IONIC,BIOCHEMICAL,GENETIC BASIS FOR CYCLOSPORIN ACTIVITY

环孢菌素A（CsA）是一种有效的免疫抑制剂， 主要是通过抑制T细胞功能，可能是通过阻止IL 2 阵 它在临床移植中的广泛而成功的应用 需要更准确地定义其作用方式。 的 CsA选择性阻断IL 2产生的假设忽略了 对早期代谢事件的抑制作用， 在B淋巴细胞中也具有抑制活性。 我们有 表明免疫抑制性环孢菌素，而不是 非免疫抑制类似物，影响离子动员， 细胞静息膜电位降低 细胞内Ca 2+浓度。 这些发现， 最早记录的药物作用，可能有助于 这组药物的总体免疫抑制活性， 特别是对IL 2产生和T细胞增殖的影响。 的目标 进一步定义离子， 环孢菌素依赖效应的生物化学和遗传基础 对膜电位、Ca 2+摄取和IL 2形成的影响，以确定 这些现象之间的相互关系，并描绘 其免疫抑制作用的要求。 特别是在 人类T和B细胞，我们将检查早期激活事件 在受体-配体相互作用之后， 在信号到达细胞核后发生， 与细胞分裂、淋巴因子产生或IG相关 分泌物 使用细胞内捕获的荧光指示剂 我们将监测细胞内Ca 2+浓度的变化， pH和膜电位。 我们将确定是否主要影响 的环孢素是通过改变K+介导的 磁导率 我们将评估提前改变的后果 包括磷酸肌醇代谢的生化事件， c-fos、c-myc、IL 2、IL 2受体和转铁蛋白的表达 受体基因mRNA并最终影响受体表达，IL 2 形成和细胞增殖或IG分泌。 为了 为了确定特定T细胞亚群的活性，我们将获得T- 细胞亚群，并将药物偶联到脂质体， 用单克隆抗体将它们靶向特定的淋巴细胞亚群， 抗体的 更精确地定义药物的作用机制 环孢菌素类药物的应用将提供进一步的了解 作为一种免疫抑制剂， 它与其他形式的治疗和可能减少剂量， 持续时间和毒性。

Cyclosporin A (CsA) is a potent immunosuppressive agent, which acts primarily by inhibiting T cell function, likely by preventing IL2 formation. Its wide and successful use in clinical transplantation requires a more accurate definition of its mode of action. The presumption that CsA selectively blocks IL2 production ignores the inhibition readily demonstrated on earlier metabolic events and its suppressive activity in B lymphocytes as well. We have demonstrated that the immunosuppressive cyclosporins, but not the non-immunosuppressive analog, affect ion mobilization and the resting membrane potential of cells, resulting in decreased intracellular Ca2+ concentrations. These findings, which are the earliest recorded effect of the drugs, likely contribute to the overall immunosuppressive activities of this group of drugs, especially on IL2 production and T-cell proliferation. The goals of the present proposal are to further define the ionic, biochemical, and genetic basis for cyclosporin-dependent effects on membrane potential, Ca2+ uptake and IL2 formation, to determine the interrelationship between these phenomena, and to delineate the requirements for their immunosuppressive action. Specifically in human T and B cells we will examine early activation events following receptor-ligand interaction, subsequent events which occur after a signal has reached the nucleus, and late events associated with cell division, lymphokine production or Ig secretion. Using intracellularly trapped fluorescent indicators we will monitor changes in cytosolic Ca2+ concentrations, cytosolic pH, and membrane potential. We will determine if the major effects of the cyclosporins are mediated through alteration of K+ permeability. We will assess the consequences of altering early biochemical events including phosphoinositide metabolism on the expression of c-fos, c-myc, IL2, IL2 receptor and transferrin receptor gene mRNA and ultimately on receptor expression, IL2 formation and cell proliferation or Ig secretion. In an attempt to define activities of specific T-cell subsets we will obtain T- cell subsets by sorting and will couple the drugs to liposomes and target them to specific lymphocyte subsets with monoclonal antibodies. More precise definition of the mechanism of action of the cyclosporins would provide further understanding for its use as an immunosuppressive agent, offering the potential for combining it with other forms of therapy and possibly reducing dosage, duration and hence toxicity.