MECHANISMS OF ALCOHOL RELATED INHIBITION OF NK ACTIVITY

酒精相关的 NK 活性抑制机制

• 批准号: 2697449
• 负责人: GARY G MEADOWS
• 金额: $ 26.99万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2697449
• 关键词: alcoholic beverage consumption; alcoholism /alcohol abuse; athymic mouse; biological signal transduction; calcium; cellular immunity; corticosterone; cytokine; cytotoxicity; enzyme linked immunosorbent assay; flow cytometry; growth inhibitors; humoral immunity; interleukin 2; interleukin 6; laboratory mouse; lymphocyte proliferation; melanoma; metastasis; natural killer cells; neoplastic cell; neoplastic growth; toxicology

It is estimated that alcohol dependency affects over 10 million Americans and that the cost of alcohol related problems totals over 100 billion dollars. Alcohol consumption impairs both humoral and cell-mediated immunity. Natural killer (NK) cells play an important role in immunosurveillance against certain infectious diseases and cancer and are especially active in preventing tumor metastasis. NK cells also control the rate of progression of aids. We showed previously that alcohol consumption specifically results in impaired NK and lymphokine activated killer (LAK) cytolytic activity when mice consume between 25-40% of their calories from ethanol administered in the drinking water. The objective of this proposal is to determine the mechanism(s) underlying this impaired cytolytic activity and to specifically test the hypothesis that alcohol consumption impairs cytolytic activity through disruption of protein kinase C cell signaling, which in turn modulates granule and cytotoxic mediator function. We will examine the effect that alcohol consumption has on NK granule cytotoxicity and the specific function of the cytolytic mediators, perforin, granzyme A, and granzyme B. We will also determine the effect of alcohol consumption on the activity and amount of these proteins in the two primary subpopulations of NK and LAK cells that contribute to inherent NK cell cytolytic activity (NK1.1+LGL1-cells) and to LAK cytolytic activity (NK1.1+LGL-cells). In vitro colorimetric enzymatic assays will characterize granzyme A and granzyme B proteolytic activity. Isolation of enriched NK1.1+ cells and the LGL1 subpopulations will utilize magnetic bead separation technology. Western blot analysis will be used to determine protein expression in granzymes, and reverse transcriptase polymerase chain reaction (RT-PCR) will be used to determine mRNA. Protein kinase C activity will be determined using standard assays. Cytolytic granules will be isolated from NK/LAK cells with nitrogen cavitation.

据估计，有1000多万美国人受到酒精依赖的影响 与酒精有关的问题的总成本超过1000亿美元 美元。饮酒损害体液和细胞介导性 豁免权。自然杀伤(NK)细胞在 对某些传染病和癌症的免疫监控，并 在预防肿瘤转移方面尤为积极。NK细胞也控制着 艾滋病的进展速度。我们之前已经证明了酒精 消费特别会导致NK受损和淋巴因子激活 杀伤(LAK)细胞活性当小鼠摄入25%-40% 来自饮用水中乙醇的卡路里。的目标是 这项建议是为了确定这种损害背后的机制(S 细胞溶解活性，并具体检验酒精 消费通过破坏蛋白质而损害细胞溶解活性 激酶C细胞信号转导，进而调节颗粒和细胞毒 调解人功能。我们将研究饮酒的影响 NK颗粒的细胞毒作用及溶细胞剂的特异性功能 介体、穿孔素、颗粒酶A和颗粒酶B。我们还将测定 饮酒对这些酶的活性和数量的影响 NK细胞和LAK细胞两个主要亚群中的蛋白质 有助于固有的NK细胞杀伤活性(NK1.1+LGL1-细胞)和 对LAK细胞的杀伤活性(NK1.1+LGL-细胞)。体外比色法 酶分析将表征颗粒酶A和颗粒酶B的蛋白分解 活动。浓缩NK1.1+细胞及其LGL1亚群的分离 将利用磁珠分离技术。Westernblot分析 将用于确定颗粒酶中的蛋白质表达，并逆转 将使用逆转录聚合酶链式反应(RT-PCR)来确定 MRNA.蛋白激酶C活性将使用标准分析方法进行测定。 用氮气从NK/LAK细胞中分离出溶细胞颗粒 空化。