Myasthenia gravis associated with penicillamine treatment for rheumatoid arthritis.

Four patients with rheumatoid arthritis (R.A.) developed myasthenia gravis after taking penicillamine. In one patient withdrawal of the drug was followed by spontaneous remission of the myasthenia, and in two the dose of anticholinesterase was subsequently reduced. In the fourth patient continuing penicillamine treatment was associated with increasingly severe myasthenic features, but on withdrawal of the drug these resolved. As myasthenia gravis rarely complicates R.A. its onset in these patients shortly after the start of penicillamine treatment suggested that penicillamine may have precipitated this condition.     

Acetylcholine receptor peptide recognition in HLA DR3-transgenic mice: in vivo responses correlate with MHC-peptide binding.

HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor alpha subunit. We find two promiscuous determinants on this region 141-160 and 171-190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor alpha subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.     

Monoclonal Anti-Acetycholine Receptor Antibodies as Probes for Human Acetylcholine-Receptor in Myasthenia Gravis

Abstract

Monoclonal antibodies have been shown to bind to five regions on human acetylcholine receptor, each probably consisting of a discrete epitope on the extracellular surface. Two of these regions are equivalent to the ‘main immunogenic region’, and the other three appear to be close to the a-Bungarotoxin binding sites. These antibodies have been used to probe differences in myasthenia gravis anti-acetylcholine receptor antibodies, to locate acetylcholine receptor in thymic tissue, and to look for naturally-occurring anti-idiotype antibodies.

Anti-acetylcholine receptor antibody specificities differ between groups of patients defined by their age of onset, thymic pathology and HLA associations. Anti-AChR synthesised by the thymus in young onset patients has similar specificity to that found in the individual's serum, and may be stimulated by the presence of AChR on thymic myoid cells. However, myoid cells (defined by staining with anti-troponin and anti-myosin antibodies) do not appear to differ between control and myasthenia gravis patients and show no obvious involvement in an immunological reaction.

There was no convincing evidence for the presence of anti-idiotype antibodies in myasthenia gravis patients.     

Survivin as a Potential Mediator to Support Autoreactive Cell Survival in Myasthenia Gravis: A Human and Animal Model Study

The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders.     

Different HLA DR-DQ associations in subgroups of idiopathic myasthenia gravis

We have investigated theHLA-DRB and -DQB gene polymorphism in 131 myasthenia gravis (MG) patients. TheHLA genotypes in these patients were assigned by means of restriction fragment length polymorphism (RFLP)-definedDR-DQ haplotypes, correlating to serologic HLA class II typing. Using this technique we could, among randomly selected non-thymomatous (NT)-MG patients, confirm the strong association to DR3, and 70% of the patients were found to carry a specific DR3-positiveDR-DQ haplotype,T-3.1. Furthermore, an analysis of T-3.1^– NT-MG patients revealed that 59 % were T-4.1^÷ (DR4, DQw8). Thymic hyperplasia was found in approximately 85 % of the T-3.1⁺ , as well as of the T-4.1⁺ /3.1^– patients. As previously observed, we found a clear dominance of females among the T-3.1⁺ NT-MG patients. However, among T-4.1⁺/3.1- patients, males were as common as females. Furthermore, the T-4.1⁺ patients were significantly older at the onset of disease than those who were T-3.1⁺. In female MG patients, the DRwl5-Dw2-positive haplotypeT-2.1 was strongly correlated with the presence of thymoma (T-MG). These data indicate that the HLA associations in early vs late onset of NT-MG are different, and that female patients with and without thymoma differ from each other with regard to HLA markers. Thus, at least three different HLA DR-DQ associations are found in subgroups of idiopathic MG.     

Heterogeneity of non-insulin-dependent diabetes mellitus in HLA types and clinical features: comparison with insulin-dependent diabetes mellitus

We determined HLA types in 110 Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM) and studied the relationship between the HLA phenotypes and clinical features. Sixty-nine patients with insulin-dependent diabetes mellitus (IDDM) and 100 healthy blood donors served as controls. Concerning HLA DR and DQ loci, frequencies of DR4, DRw9 and DQw3.2 were higher, and those of DR2, DRw8, DRw11, DRw12 and DQw1 were lower in patients with IDDM compared than in healthy controls. There were no differences between NIDDM and normal controls in the frequency of a particular HLA DR antigen except for a decreased frequency in DRw11 in the former. The frequency of DQw3.2 antigen in NIDDM was intermediate between IDDM and normal controls. There were some differences between DQw3.2-positive and -negative NIDDM patients in clinical features. Those who showed low C-peptide responses during oral glucose tolerance test were more frequently found among DQw3.2-positive NIDDM patients. These results suggest that Type 1 diabetes mellitus may have a mild clinical course and is found among the Japanese NIDDM population.     

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.     

Myasthenia gravis. T epitopes on the delta subunit of human muscle acetylcholine receptor.

Autoimmune T cell lines specific for muscle nicotinic acetylcholine receptor (AChR) were propagated from the blood of three myasthenia gravis patients by the use of a pool of synthetic peptides (delta-pool) corresponding to the complete sequence of the delta-subunit of human muscle AChR. Propagation of AChR-specific T cell lines was attempted unsuccessfully from four other myasthenia gravis patients and from four healthy controls. The lines had CD3+, CD4+, CD8- phenotype, strongly recognized the delta-pool, and cross-reacted vigorously with non-denatured AChR purified from mammalian muscle. They did not cross-react detectably with pools of similar overlapping synthetic peptides corresponding to the complete sequences of the alpha- and gamma-subunits of human muscle AChR. The sequence segments of the delta-subunit that contain T epitopes were identified by investigating the response of the three CD4+ T cell lines to the individual synthetic peptides forming the delta-pool. Each line had an individual pattern of peptide recognition. Although no immunodominant region, recognized in association with different DR haplotypes, could be identified, the sequence segments most strongly recognized by the CD4+ T cell lines were clustered within residues 121-290. One of the peptides more strongly recognized by the T cells corresponded to a sequence segment with high predicted propensity to form an amphipathic alpha-helix, a structural motif proposed to be typical of T epitopes.     

Idiotypic and antiidiotypic T and B lymphocytes in myasthenia gravis.

The prevalence of Id and anti-Id T and B cells as measured by their reactivities with two human mAb, one antiacetylcholine receptor mAb and one anti-Id mAb, was studied in 38 patients with myasthenia gravis and in 27 healthy individuals. Id and anti-Id T cells were estimated by enumerating the numbers of cells secreting IFN-gamma in response to 10 pg/ml of the human mAb. T cell stimulation, measured as numbers of IFN-gamma-secreting cells that exceeded the mean + 2 SD of controls, was induced by the Id mAb in 78.9% of the patients and in 7.4% of the controls, whereas the anti-Id mAb-stimulated T cells in 55.3% of the patients and in 3.7% of the controls. The mean value of the Id and anti-Id-reactive T cells in the patients was 18.3/10(5) and 10.1/10(5) PBMC, respectively. B cells secreting IgM antibodies binding to the human mAb were increased in patients with myasthenia gravis compared to healthy controls. Seventy-five percent of the patients and 12% of the controls had B cells secreting IgM antibodies binding to the Id mAb, although 89% of the patients and 16% of the controls had B cells secreting IgM antibodies binding to the anti-Id mAb. The mean value of B cells secreting IgM antibodies binding to Id or anti-Id mAb in the patients were 7.4 cells/10(6) and 5.5 cells/10(6) PBMC, respectively. We conclude that Id and anti-Id T and B cells are present in myasthenia gravis. These methods allow a quantitative estimation of T and B cells with defined specificities and thus a way of mapping the repertoire of lymphocytes.     

Cathepsin S is required for murine autoimmune myasthenia gravis pathogenesis

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.     

An approach to the localization of the susceptibility genes for generalized myasthenia gravis by mapping recombinant ancestral haplotypes

The association of HLA A1, B8, and DR3 with generalized myasthenia gravis (GMG) ini Caucasoids is well established, but no particular gene has been implicated and there is still no adequate explanation in functional terms. In study we have taken advantage of sequential genomic markers between B8 and DR3 so as to map the location of susceptibility gene(s) on the A1, B8, DR3 (8.1) ancestral haplotype. By studying 51 patients, we have delineated a region between HLA B and TNF which is shared by 29/29 patients with B8 and DR3, 19/19 patients with B8 but not DR3 and 2/3 patients with DR3 but not B8. The potential importance of this region was confirmed by examining a similar disease induced by D-Penicillamine (D-PenMG) and associated with different HLA class II alleles (DR1 and/or DR7). Among these patients, 7/16 (44%) have B8, often with other markers of 8.1. These results implicate at least two categories of genes in determining susceptibility to MG; one located in the region between HLA B and TNF may be immunoregulatory, whereas the second, located in the class II region, may relate to the inducing factor (e. g., DR1 or DR7 in D-PenMG).     

The role of acetylcholine receptor antibodies in myasthenia gravis.

Myasthenia gravis is an autoimmune disease of man characterized by remitting and relapsing muscle fatigability. Although the etiology and pathogenesis are incompletely understood, the presence of circulating antibodies directed against the nicotinic acetylcholine (ACh) receptor in 80--90% of patients with myasthenia gravis and the identification of immune complexes at their neuromuscular junction have helped explain the altered neuromuscular transmission. The ACh receptor antibodies do not block access of ACh to the receptor, but do decrease the number of receptors by accelerating their degradation both in rat myotube cultures and in vivo models. In vitro these antibodies play a major role in myasthenia gravis. However, correlations of antibody titers with the clinical state following thymectomy or in neonatal myasthenia suggest that host factors may be equally important in determining whether the ACh receptor antibodies will result in clinical myasthenia.     

Myasthenia Gravis: Analysis of Serum Autoantibody Reactivities to 1827 Potential Human Autoantigens by Protein Macroarrays

Background

Myasthenia gravis is a disorder of neuromuscular transmission associated with autoantibodies against the nicotinic acetylcholine receptor. We have previously developed a customized protein macroarray comprising 1827 potential human autoantigens, which permitted to discriminate sera of patients with different cancers from sera of healthy controls, but has not yet been evaluated in antibody-mediated autoimmune diseases.

Objective

To determine whether autoantibody signatures obtained by protein macroarray separate sera of patients with myasthenia gravis from healthy controls.

Methods

Sera of patients with acetylcholine receptor antibody-positive myasthenia gravis (n = 25) and healthy controls (n = 32) were analyzed by protein macroarrays comprising 1827 peptide clones.

Results

Autoantibody signatures did not separate patients with myasthenia gravis from controls with sufficient sensitivity, specificity, and accuracy. Intensity values of one antigen (poly A binding protein cytoplasmic 1, p = 0.0045) were higher in patients with myasthenia gravis, but the relevance of this and two further antigens, 40S ribosomal protein S13 (20.8% vs. 0%, p = 0.011) and proteasome subunit alpha type 1 (25% vs. 3.1%, p = 0.035), which were detected more frequently by myasthenia gravis than by control sera, currently remains uncertain.

Conclusion

Seroreactivity profiles of patients with myasthenia gravis detected by a customized protein macroarray did not allow discrimination from healthy controls, compatible with the notion that the autoantibody response in myasthenia gravis is highly focussed against the acetylcholine receptor.     

Suppression of CHRN endocytosis by carbonic anhydrase CAR3 in the pathogenesis of myasthenia gravis

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.     

Myasthenia Gravis,Autoantibodies, and HL-A Antigens

The serum of 100 patients with myasthenia gravis and 441 of their first-degree relatives was studied for the presence of autoantibodies against several antigens. Antibodies to skeletal muscle were present in 22% of the patients and in 2% of the relatives. Both these frequencies were significantly higher than those in matched control subjects. Also, antinuclear antibodies were present more often both in the patients and in the relatives. Typing for HL-A antigens had shown a positive correlation between HL-A 8 and myasthenia gravis which was significantly higher in women than in men. Antibodies to skeletal muscle and thymomas were found to be much rarer in HL-A 8-positive patients than in HL-A 8-negative patients; HL-A 8-positive patients acquired the disease at an earlier age.HL-A 2-positive patients more often had thymomas and antibodies to skeletal muscle than HL-A 2-negative patients; HL-A 2-positive patients acquired myasthenia gravis at a later age.The fact that the clinical aspects of the HL-A 8-negative and HL-A 2-positive patients were different from those of the HL-A 8-positive and HL-A 2-negative patients justifies the hypothesis that there are two forms of myasthenia gravis.     

MYASTHENIA GRAVIS—Problems in Diagnosis

The possibility of myasthenia gravis must be considered in patients persistently complaining of weakness and fatigue. There may be many difficulties and pitfalls in differentiating myasthenia gravis from other disorders in which muscular weakness is a common complaint.Observation of a group of 36 patients with myasthenia gravis, and another group of 30 cases involving the differential diagnosis of myasthenia gravis, led to a conclusion that a physician should apply criteria carefully before arriving at a diagnosis of myasthenia gravis and instituting drug therapy, since nonmyasthenics may frequently respond with subjective improvement temporarily following administration of cholinergic drugs.Myasthenia gravis may be a more common disorder than was suspected in the past.     

HLA genotypes and HLA-linked genetic markers in Italian patients with classical 21-hydroxylase deficiency

Summary HLA genotype and HLA-linked marker data for 40 unrelated patients from central Italy and 2 unrelated patients from Sardinia with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) were analyzed. The results confirm that the HLA-linked 21-OH-def gene is associated with several different HLA determinants and complete HLA haplotypes, although the only determinant with significantly increased frequency was the complement C2 allele C2B. The HLA antigens B8 and DR3 were found in significantly decreased frequencies. The haplotype A3, Cw6, Bw47, BfF, DR7, which is exceptionally rare in the general population but which has been found in many other 21-OH-def patients from diverse geographical origins, was also found in one of the Italian patients. This and other HLA haplotype associations found among the Italian patients may represent mutations that have occurred on HLA haplotypes with genetic linkage disequilibrium or, alternatively, may represent mutations that have not yet had time to become randomly associated with different HLA complex determinants. The marked negative associations with B8 and DR3 could, however, result from an interaction between the gene products of the HLA complex and the 21-OH-def phenotype.     

Myasthenial gravis; problems in diagnosis

The possibility of myasthenia gravis must be considered in patients persistently complaining of weakness and fatigue. There may be many difficulties and pitfalls in differentiating myasthenia gravis from other disorders in which muscular weakness is a common complaint. Observation of a group of 36 patients with myasthenia gravis, and another group of 30 cases involving the differential diagnosis of myasthenia gravis, led to a conclusion that a physician should apply criteria carefully before arriving at a diagnosis of myasthenia gravis and instituting drug therapy, since nonmyasthenics may frequently respond with subjective improvement temporarily following administration of cholinergic drugs.Myasthenia gravis may be a more common disorder than was suspected in the past.     

Inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera from animals with experimental autoimmune myasthenia gravis

Conditions are described for an assay that allows the percent inhibition of α-bungarotoxin binding to acetylcholine receptors by antisera and monovalent antigen-binding fragments of antibody molecules (Fab) to be determined. Anti-Torpedo californica acetylcholine-receptor antisera, prepared in New Zealand White rabbits and Lewis rats, were tested for the ability to inhibit [¹²⁵I]-α-bungarotoxin binding to membrane-associated and detergent-solubilized T californica acetylcholine receptors. Similar inhibition studies were performed using rabbit antisera and antigen-binding fragments prepared against each of the four acetylcholine receptor subunits. Antisera and antigen-binding fragments prepared against intact receptor could inhibit a maximum of 50% of the α-bungarotoxin binding to solubilized receptor. The results using monovalent antigen-binding fragments indicated that the inhibition was not due to antibody-mediated aggregation of receptor molecules. Rabbits and rats immunized with receptor denatured by sodium dodecyl sulfate all produced antisera that could bind to nondenatured receptor, but none of these animals developed experimental autoimmune myasthenia gravis. These results suggest that the antigenic determinants present on acetylcholine receptors responsible for induction of experimental auto-immune myasthenia gravis are lost with sodium dodecyl sulfate denaturation. A strong correlation was also observed between the presence of experimental autoimmune myasthenia gravis in rats and rabbits and the ability of the antisera from these animals to inhibit 50% of α-bungarotoxin binding to solubilized acetylcholine receptors.     

Analysis of families at risk for insulin-dependent diabetes mellitus reveals that HLA antigens influence progression to clinical disease.

BACKGROUND: Individuals at risk for insulin-dependent diabetes mellitus (IDDM), with an affected first-degree relative, can be identified by the presence of islet cell antibodies (ICA). ICA-positive relatives progress at variable rates to IDDM and identification of those at highest risk is a prerequisite for possible preventative treatment. Those who develop IDDM may exhibit less genetic heterogeneity than their ICA-positive or ICA-negative relatives. Specific human leucocyte antigen (HLA) genes predispose to IDDM but could also influence the rate of progression of preclinical disease. Therefore, by comparing HLA antigen frequencies between first-degree relatives, we sought to identify HLA genes that influence progression to IDDM. MATERIALS AND METHODS: HLA antigen frequencies were compared in 68 IDDM, 53 ICA-positive, and 96 ICA-negative first-degree relatives from 40 Caucasoid families. Predictions were tested in a panel of 270 unrelated IDDM subjects. HLA typing was performed serologically (HLA class I and II) and by sequence-specific oligotyping (11th International Histocompatibility Workshop protocol) (HLA class II). ICA tests were measured by an internationally standardized indirect immunofluorescence assay. RESULTS: In general, known susceptibility class II HLA antigens increased in frequency and known protective class II HLA antigens decreased in frequency, from ICA-negative to ICA-positive to IDDM relatives. Thus, DR4 and DQ8 increased whereas DR2 and DQ6 decreased; DR3 and DQ2 increased from ICA-negative to ICA-positive relatives, but not further in IDDM relatives. The high-risk DR3, 4 phenotype increased across the three groups; DR4,X was unchanged, and DR3,X and DRX,X both decreased. The HLA class I antigen, A24, occurred more frequently in ICA-positive relatives who developed IDDM and, in 270 unrelated IDDM subjects, was associated with an earlier age at diagnosis of IDDM in those with the lower risk class II phenotypes DR4,4 and DR3,X. CONCLUSIONS: HLA-DR3 and DQ2 predispose to islet autoimmunity, but not development of clinical IDDM in the absence and DR4 and DQ8. DR4 and DQ8 predispose to the development of clinical IDDM in ICA-positive relatives, in combination with DR3-DQ2 and other haplotypes but not when homozygous. HLA-A24 is significantly associated with rapid progression to IDDM in ICA-positive relatives and with an earlier age at clinical diagnosis. Analysis of IDDM families reveals that HLA genes not only predispose to islet autoimmunity but influence progression to clinical disease. The findings have implications for identifying high-risk relatives as candidates for possible preventative therapy.