Anti-acetylcholine receptor antibodies block bungarotoxin binding to native human acetylcholine receptor on the surface of TE671 cells

We assayed sera from 20 myasthenics of various clinical stages and anti-acetylcholine receptor (AChR) antibody levels for their ability to affect bungarotoxin (BGT) binding to native human AChR on the surface of TE671 cells. Thirty-five percent of sera blocked BGT binding to the AChR, some at a dilution of up to 1:1000. The 7 patients whose sera blocked toxin binding were all generalized myasthenics with particularly severe disease, 6 of whom had had myasthenic crisis at some point in their course. No ocular myasthenics had blocking antibody. Blockade of toxin binding by myasthenic antibody to TE671 cells resembled blockade produced by unlabeled toxin in being irreversible with washing. There was little correlation between ability to block toxin binding and amplitude of the AChR binding antibody. These data are consistent with the hypothesis that patients with more aggressive generalized myasthenia preferentially have anti-AChR antibody that blocks toxin binding.     

Anti-acetylcholine receptor antibodies in myasthenia gravis: Part 1. Relation to clinical parameters in 250 patients

We examined the significance of the presence or absence of anti-acetylcholine receptor (anti-AChR) antibodies in 250 myasthenia gravis (MG) patients and the relation between clinical features and anti-AChR levels.We found high anti-AChR levels in 2 out of 11 thymoma patients without MG, while 37 out of 250 MG patients had no detectable anti-AChR. The absence of these antibodies was related to purely ocular disease and to steroid therapy and/or thymectomy.Differences in anti-AChR levels did not correspond significantly to differences in disease activity when single measurements in patients were analysed. However, the results were influenced by both the presence or absence of a thymoma, the age at onset of disease and by steroid therapy. The thymic pathology and age at onset seemed to act independently. Early onset of disease was associated with high anti-AChR levels and absence of antibodies to striated muscle (anti-SM), whereas late onset was associated with low anti-AChR and the presence of anti-SM. Thymomas both have high anti-AChR and high anti-SM. The effect of steroid therapy on antibody levels was seen in all patient groups but was strongest in thymoma patients with early onset of disease.     

Complement-dependent toxicity of serum from myasthenic patients to muscle cells in culture

The toxicity of myasthenic sera to rat myotubes in monolayer culture was examined by measuring the release of [Me-3H]carnitine from pre-loaded cells. In the presence of guinea pig complement, heat-inactivated serum samples from 9 out of 13 myasthenic patients showed clear myotoxicity, in contrast to 0 out of 11 normal controls and 0 out of 6 polymyositis patients. Neither heat-inactivated sera alone nor guinea pig complement sera alone showed myotoxicity. Removal of anti-acetylcholine receptor (anti-AChR) antibodies from a myasthenic serum sample by affinity absorption led to loss of myotoxicity. Myotoxicity of myasthenic sera could, in most cases, be confirmed by light microscopy. These results support the idea that complement-mediated cell damage, initiated by anti-AChR antibodies, contributes to post-synaptic membrane degeneration in myasthenia gravis.     

A sensitive rosetting assay for detection of acetylcholine receptor antibodies using BC3H-1 cells: positive results in 'antibody-negative' myasthenia gravis

Antibodies to acetylcholine receptor (AChR) were measured in a group of patients with myasthenia gravis (MG), some of whom had previously been classified as 'antibody negative' using the standard anti-AChR radioimmunoassay (RIA). AChR antibodies were measured using the rosetting assay, a new detection method which utilizes protein A-coated red blood cells and live BC3H-1 cells, a murine cell line which expresses muscle nicotinic AChR. The results of the rosetting assay were compared with those obtained in the anti-AChR RIA. 76% of all myasthenic sera tested showed rosetting at titers higher than any of the control sera (from patients with non-myasthenic neurologic disease and normal individuals). Of the myasthenic patients previously classified as 'antibody negative' in the RIA using human AChR, 71% demonstrated positive rosetting. There was no correlation between the anti-AChR antibody titer obtained in the rosetting assay and that obtained in the RIA using either human or denervated rat AChR. The results suggest that the rosetting assay may measure a subpopulation of antibodies that differs from those detected in the RIA.     

Patient-specific anti=acetylcholine receptor antibody patterns in myasthenia gravis.

Anti-acetylcholine receptor (AChR) antibodies have been determined in the sera of ten myasthenic patients over a period of several months which in each case included a series of plasma-exchanges coupled with immunosuppressive therapy. The ratio of anti-(extra-junctional rat AChR): anti-(human AChR) antibody titres was found to vary with the patient but to be constant for a given patient over the period of study. Similar indications of a patient-specific anti-AChR antibody pattern were obtained by using junctional as well as extra-junctional rat AChR and also by precipitation of human AChR in the presence of excess antiserum. Individual anti-AChR antibody patterns may have relevance to the varying courses taken by myasthenia gravis in different patients.     

Anti-acetylcholine receptor antibody specificities in serum and in thymic cell culture supernatants from myasthenia gravis patients

We investigated the role of the thymus in myasthenia gravis by comparing the antigenic specificities of anti-acetylcholine receptor antibodies (anti-AChR), defined by competition with mouse monoclonal antibodies that bind to five different regions on human muscle AChR, in thymic culture supernatants and in serum pre- and post-thymectomy. Anti-AChR specificities present in the serum were broadly unchanged in 16 non-thymoma and six thymoma patients 7-30 months after thymectomy compared with an initial sample, although total anti-AChR frequently fell. The fine specificities of the anti-AChR synthesized in vitro by cultured lymphocytes from the thymus of ten patients (without thymoma) correlated significantly with that of the anti-AChR in the serum at the same time. We conclude that AChR-specific B cells in the thymus are representative of the total AChR-specific repertoire, and that thymectomy does not selectively deplete particular B cell clones.     

Anticorps dans la myasthénie

In autoimmune myasthenia gravis, 75 to 80% of patients have antiacetylcholine receptor antibodies (anti-AChR abs) quantified by immunoprecipitation. Anti-AChR abs are polyclonal, directed against all AChR subunits, with a major fraction against the main immunogenic region, (alpha-subunit, aminoacids 67-76); they cause AChR loss by three mechanisms: blocking of acetylcholine binding; accelerated degradation or AChR due to bridging of two adjacent AChR molecules (antigenic modulation); lysis of postsynaptic membrane induced by complement. Neither anti-AChR ab level nor antigenic repertoire are correlated with disease severity. Studies performed on rat and human myotubes have shown that capacity of myasthenic patients's sera or immunoglobulins to induce AChR loss was correlated with anti-AChR ab titer but not with severity. Highest anti-AChR ab titers are found in young women with hyperplastic thymus, lowest in older patients and atrophic thymus. Ten to fifteen percent of babies born to myasthenic mothers suffer from a transitory neonatal myasthenic syndrome due to passive transfer of maternal anti-AChR abs. There is no correlation between clinical condition of the baby (presence and severity of neonatal myasthenia) and severity of maternal myasthenia. The risk of neonatal myasthenia is high when maternal ab titer is elevated (≥ 100 nM). Very rare and severe cases of foetal neonatal myasthenia gravis with arthrogryposis, hypomobility are due to presence in maternal serum of anti-AChR ab directed against foetal (gamma) AChR. In generalized myasthenia without anti-AChR ab, antibodies directed against MuSK, a postsynaptic molecule involved in AChR aggregation, are detected in around 40% of patients. Features of MuSK+ myasthenia are the following: strong female preponderance, severity (respiratory and bulbar) requiring immunosuppressants, facial and tongue atrophy, poor response to anticholinesterase inhibitors, atrophic thymus and poor response to thymectomy. Low-affinity anti-AChR abs have been recently reported in myasthenia gravis without anti-AChR and anti-MuSK ABS.     

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.     

Anti AChR antibody: Relevance to diagnosis and clinical aspects of myasthenia gravis

227 sera from myasthenic patients were tested for the presence of anti-AChR antibodies (anti-AChR Abs) by mean of a fetal calf receptor (Fc-AChR); 73.5% of cases proved positive with this method. Significant correlations were found between presence of anti-Fc-AChr Abs and various clinical aspects such as: clinical stage and duration of disease; moreover significantly higher than the median titers were found among younger patients (i.e. age <40 yrs). This work was partially supported by the National Research Council (CNR) — Progetto Finalizzato “Medicina Preventiva e Riabilitativa” n^o 85.00513.56     

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

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

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

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

The predictive value of the presence of different antibodies and thymus pathology to the clinical outcome in patients with generalized myasthenia gravis

Objectives

To analyze the predictive value of anti-acetylcholine receptor antibodies (anti-AChR Ab) and anti-muscle specific kinase antibodies (anti-MuSK Ab), as well as the thymus pathology to the clinical outcome in patients with generalized myasthenia gravis (MG).

Methods

We analyzed 138 patients with generalized MG, who were thymectomized and assayed for anti-AChR Ab and anti-MuSK Ab.

Results

Anti-AChR Ab were detected in 84% of patients, while anti-MuSK Ab were present in 36% of the AChR Ab negative patients. Severe forms of the disease were more frequent in MuSK Ab positive, compared to the AChR Ab positive and complete seronegative patients. Thymic lymphoid follicular hyperplasia (LFH) was present in 60%, thymoma in 23%, atrophic thymus in 9% and the normal thymus in 8% of patients. LFH was more frequent among women, while thymoma and atrophic thymus were more frequent in men. The younger patients mainly had LFH and normal thymus, while thymoma and atrophic thymus were more frequent in older patients. The mildest clinical presentation was present in patients with normal thymus, while severe forms of the disease were registered in the patients with thymoma. The AChR Ab positive patients had more often LFH and thymoma, while within MuSK Ab positive patients atrophic thymus was most common.

Conclusion

The best disease outcome was observed in patients with normal thymus or LFH with anti-AChR Ab or without both types of antibodies.     

Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients

BACKGROUND: Titin is the major autoantigen recognized by anti-striated muscle antibodies, which are characteristic of generalized myasthenia gravis (MG). OBJECTIVE: To seek a correlation between anti-titin antibodies and other features of MG patients, including histopathology, age at diagnosis, anti-acetylcholine receptor (anti-AChR), autoantibody titers, and clinical severity. METHODS: A novel, highly specific radioligand assay was performed on a large group of 398 patients with generalized MG. RESULTS: Among thymectomized patients, anti-titin antibodies were present in most patients with thymoma (56/70 [80%]), contrasting with only a minority of patients with thymus atrophy or hyperplasia (17/165 [10%]). They were also present in 64 (41%) of 155 nonthymectomized patients who had a radiologically normal thymus. In these patients and in those who had a histologically normal thymus, anti-titin antibodies were associated with a later age at onset of disease and with intermediate titers of anti-AChR antibodies. After controlling for these 2 variables, disease severity was not significantly influenced by anti-titin antibodies. CONCLUSIONS: Anti-titin antibodies are a sensitive marker of thymoma associated with MG in patients 60 years and younger, justifying the insistent search for a thymoma in MG patients of this age group who have these antibodies. In nonthymoma patients, anti-titin antibodies represent an interesting marker complementary to the anti-AChR antibody titer, identifying a restricted subset of patients. These clinical correlations should prompt further studies to examine the mechanisms leading to the production of anti-titin antibodies.     

Myasthenia gravis: monoclonal antihuman acetylcholine receptor antibodies used to analyze antibody specificities and responses to treatment

We investigated the heterogeneity of serum antibodies to acetylcholine receptor (AChR) by competition with nine antihuman monoclonal antibodies in a cross-sectional study of 36 patients with myasthenia gravis (MG), and in three who showed clinical improvement associated with decrease in total anti-AChR following immunologic treatment. Two specificities were more prevalent in patients without thymoma, an done of these was more prevalent in cases beginning before age 40. Some specificities were stable during serial studies, whereas other fluctuated. We found evidence of three groups of antibody specificities that had different control mechanisms and may define different regions of the receptor.     

Heterogeneity of antibodies directed against the alpha-bungarotoxin binding site on human acetylcholine receptor and severity of myasthenia gravis

A rapid and sensitive radioimmunological method is described, using decamethonium (DC), which revealed antibodies which blocked alpha-bungarotoxin (alpha-Bgt) binding to human acetylcholine receptor (AChR) in 98% of myasthenia gravis (MG) patients' sera tested. These sera had anti-AChR antibody titres by the conventional assay. The titre of blocking antibodies (1 to 110 nM) could be measured and was found to produce from 1 to 54% inhibition of alpha-Bgt binding. No relationship was found between these titres and anti-AChR antibody titres. MG sera were divided into 2 major groups on the basis of their blocking effects, with and without DC, but there was no correlation between these and the clinical status, as defined by Osserman's classification. However, no sera from asymptomatic or ocular MG patients had the dual capacities of blocking alpha-Bgt binding, directly and in the presence of DC.     

Anti-acetylcholine receptor antibodies in myasthenia gravis. Part 2. Clinical and serological follow-up of individual patients

Circulating antibodies to acetylcholine receptor protein (anti-AChR) were measured in the sera collected from 75 patients (53 women, 22 men, ages 9-83 year, 20 with a thymoma) with myasthenia gravis (MG) during 5-44 (mean 25) months. The clinical state of each patient was graded on a 6-point scale. Anti-AChR concentrations were measured by a radioimmunoassay with human antigen. We analysed the relation between the change in clinical state and the change in anti-AChR concentration in 155 periods (1-7, mean 2.1 per patient). The change in clinical state is given as the difference in score at the onset and at the end of this period. The change in anti-AChR concentration is expressed as the percentage of the original concentration at the onset of the period. The results were analysed in relation to the therapy and to the severity of the MG at the onset of each period. A strong correlation between a change in anti-AChR concentration and a change in clinical condition existed during treatment with prednisone or immunosuppression and in the period after thymectomy, while a weaker correlation was present in periods without immunosuppression. In only 3 patients did the changes in anti-AChR concentration precede the clinical change. No changes in anti-AChR concentrations were found if improvement was due to the effect of anticholinesterases or if deterioration was caused by infection or emotion. The serial measurement of anti-AChR may be a valuable method of following the basic trend of the MG in severely affected patients.     

Antibody dependent cell-mediated cytotoxicity--an additional mechanism in human autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease characterised by serum anti-acetylcholine receptor (AChR) antibodies and several pathogenic mechanisms for the action of these antibodies have been elucidated. In this study, we have analysed a possible additional mechanism for these antibodies, namely antibody-dependent cell-mediated cytotoxicity (ADCC). Using as target cells a cell line expressing AChR, we could show an increased ADCC mediated by sera from MG patients. Sera with AChR antibodies induced a higher cytotoxicity than sera from patients without these antibodies or healthy individuals. Sera from MG patients with thymoma induced a higher cytotoxic effect than sera from other patients. There was a strong positive correlation between the concentration of AChR antibodies and cytotoxicity mediated by purified IgG fractions from thymoma patients. In addition, there was a higher cytotoxicity mediated by sera from thymoma patients with extended dinucleotide repeats, (AT)n repeats, in the CTLA-4 gene. ADCC mediated by AChR antibodies may thus be another possible pathogenic mechanism that could operate in MG patients, especially in patients with thymoma.     

Combined effects of a thymic peptide, thymopoietin and myasthenic patient sera in rat myotube culture.

We investigated in a rat myotube assay the combined effect of 26 myasthenic (MG) patient sera and a thymic peptide, thymopoietin (Tpo) which had previously been shown to bind Torpedo and human AChR and to compete with alpha-bungarotoxin (alpha-Bgt) binding. Cultures were first exposed to Tpo alone for 3 h (0.3, 7.5, 15 nM), then MG sera (5% final dilution) were added for an additional 18 h. Reduction in the amount of 125I-alpha-Bgt binding sites in the presence of various concentrations of Tpo were similar with control sera and in all the patients with low or undetectable anti-AChR Ab (11 cases). In cultures exposed to Tpo and sera with high anti-AChR Ab titre (15 cases), Tpo and anti-AChR Ab have an additive capacity to reduce the number of alpha-Bgt binding sites. The results are compatible with the hypothesis that anti-AChR Ab and Tpo could impair neuromuscular transmission by complementary mechanisms.     

Antibodies against saline-soluble components of skeletal muscle in myasthenia gravis

Summary Antibodies against phosphate-buffered-saline extracts (SE) of non-acetylcholine receptor (AChR) skeletal muscle antigens were found in patients with myasthenia gravis (MG). The antigenicity of SE was distributed in three fractions with molecular masses of over 200 kDa, 90–150 kDa and 7–14 kDa on gel filtration. These fractions shared common antigenicities. Further analysis of 90–150 kDa fractions on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed five major bands, ranging from 105 kDa to 275 kDa. The antibodies against SE were detected in 52% (58/112) of the MG patients; incidence and titres were higher in the thymoma group (n=21; 90% and 0.872 respectively) than in the non-thymoma group (n=91; 43% and 0.200, P<0.001). In patients without a thymoma, these antibodies were frequently observed in late-onset disease and the severe generalized form (P<0.01). In 4 of 7 ocular MG patients without anti-AChR antibodies, low but appreciable levels of anti-SE antibodies were found. In 73% (11/15) of generalized MG patients treated with prednisolone and thymectomy, anti-SE antibody titres changed in association with those of anti-AChR antibodies and with the clinical course. Both antibody titres increased synchronously in patients who developed crises.