HLA antigens and susceptibility to myasthenia gravis

HLA-A, -B, -C and -DR antigen frequencies determined in a group of 73 myasthenia gravis (MG) patients were compared with those of a control group of 205 subjects. The strongest positive association with MG was found antigens B8 and DR3 (relative risks 9.56 and 8.84 respectively). Analysis of our data indicates that both antigens, independently from their linkage disequilibrium, are involved in susceptibility to MG. No relationship between HLA antigens on thymic pathology was observed in our material. In male MG patients the association with DR3 was weaker than in female patients. The difference in DR3 frequency between male and female patients was statistically significant; no significant difference was found for antigen B8. It appears that DR3 contributes to development of MG only in females. In male patients aged more than 30 years at the onset of disease, MG was not associated with B8 or DR3. In contrast, in female patients aged more than 30 years at the onset of disease there was a strong association of B8 and DR3 with the disease.     

Association with HLA DQ of early onset myasthenia gravis in Southeast Texas region of the United States

Forty-four Caucasian American myasthenia gravis (MG) patients from Southeast Texas underwent high resolution HLA DQ analysis. For the majority of patients who were late onset or male, no significant associations with DQ were observed. However, associations with DQ increased in female patients and early onset patients. At the allele level, DQB1 *0503, *0604, *0502 and *0402 collectively contributed to a positive association of the DQ locus with early onset MG (EOMG), while individually failing to show significant association. At DQ level, the novel haplotype DQA1*0401:DQB1*0201 was the primary factor in the association of combined DQ loci with early onset. In addition, *0104:*0503, *0102:*0604, *0102:*0502 and *0303:*0402 collectively contributed to the positive association of the haplotype loci. DR3-DQ2.5cis, a well known risk factor for MG in Western Eurasia, was not found associated with disease in any group. For typical EOMG [early onset, no thymoma, anti-acetylcholine receptor (AChR) antibody (Ab) positive] no association with DQA1 locus was found, however DQB1*0604 demonstrated an 'uncorrected' positive association. A few DQ haplotype (DQA1:DQB1) were positively associated with typical EOMG; a positive individual association for *0401:*0201 was complimented by the contributions of *0102:*0604 and *0303:*0402 haplotypes. A small minority of patients that were atypical and EOMG had a strong genetic association with DQA1*0104:DQB1*0503, the group included an anti-MuSK Ab positive and an anti-AChR negative patient. This report finds common ground with European studies regarding MuSK association; however similarities in association for typical early onset disease resembled HLA risk factors in East Asia and Southern Europe.     

重症肌无力患者肌肉AChR亚单位基因的表达

目的：探讨重症肌无力（MG）患者眼外肌易于受累的机制。方法：采用RT-PCR对正常人眼外肌、其他骨骼肌群（8例）以及MG患者肋间肌（10例）AChRα、β、γ、ε、δ亚单位mRNA的表达进行分析，同时采用ELISA方法检测83例MG患者血清AChR抗体对不同骨骼肌群AChR的反应。结果：①正常人眼外肌与其他骨骼肌一样均可见AChRα、β、γ、ε、δ亚单位mRNA的表达；②全身型MG患者肌肉AChRα P3A^＋／β—actin较正常对照组高（P=O．028）；③眼肌型、全身型MG抗成人腓肠肌AChR抗体显著高于抗胎儿肌肉AChR抗体（P=0．001，0．016）。结论：MG患者眼外肌易受累机制可能与眼外肌AChR亚单位mRNA表达水平及眼外肌的内在易感性有关。     

HLA in myasthenia gravis: From superficial correlation to underlying mechanism

Myasthenia gravis (MG) is a rare autoimmune disease characterized by muscle weakness and abnormal fatigability. Like many other autoimmune diseases, genetic contribution to MG has been studied where the HLA system appears to play the most vital role. Although many correlations have been revealed in these studies, the underlying mechanism for them is still in the veil. Based on current evidence, we propose two synergetic mechanisms underlying the MG predisposition via HLA. In brief, the first advocates specific MHC II-peptide patterns that influence the efficacy of antigen presentation, and the second emphasizes the role of classical MHC alleles in shaping the TCR repertoire for MG predisposition. Besides, possible explanations for unresolved or controversial MG-related epidemiological phenomenon or clinical problems are addressed as well. Then, we discuss three factors influencing the effect of HLA on MG: gender discrepancy, inflammatory microenvironment, and epigenetic regulation. Lastly, from a provisional angle, we introduce several precautious treatments for people highly predisposed to MG. Although this is a review focusing on MG, the underlying mechanisms might be applicable in other autoimmune diseases as well.     

Natural peptide ligand motifs of two HLA molecules associated with myasthenia gravis

The peptide motifs of two HLA molecules, B8 and DR3(17), whichare associated with autoimmune diseases including myastheniagravls, were determined from natural peptide pools using Edmandegradation. The majority of HLA-B8 liganda are nonamere preferentiallyterminated by leucine. As a characteristic feature of the HLA-B8motif, there is a high degree of conservation of positivelycharged amlno acids at position 3 and 5, exclusively lysineat position 3, and lysine or arglnine at position 5. Additionalevidence for this allele-specific motif is the presence of thesefeatures in several viral peptldes recognized by HLA-B8 restrictedT cells. The DR3(17) motif is characterized by four conservedanchor-like positions ordered in an almost symmetrical arrangement,as has been found for DR1 and DR5 motifs. A first hydrophobic/aromaticanchor three to four residues apart from the N-terminus (atrelative position 1) appears to be a common feature of DR liganda.The second anchor is an aspartate at relative position 4, whichis likely to be the DR3(17)-speclfIc contact site in the groove.Two additional conserved positions closer to the C-terminusare occupied by charged amino acids at relative position 6 andby hydrophobic/aromatic residues at positions 8, 9, or 10. Eightindividual naturally processed DR17 ligands were sequenced andwere found to be derived from exogenousiproteins and cytoplasmicmembrane receptors. These natural peptldes conform well to thedetermined motif. A single exchange of the anchor-like positionsin a model peptide abrogated binding to DR17⁺ cells. On thebasis of the DR17 motif, peptldes from the acetylcholine receptor(AChR),the autoimmune target in myasthenia gravls, were selected thatshould contain candidate epitopes for AChR-speclflc T helpercells. Several peptide sequences which were reported to activateT cells from DR3⁺ individuals were among this collection.     

HLA class II and class I polymorphism in Venezuelan patients with myasthenia gravis

Oligotyping performed among ethnically mixed Venezuelan patients with myasthenia gravis (MG) and controls has revealed positive associations of HLA class I A*31, B*08, B*39, B*40, C*15, C*17, and class II DRB1*09 and negative associations of DQB1*06 and DQA1*02 with the disease. Sequential removal of human leukocyte antigen B (HLA-B) alleles when relative predispositional effects (RPEs) were looked for demonstrated that B*08 is the allele group with the largest contribution in the overall MG patients followed by B*39 and B*40. Several specificities (A*31, B*08, C*17, DRB1*03, DQA1*05, and DQB1*02) indicated increased frequencies among patients with thymic hyperplasia versus patients without hyperplasia or controls. Tests to identify alleles with the strongest association to MG in our patients detected DRB1*13 and B*38 as possible predisposing secondarily associated alleles in patients with hyperplasia. The associations observed disappear after Bonferoni correction of probability values and have been described in patients of Caucasian and/or Oriental ethnic background. Thus, our results reflect the heterogeneity of our population and of the patients tested and suggest a limited influence of several HLA genes in this heterogeneous disease or that these might be only markers of nearby non-HLA genes responsible for the susceptibility or resistance effect.     

Schwann cells and myasthenia gravis. Preferential uptake of soluble and membrane-bound AChR by normal and immortalized Schwann cells, and immunogenic presentation to AChR-specific T line lymphocytes.

The normal neuromuscular synapse is formed by the intimate association of nerve endings, postsynaptic end-plate foldings in the muscle fiber, and nonmyelinating Schwann cells (SC) sealing the synaptic ramifications. Because SC have been recognized recently to have an immunogenic potential inducible to present protein autoantigens to autoimmune T lymphocytes, and considering their close proximity to the acetylcholine receptor (AChR)-bearing postsynaptic membranes, presentation of soluble and membrane vesicle-bound AChR to appropriate T cells was investigated. Short-term monolayer cultures of SC isolated from neonatal rat sciatic nerves, as well as cells of an immortalized SC line of similar origin, were fully able to present the relevant molecular epitopes to major histocompatibility complex (MHC) compatible AChR-specific T line lymphocytes immunogenically. Presentation of AChR was restricted by RT1.B (I-A) MHC class II products. Both types of cultured rat SC were inducible to expression of MHC class I and II products, and they were able to phagocytose AChR-enriched membrane vesicles preferentially. In contrast, phagocytosis of latex particles by SC was negligible. These data qualify perisynaptic SC as potential presenter cells of autoimmunogenic AChR in myasthenia gravis. Thus, SC may play a critical and as-yet unpredicted regulatory role in the cellular pathogenesis of myasthenia gravis.     

HLA class I and class II polymorphisms in Saudi patients with myasthenia gravis

Myasthenia gravis (MG) is a rare autoimmune disease of the neuromuscular junction. MG has been shown to be associated with many HLA antigens in different populations. Here we have analysed the frequency of HLA‐A, B, DR and DQ in a group of Saudi MG patients and compared their results to a group of healthy controls. MG in Saudi patients is found to be associated with HLA‐A*23, B*08, B*18, DRB1*16 and DRB1*13. The strongest association was with HLA‐B*08, which was associated with young age at onset and female gender. Our results are in line with other published results from around the world and warrant fine mapping of the area using microsatellite to map the disease gene.     

AChRα211的分泌表达及其对肌无力患者血清中AChRAb的清除作用

目的在毕赤酵母(P．pastoris)中分泌表达AChRα211，研究其对重症肌无力患者血清中AChRAb的清除作用。方法以质粒pUC—AChRα211为模板，PCR扩增人AChRα21 DNA片段，插入真核表达载体pPIC9K中。重组质粒转入P．paatoris GS115后，经甲醇诱导分泌表达AChRα211，用Sepharose Q离子交换柱和Superdex 75分子排阻层析进行纯化。Western blot和ELISA进行免疫活性分析后，将目的蛋白偶联于CNBr-Sepharose 4B树脂上，研究该免疫吸附剂对肌无力患者血清中AChRAb的清除作用。结果双酶切鉴定和序列分析表明，AchRα21 DNA片段已正确插入到pPIC9K载体中。重组菌P．pastoris GS115／pPIC9K—AChRα211经甲醇诱导可分泌表达目的蛋白AChRα211，经阴离子交换柱和分子排阻层析可得95％纯AChRα211。并制备成免疫吸附剂。Western blot和ELISA分析表明，重组蛋白具有与天然AChRα亚基相似的免疫活性。免疫吸附实验表明该重组蛋白可特异性地去除肌无力患者血清中的AChRAb。结论在P．pastoris中分泌表达并纯化重组蛋白AChRα211，制备成一种新的免疫吸附剂AChRα211-Sepharose，可清除肌无力患者血清中的AChRAb。     

A variant of childhood-onset myasthenia gravis: HLA typing and clinical characteristics in Japan

To investigate the correlation between clinical features and HLA DR/DQ genetic variability in myasthenia gravis (MG), we evaluated HLA DR/DQ allele frequencies in 87 Japanese patients with childhood-onset disease. HLA genotypes DRB1*1302/DQA1*0102/DQB1*0604 and DRB1*0901/DQA1*0301/DQB1*0303 were significantly higher in patients than in healthy controls (P(c) < 0.0001, RR = 5.5; P(c) < 0.0001, RR = 8.5, for two genotypes, respectively). Patients who had a significantly higher likelihood of the HLA types DRB1*1302/DQA1*0102/DQB1*0604 or DRB1*0901/DQA1*0301/DQB1*0303 belonged to the latent general type (LG) of MG; this is clinically ocular type, but shows myasthenic electromyographic findings in extremity muscles. The LG type of MG was observed in 78% of patients exhibiting the clinically ocular type; this group comprised approximately 75% of patients with childhood-onset MG. These date suggest that LG type of MG may present a particular subset of childhood-onset MG, which is associated with the specific HLA subtypes DRB1*1302/DQA1*0102/DQB1*0604 and DRB1*0901/DQA1*0301/DQB1*0303.     

Profile of the regions on the alpha-chain of human acetylcholine receptor recognized by autoantibodies in myasthenia gravis.

Eighteen synthetic overlapping peptides encompassing the entire extracellular part (residues alpha 1-210) of the alpha-chain of human acetylcholine receptor (AChR) and a 19th peptide (residues alpha 262-276) corresponding to an extracellular connection between two transmembrane regions were prepared and used for the measurement, by solid-phase radioimmunoassay, of the binding of autoantibodies in plasma from myasthenia gravis (MG) patients. Autoantibodies were found to recognize only a limited number of the synthetic peptides. The regions recognized resided predominantly within the areas alpha 10-30, alpha 111-145 and alpha 175-198 and, less frequently, region alpha 45-77. Differences in the recognition profile of the peptides from patient to patient indicated that the autoantibody responses were under genetic control. However, by using a mixture of the appropriate peptides, it was possible to determine autoantibodies in all 15 myasthenia sera and to distinguish between these, normal human sera and other neurological or autoimmune diseases. The mapping of the continuous antigenic regions recognized by autoantibodies on the alpha-chain of human AChR has permitted a comparison of the regions recognized by autoantibodies and autoimmune T-cells from the same donor. It also provided a peptide-based direct antibody binding method for diagnosis of MG.     

Responses in vitro of peripheral blood lymphocytes from patients with myasthenia gravis to stimulation with human acetylcholine receptor alpha-chain peptides: analysis in relation to age, thymic abnormality, and ethnicity

Peripheral blood lymphocytes (PBLs) were isolated from 24 patients with myasthenia gravis of three ethnic groups (Caucasian, African American, and Hispanic) and ten healthy individuals. We determined the in vitro proliferative responses of the PBL samples to each of 18 overlapping synthetic peptides corresponding to the entire main extracellular domain (residues 1-210) of the alpha-subunit of human acetylcholine receptor. The profiles of the T-cell responses (expressed in stimulation index [SI]) to the peptides varied among the 24 patient samples. There was a significant difference in the overall patient responses relative to controls toward 17 of 18 peptides. T cells from the patients gave responses greater than control mean SI + 4 standard deviation (Z(SI) > 4) to 2 approximately 9 peptides/sample. Six peptides, alpha 23-38, alpha 34-49, alpha 78-93, alpha 122-138, alpha 146-162, and alpha 182-198, were recognized with Z > 4 level by 42% to 58% of the patients' PBLs. The grouped patient responses, divided according to age, thymic diagnosis, or ethnicity, were compared with controls and with each other. Significant differences were observed between early- and late-onset cases in recognition of residues alpha 34-49 (p = 0.015) and alpha 78-93 (p = 0.053), and in recognition of residues alpha 12-27, alpha 56-71, alpha 134-150, and alpha 146-162 (0.0072 < p < 0.064) when two ethnic groups were compared with each other.     

Differential presentation of group A streptococcal superantigens by HLA class II DQ and DR alleles

Superantigens have been implicated as pivotal mediators of severe invasive group A streptococcal (GAS) infections, by virtue of their potent immunostimulatory activity. HLA polymorphism has been suggested to influence the susceptibility to severe invasive GAS infection. Here we studied the influence of allelic and isotypic variation of HLA class II molecules on GAS superantigen-induced immune responses using cells derived from patients with bare lymphocyte syndrome, untransfected or transfected with various HLA class II alleles. Significantly higher proliferative responses were detected when streptococcal pyrogenic exotoxin (Spe) A was presented by cells expressing DQA1*0101/DQB1*0302 (DQ3.2), as compared to cells expressing DR1, DR4, or DR5 alleles (p=0.0002-0.01). In contrast to SpeA, SpeC was preferentially presented by DR4 as compared to DQB1*03 (p=0.04). In agreement with the proliferation results, a significantly higher frequency of IL-2-, TNF-alpha-, TNF-beta-, and IFN-gamma-producing cells was detected when SpeA was presented by HLA class II DQB1*03 alleles as compared to DR4 (p=0.0002-0.04). Binding experiments showed a high affinitybinding of SpeA to both class II DR4 and DQB1*0302, and there was no significant difference in SpeA binding affinity between the two alleles. The data confirm the effect of allelic polymorphism in superantigen responses and show that different superantigens are preferentially presented by distinct class II alleles.     

Antibodies to nucleic acids in myasthenia gravis.

An increased frequency of antibodies to native DNA, thymocytes, and striated muscle was found in patients with myasthenia gravis (MG). The prevalence of such antibodies lends considerable support to the concept of MG as an autoimmune disorder and militates in favor of major abnormalities in the thymic dependent immune system. There was no correlation between serum-blocking activity to acetylcholine receptor protein and antibodies to thymocytes.     

Characterization of anti-acetylcholine receptor (AChR) antibodies from mice differing in susceptibility for experimental autoimmune myasthenia gravis (EAMG).

In the murine model for EAMG we investigated the relation between disease susceptibility and fine specificity of anti-AChR antibodies obtained from high susceptible C57Bl/6 and low susceptible BALB/c mice after immunization with Torpedo acetylcholine receptor (tAChR). Anti-AChR MoAbs with fine specificity for the main immunogenic region (MIR), the alpha-bungarotoxin (alpha-BT)/acetylcholine binding sites and other extra- and intracellular epitopes were isolated from both mouse strains. In total, nine out of 38 MoAbs obtained from C57Bl/6 mice were directed against extracellular epitopes on mouse AChR in contrast to only one out of 27 MoAbs from BALB/c mice. A difference in antibody repertoire may underlie the difference in pathogenic response observed between these mouse strains. These results indicate that strain-specific differences in disease susceptibility in murine EAMG may be related to differences in the available repertoire of potential pathogenic antibodies.     

Studies on HLA antigens and cellular and humoral autoimmune phenomena in patients with myasthenia gravis.

The HLA phenotype of fifty-four patients with myasthenia gravis (MG) was compared to that of 600 controls. When male and female patients were compared separately with the control group, HLA-B12 was increased in MG males (P less than 0-023) and HLA-A1, B8 in MG females (P less than 0-023). In addition, HLA-A1, B8 was correlated with the early onset type of MG and with a more severe clinical course (Osserman scale IIb-III) in female patients. Cell-mediated immune responses were studied in a twenty-one hospitalized MG patients. Specific cell-mediated immunity to highly purified muscle proteins was investigated using the two-step-leucocyte migration agarose test and general cell-mediated immunity was studied by determining the cutaneous response to four extrinsic antigens. Cellular immune activity to muscle antigens occurred in fourteen out of twenty-one patients (67%) with MG and with one exception, in none of the controls. There was no significant difference in the LIF inducing ability of the different muscle antigens. A statistically significant correlation between HLA-A1 and B8 and either cell-mediated immune reactivity to muscle antigens or humoral autoimmune phenomena or clinical parameters was not found. Only a trend toward an association between HLA-A1 and B8 and a cell-mediated immune response to muscle antigens could be demonstrated. Positive delayed skin reactions to extrinsic antigens were observed with the same frequency as in healthy blood donors.     

Lymphorrhage localized to the muscle end-plate in myasthenia gravis

Lymphocytic infiltration of muscle (lymphorrhage) is occasionally observed in myasthenia gravis. We describe a 56-year-old woman with autoimmune myasthenia gravis, whose muscle biopsy specimen demonstrates acute cellular reaction localized to the end-plate region. This observation raises the possibility of a more direct role of the cellular immune system in the pathogenesis of the disease, and provides a morphologic link between the human and the experimental myasthenia gravis.     

Update in immunosuppressive therapy of myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction. Immunosuppressive treatments are part of the therapeutic armamentarium in MG. Long-term systemic steroid administration carry considerable risks and adverse events. Consequently, steroid-free immunosuppressive therapy is necessary to reduce the dose or discontinue steroids. First immunosuppressive drug trials in MG were performed in the mid-60s using standard and nonspecific immunosuppression. Since then, only few randomized controlled clinical trials were conducted in MG and assesed drug efficacy in terms of its steroid-sparing capacity and the ability to reduce myasthenic signs and symptoms. Treatment strategy in MG is quite challenging, mainly due to the disease heterogeneity in terms of clinical presentation, immunopathogenesis and drug response. To solve this dilemma, emerging treatment are based on biological drugs and use new targets of the immune pathway.