STUDY OF GAMMA GLOBULIN STRUCTURE AND HYPERSENSITIVITY

丙种球蛋白结构和超敏反应的研究

• 批准号: 3124627
• 负责人: KIMISHIGE ISHIZAKA
• 金额: $ 22.4万
• 依托单位: LA JOLLA INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1974
• 资助国家: 美国
• 起止时间: 1974-09-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124627
• 关键词: DNA binding protein; Escherichia coli; SDS polyacrylamide gel electrophoresis; T lymphocyte; allergens; arachidonate; biological signal transduction; bone marrow disorder; cell differentiation; cell growth regulation; enzyme inhibitors; gamma globulin; histamine; histamine release; human tissue; hypersensitivity; immunochemistry; immunoglobulin E; immunoglobulin structure; laboratory mouse; lymphokines; mast cell; phosphatidylethanolamines; phosphatidylinositols; radioimmunoassay; receptor binding; tissue /cell culture

(Adapted from applicant's abstract): It has been established that IgE molecules bind to Fc delta receptors on mast cells with high affinity, and that cross linking of the cell bound IgE antibodies by antigen trigger the cells for the release of mediators which cause allergic diseases. The purpose of this research project is to elucidate the immunological and biochemical mechanisms of IgE-dependent mediator release. Previous work indicated that cross-linking of cell-bound IgE antibody molecules by multivalent ligand induced activation of phospholipase C (PLC) for hydrolysis of inositol phospholipids, and that this process may play a major role in the transduction of triggering signals for mediator release. In this proposal, i) the applicant shall study whether guanidine- nucleotide binding protein(s) is involved in the IgE-mediated activation of PLC and histamine release. As the investigators have established the method to permeabilize rat mast cells and bone marrow-derived mouse mast cells (BMMC), the investigators shall introduce non-hydrolyzable GTP analogue, such as GTP gammaS or GDPbetaS, into the permeabilized cells and determine the effect of the GTP analogue, on antigen-induced hydrolysis of inositol phospholipids and mediator release. ii) The investigator's recent experiment indicated that a newly synthesized inhibitor of phospholipase A2 (PLA2) inhibited the antigen-induced histamine release and arachidonic acid release from sensitized BMMC without affecting the formation of inositol phosphates. The investigators shall confirm that the inhibitor does not affect the antigen-induced activation of PLC, and determine whether the inhibitor may affect other enzymes involved in phosphatidylinositol turnover. The investigators expect that the experiments will establish a possible role of PLA2 in IgE-dependent mediator release. iii) The investigators shall try to establish culture conditions for selective differentiation and growth of human basophil granulocytes and mast cells from umbilical cord blood cells and bone marrow cells. Recombinant interleukins from human T-cells or non-T-cells will be employed. Fibroblast monolayers will also be employed for the differentiation of mast cells. iv) The investigators shall also continue their collaboration with molecular biologists to determine the binding site on human IgE molecules to Fc delta receptors on basophils. Recombinant peptides and synthetic peptides representing small segments of the Fc portion of delta chain will be tested for the ability to block passive sensitization of human basophils with IgE antibodies, and for the ability to block the binding of 125 I-labeled IgE to Fc delta receptors on cultured human basophils.

（改编自申请人摘要）：已确定IgE 分子以高亲和力与肥大细胞上的Fc δ受体结合，并且 抗原与细胞结合的IgE抗体的交联触发了免疫反应， 细胞释放引起过敏性疾病的介质。 的 本研究项目的目的是阐明免疫学和 IgE依赖性介质释放的生化机制。 以前的工作 表明细胞结合的IgE抗体分子通过 多价配体诱导的磷脂酶C（PLC）活化， 肌醇磷脂的水解，这一过程可能发挥 在介体释放触发信号的传导中发挥主要作用。 在本建议中，i）申请人须研究胍- 核苷酸结合蛋白参与IgE介导的激活 PLC和组胺释放。 正如调查人员所确定的那样， 透化大鼠肥大细胞和骨髓来源的小鼠肥大细胞的方法 细胞（BMMC），研究者应引入不可水解的GTP 将GTP γ S或GDP β S等类似物导入透化细胞， 确定GTP类似物对抗原诱导的 肌醇磷脂和介质释放。 （二）研究者 最近的实验表明，一种新合成的抑制剂 磷脂酶A2（PLA 2）抑制抗原诱导的组胺释放 和花生四烯酸从致敏的BMMC释放，而不影响 肌醇磷酸的形成。 研究者应确认， 所述抑制剂不影响抗原诱导的PLC活化，和 确定该抑制剂是否可能影响其他酶参与 磷脂酰肌醇周转 调查人员预计， 实验将确定PLA 2在IgE依赖性免疫应答中的可能作用。 释放介质。 （三）研究者应努力建立文化 人嗜碱性粒细胞选择性分化和生长的条件 来自脐带血细胞和骨的粒细胞和肥大细胞 骨髓细胞 来自人T细胞或非T细胞的重组白细胞介素 将被雇用。 成纤维细胞单层也将用于 肥大细胞的分化。 （四）调查人员还应继续 他们与分子生物学家合作， 与嗜碱性粒细胞上的Fc δ受体结合。 重组肽和合成肽代表小片段 将测试δ链的Fc部分阻断 被动致敏的人嗜碱性粒细胞与IgE抗体，并为 阻断125 I标记的IgE与Fc δ受体结合的能力 培养的人嗜碱性粒细胞。