Analysis of B Cell Responses in Multiple Sclerosis

多发性硬化症中 B 细胞反应的分析

• 批准号: 6745999
• 负责人: DONALD GILDEN
• 金额: $ 53.02万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745999
• 关键词: B lymphocyte; antibody formation; autoantigens; brain; cerebrospinal fluid; complementary DNA; flow cytometry; gene expression; genetic library; human subject; immunoglobulin G; leukocyte activation /transformation; molecular cloning; monoclonal antibody; multiple sclerosis; patient oriented research; protein sequence; recombinant proteins; tissue /cell culture; transfection /expression vector

Multiple sclerosis (MS) is the most common demyelinating disease of humans. Although its cause is unknown, clues to the nature of disease may be found in the increased IgG and oligoclonal bands (OGBs) present in the CSF and brain of MS patients, an phenomenon almost exclusively seen with infectious and inflammatory diseases of the CNS. Although the specificity of OGBs in MS is unknown, in infectious diseases of the CNS, OGBs are specific for the agent that causes disease, thus providing a rationale for our hypothesis that OGBs in MS brain and CSF are directed against the antigen that causes disease. In the past funding period, analysis of the lgG heavy (VH) and light (VL) chain variable region sequences expressed in acute MS brains and CSFs revealed a restricted IgG repertoire indicative of an antigen-driven B cell response. To produce recombinant antibodies from prominent MS Ig sequences that accurately duplicate the B cell heavy- and light- antibody chain pairings found in vivo, we developed an RT-PCR protocol to amplify the expressed V-region sequences of single B cells and plasma cells isolated from MS CSF by fluorescence-activated cell sorting. The paired heavy and light chains from CSF clonal B cell populations will now be used to produce a panel of recombinant mAbs whose specificity will be determined. Once specificity is established, cDNAs whose protein products react with MS-specific Abs will be cloned and characterized. Also, to better understand the dynamics of the B cell response in MS and to further characterize the nature of the humoral response in MS, changes in the IgG repertoire of MS CSF with disease progression will be analyzed. We are well-prepared to conduct these studies because we have: (a) a well-characterized pool of CSF from MS patients and patients with other CNS inflammatory disease; (b) a demonstrated ability to amplify disease-relevant IgG sequences from human CSF and brain, and to use these sequences to generate recombinant IgG; and (c) the expertise in molecular biology and immunology to identify the antigenic target of the recombinant Abs. Identification of an MS-specific antigen(s) will have wide application, not only for early definitive diagnosis, but also for developing strategies to modulate and possibly prevent disease.

多发性硬化（MS）是人类最常见的脱髓鞘疾病。虽然其原因尚不清楚，但在MS患者的CSF和脑中存在的IgG和寡克隆条带（OGB）增加中可以发现疾病性质的线索，这种现象几乎仅见于CNS的感染性和炎性疾病。尽管MS中OGB的特异性尚不清楚，但在CNS感染性疾病中，OGB对致病因子具有特异性，因此为我们的假设提供了理论基础，即MS脑和CSF中的OGB是 直接对抗致病抗原在过去的资助期内，对急性MS脑和CSF中表达的IgG重链（VH）和轻链（VL）可变区序列的分析揭示了指示抗原驱动的B细胞应答的受限IgG库。为了从突出的MS IG序列产生重组抗体，其精确地复制体内发现的B细胞重链和轻链抗体配对，我们开发了RT-PCR方案来扩增从MS分离的单个B细胞和浆细胞的表达的V区序列 通过荧光激活细胞分选的CSF。来自CSF克隆B细胞群的成对重链和轻链现在将用于产生一组重组mAb，其特异性将被确定。一旦建立特异性，其蛋白产物与MS特异性抗体反应的cDNA将被克隆和表征。此外，为了更好地了解MS中B细胞应答的动力学并进一步表征MS中体液应答的性质，将分析MS CSF的IgG库随疾病进展的变化。我们已为开展这些研究做好充分准备，因为我们拥有：（a）来自MS患者和其他CNS炎性疾病患者的CSF样本库已得到充分表征;（B）已证明能够扩增来自人CSF和脑的疾病相关IgG序列，并使用这些序列生成重组IgG;以及（c）分子生物学和免疫学方面的专业知识，以鉴定重组Ab的抗原靶标。MS特异性抗原的鉴定将具有广泛的应用，不仅用于早期明确诊断，而且用于开发调节和可能预防疾病的策略。