MECHANISMS OF ALCOHOL RELATED INHIBITION OF NK ACTIVITY

据估计，酒精依赖影响着超过1000万美国人 与酒精有关的问题的费用总计超过1000亿美元 美元.饮酒损害体液和细胞介导的 免疫力自然杀伤（NK）细胞在免疫系统中起着重要作用。 针对某些传染病和癌症的免疫监视， 特别是在预防肿瘤转移方面具有活性。NK细胞也控制 艾滋病的发展速度。我们之前的研究表明， 消耗特异性地导致NK和淋巴因子活化受损， 当小鼠消耗25-40%的杀伤细胞（LAK）细胞溶解活性时， 从饮用水中摄入的乙醇中获得的热量。的目标 该建议旨在确定这种受损的潜在机制， 细胞溶解活性，并专门测试酒精 消耗通过破坏蛋白质损害细胞溶解活性 激酶C细胞信号传导，其反过来调节颗粒和细胞毒性 中介功能我们将研究酒精消费的影响 对NK颗粒细胞毒活性和细胞溶解因子特异性功能的影响 介质、穿孔素、颗粒酶A和颗粒酶B。我们还将确定 酒精消费对这些活动和数量的影响 NK和LAK细胞两个主要亚群中的蛋白质， 有助于固有的NK细胞溶细胞活性（NK1.1+ LGL 1-细胞）， LAK细胞溶解活性（NK1.1+ LGL-细胞）。体外比色法 酶测定将表征颗粒酶A和颗粒酶B的蛋白水解 活动富集的NK1.1+细胞和LGL 1亚群的分离 将采用磁珠分离技术。蛋白质印迹分析 将用于确定颗粒酶中的蛋白质表达，并逆转 将使用转录酶聚合酶链反应（RT-PCR）来确定 mRNA。将使用标准测定法测定蛋白激酶C活性。 将用氮气从NK/LAK细胞中分离溶细胞颗粒 气穴现象。

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.