STUDIES OF PRIMARY IMMUNODEFICIENCY DISEASES

原发性免疫缺陷疾病的研究

• 批准号: 6160653
• 负责人: WARREN STROBER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6160653
• 关键词: B lymphocyte; T lymphocyte; biological signal transduction; cell cell interaction; clinical research; gene expression; helper T lymphocyte; human subject; hypogammaglobulinemia; immunodeficiency; immunogenetics; immunoglobulin genes; immunopathology; leukocyte activation disorder; leukopoiesis; lymphocyte proliferation; protein tyrosine kinase; tissue /cell culture

Studies in our laboratory have focused on defining the nature of the B cell defects in the disease, Common Variable Immunodeficiency (CVI), a primary acquired human immunodeficiency state characterized by hypogammaglobulinemia (low levels of immunoglobulin in the serum), and impaired functional antibody responses. Our previous studies of purified B cells from CVI patients show that although the cells have a normal capacity to proliferate, they manifest differentiation defects at multiple levels. For example, when compared to normal B cells, circulating CVI B cells contain reduced numbers of surface bearing IgG and IgA cells with a commensurate increase in surface bearing IgM B cells, suggesting an in vivo defect in isotype switch. In addition, these cells fail to undergo differentiation into immunoglobulin producing cells. During this period we have initiated the study of CVI B cells with respect to their expression of transcription factors and cofactors known to be necessary for B cell switching and terminal differentiation. In particular, we have focused on the B cell-specific transcription factor, Oct-2, sine mice lacking this protein as a result of gene targeting have a phenotype similar to that of patients with CVI. In initial studies, we prepared RNA from purified B cells of patients and controls following stimulation with SAC plus IL-2 and amplified the RNA by RT-PCR using primers specific for the Oct-2 gene. CVI patient and control RNA gave rise to identical signals indicating no gross abnormality in the expression of Oct-2 RNA. We then evaluated Oct-2 protein function by performing electrophoretic mobility shift assays using the nuclear extracts of stimulated fresh B cells from patients and controls, as well as EBV-transformed B cell lines from patients and controls. The target oligonucleotide utilized was that of the core Oct-2 promoter site. We found that extracts from both patients and controls yielded an EMSA signal of appropriate size and identical intensity. These results indicate that CVI B cells do not manifest a gross deletion of the Oct-2 gene, nor do they manifest an abnormality of Oct-2 binding to the core Oct-2 promoter site.

我们实验室的研究重点是确定B细胞的性质 疾病中的细胞缺陷，常见变量免疫缺陷(CVI)，a 原发性获得性人类免疫缺陷状态的特征是 低丙种球蛋白血症(血清中免疫球蛋白水平低)，以及 功能性抗体反应受损。我们之前的研究 从CVI患者中纯化的B细胞显示，尽管这些细胞具有 正常的增殖能力，它们表现出分化缺陷 在多个层面上。例如，与正常的B细胞相比， 循环CVIB细胞表面携带免疫球蛋白的数量减少 和表面承载IgM B的IgA细胞相应增加 细胞，提示同型转换在体内存在缺陷。此外, 这些细胞不能分化为免疫球蛋白 生产细胞。 在此期间，我们已经开始了对CVIB细胞的研究 已知的转录因子和辅因子的表达 是B细胞交换和终末分化所必需的。在……里面 特别是，我们关注的是B细胞特异的转录因子， OCT-2，由于基因打靶而导致缺乏该蛋白的Sine小鼠 一种与CVI患者相似的表型。 在最初的研究中，我们从患者纯化的B细胞中制备了RNA 和对照组在SAC+IL-2刺激后，并放大 用针对Oct-2基因的特异性引物进行RT-PCR。CVI患者 和对照RNA产生了相同的信号，表明没有总 OCT-2RNA表达异常。然后我们评估了OCT-2 通过进行电泳迁移率改变分析来实现蛋白质的功能 利用患者刺激的新鲜B细胞的核提取液和 对照，以及EBV转化的B细胞系从患者和 控制。所使用的靶向寡核苷酸是核心的寡核苷酸 OCT-2启动子位点。我们发现患者和患者的提取物 控制组产生了大小合适且相同的EMSA信号 强度。这些结果表明，CVIB细胞不表现为 Oct-2基因的完全缺失，也没有表现出异常 OCT-2与核心OCT-2启动子结合位点。