THYMIC B-CELL ACTIVATION IN MYASTHENIA GRAVIS

取自胸腺组织的胸腺细胞(TC)悬液 重症肌无力(MG)患者在胸腺切除术中经常分泌 免疫球蛋白和抗乙酰胆碱受体 在体外培养时抗体显著，尽管缺乏 表达表面IgM的B淋巴细胞。目前在这方面的发现 实验室表明，这些突出的自发和商陆 有丝分裂原诱导的体液反应可能已经来自B淋巴细胞 由于体内先前的原因，与同型转换有关 激活。该项目的总体目标是扩展这些 进行研究，以加强我们对这些特点和 这些胸腺B淋巴细胞体液的潜在机制 回应。具体目标是：1)进一步澄清 这些TC中B细胞的分化/激活状态；2) 确定这些TC中B细胞所占的比例 生发中心池；3)表型与功能的相关性 MG胸腺辅助T细胞在mRNA水平的能力；5)产生 MG胸腺B细胞分泌抗AChR杂交瘤；6)确定 霍乱弧菌分泌的抗乙酰胆碱受体可变区(V基因)序列 胸腺B细胞。这些目标将通过胸腺和 重症肌无力患者胸腺切除时采集的血细胞。表型 将使用标记和流式细胞术进行分析和分离 细胞亚群的数量。这些细胞亚群将成为 单独培养或与其他细胞亚群重组培养。这个 在培养中分泌的物质将被检测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患者细胞的反应 在胸腺切除前，自身免疫(AChR)与 外源性(破伤风类毒素)抗原刺激可以比较。 合适的杂交瘤和分子生物技术将是 用于鉴定TC分泌的抗AChR。这些 这些发现将具有巨大的潜在临床重要性，因为 抗AChR抗体可能是MG的致病因素，胸腺切除术可能是 通常对这种疾病有益。此外，关于 TC分泌的抗AChR抗体是否由：a)编码 胚系基因；b)体细胞突变；或3)独特的基因片段 是可以接近的。

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.