SEROLOGIC, CELLULAR, MOLECULAR STUDIES OF AUTOANTIBODIES

自身抗体的血清学、细胞、分子研究

• 批准号: 2059755
• 负责人: Nicholas Chiorazzi
• 金额: $ 25.04万
• 依托单位: NORTH SHORE UNIVERSITY HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2059755
• 关键词: B lymphocyte; CD5 molecule; Epstein Barr virus; T lymphocyte; adult human (21+); antibody formation; antigen antibody reaction; autoantibody; cell transformation; flow cytometry; gene expression; gene rearrangement; human subject; hybrid cells; immunoglobulin G; immunoglobulin M; immunoglobulin genes; immunoglobulin idiotypes; in situ hybridization; laboratory mouse; laboratory rabbit; leukocyte activation /transformation; newborn human (0-6 weeks); nucleic acid sequence; nucleic acid structure; polymerase chain reaction; purpura; rheumatoid arthritis; rheumatoid factor; serology /serodiagnosis

The long-term goal of this proposal is to understand the relationship between "naturally-occurring" and "disease-related" autoantibodies in man. The former are polyreactive, low affinity, IgM antibodies produced by CD5+ B cells that have non-mutated Ig V genes, presumably because of a lack of T cell-stimulated, antigen-driven maturation. In contrast, disease-related autoantibodies are monoreactive, higher affinity IgM or IgG antibodies whose cellular origin has not been exclusively assigned to either the CD5+ or CD5- subsets. Based on animal experiments, these antibodies are products of cells that are clonally selected and have highly mutated V genes. However, human experiments have failed to demonstrate evidence for V gene somatic mutation. This may relate to the lack of availability of B cell lines making true disease-related autoantibodies. We will try to solve this dilemma by studying the B cells and IgM and IgG rheumatoid factors (RF) from rheumatoid arthritis (RA) and hypergammaglobulinemic purpura (HGGP) patients and comparing these with comparable cells and antibodies from normal neonates and adults. B cells will be taken from umbilical cord blood, and from the major sites of autoantibody synthesis in these diseases: the synovium in RA and the spleen in HGGP. CD5+ and CD5- B cells from these sources will be subcategorized: phenotypically by the expression of mutation and activation antigens; functionally by their T cell dependencies, cytokine responsiveness, and autoantibody synthesis; and genetically by Ig V/H gene family usage and RF cross-reactive idiotype (CRI) expression. Based on these subcategorization data, we will expand those B cell subsets that make disease-related autoantibodies, some of which will be immortalized by EBV transformation and/or somatic cell hybridization. These populations, and those generated during the previous granting period that make natural autoantibodies, will be used for Ig V/H and V/L gene DNA sequencing studies. Sequencing will be facilitated by suing a PCR technique that employs a series of oligonucleotide primers spanning the known upstream leader and downstream C region sequence. Finally, the autoantibodies produced by these cell lines will be analyzed for their antigen-binding specificities, affinities, and CRIs, and these immunologic characteristics compared with the DNA sequences to determine the molecular bases for their expression.

该提案的长期目标是了解两者之间的关系 人类“天然存在”和“疾病相关”自身抗体之间的关系。 前者是由 CD5+ 产生的多反应性、低亲和力 IgM 抗体 B 细胞具有未突变的 Ig V 基因，可能是由于缺乏 T 细胞刺激、抗原驱动的成熟。 相比之下，与疾病相关的 自身抗体是单反应性、亲和力较高的 IgM 或 IgG 抗体 其细胞起源尚未完全指定为 CD5+ 或CD5-子集。 根据动物实验，这些抗体是 经过克隆选择并具有高度突变 V 的细胞的产物 基因。 然而，人体实验未能证明 V基因体细胞突变。 这可能与 B 的可用性不足有关 产生真正的疾病相关自身抗体的细胞系。 我们会尽力 通过研究 B 细胞以及类风湿 IgM 和 IgG 来解决这个困境 类风湿性关节炎 (RA) 和高丙种球蛋白血症的因子 (RF) 紫癜 (HGGP) 患者并将其与可比较的细胞进行比较 来自正常新生儿和成人的抗体。 B细胞将取自 脐带血，以及来自自身抗体合成的主要部位 这些疾病：RA 中的滑膜疾病和 HGGP 中的脾脏疾病。 来自这些来源的 CD5+ 和 CD5- B 细胞将被细分为： 表型上由突变和激活抗原的表达决定； 通过其 T 细胞依赖性、细胞因子反应性和 自身抗体合成；遗传上通过 Ig V/H 基因家族使用和 RF 交叉反应独特型（CRI）表达。 基于这些子分类 数据，我们将扩展那些与疾病相关的 B 细胞亚群 自身抗体，其中一些将通过 EBV 转化而永生化 和/或体细胞杂交。 这些人口以及产生的人口 在先前的授予期内产生天然自身抗体，将 用于 Ig V/H 和 V/L 基因 DNA 测序研究。 测序将是 通过使用 PCR 技术来促进，该技术采用了一系列 跨越已知上游前导序列和下游序列的寡核苷酸引物 C区序列。 最后，这些细胞产生的自身抗体 将分析品系的抗原结合特异性、亲和力、 和 CRI，以及与 DNA 相比的这些免疫学特征 序列以确定其表达的分子碱基。