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.

多发性硬化症（MS）是人类最常见的脱髓鞘疾病。尽管其原因尚不清楚，但疾病本质的线索可能会在多发性硬化症患者的脑脊液和大脑中出现的 IgG 和寡克隆带 (OGB) 增加中找到，这种现象几乎只出现在中枢神经系统感染性和炎症性疾病中。尽管多发性硬化症中 OGB 的特异性尚不清楚，但在中枢神经系统传染病中，OGB 对引起疾病的病原体具有特异性，从而为我们的假设提供了理论基础，即多发性硬化症大脑和脑脊液中的 OGB 针对引起疾病的抗原。在过去的资助期间，对急性多发性硬化症大脑和脑脊液中表达的 IgG 重链 (VH) 和轻链 (VL) 可变区序列的分析揭示了表明抗原驱动的 B 细胞反应的受限 IgG 库。为了从显着的 MS Ig 序列生产重组抗体，准确复制体内发现的 B 细胞重链和轻链抗体配对，我们开发了一种 RT-PCR 方案来扩​​增分离的单个 B 细胞和浆细胞的表达 V 区序列来自微软 通过荧光激活细胞分选进行脑脊液分离。来自 CSF 克隆 B 细胞群的配对重链和轻链现在将用于生产一组重组 mAb，其特异性将被确定。一旦确定了特异性，其蛋白质产物与 MS 特异性抗体发生反应的 cDNA 将被克隆和表征。此外，为了更好地了解 MS 中 B 细胞反应的动态并进一步表征 MS 中体液反应的性质，我们将分析 MS CSF 的 IgG 库随疾病进展的变化。我们为开展这些研究做好了充分准备，因为我们拥有： (a) 来自多发性硬化症患者和其他中枢神经系统炎症性疾病患者的特征明确的脑脊液库； (b) 具有从人脑脊液和脑中扩增与疾病相关的 IgG 序列并使用这些序列产生重组 IgG 的能力； (c) 分子生物学和免疫学方面的专业知识，以确定重组抗体的抗原靶标。 MS 特异性抗原的鉴定将具有广泛的应用，不仅可以用于早期明确诊断，还可以用于制定调节和可能预防疾病的策略。