THYMIC B-CELL ACTIVATION IN MYASTHENIA GRAVIS

从移除的胸腺组织获得的胸腺细胞（TC）悬液 重症肌无力（MG）患者胸腺切除术后， 免疫球蛋白（IG）和抗乙酰胆碱受体（抗AChR） 抗体显着时，在体外培养，尽管缺乏 表达表面IgM的B淋巴细胞。 目前在这方面的发现 实验表明，这些突出自发和商陆 有丝分裂原诱导的体液应答可能已经来自B淋巴细胞 由于先前的体内转化， activation. 该项目的总体目标是扩展这些 进行研究，以加深我们对 这些胸腺B淋巴细胞体液免疫的潜在机制 应答 具体目标是：1）进一步明确 这些TC中B细胞的分化/活化状态; 2） 确定这些TC中属于以下类型的B细胞的比例： 生殖中心库; 3）将表型和功能 MG胸腺辅助性T细胞在mRNA水平上的能力; 5）产生 从MG胸腺B细胞分泌抗AChR的杂交瘤; 6）确定 抗AChR的可变区（V基因）序列， 胸腺B细胞。 这些目标将通过使用胸腺和 MG患者胸腺切除术时获得的血细胞。 表型 将使用标记物和流式细胞术进行分析和分离 细胞亚群。 这些细胞亚群将比 单独培养或与其它细胞亚群重新组合培养。 的 将测定培养物中分泌的物质的IG和抗AChR 和抗破伤风（MG无关）抗体。 通过分析这些 破伤风类毒素免疫MG患者的细胞应答 在胸腺切除术前，自身免疫（AChR）与 可以比较外源性（破伤风类毒素）抗原刺激。 适当的杂交瘤和分子生物学技术将 用于表征TC分泌的抗AChR。 这些 研究结果将具有巨大的潜在临床意义，因为 抗AChR抗体可能是MG的致病因素， 通常对这种疾病有益。 此外， TC分泌的抗AChR抗体是否由以下编码：a） 生殖系基因; B）体细胞突变;或3）独特的基因片段 可以接近。

Thymus cell (TC) suspensions obtained from thymic tissue removed at thymectomy in myasthenia gravis (MG) patients frequently secrete immunoglobulin (Ig) and anti-acetyl-choline receptor (anti-AChR) antibodies prominently when cultured in vitro despite a paucity of B lymphocytes expressing surface IgM. Current findings in this laboratory suggest that these prominent spontaneous and pokeweed mitogen-induced humoral responses may be from B lymphocytes already differentiated with an isotype switch due to prior in vivo activation. The overall goal of this project is to extend these studies to enhance our understanding of the characteristics and underlying mechanisms in these thymic B lymphocyte humoral responses. Specific aims are to: 1) further clarify the differentiation/activation status of the B cells in these TC; 2) determine the proportion of the B cells in these TC that belong to the germinal center pool; 3) correlate the phenotype and functional capacity of MG thymic helper T cells at the mRNA level; 5) generate anti-AChR secreting hybridomas from MG thymic B cells; 6) define the variable region (V-gene) sequence of anti-AChR secreted by thymic B cells. These aims will be approached using thymic and blood cells obtained at thymectomy in MG patients. Phenotypic markers and flow cytometry will be used for analysis and separation of cell subpopulations. These cell sub-populations will than be cultured either alone or re-combined with other cell subsets. The material secreted in culture will be assayed for Ig and anti-AChR and anti-tetanus (MG irrelevant) antibodies. By analyzing these responses in cells from MG patients immunized with tetanus toxoid prior to thymectomy, the role of the auto-immune (AChR) vs. exogenous (tetanus toxoid) antigenic stimulus can be compared. Appropriate hybridoma and molecular biology technology will be applied to characterize the anti-AChR secreted by TC. These findings will be of great potential clinical importance since the anti-AChR antibody is likely pathogenic in MG and thymectomy is often beneficial in this disorder. In addition, the question of whether anti-AChR antibodies secreted by TC are encoded by: a) germ line genes; b) somatic mutation; or 3) unique gene segments can be approached.