CONTROL OF EOSINOPHIL APOPTOSIS BY INTRACELLULAR SIGNALS

描述(改编自申请者的摘要)：最 吸入糖皮质激素是治疗慢性哮喘的有效方法 它通过抑制细胞因子的产生来减少呼吸道的炎症 诱导嗜酸性粒细胞凋亡。尽管糖皮质激素疗法已被证明是 对大多数哮喘患者有效，有一系列的患者反应， 这表明可变性可能是由于 细胞内信号。哮喘患者嗜酸性粒细胞减少 接受糖皮质激素治疗与改善呼吸道状况密切相关 功能，糖皮质激素已被证明可诱导人类细胞凋亡 体外和体内的嗜酸性粒细胞。基于这些观察， 研究人员假设，细胞因子产生的质量和数量 以及嗜酸性粒细胞基因表达谱的差异 个体，是调节糖皮质激素效力的两个关键因素 心理治疗。 为了检验这一普遍假设，本文提出了三个具体目标。1)他们有 发现糖皮质激素诱导原代细胞中的人嗜酸性粒细胞凋亡 含有低水平细胞因子的培养物，但实际上增强了嗜酸性粒细胞 在高浓度细胞因子的培养中存活。他们会 探讨糖皮质激素不同作用的分子基础 用细胞提取液监测线粒体的纯化人嗜酸性粒细胞 功能障碍和激酶特异性抑制物来确定哪些途径 有助于实现这种增强效果。2)他们将识别关键信令 人嗜酸性粒细胞抗和促凋亡信号转导途径的研究 细胞模型AML14.3D10。这将使用分子遗传学来完成 利用含有绿色荧光的共转染试验的方法 蛋白质(GFP)基因和显性负基因突变体的选择信号 分子。3)他们将使用20,000个人类基因的cDNA微阵列表型 识别和表征与细胞凋亡相关的表达模式 原代人嗜酸性粒细胞的敏感性及其与细胞内信号的关系 不同培养条件下AML14.3D10细胞的生长途径。这 微阵列分析利用提供的低成本服务和资源 由亚利桑那大学的微阵列核心设施。

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.