Epitope-specific suppression of antibody response in experimental autoimmune myasthenia gravis by a monomethoxypolyethylene glycol conjugate of a myasthenogenic synthetic peptide.

A synthetic peptide corresponding to a myasthenogenic region of Torpedo californica acetylcholine (AcCho) receptor (AcChoR) alpha subunit, AcChoR alpha-(125-148), was conjugated to monomethoxypolyethylene glycol (mPEG). Injection of mice with the mPEG-AcChoR alpha-(125-148) conjugate and subsequent immunization with whole Torpedo AcChoR suppressed the development of experimental autoimmune myasthenia gravis (EAMG) by electrophysiological criteria. In anti-AcChoR sera from these animals, the antibody response against unconjugated AcChoR alpha-(125-148) was decreased, while the antibody responses against whole AcChoR and other epitopes were not altered. There were no detectable changes in T-cell proliferation responses to AcChoR alpha-(125-148) or to whole AcChoR in these animals. Prior injections with a "nonsense" peptide-mPEG conjugate had no effect on responses to the subsequent immunization with whole Torpedo AcChoR. The results indicate that the mPEG-AcChoR alpha-(125-148) conjugate has epitope-specific tolerogenicity for antibody responses in EAMG and that the AcChoR alpha-subunit region comprising residues 125-148 plays an important pathophysiological role in EAMG. The epitope-directed tolerogenic conjugates may be useful for future immunotherapies of human myasthenia gravis. The strategy of specific suppression of the antibody response to a predetermined epitope by using a synthetic mPEG-peptide conjugate may prove useful in manipulation and suppression of unwanted immune responses such as autoimmunity and allergy.     

Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide

Many pathogenic antibodies in myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are directed against the main immunogenic region (MIR) of the acetylcholine receptor (AcChoR). These antibodies are highly conformation dependent; hence, linear peptides derived from native receptor sequences are poor candidates for their immunoneutralization. We employed a phage-epitope library to identify peptide-mimotopes capable of preventing the pathogenicity of the anti-MIR mAb 198. We identified a 15-mer peptide (PMTLPENYFSERPYH) that binds specifically to mAb 198 and inhibits its binding to AcChoR. A 10-fold increase in the affinity of this peptide was achieved by incorporating flanking amino acid residues from the coat protein as present in the original phage library. This extended peptide (AEPMTLPENYFSERPYHPPPP) was constrained by the addition of cysteine residues on both ends of the peptide, thus generating a cyclic peptide that inhibited the binding of mAb 198 to AcChoR with a potency that is three orders of magnitude higher when compared with the parent library peptide. This cyclic peptide inhibited the in vitro binding of mAb 198 to AcChoR and prevented the antigenic modulation of AcChoR caused by mAb 198 in human muscle cell cultures. The cyclic peptide also reacted with several other anti-MIR mAbs and the sera of EAMG rats. In addition, this peptide blocked the ability of mAb 198 to passively transfer EAMG in rats. Further derivatization of the cyclic peptide may aid in the design of suitable synthetic mimotopes for modulation of MG.     

Oral administration of a dual analog of two myasthenogenic T cell epitopes down-regulates experimental autoimmune myasthenia gravis in mice

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-regulated, antibody-mediated autoimmune diseases. The major autoantigen in MG is the nicotinic acetylcholine receptor (AChR). Two peptides, representing sequences of the human AChR alpha-subunit, p195-212 and p259-271, were previously shown to be immunodominant T cell epitopes in MG patients as well as, respectively, in SJL and BALB/c mice. A dual analog (termed Lys-262-Ala-207) composed of the tandemly arranged two single amino acid analogs of p195-212 and p259-271 was shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Furthermore, the dual analog could down-regulate myasthenogenic manifestations in mice with EAMG that was induced by inoculation of a pathogenic T cell line. In the present study, the ability of the dual analog to treat EAMG induced in susceptible C57BL/6 mice by native Torpedo AChR was evaluated. Mice that were diagnosed to have clinical symptoms of EAMG were treated with the dual analog by oral administration, 500 microg per mouse three times a week for 5-8 weeks. Treatment with the dual analog down-regulated the clinical manifestations of the ongoing disease as assessed by the clinical score, grip strength (measured by a grip strength meter), and electromyography. The effects on the clinical EAMG correlated with a reduced production of anti-AChR antibody as well as a decrease in the secretion of interleukin-2 and, more dramatically, interferon-gamma, in response to AChR triggering. Thus, the dual analog is an efficient immunomodulator of EAMG in mice and might be of specific therapeutic potential for MG.     

Human T-helper lymphocytes in myasthenia gravis recognize the nicotinic receptor alpha subunit.

Myasthenia gravis is a human disease caused by an autoimmune response against the nicotinic acetylcholine receptor (AcChoR). Since the molecular structure of AcChoR is well known, myasthenia gravis is an excellent system for studying the recognition of a complex membrane antigen in the human immune system. Human T-helper (TH) cell lines reactive to the AcChoR were isolated from four myasthenic patients by selection with native AcChoR from Torpedo californica. The selected TH cells could efficiently recognize native and fully denatured AcChoR. The vast majority of the TH-stimulating AcChoR epitopes were located on the denatured alpha subunit of AcChoR. Antibody competition experiments using a panel of rat anti-AcChoR monoclonal antibodies showed that 39-45% of the autoantibodies present in the sera of these same patients bound to the conformation-sensitive "main immunogenic region" (MIR), also located on the alpha subunit. However, AcChoR-induced stimulation of the T cells could not be inhibited with up to 20-fold molar excess of different rat anti-MIR monoclonal antibodies. These results suggest that the Torpedo AcChoR alpha subunit contains conformation-insensitive epitopes that play a role in the autosensitization of TH cells and that seem to be physically separated from the MIR. The specificity of the TH cell response may contribute to directing the B-cell response to other alpha-subunit determinants, such as the MIR itself.     

Specific immunosuppression of experimental autoimmune myasthenia gravis by denatured acetylcholine receptor.

Specific immunosuppression of experimental autoimmune myasthenia gravis (EAMG) was achieved by the use of a denatured preparation of the acetylcholine receptor (AcChoR) that did not in itself induce the disease. Torpedo californica AcChoR was irreversibly denatured by complete reduction and carboxymethylation in 6 M guanidine hydrochloride. Rabbits immunized with reduced carboxymethylated receptor (RCM-AcChoR) produced antibodies that reacted with both RCM-AcChoR and intact AcChoR. The specificity of anti-RCM-AcChoR antibodies is different from that of anti-AcChoR antibodies because the former are directed to only part of the antigenic determinants present in the intact receptor. RCM-AcChoR, which by itself is completely nonmyasthenic, was shown to be capable of both preventing the onset of EAMG and of reversing the clinical symptoms in myasthenic rabbits. In all cases the therapeutic effect of RCM-AcChoR administration on EAMG was accompanied by a change in the immunological specificity of the antibodies. The crossreactivity between AcChoR and RCM-AcChoR and the nonpathogenicity of RCM-AcChoR appear to be crucial in governing the specific immunosuppressive effects of RCM-AcChoR on EAMG.     

The nature of the active suppression of responses associated with experimental autoimmune myasthenia gravis by a dual altered peptide ligand administered by different routes

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T-cell regulated, antibody-mediated diseases. Peptides p195-212 and p259-271 of the human acetylcholine receptor (AChR) alpha-subunit, were previously shown to be immunodominant T cell epitopes in MG patients as well as in SJL and BALB/c mice, respectively. A dual altered peptide ligand (APL) composed of the two single amino acid analogs of the myasthenogenic peptides was shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Furthermore, the dual APL was shown to down-regulate the clinical manifestations of an established EAMG in C57BL/6 mice injected with Torpedo AChR (TAChR). In the present study we attempted the elucidation of the mechanism(s) by which the dual APL down-regulates EAMG-associated responses. It is shown here that the dual APL acts by actively suppressing, in a specific manner, myasthenogenic T cell responses. The active suppression is mediated, at least partially, by the up-regulation of the secretion of TGF-beta following administration of the dual APL. The up-regulated secretion of TGF-beta is accompanied by down-regulation of IFN-gamma and IL-2 [T helper (Th) 1-type cytokine] secretion and by an up-regulation of IL-10 secretion (Th2-type cytokine). Furthermore, the inhibitory effect of the dual APL could be adoptively transferred to p195-212 or TAChR-immunized mice. The down-regulation of IL-2 secretion and the ability of recombinant IL-2 to rescue lymph node cells of mice treated with the dual APL from a state of unresponsiveness suggests that the dual APL acts also, at least partially, by causing the cells to undergo anergy.     

Prevention of experimental autoimmune myasthenia gravis by manipulation of the immune network with a complementary peptide for the acetylcholine receptor.

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused, in part, by the production of autoantibodies against the main immunogenic region, amino acids 61-76, of the alpha chain of the acetylcholine receptor (AChR). Theoretically, induction of anti-idiotypic (Id) antibodies (Abs) should be a highly specific treatment for the disease by virtue of their potential ability to neutralize Abs to the AChR. We have tested this idea by attempting to evoke such anti-Id Abs by immunization with a peptide (termed RhCA 67-16) encoded by RNA complementary to the Torpedo AChR main immunogenic region and determining whether such treatment will prevent the development of EAMG. Immunization with RhCA 67-16, but not a control peptide termed PBM 9-1, was found to elicit the production of anti-Id Abs that blocked recognition of native Torpedo AChR by its Ab. This anti-Id Ab activity was ablated by incubation of the anti-RhCA 67-16 serum with RhCA 67-16, but PBM 9-1, prior to the assay for Ab binding to AChR. The anti-Id Ab-inducing activity of RhCA 67-16 was confirmed by the ability to produce a rat monoclonal Ab to RhCA 67-16 that showed anti-Id activity for polyclonal rat Ab reactive with AChR residues 67-76. Most importantly, RhCA 67-16 immunization also prevented the development of EAMG in Lewis rats challenged with Torpedo AChR (25% incidence versus 90% in the controls) and diminished the AChR Ab levels in animals injected with low doses of AChR. Our results suggest a therapy for MG and perhaps other autoimmune diseases through the induction of anti-Id Abs by peptide immunogens.     

Region of peptide 125-147 of acetylcholine receptor alpha subunit is exposed at neuromuscular junction and induces experimental autoimmune myasthenia gravis, T-cell immunity, and modulating autoantibodies.

A major antigenic region of native nicotinic acetylcholine receptors (AcChoR) has been identified by using a synthetic disulfide-looped peptide corresponding to alpha-subunit residues 125-147 of Torpedo electric organ AcChoR: Lys-Ser-Tyr-Cys-Glu-Ile-Ile-Val-Thr-His-Phe- Pro-Phe-Asp-Gln-Gln-Asn-Cys-Thr-Met-Lys-Leu-Gly. The peptide bound 26-56% of polyclonal antibodies induced in rat, rabbit, and dog by immunization with native AcChoR. Rats inoculated with 50 micrograms of unconjugated peptide developed helper T-cell responses, delayed hypersensitivity, and antibodies to native AcChoR. Anti-peptide antibodies were more reactive with native than denatured AcChoR and bound to the alpha subunit. Some reacted exclusively with mammalian muscle AcChoR, some induced modulation of AcChoR on cultured myotubes, but none inhibited binding of alpha-bungarotoxin to solubilized or membrane-associated AcChoR. Repeated immunization induced experimental autoimmune myasthenia gravis: clinical signs in one rat and electrophysiologic and/or biochemical signs in 10 of 11 rats. Thus, at least part of the corresponding region of the mammalian AcChoR alpha subunit is extracellular at the neuromuscular junction and a potential target for pathogenic autoantibodies in patients with acquired myasthenia gravis.     

重症肌无力全身表现及发病机制探讨

６４４例重症肌无力（ＭＧ）患者合并多种免疫有关性疾病，并且有多种自身抗体阳性，同时发现了一些骨骼肌以外受损害的表现，伴锥体束征者（ＰＳ）１１例，伴发癫痫４例，原因未明的周围神经病变３例，部分重症肌无力病人伴有血清ＡＬＴ增高，１４例ＭＧ患者的超声心动图，射血指数，２例心脏核素扫描，均未见明显异常。ＭＧ病人的骨骼肌以外损伤的表现，一般不需要特殊处理，随着重症肌无力的好转，绝大部分能够好转，其发病机制可能与乙酰胆碱受体抗体（ＡＣｈＲＡｂ）有关，或者是免疫泛化产生的其他免疫因素的作用，由此可见ＭＧ可能是一种主要累及神经肌肉接头处突触后膜上ＡＣｈＲ的全身性自身免疫性疾病。     

Anti-Sm-RNP activity in sera of patients with rheumatic and autoimmune diseases

Summary The sera of various rheumatic and autoimmune diseases were examined for the presence of anti- RNP/Sm activity. An enzymelinked immunosorbent assay (ELISA) was employed. Anti- RNP Ab's were detected in 18%, 20%, 28%, 16% of the sera of SLE, myasthenia gravis (MG), rheumatoid arthritis (RA) and thyroid diseases respectively. The anti- RNP Ab's belonged to the IgG and IgM isotypes. Most of the IgG anti- Sm antibodies were detected in SLE sera, but they were found also in two sera of MG and in one sera of RA patients. IgM anti- Sm antibodies were not found in SLE sera, but they were detected in low titer in MG, RA and autoimmune thyroid diseases. The activity against RNP and/or Sm was further confirmed by employing immunoblotting assays. In none of the patients, except those with SLE, was any clinical manifestation of SLE noted. The mere presence of anti- Sm antibodies of the IgG isotypes is not sufficient for the development of SLE, however, its presence is highly specific for SLE.     

Therapeutic vaccines in autoimmunity

Similarly to prophylactic vaccines whose purpose is to prevent infectious diseases, therapeutic vaccines against autoimmune diseases are based on their similarity to the putative causes of the disease. We shall describe here two such examples: a copolymer of amino acids related to myelin basic protein, in the case of multiple sclerosis, and a peptide derived from the nicotinic acetylcholine receptor (AChR), in the case of myasthenia gravis (MG). Copolymer 1 (Cop 1, glatiramer acetate, Copaxone) is a synthetic amino acid random copolymer, immunologically cross-reactive with myelin basic protein and suppresses experimental allergic encephalomyelitis in several animal species. Cop 1 slows the progression of disability and reduces relapse rate in exacerbating-remitting multiple sclerosis patients. It was approved by the Food and Drug Administration in 1996, and today is used by tens of thousands of patients. Cop 1 is a potent inducer of T helper 2 (Th2) regulatory cells in mice and humans, and Th2 cells are found both in the brains and spinal cords of Cop 1-treated mice. MG and experimental autoimmune MG are T cell-regulated, antibody-mediated autoimmune diseases. Two peptides, representing sequences of the human AChR alpha-subunit, p195-212 and p259-271, are immunodominant T cell epitopes in MG patients and in two strains of mice. Altered peptide ligand, composed of the tandemly arranged two single amino acid analogs, inhibits in vitro and in vivo MG-associated autoimmune responses. The active suppression is mediated by the CD4(+)CD25(+) immunoregulatory cells and is associated with the down-regulation of Th1-type cytokines and the up-regulation of the secretion of IL-10 and the immunosuppressive cytokine, transforming growth factor beta.     

Resolution of autoimmune oophoritis after thymectomy in a myasthenia gravis patient

Myasthenia gravis (MG) is an autoimmune disorder characterized by autoantibodies against acetylcholine receptors. MG is generally an isolated disorder but may occur concomitantly with other autoimmune diseases. We describe an eighteen-year-old girl with MG who was admitted to our clinic with secondary amenorrhea and diagnosed as autoimmune oophoritis. Since her myasthenic symptoms did not resolve with anticholinesterase therapy, thymectomy was performed. After thymectomy, her menses have been regular without any hormonal replacement therapy. To our knowledge, this is the first report on a patient with autoimmune ovarian insufficiency and MG in whom premature ovarian insufficiency resolved after thymectomy, without hormonal therapy.     

A 50-kDa ERK-like protein is up-regulated by a dual altered peptide ligand that suppresses myasthenia gravis-associated responses

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent antibody-mediated autoimmune diseases. A dual altered peptide ligand (APL) that is composed of the tandemly arranged two single amino acid analogues of two myasthenogenic peptides, p195-212 and p259-271, down-regulated in vitro and in vivo MG-associated autoreactive responses. The dual APL was shown to exert its beneficial effects by up-regulating ERK1,2 in CD4(+)CD25(+) regulatory cells. In this study, we investigated a novel 50-kDa ERK-like protein (ERK-50) that is up-regulated significantly in addition to ERK1,2 after treatment with the dual APL. We report here that ERK-50 was up-regulated in LN cells and in LN-derived T cells of mice that were immunized with the myasthenogenic peptides and treated with the dual APL. Moreover, ERK-50 was up-regulated in dual-APL- treated mice that were immunized with the Torpedo acetylcholine receptor. ERK-50 was demonstrated to be recognized by antibodies directed against the C and N termini of ERK1, against the C terminus of ERK2, and against general ERK. The 50-kDa ERK was shown to be stimulated by Con A, and inhibition of MEK1 down-regulated the 50-kDa ERK as was shown for ERK1,2. However, 4beta-phorbol 12-myristate 13-acetate (TPA) did not stimulate ERK-50. Finally, the activated ERK-50 was up-regulated in the dual-APL-induced CD4(+)CD25(+) regulatory cells. Thus, ERK-50 is suggested to be a novel ERK isoform, being up-regulated in response to treatment with the dual APL.     

Concomitant polymyositis and myasthenia gravis reveal malignant thymoma. A case report and review of the literature.

The frequency of thymona associated with autoimmune diseases has been reported to be increased, with 50% of thymoma patients also having myasthenia gravis (MG). Other autoimmune disorders, such as autoimmune erythroblastopenia and polymyositis (PM), have been less frequently associated with thymoma. The association of MG and PM with thymoma is rare. We here report the case of a 66-year-old woman whose concomitant MG and PM revealed malignant thymoma and review the other published cases of this association.     

Seronegative myasthenia gravis and Graves' disease. Is there a link?

Graves' disease (GD) and myasthenia gravis (MG) are common autoimmune diseases but their coexistence is very rare. They may possibly share the same pathogenetic mechanisms. Recent research has shown the involvement of autoantibodies, lymphocytes, cytokines and chemokines in the pathogenesis of MG and GD. It appears that Th17 cell lineage is involved in autoimmune thyroid disease (AITD) and seems to be key factor in the development of both MG and GD.A 34-year-old male with seronegative myasthenia gravis due to thymic hyperplasia was diagnosed with also GD and opthalmopathy. Several diagnostic and therapeutic issues regarding the relevant literature are discussed.     

A Prospective Assessment of the Characteristics of Dysphagia in Myasthenia Gravis

Fatigable muscle weakness is the clinical hallmark of the human autoimmune disease myasthenia gravis (MG). Weakness of the oropharyngeal muscles produces dysphagia, which continues to be a major source of morbidity in MG. In this study we prospectively assessed 20 patients with myasthenia gravis who described difficulties with swallowing. Videofluoroscopic assessment showed disordered swallowing in all, with abnormalities in oral, pharyngeal, and, to a lesser extent, oral preparatory phases. Of the 20 studied, 7 aspirated, most of whom did so silently. Laryngeal penetration occurred in many more patients. The characteristics of dysphagia in MG are described and compared with other neurological disorders that can produce dysphagia.     

Acetylcholine receptors and myasthenia gravis

Recent years have seen considerable progress in understanding the nature of the molecular events involved in neuromuscular transmission. The acetylcholine receptor (AChR) has been purified to homogeneity and acetylcholine-induced ion transport has been reconstituted by incorporation of pure AChR into artificial membranes. Immunization against purified AChR induces a condition, clinically and physiologically similar to the human disease myasthenia gravis, which is due to circulating anti-AChR antibodies. This model, experimental autoimmune myasthenia gravis, is proving useful for investigating the role of genetic factors in determining the immune response to AChRs and for testing various experimental approaches to specific treatment. Myasthenia gravis is an autoimmune disease in which there is loss of acetylcholine receptors at the neuromuscular junction. Anti-AChR antibodies can be detected in the majority of patients and they cause loss of AChR by a variety of mechanisms. Anti-AChR antibody is heterogeneous and not restricted in idiotype. The role of the thymus in MG is still uncertain, but recent experiments implicate the presence of a cell type in MG thymus which may be involved in autosensitization to AChR.     

Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the alpha subunit.

The majority of antibodies to the acetylcholine receptor (AcChoR), both in the human disease myasthenia gravis and in its experimental models, are directed against an extracellular area of the AcChoR alpha subunit called the main immunogenic region (MIR). We have studied the binding of anti-AcChoR monoclonal antibodies (mAbs) to 26 synthetic peptides corresponding to the hydrophilic parts of the human AcChoR alpha subunit. The binding sites for eight anti-MIR mAbs and for eight anti-alpha-subunit, non-anti-MIR mAbs were localized. Anti-MIR mAbs bound to one peptide corresponding to residues 63-80 of the human alpha subunit. A second panel of peptides corresponding to the various parts of the alpha-subunit segment 63-80 was synthesized. Anti-MIR antibodies bound to a peptide that contained the alpha-subunit sequence 67-76. Thus, a main constituent loop of the MIR is localized between residues 67 and 76 of the alpha subunit.     

Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits.

Seventeen cell lines producing monoclonal antibodies against Torpedo californica (torpedo) acetylcholine receptor (AcChoR) and its subunits were established. By using these antibodies as probes, we identified: (i) a similar antigenic determinant on alpha and beta torpedo subunits, (ii) a similar antigenic determinant on gamma and delta subunits, (iii) antigenic determinants unique for alpha or beta torpedo AcChoR subunits, (iv) a small region on the alpha subunit that dominates the immunogenicity of native torpedo AcChoR in rats (a monoclonal antibody directed at this region could bind to rat AcChoR in vivo and cause passive experimental autoimmune myasthenia gravis), and (v) antigenic determinants on torpedo subunits recognized in AcChoR from other species. The unexpected similarities between alpha and beta and between gamma and delta subunits raise the possibility that the complex four-subunit structure of AcChoR was derived from a simpler precursor and suggests that these antigenic similarities might reflect some structural and functional homologies.