Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies that are either directed to the muscle nicotinic acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase ...(MuSK). These autoantibodies define two distinct subforms of the disease-AChR-MG and MuSK-MG. Both AChR and MuSK are expressed on the postsynaptic membrane of the neuromuscular junction (NMJ), which is a highly specialized region of the muscle dedicated to receive and process signals from the motor nerve. Autoantibody binding to proteins of the postsynaptic membrane leads to impaired neuromuscular transmission and muscle weakness. Pro-inflammatory antibodies of the human IgG1 and IgG3 subclass modulate the AChR, cause complement activation, and attract lymphocytes; together acting to decrease levels of the AChR and AChR-associated proteins and to reduce postsynaptic folding. In patients with anti-MuSK antibodies, there is no evidence of loss of junctional folds and no apparent loss of AChR density. Anti-MuSK antibodies are predominantly of the IgG4 isotype, which functionally differs from other IgG subclasses in its anti-inflammatory activity. Moreover, IgG4 undergoes a posttranslational modification termed Fab arm exchange that prevents cross-linking of antigens. These findings suggest that MuSK-MG may be different in etiological and pathological mechanisms from AChR-MG. The effector functions of IgG subclasses on synapse structure and function are discussed in this review.
We have combined major histocompatibility complex-binding assays with immunization and tolerance induction experiments in HLA-DR3 transgenic mice to design apitopes (antigen-processing independent ...epitopes) derived from thyrotropin receptor (TSHR) for treatment of patients with Graves' disease (GD). A challenge model was created by using an adenovirus-expressing part of the extracellular domain of the thyrotropin receptor (TSHR289). This model was used to test whether current drug treatments for GD would have an impact on effective antigen-specific immunotherapy using the apitope approach. Furthermore, selected peptides were assessed for their antigenicity using peripheral blood mononuclear cell samples from patients with GD. A mixture of two immunodominant apitopes was sufficient to suppress both the T-cell and antibody response to TSHR when administered in soluble form to HLA-DR transgenic mice. Tolerance induction was not disrupted by current drug treatments. These results demonstrate that antigen-specific immunotherapy with apitopes from TSHR is a suitable approach for treatment of GD.
Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- ...and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.
Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in ...acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.
We report here the sequence and functional characterization of a recombinantly expressed autoantibody (mAb 131) previously isolated from a myasthenia gravis patient by immortalization of thymic B ...cells using Epstein-Barr virus and TLR9 activation. The antibody is characterized by a high degree of somatic mutations as well as a 6 amino acid insertion within the VHCDR2. The recombinant mAb 131 is specific for the γ-subunit of the fetal AChR to which it bound with sub-nanomolar apparent affinity, and detected the presence of fetal AChR on a number of rhabdomyosarcoma cell lines. Mab 131 blocked one of the two α-bungarotoxin binding sites on the fetal AChR, and partially blocked the binding of an antibody (mAb 637) to the α-subunit of the AChR, suggesting that both antibodies bind at or near one ACh binding site at the α/γ subunit interface. However, mAb 131 did not reduce fetal AChR ion channel currents in electrophysiological experiments. These results indicate that mAb 131, although generated from an MG patient, is unlikely to be pathogenic and may make it a potentially useful reagent for studies of myasthenia gravis, rhabdomyosarcoma and arthrogryposis multiplex congenita which can be caused by fetal-specific AChR-blocking autoantibodies.
Abstract Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR–MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the ...disease since thymus hyperplasia is a characteristic of early-onset AChR–MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR–MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein–Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR–MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR–MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR–MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected.
The auto-antigen repertoire in myasthenia gravis Vrolix, Kathleen; Fraussen, Judith; Molenaar, Peter C. ...
Autoimmunity (Chur, Switzerland),
2010-August, 8/1/2010, 2010-Aug, 2010-08-00, 20100801, Volume:
43, Issue:
5-6
Journal Article
Peer reviewed
Myasthenia Gravis (MG) is an antibody-mediated autoimmune disorder affecting the postsynaptic membrane of the neuromuscular junction (NMJ). MG is characterized by an impaired signal transmission ...between the motor neuron and the skeletal muscle cell, caused by auto-antibodies directed against NMJ proteins. The auto-antibodies target the nicotinic acetylcholine receptor (nAChR) in about 90% of MG patients. In approximately 5% of MG patients, the muscle specific kinase (MuSK) is the auto-antigen. In the remaining 5% of MG patients, however, antibodies against the nAChR or MuSK are not detectable (idiopathic MG, iMG). Although only the anti-nAChR and anti-MuSK auto-antibodies have been demonstrated to be pathogenic, several other antibodies recognizing self-antigens can also be found in MG patients. Various auto-antibodies associated with thymic abnormalities have been reported, as well as many non-MG-specific auto-antibodies. However, their contribution to the cause, pathology and severity of the disease is still poorly understood. Here, we comprehensively review the reported auto-antibodies in MG patients and discuss their role in the pathology of this autoimmune disease.
Abstract
In the autoimmune disease myasthenia gravis (MG), autoantibodies against the muscle AChR are mainly produced by both short- and long-lived plasma cells, which are resistant to standard ...immunosuppressive drugs (i.e. glucocorticoids). A novel therapy to eliminate plasma cells is the proteasome inhibitor bortezomib, which is used to treat patients with multiple myeloma (MM, a plasma cell malignancy). Previously, we demonstrated that bortezomib also reduced autoantibody titers in an animal model of MG (Gomez, A. M. J. Immunol. 2011). The thymus of MG patients is frequently enriched in germinal centers and contains plasma cells that produce autoantibodies in vitro, even after irradiation (which depletes B and T lymphocytes). We studied the in vitro effects of bortezomib in cultured thymus cells from MG patients undergoing therapeutic thymectomy. Treatment with a single dose of bortezomib blocked the production of these pathogenic autoantibodies, reduced the total IgG levels and eliminated plasma cells. Ultrastructural signs of apoptosis were detected in plasma cells as early as 8 h after addition of bortezomib; at 24 h, no plasma cells could be detected. Moreover, we demonstrated that the minimum concentration of bortezomib that eliminates plasma cells in vitro is 60-fold lower than the peak concentration found in MM patients treated with bortezomib, suggesting that low doses might be effective for eliminating plasma cells in patients with antibody-mediated autoimmune diseases.
Abstract We studied Ig heavy chain (VDJ) sequences and antigen reactivity of 412 immortalized B cell lines from the peripheral blood of 10 multiple sclerosis (MS) patients, 4 clinically isolated ...syndrome (CIS) patients and 6 healthy controls (HCs). 78/238 (32.8%) MS and CIS B cell lines were part of 9 clonally expanded B cell populations, of which 5 were present in multiple patients. Increased VH1 gene family usage was evidenced for MS B cells, with 29.2% expressing VH1–69. Affinity maturation in MS and CIS was indicated by increased Ig VDJ mutations. Autoantibody producing B cells reactive to intracellular antigens were significantly higher in MS (25%) and CIS (28%) patients than in HCs (5%), including 3/9 expanded B cell clones. Specificity for phosphatidylcholine was observed for 1/9 B cell clones. These findings indicate clonally expanded autoreactive B cells with affinity maturation in the peripheral blood in MS and CIS.
Abstract In myasthenia gravis (MG), the neuromuscular transmission is impaired mainly by auto-antibodies against the acetylcholine receptor (AChR) or MuSK. In about 5% of the MG patients, however, ...the auto-antigen is still unknown. We investigated whether these idiopathic MG patients (iMG) have auto-antibodies against ErbB proteins, which influence the AChR density at the NMJ. Our results show reduced mRNA expression levels of ErbB4 in thymus tissue of iMG patients compared to AChR-MG and non-MG patients, but we could not detect anti-ErbB antibodies in sera of iMG patients. Therefore, our results do not support a role for ErbB receptors as auto-antigens in iMG patients.
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