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    • Our studies also reveal the normal functional roles

    2022-12-01

    Our studies also reveal the normal functional roles of the MIR. Interaction between the MIR loop and the N-terminal α helix nucleates conformational maturation of AChR subunits, thereby promoting assembly of mature AChRs [52], [55]. This interaction in homologous parts of all AChR subunits appears to be critical to their conformational maturation [62]. The interaction between the two structural elements greatly influences the sensitivity to activation of chimeric AChRs by ACh [52], [55]. This suggests that the MIR may influence conformational changes in this region associated with activation.
    AChR-specific mucosal tolerance MG is an ideal candidate disease for antigen-specific immunotherapies because the autoantigen is clearly defined. The first attempt to suppress EAMG through an antigen-specific immunotherapy dates back to 1978, at which time a denatured Torpedo AChR was used to treat EAMG in rabbits [63]. The observation that mucosal exposure to Celecoxib receptor can induce a state of systemic immunological tolerance to the same antigen leads to some studies on mucosal delivery of Torpedo AChR for immunosuppression of EAMG. Oral and nasal administration of native Torpedo AChR prevents the onset of EAMG in rats [64], [65], [66]. However, suppression of ongoing EAMG, which is the relevant model for treating MG, is much more difficult. Nasal administration of ten-fold higher amounts of Torpedo AChR is required to suppress ongoing EAMG compared to the amounts required for prevention of EAMG [67]. It is accompanied by a selective suppression of T helper 1 (Th1) regulated responses and a shift in anti-AChR antibody isotype from IgG2b to IgG1. Xenogeneicity and high immunogenicity of Torpedo AChR and its limited availability hamper its therapeutic application for human MG. The extracellular domain of AChR α1 subunit contains many T and B cell epitopes (including the MIR) and is the main target of pathogenic autoantibodies in MG sera [45], [68]. Based on these findings, recombinant fragments corresponding to the extracellular domain of human AChR α1 subunit, which can be expressed in large amounts in bacteria, were used as a substitute for the entire AChR to induce mucosal tolerance [69], [70]. Nasal or oral administration of a recombinant human α1 subunit extracellular domain (human α1 1-205) to rats prior to immunization with Torpedo AChR prevents EAMG and suppresses ongoing EAMG when treatment is initiated during the acute phase or during the chronic phase [71], [72]. Prevention and suppression of EAMG are attributed to active T cell suppression mediated by a shift from Th1 to Th2/Th3-regulatory AChR-specific responses, as reflected by changes in the cytokine profile, an AChR-specific antibody isotype switch from IgG2 to IgG1, and down-regulation of co-stimulatory factors. Syngeneic rat α1 extracellular domain (rat α1 1–205) is equally effective as the human α1 1–205 at suppressing ongoing EAMG [73]. The syngeneic rat α1 1–205 induces a shift from Th1 to Th2 but does not cause up-regulation of the Th3-type cytokine TGF-β and activation of regulatory T cells as does the xenogeneic human α1 1–205. However, oral administration of a more native fragment (thioredoxin-fused α1 1–210) exacerbates rather than suppresses EAMG because of renaturation of the MIR [74]. Removing two major B-cell epitopes (the MIR and α1 129–145) from the thioredoxin-fused human α1 1–210 converts the myasthenogenic fragment into a therapeutic one [75]. These results show that AChR-specific immunosuppressive therapy is possible in principle, but are not sufficient to lay the foundation for translation to therapy for human MG. In our rat EAMG model, oral administration of a recombinant protein corresponding to residues 1–209 of human α1 subunit (human α1 1–209) is not fully efficient in immunosuppression of EAMG probably due to renaturation of some conformation-dependent epitopes [76]. Thus, we used a 2:1:1:1:1 mixture of subunit constructs containing both extracellular and cytoplasmic domains for human AChR α1, β1, γ, δ and ϵ subunits, seeking a robust therapeutic response that would be impervious to genetic or immunological heterogeneity of EAMG animals or MG patients (Table 1). These subunit constructs were engineered to remove the transmembrane domains because the hydrophobic transmembrane domains impair bacterial expression and these sequences encompass few T-cell epitopes [77]. Oral administration of this AChR subunit mixture to rats prevents induction of EAMG, and reduces the severity of ongoing EAMG [76], [78]. Nasal administration of the subunit mixture is highly efficient in preventing EAMG, but ineffective at treating ongoing EAMG [78], [79]. Intraperitoneal (i.p.) administration of the subunit mixture is more potent than oral administration. This will be discussed in detail in a subsequent section.