IBD--MUCOSA-SPECIFIC REGULATION OF IFN-GAMMA PRODUCTION

描述：（改编自申请人的摘要）： IFN-γ在疾病的发生和疾病的严重程度中起重要作用 在动物模型和克罗恩病中的作用。粘液T细胞不同于 外周血T细胞在几个方面。粘液T细胞是沉默的应答者， 通过T细胞受体活化，并且是CD 2途径占主导地位。期间 在上一个授予期，研究人员利用他们的能力， T细胞显示出独特的粘膜特异性机制，T细胞通过该机制产生Th 1 细胞因子他们证明了AP 1在IL-2的CD 2反式激活中的作用。 基因他们表明，通过CD 2/CD 28共激活的IL-2产生的增强 外周血T细胞通过mRNA稳定和反式激活发生， 然而，在粘膜T细胞中，这种作用可能仅仅是外周T细胞的作用。 mRNA稳定化的结果。这种差异是由于缺乏激活 在粘膜细胞中IL-2启动子中的AP 1和CD 28应答元件。 他们证明了IFN-γ调节的几种粘膜特异性机制 并确定了TNF-α在粘膜T细胞IFN-γ中的独特作用， 调控他们定义了固有层单核细胞因子， 使T细胞对TNF-α产生应答。此外，他们表现出不同的 IFN-γ启动子中的顺式元件，其用于转录激活 IFN-γ基因和粘膜T细胞与外周T细胞相比。最后， 他们第一次证明了第一个基因的增强子功能， IFN-γ基因的内含子区，涉及JAK/STAT途径， 粘膜T细胞，而不是外周T细胞。因为这是至关重要的 IFN-γ在疾病相关的粘膜炎症中的作用， 宿主防御，粘膜特异性靶点的知识，这将选择性地 减弱粘膜IFN-γ的产生而不消除，将是一种 重大成就。本续设提案的总体目标是 是为了更精确地定义粘膜特异性调节途径， 对于T细胞中IFN-γ调节很重要，并利用这些知识 设计以粘膜特异性方式改变IFN-γ产生的方法。 这将通过以下具体目标来实现： 精细启动子分析内的阳性和阴性顺式调节区 IFN-γ启动子，其中固有层中的转录调节 单核细胞与外周血淋巴细胞不同， 先前鉴定的-204至-108（增强子）和-528至-204（增强子）区域 碱基对（阻遏物）。为了确定粘膜特异性机制， 调节IFN-γ基因的第一内含子区。结果所 目的1和目的2设计包括转染反义核酸的分子方法， 寡核苷酸和/或显性负反式作用因子，以减弱 体外IFN-γ启动子以粘膜特异性方式表达。

DESCRIPTION: (Adapted from the Applicant's Abstract): T-cell production of IFN-gamma plays an essential role in disease initiation and severity of disease in animal models and Crohn's disease. Mucosal T-cells are different from peripheral T-cells in several ways. Mucosal T-cells are muted responders to activation via the T-cell receptor and are CD2 pathway dominant. During the previous award period the investigators used their ability to transfect primary T-cells to show unique mucosa-specific mechanisms by which T-cells produced Th1 cytokines. They demonstrated a role for AP1 in CD2 transactivation of the IL-2 gene. They showed that enhanced IL-2 production by CD2/CD28 co-activated peripheral T-cells occurs through mRNA stabilization and trans-activation in peripheral T-cells, however, in mucosal T-cells this effect can be solely the result of mRNA stabilization. This difference is due to the lack of activation of the AP1 and CD28 response elements in the IL-2 promotor in mucosal cells. They demonstrated several mucosal-specific mechanisms of IFN-gamma regulation and identified a unique role for TNF-alpha in mucosal T-cell IFN-gamma regulation. They defined the lamina propria mononuclear cell factor which can prime T-cells to respond to TNF-alpha. Furthermore, they demonstrated different cis elements in the IFN-gamma promotor that are used for transactivation of the IFN-gamma gene and mucosal T-cells as compared to peripheral T-cells. Finally, they demonstrated for the first time the enhancer function of the first intronic region of the IFN-gamma gene that involves the JAK/STAT pathway in mucosal T-cells but not in peripheral T-cells. Because of the vital importance of IFN-gamma in disease related mucosal inflammation yet its critical role for host defense, the knowledge of mucosa-specific targets, which would selectively attenuate mucosal IFN-gamma production without eliminating, would be a significant accomplishment. The overall objective of this continuation proposal is to more precisely define the mucosal specific regulatory pathways that are important for IFN-gamma regulation in T-cells and to use this knowledge to design approaches to modify IFN-gamma production in a mucosa-specific manner. This will be accomplished by the following specific aims: To identify through fine promotor analysis positive and negative cis-regulatory regions within the IFN-gamma promotor in which transcriptional regulation in lamina propria mononuclear cells differ from that in peripheral blood lymphocytes focusing on the previously identified regions of -204 to -108 (enhancer) and -528 to -204 base pair (repressor). To determine the mucosa-specific mechanisms of regulating the first intronic region of the IFN-gamma gene. From the results of Aim 1 and Aim 2 design molecular approaches including transfection of antisense oligonucleotides and or dominant negative transacting factors to attenuate in vitro IFN-gamma promotor expression in a mucosal-specific manner.