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    • Both subcutaneous s c and mucosal administration of s

    2022-12-01

    Both subcutaneous (s.c.) and mucosal administration of short synthetic peptide derivatives of AChR prevent the onset of EAMG, but do not treat ongoing EAMG [80], [81], [82], [83], [84], [85]. The selection of peptides for treatment is mainly based on the identification of immunodominant T- and B-cell epitopes in AChR. It should be noted that the autoimmune response to AChR in MG patients is highly heterogeneous [77], [86]. The heterogeneity of T- and B-cell epitopes of AChR makes it difficult to treat the disease using synthetic peptides that depend on one or two particular epitopes. The rationale behind the use of recombinant fragments and peptides from the extracellular portion of the AChR for suppression of EAMG is based on an assumption that specific immunosuppression of myasthenogenic autoimmune responses requires the use of therapeutic 1602 that encompass disease-inducing epitopes [87], [88]. Mucosal administration of an antigen can exacerbate, rather than suppress, the pathogenesis of autoimmune diseases when the antigen is exposed to compartments containing more helper than regulatory lymphocytes [89], [90]. This is especially a problem if the therapeutic antigen also contains pathogenic B cell epitopes that could provoke the production of pathological autoantibodies. Immunization of rats with either denatured subunits from Torpedo AChR or recombinant human α1 subunit extracellular domain 1–210 can induce EAMG, if sufficiently high doses are given [91], [92]. Oral administration of human α1 1–205 suppresses development of EAMG, but feeding with a more native fragment that corresponds to the same region in human AChR α1 subunit exacerbates EAMG due to renaturation of the MIR [74]. This suggests that traces of pathogenic B cell epitopes in bacterially-expressed AChR extracellular fragments can stimulate already activated B cells and overcome the therapeutic regulatory effect. Removal of two major B-cell epitopes from the thioredoxin-fused human α1 1–210 turns it from an exacerbator of EAMG into an effective tolerogen [75]. However, any extracellular antibody epitope is potentially pathogenic [93]. Thus, immunosuppression of EAMG using disease-inducing sequences is thought to have insurmountable difficulties [87], [93].
    Specific immunotherapy with AChR cytoplasmic domains We found that i.p. administration of the mixture of extracellular and cytoplasmic domain constructs for human AChR α1, β1, γ, δ and ϵ subunits is more effective at suppressing ongoing EAMG than is oral administration [78], [79]. Administration of 5mg/dose of subunit mixture i.p. weekly, starting after the acute phase and before the onset of the chronic phase, substantially suppresses ongoing EAMG (Fig. 2A). This treatment suppresses ongoing EAMG in a dose-dependent manner. By contrast, little benefit was observed after similar treatment with an irrelevant protein (ovalbumin), an α4 neuronal AChR subunit extracellular and cytoplasmic domain construct, or human α1 1–209 (Fig. 2B). Treatment with the subunit mixture reduces autoantibody level by half and greatly increases antibody response to AChR cytoplasmic domains. This indicates that autoantibody specificity is diverted away from pathologically significant epitopes on the extracellular portion of muscle AChR and toward pathologically irrelevant epitopes on the cytoplasmic domain. Loss of pathogenicity of serum autoantibodies from successfully treated rats is revealed by failure of passive transfer of EAMG by these antibodies (Fig. 2C). In order to test the efficiency and potency of therapy with the AChR cytoplasmic domains, we have employed two contiguous cytoplasmic domain constructs that encompass the large cytoplasmic domains of human muscle AChR β1, α1, and γ subunits and those of human muscle AChR ϵ, α1, and δ subunits respectively (Table 1) [79], [94]. Together, these two constructs reflect the order of subunits around the ion channel (Fig. 1). Indeed, we have demonstrated that potency of therapy with the mixture of the cytoplasmic domains was incrementally better than that of therapy with the subunit mixture by measures of clinical state, weight loss, death rate, EAMG incidence, reduction in titer to muscle AChR, and reduction in ability to cause antigenic modulation (Fig. 3 and Table 2) [78], [79]. Both therapies are accompanied by a shift of the anti-AChR IgG isotype profile from IgG2b to IgG1. Therapy with the cytoplasmic domains results in increased antibodies to cytoplasmic domains and decreased antibodies to α1 extracellular domain, while therapy with the subunit mixture increases the antibody response to both extracellular and cytoplasmic domains. This indicates that in rats treated with cytoplasmic domains there is no competing stimulation of responses to pathogenic extracellular epitopes observed in rats treated with the subunit mixture. 1602 Successful suppression of ongoing EAMG with human AChR cytoplasmic domain sequences indicates that the extracellular sequences, which in native form induce EAMG, are not required for successful therapy. This process is antigen-specific, but not epitope-specific.