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

描述：(改编自申请人的摘要)：T细胞的生产 干扰素-γ在疾病的发生和严重程度中起着重要作用。 在动物模型和克罗恩病中。粘膜T细胞不同于 外周T细胞以几种方式。粘膜T细胞是沉默的反应者 激活途径主要通过T细胞受体和CD2途径。在.期间 在之前的获奖期间，研究人员利用他们的能力将 T细胞显示独特的粘膜特异性机制，通过T细胞产生Th1 细胞因子。他们证明了AP1在CD2反式激活IL-2中的作用 吉恩。他们表明，CD2/CD28共同激活可增强IL-2的产生 外周T细胞通过mRNA稳定和反式激活而发生 然而，在粘膜T细胞中，外周T细胞的这种作用可能仅仅是 MRNA稳定的结果。这种差异是由于缺乏激活造成的 黏膜细胞IL-2启动子中的AP1和CD28反应元件。 他们展示了几种黏膜特异性的干扰素-γ调节机制。 并确定了肿瘤坏死因子-α在粘膜T细胞干扰素-γ中的独特作用 监管。他们定义了固有层单个核细胞因子，它可以 激发T细胞对肿瘤坏死因子-α的反应。此外，他们表现出了不同的 干扰素-γ启动子中用于反式激活的顺式元件 干扰素-γ基因与粘膜T细胞和外周T细胞的比较。最后， 他们首次展示了第一个 干扰素-γ基因内含子区域，涉及JAK/STAT途径 粘膜T细胞，而外周T细胞不表达。因为至关重要的是 干扰素-γ在疾病相关性粘膜炎症中的作用 宿主防御，粘膜特异性靶点的知识，这将选择性地 在不消除的情况下，减弱粘膜中干扰素-γ的产生，将是一种 取得了重大成就。这项延续提案的总体目标是 是更准确地定义黏膜特异性调节通路，这些调节通路是 对T细胞中干扰素-γ的调节很重要，并利用这一知识 设计以黏膜特异性方式修饰干扰素-γ产生的方法。 这将通过以下具体目标来实现：通过 精细的启动子分析正面和负面顺式调控区域 干扰素-γ启动子在固有层转录调控中的作用 单个核细胞与外周血淋巴细胞不同，主要集中在 先前确定的-204至-108(增强子)和-528至-204区域 碱基对(抑制子)。为了确定粘膜特异性的机制， 调节干扰素-γ基因的第一内含子区域。从以下结果来看 目标1和目标2设计包括反义基因转染的分子方法 寡核苷酸和或显性负性交易因子在 体外以黏膜特异性方式表达干扰素-γ启动子。

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.