We recently demonstrated that oral or nasal administration of recombinant fragments of the acetylcholine receptor (AChR) prevents the induction of experimental autoimmune myasthenia gravis (EAMG) and suppresses ongoing EAMG in rats. We have now studied the role of spatial conformation of these recombinant fragments in determining their tolerogenicity. Two fragments corresponding to the extracellular domain of the human AChR alpha-subunit and differing in conformation were tested: Halpha1-205 expressed with no fusion partner and Halpha1-210 fused to thioredoxin (Trx), and designated Trx-Halpha1-210. The conformational similarity of the fragments to intact AChR was assessed by their reactivity with alpha-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes. Oral administration of the more native fragment, Trx-Halpha1-210, at the acute phase of disease led to exacerbation of EAMG, accompanied by an elevation of AChR-specific humoral and cellular reactivity, increased levels of Th1-type cytokines (IL-2, IL-12), decreased levels of Th2 (IL-10)- or Th3 (TGF-beta)-type cytokines, and higher expression of costimulatory factors (CD28, CTLA4, B7-1, B7-2, CD40L, and CD40). On the other hand, oral administration of the less native fragments Halpha1-205 or denatured Trx-Halpha1-210 suppressed ongoing EAMG and led to opposite changes in the immunological parameters. It thus seems that native conformation of AChR-derived fragments renders them immunogenic and immunopathogenic and therefore not suitable for treatment of myasthenia gravis. Conformation of tolerogens should therefore be given careful attention when considering oral tolerance for treatment of autoimmune diseases.

译文

我们最近证明,口服或鼻腔给予乙酰胆碱受体 (AChR) 的重组片段可防止实验性自身免疫性重症肌无力 (EAMG) 的诱导,并抑制大鼠正在进行的EAMG。我们现在已经研究了这些重组片段的空间构象在确定其耐受性中的作用。测试了与人achrα 亚基的胞外域相对应且构象不同的两个片段: Halpha1-205在没有融合伴侣的情况下表达,Halpha1-210与硫氧还蛋白 (Trx) 融合,并指定为Trx-Halpha1-210。片段与完整AChR的构象相似性通过其与 α-bungarotoxin和抗AChR mab的反应性来评估,这些抗体对构象依赖性表位具有特异性。Trx-Halpha1-210,在疾病的急性期口服更天然的片段导致EAMG恶化,伴随着AChR特异性体液和细胞反应性的升高,Th1-type细胞因子 (IL-2,IL-12) 的水平升高,th2 (IL-10) 或Th3 (TGF-β) 型细胞因子水平降低,共刺激因子 (CD28,CTLA4,B7-1,B7-2,CD40L和CD40) 表达升高。另一方面,Halpha1-205或变性Trx-Halpha1-210的较低天然片段的口服给药抑制了正在进行的EAMG,并导致免疫学参数的相反变化。因此,似乎AChR衍生片段的天然构象使它们具有免疫原性和免疫致病性,因此不适合治疗重症肌无力。因此,在考虑口服耐受性治疗自身免疫性疾病时,应特别注意耐受性的构象。

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