Direct Proof of the In Vivo Pathogenic Role of the AChR Autoantibodies from Myasthenia Gravis Patients

Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG.     

Myasthenia Gravis: Effect on Antibody Binding of Conservative Substitutions of Amino Acid Residues Forming the Main Immunogenic Region of the Nicotinic Acetylcholine Receptor

Abstract

In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR α-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence α67–76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence α67–76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure.

Conservative substitutions of residues Asn₆₈ and Asp₇₁ greatly diminished mAb binding, identifying them as critical contact residues for anti-MIR mAbs. Substitutions at Asp₇₀ and Tyr₇₂ moderately affected binding. Cross-reactive mAbs originally raised against Electrophorus AChR bound single residue-substituted synthetic peptides in a manner consistent with the possibility that Electrophorus AChR may have a glutamic acid residue at position α70 or α71. Substitutions at residues Asp/Ala₇₀ and Val/Ile₇₀ between human and Torpedo α-subunits may be size-compensating, suggesting these amino acids in the native AChR may be in closer proximity than proposed in previous models of the MIR.     

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

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.     

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.     

Antibodies to acetylcholine receptor and tetanus toxoid: in vitro synthesis by thymic lymphocytes

Thymic lymphocytes (TL) of patients with myasthenia gravis (MG) have been reported to synthesize antibodies to acetylcholine receptors (anti-AChR). Incubation of TL with pokeweed mitogen (PWM), a polyclonal T cell- and monocyte-dependent activator of B cell differentiation, was reported to inhibit TL in vitro synthesis of anti-AChR. We studied the TL of 16 patients with MG without thymoma. TL of 10 of 16 patients synthesized anti-AChR in vitro without stimulation. In the presence of PWM, the amount of anti-AChR synthesized by the TL of these 10 patients increased in five, decreased in three, and was unaffected in two. There was a correlation between serum anti-AChR titer and PWM-stimulated synthesis (r = 0.87), but not with unstimulated synthesis (r = 0.33) of anti-AChR by TL. There was no correlation between the amount of synthesized anti-AChR and the amount of secreted IgG or with the percentage of B cells (surface Ig+) in the TL suspensions. TL of three age-matched cardiac surgery controls failed to synthesize detectable anti-AChR, although two of three synthesized IgG. Four patients with MG were booster immunized with tetanus toxoid (TT) 3 to 4 wk prior to thymectomy. TL of three synthesized anti-TT in vitro, whereas TL of three nonboosted MG patients failed to synthesize anti-TT. Thus, we have shown that TL of some patients with MG are capable of anti-AChR synthesis, with evidence of heterogeneity of the in vitro response, and the B cell repertoire in the thymus may, in part, reflect recent systemic immune events of the host.     

Amino Acid Residues Within the Sequence Region α55–74 of Torpedo Nicotinic Acetylcholine Receptor Interacting with Antibodies to the Main Immunogenic Region and with Snake α-Neurotoxins

Abstract

The sequence region 55–74 of the α-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment—residues α67–76—forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence α55–74 of Torpedo AChR binds α-bungarotoxin (αBTX), suggesting that amino acid residues within this sequence region may contribute to formation of an αBTX binding site.

Using single-residue substituted synthetic analogues of the sequence α55–74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with α-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg₅₅, Arg₅₇, Trp₆₀, Arg₆₄, Leu₆₅, Arg₆₆, Trp₆₇, or Asn₆₈ strongly inhibited α-toxin binding, whereas substitutions of Ile₆₁, Val₆₃, Pro₆₉, Ala₇₀, Asp₇₁, or Tyr₇₂ had marginal effects. Substitutions within the region α68–72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp₆₀ substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp₆₂ slightly but consistently affected binding of mAbs 6 and 22.     

A systematic review of population based epidemiological studies in Myasthenia Gravis

Background  

The aim was to collate all myasthenia gravis (MG) epidemiological studies including AChR MG and MuSK MG specific studies. To synthesize data on incidence rate (IR), prevalence rate (PR) and mortality rate (MR) of the condition and investigate the influence of environmental and technical factors on any trends or variation observed.     

Association of the DNMT3B -579G>T Polymorphism with Risk of Thymomas in Patients with Myasthenia Gravis

Increasing evidence suggests a contribution of epigenetic processes in promoting cancer and autoimmunity. Myasthenia gravis (MG) is an autoimmune disease mediated, in approximately 80% of the patients, by antibodies against the nicotinic acetylcholine receptor (AChR+). Moreover, epithelial tumours (thymomas) are present in about 10-20% of the patients, and there is indication that changes in DNA methylation might contribute to the risk and progression of thymomas. However, the role of epigenetics in MG is still not completely clarified. In the present study we investigated if a common polymorphism (-579G>T: rs1569686) in the promoter of the DNMT3B gene coding for the DNA methyltransferase 3B, an enzyme that mediates DNA methylation, increases the risk to develop MG or MG-associated thymomas. The study polymorphism was selected based on recent reports and a literature meta-analysis suggesting association with increased risk of various types of cancer. We screened 324 AChR+ MG patients (140 males and 184 females, mean age 56.0 ± 16.5 years) and 735 healthy matched controls (294 males and 441 females, mean age 57.3 ± 15.6 years). 94 of the total MG patients had a thymoma. While there was no association with the whole cohort of MG patients, we found a statistically significant association of the DNMT3B -579T allele (OR = 1.51; 95% CI=1.1-2.1, P = 0.01) and the TT homozygous genotype (OR = 2.59; 95% CI=1.4-4.9, P = 0.006) with the risk of thymoma. No association was observed in MG patients without thymoma, even after stratification into clinical subtypes. Present results suggest that the DNMT3B -579T allele might contribute to the risk of developing thymoma in MG patients, particularly in homozygous TT subjects.     

Prophylactic Effect of Probiotics on the Development of Experimental Autoimmune Myasthenia Gravis

Probiotics are live bacteria that confer health benefits to the host physiology. Although protective role of probiotics have been reported in diverse diseases, no information is available whether probiotics can modulate neuromuscular immune disorders. We have recently demonstrated that IRT5 probiotics, a mixture of 5 probiotics, could suppress diverse experimental disorders in mice model. In this study we further investigated whether IRT5 probiotics could modulate the progression of experimental autoimmune myasthenia gravis (EAMG). Myasthenia gravis (MG) is a T cell dependent antibody mediated autoimmune disorder in which acetylcholine receptor (AChR) at the neuromuscular junction is the major auto-antigen. Oral administration of IRT5 probiotics significantly reduced clinical symptoms of EAMG such as weight loss, body trembling and grip strength. Prophylactic effect of IRT5 probiotics on EMAG is mediated by down-regulation of effector function of AChR-reactive T cells and B cells. Administration of IRT5 probiotics decreased AChR-reactive lymphocyte proliferation, anti-AChR reactive IgG levels and inflammatory cytokine levels such as IFN-γ, TNF-α, IL-6 and IL-17. Down-regulation of inflammatory mediators in AChR-reactive lymphocytes by IRT5 probiotics is mediated by the generation of regulatory dendritic cells (rDCs) that express increased levels of IL-10, TGF-β, arginase 1 and aldh1a2. Furthermore, DCs isolated from IRT5 probiotics-fed group effectively converted CD4⁺ T cells into CD4⁺Foxp3⁺ regulatory T cells compared with control DCs. Our data suggest that IRT5 probiotics could be applicable to modulate antibody mediated autoimmune diseases including myasthenia gravis.     

Specificity of the T cell immune response to acetylcholine receptor in experimental autoimmune myasthenia gravis. Response to subunits and synthetic peptides

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent diseases mediated by antibodies against acetylcholine receptor (AChR) on skeletal muscle. Most of the antibodies are directed toward conformation-dependent epitopes on the AChR, whereas T cells recognize denatured AChR. In search of T cell epitopes in EAMG, we tested 24 synthetic peptides covering 62% of the alpha-subunit sequence of Torpedo californica electric organ AChR in the T cell proliferation assay with lymph node cells from rats immunized with AChR. In Lewis rats, 2 of these peptides, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90, strongly stimulated T cells and, of these, [Tyr 100]alpha 100-116 was much more potent; 4 other peptides were weakly mitogenic and 18 were ineffective. None of the 24 synthetic peptides alone stimulated anti-AChR production and, when added to cultures along with AChR, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90 suppressed antibody production. Of twelve cloned T cell lines specific to AChR, 4 responded to [Tyr 100]alpha 100-116, indicating the importance of the epitope in alpha 101-116 in Lewis rats. In three other strains of rats whose responses to AChR and its subunits were similar to those in the Lewis rat, neither [Tyr 100]alpha 100-116 nor [Gly 89, Tyr 90]alpha 73-90 was stimulatory. Instead, completely different sets of peptides stimulated their T cells. When peptides were used as immunogens, each strain (except Lewis rats) responded only to the peptides that stimulated AChR-immune T cells from the same strain. Genetically restricted T cell recognition of AChR peptides in rats suggests that T cells from MG patients with different major histocompatibility haplotypes may recognize different AChR peptides.     

Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

Background

Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG.

Methods

The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system.

Results

We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals.

Conclusion

Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis.     

MuSK Myasthenia Gravis IgG4 Disrupts the Interaction of LRP4 with MuSK but Both IgG4 and IgG1-3 Can Disperse Preformed Agrin-Independent AChR Clusters

A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 MuSK antibodies will also contribute to the reduced AChR density and neuromuscular dysfunction in myasthenia patients with MuSK antibodies.     

Monoclonal anti-idiotopic antibodies against myasthenia-inducing anti-acetylcholine receptor monoclonal antibodies. Preponderance of nonparatope-directed antibodies affecting antigen binding

To analyze components of the idiotypic network in experimental autoimmune disease, we produced 17 isogeneic anti-idiotopic monoclonal antibodies (anti-Id) against two experimental autoimmune myasthenia gravis-producing anti-acetylcholine receptor (anti-AChR) monoclonal antibodies. We studied the binding of five of the anti-Id to the anti-AChR monoclonal antibodies bearing the complementary idiotopes (Id-mAb). They bound with Kd values ranging from 0.06 to 0.86 nM, values comparable to those of Id-mAb:AChR complexes (0.26 and 0.34 nM). All of the anti-Id tested moderately inhibited the binding of AChR to Id-mAb, whereas for each anti-Id, AChR either strongly inhibited anti-Id binding or had no effect on anti-Id binding. Hence, the inhibition of Id-mAb:AChR binding by anti-Id was not reciprocal with the inhibition of anti-Id:Id-mAb binding by AChR. For each anti-Id, the relative affinities of anti-Id and AChR for Id-mAb together with the lack of symmetry of inhibition by anti-Id compared to inhibition by AChR indicate that these two "ligands" are not competitive inhibitors. Consequently, anti-Id and AChR do not bind to overlapping sites on the Id-mAb, suggesting that the observed inhibition is mediated allosterically. This may be a common mechanism of anti-Id:Id binding, which would have important implications for the mechanism of anti-Id-induced suppression.     

Split tolerance in a novel transgenic model of autoimmune myasthenia gravis

Because it is one of the few autoimmune disorders in which the target autoantigen has been definitively identified, myasthenia gravis (MG) provides a unique opportunity for testing basic concepts of immune tolerance. In most MG patients, Abs against the acetylcholine receptors (AChR) at the neuromuscular junction can be readily identified and have been directly shown to cause muscle weakness. T cells have also been implicated and appear to play a role in regulating the pathogenic B cells. A murine MG model, generated by immunizing mice with heterologous AChR from the electric fish Torpedo californica, has been used extensively. In these animals, Abs cross-react with murine AChR; however, the T cells do not. Thus, to study tolerance to AChR, a transgenic mouse model was generated in which the immunodominant Torpedo AChR (T-AChR) alpha subunit is expressed in appropriate tissues. Upon immunization, these mice showed greatly reduced T cell responses to T-AChR and the immunodominant alpha-chain peptide. Limiting dilution assays suggest the likely mechanism of tolerance is deletion or anergy. Despite this tolerance, immunization with intact T-AChR induced anti-AChR Abs, including Abs against the alpha subunit, and the incidence of MG-like symptoms was similar to that of wild-type animals. Furthermore, evidence suggests that this B cell response to the alpha-chain receives help from T cells directed against the other AChR polypeptides (beta, gamma, or delta). This model offers a novel opportunity to elucidate mechanisms of tolerance regulation to muscle AChR and to clarify the role of T cells in MG.     

Chronic experimental autoimmune myasthenia gravis induced by monoclonal antibody to acetylcholine receptor: biochemical and electrophysiologic criteria

To determine whether the chronic presence of antibody to acetylcholine receptor (AChR) can account for the neuromuscular abnormalities in myasthenia gravis (MG), rats injected repeatedly with monoclonal antibody (mAb) to AChR were compared with those injected with control mAb. In a previous report, those receiving anti-AChR mAb, studied ultrastructurally, had grossly simplified endplates when compared with normal controls. In this report, animals injected once or chronically for 9 to 12 wk had reduced content of muscle AChR. The chronically injected animals also had diminished miniature endplate potential amplitudes, but to a lesser extent than the reduction in AChR content. These studies establish the pathogenetic role of antibody to AChR in the induction of the ultrastructural, biochemical, and electrophysiologic hallmarks of MG.     

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.     

Clonotypic analysis of anti-acetylcholine receptor antibodies from experimental autoimmune myasthenia gravis-sensitive Lewis rats and experimental autoimmune myasthenia gravis-resistant Wistar Furth rats

A single immunization of Lewis rats with purified acetylcholine receptor (AChR) emulsified in adjuvant typically stimulates the production of oligoclonal AChR-reactive antibodies (as demonstrated by IEF) dominated by the IgG2a subclass, of moderate but clonotypically heterogeneous relative Ag-binding avidity, and capable of inducing symptoms of experimental autoimmune myasthenia gravis. Although similar immunization of Wistar Furth rats produces AChR-reactive antibodies with similar characteristics of clonotypic heterogeneity, avidity, and isotype expression, no detectable signs of AChR-dependent muscle impairment is observed. This contrasts the ability to induce impaired AChR function upon the passive transfer of pre-formed Lewis anti-AChR antibodies into naive Wistar Furth rats, suggesting that disease resistance in this model is not conferred at the level of the AChR itself. Moreover, if more aggressive immunization protocols are used (i.e., multiple injections of AChR), a transient breakthrough of AChR-dependent muscle dysfunction can be induced directly in the Wistar Furth strain indicating that the potential for the production of disease-causing antibodies does exist in the Wistar Furth repertoire. IEF analysis of Wistar Furth anti-AChR antibodies has revealed that hyperimmunization results in modified antibody clonotype expression that might explain changing expression of disease symptoms; however, explanations for the apparent "resistance" of Wistar Furth rats to disease induction are likely to be complex.     

Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis

CTLA-4 appears to be a negative regulator of T cell activation and is implicated in T cell-mediated autoimmune diseases. Experimental autoimmune myasthenia gravis (EAMG), induced by immunization of C57BL/6 mice with acetylcholine receptor (AChR) in adjuvant, is an autoantibody-mediated disease model for human myasthenia gravis (MG). The production of anti-AChR Abs in MG and EAMG is T cell dependent. In the present study, we demonstrate that anti-CTLA-4 Ab treatment enhances T cell responses to AChR, increases anti-AChR Ab production, and provokes a rapid onset and severe EAMG. To address possible mechanisms underlying the enhanced autoreactive T cell responses after anti-CTLA-4 Ab treatment, mice were immunized with the immunodominant peptide alpha(146-162) representing an extracellular sequence of the ACHR: Anti-CTLA-4 Ab, but not control Ab, treatment subsequent to peptide immunization results in clinical EAMG with diversification of the autoantibody repertoire as well as enhanced T cell proliferation against not only the immunizing alpha(146-162) peptide, but also against other subdominant epitopes. Thus, treatment with anti-CTLA-4 Ab appears to induce determinant spreading, diversify the autoantibody repertoire, and enhance B cell-mediated autoimmune disease in this murine model of MG.     

Mapping the extracellular topography of the α-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain

The extracellular surface of theα-chain ofTorpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR. was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptidesα1–16,α89–104,α158–174,α262–276, andα388–408. Lower, but significant, levels of reactivity were obtained with antibodies against peptidesα67–82,α78–93,α100–115, andα111–126. On the other hand, free AChR bound high levels of antibodies against peptidesα34–49,α78–93,α134–150,α170–186, andα194–210. It also bound moderate levels of antibodies against peptidesα262–276 andα388–408. Low, yet significant, levels of binding were exhibited by antibodies against peptidesα45–60,α111–126, andα122–138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).