CONTROL OF EOSINOPHIL APOPTOSIS BY INTRACELLULAR SIGNALS

描述（改编自申请人的摘要）： 慢性哮喘的有效治疗是吸入糖皮质激素治疗 其通过抑制细胞因子的产生来减少气道中的炎症， 诱导嗜酸性粒细胞凋亡。虽然糖皮质激素治疗已被证明是 对大多数哮喘患者有效，有一系列患者反应性， 这表明，变异性可能是由于潜在的差异， 胞内信号传导哮喘患者嗜酸性粒细胞减少 接受糖皮质激素治疗与气道改善密切相关 功能，糖皮质激素已被证明可诱导人类细胞凋亡 嗜酸性粒细胞在体外和体内。根据这些观察， 研究者假设细胞因子产生的质量和数量 在肺中，嗜酸性粒细胞基因表达谱的差异， 是调节糖皮质激素效力的两个关键因素 疗法 提出了三个具体目标来检验这一一般假设。1)他们有 发现糖皮质激素诱导人原代细胞嗜酸性粒细胞凋亡 含有低水平细胞因子的培养物，但实际上增加了嗜酸性粒细胞 在具有高浓度细胞因子的培养物中存活。他们将 研究不同糖皮质激素作用的分子基础， 使用细胞提取物监测线粒体的纯化的人嗜酸性粒细胞 功能障碍和激酶特异性抑制剂，以确定哪些途径 有助于这种增强效果。2)他们将识别关键信号 人嗜酸性粒细胞中抗凋亡和促凋亡信号传导所需的途径 细胞系模型AML 14.3D10。这将通过分子遗传学 利用含有绿色荧光的共转染测定的方法 蛋白质（GFP）基因和选择的信号传导的显性负性基因变体 分子。3)他们将使用cDNA微阵列对2万个人类基因进行表型分析 鉴定和表征与凋亡相关的表达模式， 原代人嗜酸性粒细胞的敏感性，并与细胞内信号 在各种培养条件下生长的AML14.3D10细胞中的细胞凋亡途径。这 微阵列分析利用低成本的服务和资源提供 由全美大学联合会（U.亚利桑那微阵列核心实验室

DESCRIPTION (Adapted from the Applicant's Abstract): One of the most efficacious treatments for chronic asthma is inhaled glucocorticoid therapy which reduces inflammation in the airways by inhibiting cytokine production and inducing eosinophil apoptosis. Although glucocorticoid therapy has proven to be effective for most asthmatics, there is a range of patient responsiveness, suggesting that variability may be due to underlying differences in intracellular signaling. The reduction for eosinophilia in asthmatics undergoing glucocorticoid therapy is strongly associated with improved airway functions, and glucocorticoids have been shown to induce apoptosis in human eosinophils in vitro and in vivo. Based on these observations, the investigators hypothesize that the quality and quantity of cytokine production in the lung, and differences in the eosinophil gene expression profile between individuals, are two key factors that modulate the potency of glucocorticoid therapy. Three specific aims are proposed to test this general hypothesis. 1) They have found that glucocorticoids induce human eosinophil apoptosis in primary cell cultures containing low levels of cytokines, but actually enhance eosinophil survival in cultures with high concentrations of cytokines. They will investigate the molecular basis for differential glucocorticoid effects in purified human eosinophils using cell extracts to monitor mitochondrial dysfunction, and kinase-specific inhibitors to determine which pathways contribute to this enhancement effect. 2) They will identify key signaling pathways required for anti- and pro-apoptotic signaling in the human eosinophil cell line model AML14.3D10. This will be done using molecular genetic approaches that exploit co-transfection assays containing the green fluorescent protein (GFP) gene and dominant negative gene variants of selected signaling molecules. 3) They will use cDNA microarray phenotyping of 20,000 human genes to identify and characterize expression patterns that associate with apoptotic sensitivity of primary human eosinophils, and with intracellular signaling pathways in AML14.3D10 cells grown under various culture conditions. This microarray analysis takes advantage of low-cost services and resources provided by the university-wide U. of Arizona Microarray Core Facility.