previously cloned from non-thymoma patients, show that thymomas generate and actively induce specific T cells rather than merely failing to tolerize them against self antigens.

A new autoimmune disease affecting the neuromuscular junction has been defined. Acquired neuromyotonia is associated with antibodies to voltage-gated potassium channels that act, at least in part, by reducing potassium channel function with resulting neuronal hyperactivity. This condition is quite frequently associated with thymoma and, in many cases, antibodies to acetylcholine receptors are present as well as antibodies to VGKC. Improvements in techniques and the availability of cloned DNA and recombinant forms of the AChR subunits have led to new observations concerning the specificity and roles of antibodies in myasthenia gravis. The transfection of a cell line with the epsilon subunit means that we can now accurately compare antibodies reactive with adult and fetal human AChR. This may help to determine the relationship between AChR subunit expression in different tissues and the induction of antibodies that bind specifically to the two forms, as well as to clarify the role of antibodies to fetal or adult AChR in causing ocular muscle symptoms. Serum antibodies from a few mothers with obstetric histories of recurrent arthrogryposis multiplex congenita in their babies specifically inhibit the function of fetal AChR. These observations not only explain the cause of some cases of arthrogryposis multiplex congenita, but also suggest that other fetal-specific antibodies might be responsible for other fetal or neonatal conditions. An animal model has been established to enable us to investigate the role of maternal serum factors in causing such disorders. Seronegative MG has been the subject of many studies from our laboratory over the last ten years. The transience of the effects of SNMG plasmas on AChR function strongly suggests that the plasma antibodies do not bind directly to the AChR, but inhibit function by some indirect mechanism. They do not appear to act via the cAMP-dependent protein kinase pathway, and studies are in progress to investigate the involvement of other second messenger systems.

Using an acetylcholine receptor-specific T-cell clone (TCC) from a myasthenia gravis patient, we have compared the induction of unresponsiveness by three agents: an anti-V beta monoclonal antibody (mAb), complexes of MHC class II with specific peptide (DR4:peptide) and free peptide. Pretreatment with each agent without antigen-presenting cells specifically rendered the TCC unresponsive to a subsequent optimal stimulus. A substantial proportion of the DR4:peptide pretreated cells underwent apoptosis and the remainder were profoundly unresponsive. Apoptosis was less prominent after pretreatment with free peptide and was not significant with anti-V beta mab; with both, the unresponsiveness was transient in the survivors. These diverse effects of engaging the T-cell receptor in the absence of costimulation suggest that these agents act via different mechanisms, and give insights to the potential for specific immunotherapy.