INTRACELLULAR SIGNALLING IN AIDS AND TUBERCULOSIS

这项提案中描述的实验试图确定 人类感染结核分枝杆菌和/或HIV-1导致 导致巨噬细胞的细胞内信号通路的扰动 活化与NK细胞和T细胞分化。两个主要的见解将 从这条调查路线衍生出来的。首先，我们将确定是否 自然杀伤细胞和T细胞对IL-2仍有反应，以及 单核细胞和巨噬细胞保持被激活的能力 感染患者在疾病进展过程中的干扰素-γ 携带结核分枝杆菌和HIV-1。第二，这些因素的结构性激活 从患者分离的细胞中的通路，以及从患者体内分离的 细胞因子治疗，将作为这些细胞产生的指标 体内的细胞因子。IL-2诱导的NK和T细胞信号转导 通过测量蛋白质酪氨酸磷酸化来评估，激活 蛋白酪氨酸激酶p56lck，蛋白激酶C的激活 (PKC)，丝氨酸/苏氨酸激酶Raf-1的激活和诱导 IL-2特异性即刻早期基因。中的IFNGamma响应 巨噬细胞和单核细胞将通过测定水平进行评估 干扰素-γ诱导的基因产物，包括48K肉豆蔻酸化的PKC 底物、IP-10和高亲和力FcGamma受体。我们还将 检测结核分枝杆菌的细胞壁产物，如MDP和LAMB 原单核细胞促进PKC依赖的增强反应 花生四烯酸代谢增加。将确定是否 启动与MARCKS的表达和磷酸化有关 42K PKC底物。我们还将确定结核分枝杆菌和 它的细胞壁产物激活了核因子-kB转录途径，并且 这是否会导致潜伏的HIV-1被激活。 最后，我们将鉴定和鉴定一种质膜受体 HIV-1在T细胞和巨噬细胞中的Pr55gag蛋白。为此， Pr55gag的精确膜结合域将通过缺失来定义 突变和一种基于该结构域的合成肽将用于 交联性实验鉴定Pr55gag结合的膜受体 捆绑。这种受体的鉴定将提供新的见解。 深入研究HIV-1的生物学并确定合理药物的潜在靶点 可能导致成功的治疗干预的设计。

The experiments described in this proposal seek to establish whether infection of humans with M. tuberculosis and/or HIV-1 result in perturbations of intracellular signalling pathways leading to macrophage activation and NK- and T-cell differentiation. Two major insights will derive from this line of investigation. First, we will determine whether natural killer cells and T cells remain responsive to IL-2, and whether monocytes and macrophages retain the capacity to be activated by interferon-gamma, during the progression of disease in patients infected with M. tuberculosis and HIV-1. Second, constitutive activation of these pathways in cells isolated from patients, and from patients undergoing cytokine therapy, will serve as an index of the production of these cytokines in vivo. IL-2-induced signaling in NK- and T-cells will be assessed by measuring protein tyrosine phosphorylation, the activation of the protein tyrosine kinase p56lck, the activation of protein kinase C (PKC), the activation of serine/threonine kinase Raf-1 and the induction of IL-2-specific immediate early genes. IFNgamma responsiveness in macrophages and monocytes will be assessed by determining the levels IFNgamma induced gene products including the 48K myristoylated PKC substrate, IP-10 and the high affinity Fcgamma-receptor. We will also examine whether cell wall products of M. tuberculosis such as MDP and LAM B prime monocytes for enhanced PKC-dependent responses such as those leading to increased arachidonic acid metabolism. It will be determined whether priming correlates with the expression and phosphorylation of the MARCKS and 42K PKC substrates. We will also determine whether M. tuberculosis and its cell wall products activate the NF-kB transcriptional pathway, and whether this results in the activation of latent HIV-1. Finally, we will identify and characterize a plasma membrane receptor for the Pr55gag protein of HIV-1 in T-cells and macrophages. To this end, the precise membrane binding domain of Pr55gag will be defined by deletional mutagenesis and a synthetic peptide, based on this domain, will be used in crosslinking experiments to identify the membrane receptor to which Pr55gag binds. The identification of such a receptor will provide new insights into the biology of HIV-1 and identify a potential target for rational drug design which could lead to successful therapeutic intervention.