'Split tolerance' induction by intrathymic injection of acetylcholine receptor in a rat model of autoimmune myasthenia gravis; implications for the design of specific immunotherapies.

Experimental autoimmune myasthenia gravis (EAMG) in the Lewis rat, induced by a single injection of acetylcholine receptor (AChR) protein, is a model used to study human myasthenia gravis (MG). The production of anti-AChR antibodies in the animal model and human MG is T cell-dependent, and AChR-specific T cells have been considered as a potential target for specific immunotherapy. Intrathymic injection of antigens induces antigen-specific tolerance in several T cell-mediated autoimmune models. We examined the effect of intrathymic injection of AChR on T cell responses and the production of antibodies to AChR in EAMG rats. Primed lymph node cells from rats receiving intrathymic injection of AChR exhibited reduced proliferation to AChR with marked suppression of interferon-gamma (IFN-gamma) secretion in the antigen-stimulated culture, compared with those of rats injected with PBS. However, neither anti-Narke AChR nor anti-rat AChR antibody production was suppressed or enhanced in intrathymically AChR-injected animals compared with that of animals injected intrathymically with PBS or perithymically with AChR. This 'split tolerance' may be attributable to the suppression of type-1 T helper cells (Th1). Our results suggest that the suppression of Th1 function alone may not be sufficient for the prevention of antibody-mediated autoimmune diseases.     

Construction and Characterization of a Single-chain Antibody Fragment Derived from Thymus of a Patient with Myasthenia Gravis

Pathogenic anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG) and the corresponding animal model, experimental autoimmune myasthenia gravis (EAMG), principally recognize the main immunogenic region (MIR) of the AChR. Bivalent anti-MIR antibodies binding to the &#102 -subunits of AChR result in AChR loss by antigenic modulation and complement activation. Monovalent Fab and single-chain variable fragments (scFv) of pathogenic anti-AChR antibodies can interfere with AChR binding of the pathogenic antibodies. In the present study, scFv637 was constructed from its parental Fab637, previously isolated from a thymus-derived phage display library with specificity toward anti-MIR of human AChR (hAChR), by PCR amplification. Bacterial produced scFv637 was able to bind to hAChR in standard precipitation radioimmunoassay (RIA). ScFv637 also bound to monkey AChR in situ on monkey neuromuscular junctions as showed in immunohistochemical staining. Furthermore, scFv637 was capable of inhibiting the binding of its intact IgG637 and anti-MIR mAb35 binding to hAChR up to 32.9 and 73.0%, respectively demonstrated in a competitive ELISA, and of MG patient sera from up to 45.5% in a competitive RIA. Therefore, scFv637, easier for manipulation in improvement of affinity and stability compared with its parental Fab637, may serve as an alternative candidate for specific immunotherapy in MG.     

Construction and characterization of a single-chain antibody fragment derived from thymus of a patient with myasthenia gravis

Pathogenic anti-acetylcholine receptor (AChR) antibodies in myasthenia gravis (MG) and the corresponding animal model, experimental autoimmune myasthenia gravis (EAMG), principally recognize the main immunogenic region (MIR) of the AChR. Bivalent anti-MIR antibodies binding to the alpha-subunits of AChR result in AChR loss by antigenic modulation and complement activation. Monovalent Fab and single-chain variable fragments (scFv) of pathogenic anti-AChR antibodies can interfere with AChR binding of the pathogenic antibodies. In the present study, scFv637 was constructed from its parental Fab637, previously isolated from a thymus-derived phage display library with specificity toward anti-MIR of human AChR (hAChR), by PCR amplification. Bacterial produced scFv637 was able to bind to hAChR in standard precipitation radioimmunoassay (RIA). ScFv637 also bound to monkey AChR in situ on monkey neuromuscular junctions as showed in immunohistochemical staining. Furthermore, scFv637 was capable of inhibiting the binding of its intact IgG637 and anti-MIR mAb35 binding to hAChR up to 32.9 and 73.0%, respectively demonstrated in a competitive ELISA, and of MG patient sera from up to 45.5% in a competitive RIA. Therefore, scFv637, easier for manipulation in improvement of affinity and stability compared with its parental Fab637, may serve as an alternative candidate for specific immunotherapy in MG.     

Experimental autoimmune myasthenia gravis induction in B cell-deficient mice

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG). Autoantibody-induced functional loss of nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane is an important pathogenic feature of both MG and EAMG. To evaluate the extent at which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (muMT) and wild- type C57BL/6 mice with AChR and complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha146-162 were intact in muMT mice as in wild-type mice. Similar amounts of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in muMT and wild-type mice. However, muMT mice had no detectable anti-AChR antibodies and remained completely free from clinical EAMG. We conclude that B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming.      

An immunodominant site of acetylcholine receptor in experimental myasthenia mapped with T lymphocyte clones and synthetic peptides

Myasthenia gravis (MG) is an autoimmune disease of man caused by antibodies directed against the acetylcholine receptor (AChR). In the experimental model of MG in mice, murine experimental autoimmune myasthenia gravis (EAMG), an anti-AChR immune response is induced by immunization with Torpedo AChR, and anti-AChR antibodies. AChR-sensitized T cells, and neuromuscular dysfunction result. The production of antibodies to AChR is thymus-dependent. In order to define the epitopes of the AChR identified by AChR-specific T cells, we generated T cell populations and T cell hybridoma clones and tested their reactivity to synthetic uniform-sized overlapping peptides representing the entire extracellular portion of the alpha-chain of the AChR. The predominant reactivity of the T cell clones and the parent lines was to a peptide corresponding to residues 146-162 of Torpedo AChR. This data is consistent with a highly limited recognition of AChR determinants in murine EAMG by AChR-specific T cells.     

Neuronal acetylcholine receptor alpha9-subunit: a possible central nervous system autoantigen

Nicotinic acetylcholine receptor (AChR) is a membrane glycoprotein composed of five subunits. Muscle AChR is consist of two alpha1 and one each beta, delta, and epsilon subunits, whereas the neuronal AChR molecules are made up of various combinations of alpha (alpha2-alpha10) and beta (beta1-beta4) subunits. Myasthenia gravis (MG) develops as a result of an autoimmune attack against muscular AChR. While the prevailing symptom is muscle weakness, very rarely MG patients may develop additional central nervous system (CNS) symptoms. The majority of the anti-AChR antibodies responsible from disease induction is directed against alpha1 subunit of AChR. There is considerable identity between muscular alpha1 and neuronal alpha9 subunits. Preliminary studies showed antibodies reactive with the CNS antigens in the serum samples of mice with experimental autoimmune myasthenia gravis (EAMG). Also, alpha9 was present in the CNS in widespread locations and the binding pattern of anti-alpha9 antibody was reminiscent of that of serum samples of some of the mice with EAMG. Serum anti-AChR antibodies of myasthenic patients might be cross-reacting with CNS AChR subunits and thus inducing CNS symptoms. Neuronal AChR alpha9-subunit might be a major target antigen in this process.     

Animal models of myasthenia gravis

Myasthenia gravis (MG) is an antibody-mediated, autoimmune neuromuscular disease. Animal models of experimental autoimmune myasthenia gravis (EAMG) can be induced in vertebrates by immunization with Torpedo californica acetylcholine receptors (AChR) in complete Freund's adjuvant. The MHC class II genes influence the cellular and humoral immune response to AChR and are involved in the development of clinical EAMG in mice. A dominant epitope within the AChR alpha146-162 region activates MHC class II-restricted CD4 cells and is involved in the production of pathogenic anti-AChR antibodies by B cells. Neonatal or adult tolerance to this T-cell epitope could prevent EAMG. During an immune response to AChR in vivo, multiple TCR genes are used. The CD28-B7 and CD40L-CD40 interaction is required during the primary immune response to AChR. However, CTLA-4 blockade augmented T- and B-cell immune response to AChR and disease. Cytokines IFN-gamma and IL-12 upregulate, while IFN-alpha downregulates, EAMG pathogenesis. However, the Th2 cytokine IL-4 fails to play a significant role in the development of antibody-mediated EAMG. Systemic or mucosal tolerance to AChR or its dominant peptide(s) has prevented EAMG in an antigen-specific manner. Antigen-specific tolerance and downregulation of pathogenic cytokines could achieve effective therapy of EAMG and probably MG.     

Passive transfer of experimental myasthenia gravis via antigenic modulation of acetylcholine receptor.

Antigenic modulation of acetylcholine receptor (AChR) is considered to contribute to the reduction of endplate AChR in myasthenia gravis (MG). Yet, the pathogenic significance of this mechanism is unclear. To investigate the in vivo role of AChR antigenic modulation we examined the ability of bivalent F(ab')2 and monovalent Fab fragments of monoclonal antibody (mAb) 35 to passively transfer experimental autoimmune MG (EAMG) in rats. mAb 35 which binds at the main immunogenic region (MIR) of the AChR causes severe EAMG without being involved in channel function. Compared to the intact mAb, F(ab')2 35 proved to be less potent but still capable of inducing moderate EAMG, whereas Fab 35 were totally ineffective. Furthermore, both intact and F(ab')2 35 induced mild EAMG in complement-depleted rats. These results (a) provide evidence that antigenic modulation of endplate AChR is sufficient to generate passive transfer of EAMG and (b) further support the pathogenic potential of the anti-MIR antibodies in MG.     

Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR α subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR α subunit (Hα1 – 210), and to portions of it that encompass either the MIR (Hα1 – 121) or the ligand binding site of AChR (Hα122 – 210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments Hα1 – 121 and Hα1 – 210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments Hα1 – 121 and Hα1 – 210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment Hα122 – 210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.     

Pathogenic autoimmunity to affinity-purified mouse acetylcholine receptor induced without adjuvant in BALB/c mice

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated disorders in which anti-acetylcholine receptor (anti-AChR) antibodies cause loss of muscle AChR and subsequent weakness. Many species are susceptible to induction of EAMG with purified xenogeneic AChR in adjuvant, but injection of Torpedo AChR without adjuvants can also induce evidence of EAMG. To see whether pathogenic autoimmunity could be induced in mice by isolated mouse AChR we injected BALB/c mice with several doses (1 pmole; about 0.1 ug) of affinity-purified AChR (from the BC3H1 cell line but thought to be identical with denervated mouse muscle) intraperitoneally, without adjuvant, over a period of 10-22 weeks. Some of the mice became ill and died. High levels of serum anti-mouse AChR, directed mainly towards the main immunogenic region, were found and, in the survivors, correlated with loss of muscle AChR. Thus BALB/c mice can mount an autoimmune response to minute amounts of mouse AChR, without the use of adjuvants, and this response is very similar to that found in MG. This novel finding has implications regarding the etiology of the human disease.     

Myasthenia gravis as a prototype autoimmune receptor disease

Myasthenia gravis (MG) is an organ-specific autoimmune disease in which autoantibodies against nicotinic acetylcholine receptors (AChR) at the postsynaptic membrane cause loss of functional AChR and disturbed neuromuscular transmission. The immunopathogenic mechanisms responsible for loss of functional AChR include antigenic modulation by anti-AChR antibodies, complement-mediated focal lysis of the postsynaptic membrane, and direct interference with binding of acetylcholine to the AChR or with ion channel function. The loss of AChR and subsequent defective neuromuscular transmission is accompanied by increased expression of the different AChR subunit genes, suggesting a role for the target organ itself in determining susceptibility and severity of disease. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for the disease MG, and is very suitable to study the immunopathogenic mechanisms leading to AChR loss and the response of the AChR to this attack. In this article the current concepts of the structure and function of the AChR and the immunopathological mechanisms in MG and EAMG are reviewed.     

Role for interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular postsynaptic membrane and represents an animal model of myasthenia gravis in human. Recent studies highlighted the roles of TH1 cytokines (IFN-gamma, IL-12), rather than TH2 cytokines (IL-4), in the pathogenesis of EAMG by using homozygous (-/-) knockout mice with an EAMG-susceptible genetic background. To further evaluate a role for IFN-gamma, we injected recombinant rat IFN-gamma (rrIFN-gamma) at the time of immunization with AChR in complete Freund's adjuvant to EAMG-susceptible Lewis rats and EAMG-resistant Wistar Furth (WF) rats. RrIFN-gamma enhanced Lewis rat EAMG. The exacerbated muscular weakness was associated with higher levels of anti-AChR IgG and enhanced TNF-alpha responses. Anti-AChR IgG antibody levels were augmented to a similar extent as in Lewis rats, however, the identical immunization and IFN-gamma injection induced only mild and transient EAMG in WF rats due to the default TH3 phenotype development and inherent low TH1 responses. We conclude that IFN-gamma plays a major role in the pathogenesis of EAMG in the Lewis rat, but fails to break disease resistance in the WF rat.     

Complement and cytokine based therapeutic strategies in myasthenia gravis

Myasthenia gravis (MG) is a T cell–dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.     

Spectrotypic analysis of antibodies to acetylcholine receptors in experimental autoimmune myasthenia gravis

We have studied the isoelectric focusing pattern of antibodies expressed in rats with experimental autoimmune myasthenia gravis (EAMG) induced by immunization with acetylcholine receptors (AChR) purified from Torpedo californica. Sera or tissue eluates were obtained at intervals in the course of disease and subjected to isoelectric focusing. Subsequently, the focused antibodies were detected by autoradiography of gels labelled with 125I-alpha-bungarotoxin conjugated AChR. Reverse electrofocusing was used to separate complexes of antibody and AChR formed in vivo, thereby allowing detection of the full spectrotype (banding pattern). As little as 1.1 X 10(-12) moles of monoclonal antibodies (MoAbs) to AChR yielded distinct bands of radiolabelled antigen binding by this technique. The anti-AChR MoAbs studied showed a multitude of bands localized in neutral to alkaline position. The clonotypes expressed in late post-immunization sera were compared to early sera. The spectrotypes of immunized Lewis and Brown Norway rats were not identical. In early sera most of the isoelectric focusing bands were specific for T. californica AChR, whereas in late sera further expansion of the repertoire produced bands that reacted with rat muscle AChR as well. The focused bands that bound rat AChR also bound T. californica AChR. The anti-AChR antibodies eluted from muscles of rats with EAMG showed similar binding patterns to anti-receptor antibodies in rats' sera. These results indicate that the antibody specificities detected in serum are the same specificities which are effective in binding to muscle AChR in vivo. Minor specificities of serum anti-receptor antibodies are not disproportionally represented in the antibodies actually bound at the neuromuscular junction in EAMG.     

Determinant spreading and immune responses to acetylcholine receptors in myasthenia gravis

Summary: In myasthenia gravis (MG), antibodies to the muscle acetylcholine receptor (AChR) cause muscle weakness. Experimental autoimmune myaschenia gravis (EAMG) can be induced by immunisation against purified AChR; the main immunogenic region (MIR) is a conformation-dependent site that includes α67-76, EAMG can also occur after immunisation against extracellular AChR sequences, but this probably involves intramolecular determinant spreading.
In MG patients, thymic hyperplasia and germinal centres are found in about 50%, and thymoma in 10–15%. The heterogeneous, high affinity, IgG anti-AChR antibodies appear to be end-products of germinal centre responses, and react mainly with the MIR or a site on fetal AChR; the latter contains a y subunit and is mainly expressed on myoid cells in the thymic medulla, T cells cloned against recombinant AChR subunits recognise principally two naturally processed epitopes: ɛ201 -219 derived from adult AChR which is expressed in muscle, and sometimes in thymic epithelium, and α 146–160, common to fetal and adult AChR. Since AChR is not normally co-expressed with class II, it is unclear how CD4* responses to AChR a and E subunits are initiated, and how and where these spread to induce antibodies against fetal AChR, Various possibilities, including upregulation of class II on muscle/myoid cells and involvement of CD8+ responses to AChR and other muscle antigens, are discussed.     

Acetylcholine receptor antibody characteristics in myasthenia gravis. II. Patients with penicillamine-induced myasthenia or idiopathic myasthenia of recent onset.

Anti-acetylcholine receptor (anti-AChR) antibody characteristics including light chain, IgG subclass, avidity for denervated human acetylcholine receptor and reaction with various human and mammalian AChR preparations were examined in 11 patients who developed myasthenia during penicillamine treatment of rheumatoid arthritis. Results were compared with those already reported in 35 patients with generalized idiopathic myasthenia gravis (MG). We found significant differences in the avidity and the light chain of the anti-AChR. However, anti-AChR characteristics in 12 patients with recent onset (less than 4 months'' duration) idiopathic MG did not differ significantly from those in patients with penicillamine-induced MG. In the patients with generalized MG a trend was found towards higher percentage of kappa light chain and higher anti-AChR avidity with duration of disease. Anti-acetylcholine receptor antibodies in penicillamine-induced myasthenia gravis therefore appear to be similar to those of idiopathic myasthenia gravis of recent onset.     

Treatment of passively transferred experimental autoimmune myasthenia gravis using papain

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.     

The thymus in myasthenia gravis. Changes typical for the human disease are absent in experimental autoimmune myasthenia gravis of the Lewis rat.

In human myasthenia gravis (MG) formation of autoantibodies against acetylcholine receptor (AChR) is commonly associated with thymic changes termed lymphofollicular hyperplasia (LFH). To learn whether the thymic lesions of human MG are primary changes in the autoimmune pathogenesis, or rather secondary events caused by peripheral autoimmunization, the authors compared the pathologic changes of MG thymuses with the thymuses of Lewis rats with experimental autoimmune myasthenia gravis (EAMG). EAMG was induced either actively by immunization with AChR, or transferred passively with monoclonal antibodies (mAb) binding to AChR. The clinical diagnosis of EAMG was confirmed by electromyography. Germinal centers, which are typical for human MG thymuses, were not detectable in the thymus of EAMG rats. Scattered B cells were seen as normal components of the thymic medulla. In EAMG their number was not augmented, nor were they accumulated focally. The perivascular spaces (PVS) were not distended and the amount of reticulin was not increased. Thymic myoid cells were identified in EAMG as well as in control thymuses; their cellular microenvironment was inconspicuous. Both in normal and in EAMG thymuses, a subpopulation of myoid cells expressed the main immunogenic region of the AChR. Heavily affected rats showed a severe cortical involution, but no specific changes of the medulla. The fact that none of the thymic lesions characteristic for human MG was found in EAMG is compatible with the concept that the thymic changes in MG are primary events in the autoimmune pathogenesis of this disease.     

Dendritic cells exposed in vitro to TGF-beta1 ameliorate experimental autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG), characterized by an autoaggressive T-cell-dependent antibody-mediated immune response directed against the acetylcholine receptor (AChR) of the neuromuscular junction. Dendritic cells (DC) are unique antigen-presenting cells which control T- and B-cell functions and induce immunity or tolerance. Here, we demonstrate that DC exposed to TGF-beta1 in vitro mediate protection against EAMG. Freshly prepared DC from spleen of healthy rats were exposed to TGF-beta1 in vitro for 48 h, and administered subcutaneously to Lewis rats (2 x 10(6)DC/rat) on day 5 post immunization with AChR in Freund's complete adjuvant. Control EAMG rats were injected in parallel with untreated DC (naive DC) or PBS. Lewis rats receiving TGF-beta1-exposed DC developed very mild symptoms of EAMG without loss of body weight compared with control EAMG rats receiving naive DC or PBS. This effect of TGF-beta1-exposed DC was associated with augmented spontaneous and AChR-induced proliferation, IFN-gamma and NO production, and decreased levels of anti-AChR antibody-secreting cells. Autologous DC exposed in vitro to TGF-beta1 could represent a new opportunity for DC-based immunotherapy of antibody-mediated autoimmune diseases.     

Anti-TNF-alpha antibodies suppress the development of experimental autoimmune myasthenia gravis

To understand the role of TNF-alpha in the induction of experimental autoimmune myasthenia gravis (EAMG) and detect a possible effect of anti-TNF-alpha antibodies in the treatment of EAMG, anti-TNF-alpha antibodies were administrated intraperitoneally to Lewis rats twice per week for 5 weeks from the day of immunization with Torpedo AChR and complete Freund's adjuvant (CFA). Administration of anti-TNF-alpha antibodies resulted in lower incidence of EAMG, and in delayed onset and only mild muscle weakness compared with control EAMG rats. These mild clinical signs were accompanied by lower AChR-specific lymphocyte proliferation, down-regulated IFN-gamma and IL-10, and up-regulated TGF-beta. The lower levels of anti-AChR IgG, Ig2a and IgG2b and decreased anti-AChR IgG affinity were found in rats treated with anti-TNF-alpha antibodies. These results demonstrate that anti-TNF-alpha antibodies can suppress the induction and development of EAMG.