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.     

Specific activation of lymphocytes against acetylcholine receptor in the thymus in myasthenia gravis

In 13 patients with myasthenia gravis, spontaneous in vitro production of antibody to acetylcholine receptor (AChR) by thymic cells was observed in seven patients, by bone marrow cells in nine, by peripheral blood cells (PBL) in six, and by lymph node cells in nine. The rate of anti-AChR production in culture closely correlated with the serum anti-AChR level. Specific activity of the immunoglobulin (Ig) G spontaneously produced (anti-AChR/total IgG) was about 10-fold higher in the thymus than in bone marrow, peripheral blood, or lymph node cultures. Pokeweed mitogen (PWM) enhanced anti-AChR production only by PBL. With neither thymus nor lymph node cells did PWM stimulate anti-AChR production, although it greatly enhanced total IgG production. In bone marrow, it depressed both, and it appeared that the anti-AChR was derived from long-lived plasma cells that may be responsible for delaying the fall of serum anti-AChR levels after thymectomy. The results suggest that AChR-specific cells are selectively activated in the thymus, and this may help to explain the benefits of thymectomy in myasthenia gravis.     

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.     

Ocular myasthenia gravis secondary to thymoma

Thymoma is a neoplasm that originates from the epithelial cells of the thymus gland. Approximately 15% of patients with thymoma will present with symptoms of myasthenia gravis (MG),an autoimmune disorder involving the neuromuscular junction. Approximately 50% of patients with symptoms of myasthenia gravis will have thymoma. This paper presents a case report of thymoma and discusses the ocular and systemic manifestations of this uncommon neoplasm. Special emphasis is placed on the association between thymoma and myasthenia gravis.     

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.     

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 antigens and acetylcholine receptor antibodies in penicillamine induced myasthenia gravis.

Antibodies to the acetylcholine receptor and HLA antigens have been studied in patients with myasthenia gravis occurring in association with penicillamine treatment. The properties of the antiacetylcholine receptor in these patients differed from those in patients with idiopathic myasthenia gravis in terms of specificity and affinity. These patients had an increased prevalence of HLA Bw35 and DR1 compared to controls and a decreased frequency of B8 and DR3 compared to patients with idiopathic myasthenia gravis. Likewise, they had a decreased frequency of DR4 compared to patients with rheumatoid arthritis. These data provide supportive evidence for a role for penicillamine in the induction of myasthenia gravis in genetically predisposed individuals.     

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.     

Antibodies to an antigen of human thymus epithelial tissue common to the epidermis of the skin in malignant myasthenia]

It has been demonstrated by the indirect immunofluorecent technique that many of the sera of patients with myasthenia gravis react with the anticells of the human thymus epithelial tissue. Sorption of the sera with the suspension of the epidermis cells and the homogenates of the tissues of other human organs showed that the epithelial cell antigen with which the sera of patients with myasthenia reacted were epidermal heteroorganic thymus antigens, i.e. common for the thymus epithelium and skin epidermis. The presence of antibodies to the cells of the epithelial tissue of the thymus in the sera of patients suffering from myasthenia gravis permits to suppose the existence of an immunopathological process against the thymus tissue antigens (including the heteroorganic structures of its epithelium) in this disease.     

Using swallow sound and surface electromyography to determine the severity of dysphagia in patients with myasthenia gravis

One of the main symptoms of patients with myasthenia gravis is dysphagia, which will reduce the patients’ nutritional absorption and influences the quality of living. The activity of swallowing involves interaction and coordination between a variety of muscles and the nervous system. It is, therefore, difficult for clinicians to detect dysphagia. Furthermore, the symptoms of myasthenia gravis are unstable, making clinical judgment troublesome. How to accurately diagnose the severity of dysphagia in the clinic has become an important research topic. This paper proposes a dysphagia severity discrimination system for patients with myasthenia gravis. It uses a non-invasive Adam's apple microphone and surface electromyography to collect the swallow signal generated by sound and muscle activity when the patient swallows water. The system then extracts features to discriminate the severity of dysphagia during each swallow phase. The experimental results show that the system can provide concrete features for clinicians to diagnose dysphagia in myasthenia gravis patients.     

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.     

Identification of autoantibodies associated with rippling muscles and myasthenia gravis that recognize skeletal muscle proteins: possible relationship of antigens and stretch-activated ion channels.

The role of mechanosensitive calcium channels in skeletal muscle physiology is not understood. This study takes advantage of an autoimmune neuromuscular disorder (myasthenia gravis associated with rippling muscles) to identify components in the skeletal muscle myocyte that may play a role in mechanosensitive calcium channel activity. Rippling muscles are characterized by stretch or percussion activated wave-like muscle contractions that do not require motor unit action potentials for propagation. Autoantibodies from the sera of patients with autoimmune rippling muscles (associated with myasthenia gravis) are directed against high molecular weight muscle proteins. Some of these proteins are uniquely recognized by antisera from patients with autoimmune rippling muscles. This suggests these autoantigens are distinct from those normally associated with myasthenia gravis, and may play a role in the mechanosensitive activation of muscle contraction.     

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.     

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.     

Myasthenia Gravis: Anesthetic and Surgical Management of the Patient Undergoing Thymectomy

Experience in the anesthetic and surgical management of 25 patients with myasthenia gravis is recorded. These are subdivided into two groups: those operated on during the period 1950-1958 and those operated on during the period 1959-1964. The purpose of this paper is to indicate improvement in mortality and morbidity due to three major advances: (1) use of the decamethonium diagnostic test in a myasthenia gravis clinic; (2) improvements in assessment and management of respiratory insufficiency; and (3) avoidance of anticholinesterase treatment in the immediate and early postoperative recovery period.Fourteen patients with myasthenia gravis, including five with thymoma and two who were refractory to medication, were in the second (1959-1964) group. There were no deaths and no myasthenic or cholinergic crises. Three prophylactic tracheostomies were performed. No emergency bronchoscopies or tracheostomies were required.     

Galectin-1 is expressed by thymic epithelial cells in myasthenia gravis

Galectin-1, a member of a family of carbohydrate binding proteins, is synthesized by thymic epithelial cells in normal juvenile thymus, and mediates adhesion of immature T cells to thymic epithelium. Because cell adhesion molecules are proposed to play a role in the thymic hyperplasia and neoplasia seen in the autoimmune disease myasthenia gravis, we examined the expression of galectin-1 in myasthenic thymi. We detected abundant galectin-1 expression in thymic epithelial cells in 27 hyperplastic and neoplastic thymi from patients with myasthenia gravis. Primary cultures of neoplastic epithelial cells from a thymoma continued to express galectin-1, and bound immature T cells; T cell binding was inhibited by the addition of the -galactosides lactose and thiodigalactoside, suggesting that galectin-1 on the thymoma cells and a saccharide ligand on the T cells participated in cell-cell adhesion. Expression of galectin-1 by thymic epithelial cells may play a role in the thymic pathology seen in myasthenia gravis.     

重症肌无力风险基因挖掘

目的:挖掘重症肌无力(Myasthenia gravis,MG)可能的风险基因。方法:通过人工挖掘在Pub Med数据库收集重症肌无力风险基因,通过Gene数据库获取重症肌无力风险基因编号,用以表示基因或者其相应的蛋白。应用基因功能分析软件DAVID(http://david.abcc.ncifcrf.gov/)对重症肌无力风险基因进行KEGG通路富集分析,挖掘重症肌无力风险通路,进而对任意两个通路进行关联分析。应用基因功能分析软件DAVID的Gene Ontology,对MG风险基因进行功能注释,以P0.01来判定注释是否有显著意义。结果:(1)本研究挖掘出97个重症肌无力的风险基因,KEGG基因富集分析共筛选出44条与重症肌无力显著相关的通路,主要包括多种自身免疫性疾病相关通路、信号转导相关通路、肿瘤相关通路、抗原的加工提呈通路等等。(2)以上44条风险通路两两通路间均具有相关性(P.0.01)。结论:本研究共挖掘出44条重症肌无力风险通路,8个重症肌无力风险基因,分别为:NF-kB、TNFR、MEK、AP-1、Raf、MEK1/2、MSK1、TAPBP。其中,MEK同时出现在多个风险通路中,考虑其风险性更高。     

Experimental models of myasthenia gravis: lessons in autoimmunity and progress toward better forms of treatment

The nicotinic acetylcholine receptor (AChR) is a large membrane protein found in muscle cells. It is involved in the transformation of acetylcholine packets into a membrane depolarization, which thereby leads to a muscle twitch. This large, complex molecule is the target of the autoimmune attack in myasthenia gravis, and much has been learned in the past decade about myasthenia by the induction of autoimmunity to AChR in experimental animals. Experimental autoimmune myasthenia gravis (EAMG) has been produced in a variety of animals by immunization with AChR or AChR-like material, or by the passive transfer of anti-AChR antibodies or lymphocytes from afflicted animals into normal animals. EAMG is a remarkably faithful model of human myasthenia and has provided much information about how the immune response to AChR progresses and how weakness and damage to the neuromuscular junction ensure. EAMG has also allowed the development of a number of revolutionary forms of treatment in which only the abnormal response to AChR is restrained, and other necessary immune functions are left intact. These advances in treatment are not far from being tested in human myasthenia gravis. The experience gained in applying these concepts in EAMG and human myasthenia will be helpful in developing similar forms of treatment for other autoimmune diseases.     

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.     

Somatic hypermutation and selection of B cells in thymic germinal centers responding to acetylcholine receptor in myasthenia gravis

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig V(H) genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.