IONIC,BIOCHEMICAL,GENETIC BASIS FOR CYCLOSPORIN ACTIVITY

环孢素A(CsA)是一种有效的免疫抑制剂，其作用 主要是通过抑制T细胞功能，可能是通过预防IL2 队形。它在临床移植中的广泛而成功的应用 需要对其行动模式有更准确的定义。这个 假设CSA选择性地阻止IL2的产生，则忽略了 对早期代谢事件的抑制作用及其机制 B淋巴细胞的抑制活性也是如此。我们有 证明了免疫抑制环孢素，但不是 非免疫抑制类似物，影响离子动员和 静息细胞膜电位，导致细胞膜电位下降 细胞内钙离子浓度。这些发现是 最早记录到的药物效果，可能有助于 这组药物的总体免疫抑制活性， 尤其是对IL-2的产生和T细胞的增殖。目标 本提案的目的是进一步定义离子， 环孢素依赖效应的生化和遗传学基础 对膜电位、钙摄取和IL_2形成的影响 这些现象之间的相互关系，并描绘出 对其免疫抑制作用的要求。特别是在 我们将检测人类T和B细胞的早期激活事件 在受体-配体相互作用之后，随后发生的事件 发生在信号到达原子核之后，以及后期事件 与细胞分裂、淋巴因子产生或Ig有关 分泌物。使用细胞内捕获的荧光指示剂 我们将监测胞浆钙浓度、胞浆钙浓度的变化 PH和膜电位。我们将确定是否会产生重大影响 的环孢素类药物是通过K的改变来调节的 渗透性。我们将评估早期改变的后果 包括磷脂酰肌醇代谢在内的生化事件 C-fos、c-myc、IL-2、IL-2受体和转铁蛋白的表达 受体基因mRNA和最终对受体表达的影响，IL-2 形成和细胞增殖或Ig分泌。在尝试中 为了定义特定T细胞亚群的活动，我们将获得T- 通过分选细胞亚群，并将药物偶联到脂质体和 用单克隆靶向特定的淋巴细胞亚群 抗体。更准确的定义作用机制 环孢菌素将为其使用提供进一步的理解。 作为一种免疫抑制剂，提供了结合 与其他形式的治疗相结合，并可能减少剂量， 持续时间，因此毒性。

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.