INDUCTION OF APOPTOSIS BY HIV-1 INFECTED MONOCYTIC CELLS

我们利用人类巨噬细胞杂交瘤系统来分析HIV-1/巨噬细胞相互作用，特别关注HIV-1感染对APC功能的影响。我们在HIV-1感染过程的早期发现了许多缺陷，但在研究慢性HIV-1感染系时发现了一个有趣的发现。T细胞与这些细胞共培养可诱导细胞凋亡。有趣的是，CD-4和CD-8+ T细胞以及B细胞都出现了凋亡。gp120和FasL的表面表达可以解释CD-4+ T细胞的凋亡，但在CD-8 T细胞和B细胞中不存在。在CD-4+ T细胞和CD-4+ T细胞以及B细胞中，我们没有发现诱导凋亡的可溶性因子。为了确保我们的因子是新颖的，我们比较了HIV-1感染和未感染细胞中与凋亡相关的不同趋化因子和细胞因子，包括SDF-1、Ltn、Rantes、IP-10、mcp -1- β、I-309、L32、IL-8、tnf - β、lt - β、tnf - α、γ -IFN、β -IFN、tgf - β 3、tgf - β 2和tgf - β 1，发现RNA的产生在HIV-1感染和未感染细胞之间没有差异。我们纯化了该因子的3个候选肽，并对其进行了部分测序，并生成了一组体外中和活性的多克隆和单克隆抗体。使用这些抗体，我们在HIV-1感染患者的PBMC中检测到该因子，但在未感染的对照组中未检测到。虽然我们鉴定了3个多肽，但可能还有其他可溶性因子具有活性。我们将扩展这些初步观察，对部分测序的肽和其他促凋亡肽进行完全测序和分离特定的cDNA克隆，筛选不同的表达文库，如果失败，则通过减法杂交。在授权期的第二部分，我们将确定该因子产生的多样性，以及促凋亡作用是普遍的还是有限的。我们还将鉴定靶细胞上的受体。我们将尝试通过量化颈部患者的生物体液（血清和脑脊液）和组织（淋巴结和脑）中的水平来确定促凋亡因子在HIV-1感染患者中的作用。在疾病的不同阶段。该因子的鉴定可能有助于解释艾滋病中通过细胞凋亡发生的一些细胞损失。

We have utilized a system of human macrophage hybridomas to analyze HIV-1/macrophage interactions with a specific focus upon the effects of HIV-1 infection on APC function. We identified numerous defects early in the course of HIV-1 infection but one intriguing finding was made when studying chronically HIV 1 infected lines. Co-culture of T cells with these cells resulted in the induction of apoptosis. Interestingly, apoptosis was noted in both CD-4 and CD-8+ T cells as well as B cells. The surface expression of gp120 and FasL could account for the apoptosis observed in the CD-4+ T cells but not in the CD-8 T cells or B cells. We could not identify soluble factors that induced apoptosis in CD-4+ T cells and CD-4+ T cells as well as B cells. To ensure that our factors were novel we compared and found no difference in RNA production between the HIV-1 infected and uninfected cells for different chemokines and cytokines associated with apoptosis including SDF-1, Ltn, Rantes, IP-10, MCP-1-beta, I-309, L32, IL-8, TNF-beta, LT-beta, TNF-alpha, gamma- IFN, beta-IFN, TGF-beta3, TGF-beta2, and TGF-beta1. We purified and partially sequenced 3 candidates peptides for this factor and generated a panel of polyclonal and monoclonal antibodies which neutralize activity in vitro. Using these antibodies we detected this factor in the PBMC of HIV-1 infected patients but not in uninfected controls. Although we characterized 3 peptides, there may be other soluble factors which have activity. We will extend these preliminary observations to fully sequence and isolate specific cDNA clones for the partially sequenced peptides and the other pro-apoptotic peptides screening different expression libraries and if this fails by subtractive hybridization. In the second part of the granting period we will determine the diversity of production of the factor and if the pro-apoptotic effect is generalized or restricted. We will also identify receptor(s) on target cells. We will attempt to define a role for the pro-apoptotic factor in HIV-1 infected patients by quantifying levels in biological fluids (serum and cerebral spinal fluid) and tissues (lymph node and brain) from necked patients. at different stages of disease. Identification of this factor may help explain some of the cell loss that occurs through apoptosis in AIDS.