CELLULAR GENES REQUIRED FOR HIV1 INFECTION AS TARGETS FOR ANTIVIRAL THERAPY

本课题的主要目的是：（1）鉴定细胞基因， （2）为了鉴定细胞基因， 参与HIV-1感染的发病机制（例如增强的 程序性细胞死亡）（3）设计分子方法来抑制 这些细胞基因在HIV感染中的表达。 (4)目的研究HIV-1在SCID-Hu动物模型中的感染及发病机制。 我们产生了一组HIV-1抗性T细胞克隆，它们来自于 艾滋病易感株系CEM。 我们通过细胞和 分子生物学方法，并确定了一个NF-κ B p50缺陷克隆。 对HIV-1进入和再激活有抵抗力。其中一个克隆人 显示新发现的艾滋病毒的表面表达减少 辅助受体CXCR 4。 在SCID/Hu模型的研究中，我们发现胸腺内HIV-1感染 导致一小部分成熟的CD 3 hiCD 8+细胞的生产性感染， 胸腺细胞这些细胞在早期分化阶段受到感染 同时表达CD 4和CD 8（DP）。这些细胞在胸腺内死亡， 而不是在外围播种。这项研究解释了 在HIV感染个体中观察到的外周“初始”CD 8细胞， 尤其是孩子们 在HIV-1辅助受体CXCR 4和 CCR 5，在原代细胞上： (a)CXCr 4在未成熟的人胸腺细胞上有很高的表达 (from婴儿），包括CD 34+胸腺内复发。胸腺细胞 对α趋化因子SDF 1（但不对任何β趋化因子）有反应， 趋化性和Ca++动员。胸腺细胞容易与嗜T细胞融合 用兔抗CXCR 4的IgG阻断融合 （在我们的实验室里）。此外，我们发现证据表明， 胸腺细胞表达额外的趋化因子受体（I309 R）， 作为HIV-1共受体并支持与T-嗜性包膜融合。 本研究揭示了新生儿胸腺的敏感性 T嗜性HIV病毒株感染，经常导致严重的胸腺炎， 耗尽 (b)正在进行广泛的研究，以了解 HIV-1辅助受体在多种细胞表面的表达和功能 细胞特别是单核细胞与巨噬细胞，以及几种细胞系。两 在这些研究中使用了生物化学和生物学方法。在 另外我们检测了促炎细胞因子（IFN γ， TNF α，IL 10，IL-1 β）对辅助受体表达和功能的影响 巨噬细胞、树突细胞、朗格汉斯细胞和T细胞。 开发的试验将用于评价 直接阻断HIV-1细胞进入的抗病毒剂， 重新激活 积累的知识将直接应用于 基于细胞因子、基于核酶和基于反义的评价 已经处于临床早期阶段的治疗方法 审判

The goals of this project are: (1) To identify cellular genes which are required for optimal HIV infection (2) To identify cellular genes which are involved in the pathogenesis of HIV-1 infection (e.g enhanced programmed cell death ) (3) To design molecular approaches to suppress the expression of these cellular genes in HIV infection. (4) To study HIV-1 infection and pathogenesis in SCID-Hu animal model. We generated a panel of HIV-1 resistant T cell clones derived from the HIV-susceptible line CEM. We analyzed these clones by cellular and molecular approaches, and identified a n NF-kB p50 defficient clone s that are resistant to HIV-1 entry and reactivation. One of these clones were shwon to have reduced surface expression of the newly discovered hiv co-receptor CXCR4. In studies with SCID/Hu model we found that intrathymic HIV-1 infection results in a productive infection in a small fraction of mature CD3hiCD8+ thymocytes. These cells got ifected at an earlier differentiation stage expressing both CD4 and CD8 (DP). These cells die intrathymically and do not seed the periphery. This study explains the gradual loss of peripheral "naive" CD8 cells observed in HIV infected individual, especially children. Significant progress was done in studies of HIV-1 coreceptors, CXCR4 and CCR5, on primary cells: (a) Very high expression of CXCr4 was found on immature human thymocytes (from infants) including the CD34+ pintrathymic recursors. Thymocytes responded to the alpha chemokine SDF1 (but not to any beta chemokines) by chemotaxis and Ca++ mobilization. Thymocytes fused readily with T-tropic envelope and the fusion was blocked with rabbit IgG against CXCR4 (generated in our laboratory). In addition we found evidence that thymocytes express additional chemokine receptor (I309R) that also behave as a n HIV-1 co-receptor and support fusion with T-tropic envelopes. This study sheds light on the exquisite sensistivity of neonatal thymuse to infection with T-tropic HIV strains that often results in sever thymic depletion. (b) Extensive studies are under way to understand the correlation between surface expression and function of the hiv-1 coreceptors on a variety of cells. Especially monocytes Vs. macrophages, and several cell lines. Both biochemical and biological approaches are used in these studies. In addition we examine the effects of proinflamatory cytokines (IFNgamma, TNFalpha, IL10, IL-1beta) on coreceptor expression and function in macrophages, dendritic cells, Langerhans cells, and T cells. Assays developed will be used in the evaluation of the potency of anti-viral agents directed at blocking of HIV-1 cell entry and reactivation. Knowledge accumulated will be directly applied to evaluation of Cytokine-based, Ribozyme-based, and anti-sense-based therapeutic approaches which are already in early phases of clinical trials.