HIV-1 INFECTION IN FETAL BRAIN CELL CULTURES AND PEDIATRIC AIDS BRAIN TISSUE

我们建立了人类潜伏感染HIV-1的体外模型 胎儿星形胶质细胞。在感染或转基因几周后， 与未感染的淋巴细胞共培养或与 细胞因子TNF-α和IL1-β将增加由此产生的病毒产量 单元类型。我们已经证明了佛波醇12-肉豆蔻酸酯13-乙酸酯 (PMA)还增加了原发人类HIV-1 p24的产量 星形细胞。应用凝胶迁移率改变分析(EMSA)检测 结合使用特定抗体的SuperShift研究，我们 证明PMA与肿瘤坏死因子-α一样，增加了p50/p65形式的核因子- KB。此外，我们还表明，蛋白激酶抑制剂H7 抑制PMA和TNF-α相关的HIV-1表达增加 一段时间，当它几乎没有抑制作用的相关 P50/p65核因子-kB表达增加。因此，除非p50/p65核因子-kB或其结合 以EMSA无法解决的方式受H7影响，以及 这种形式的核因子-kB的增加并不总是足以增加HIV-1 来自星形胶质细胞的表达。 该实验室还在研究特定的核糖核酸酶是否能够 抑制HIV-1在淋巴细胞系中的增殖。Onconase 和牛精液核糖核酸酶被证明可以阻断HIV-1的感染 生产力感染了细胞系。此块显示在 核糖核酸酶活性的细胞内机制。 除了对感染的星形胶质细胞培养中的核因子-kB进行调节外， 病毒的rev蛋白似乎也是细胞控制的目标。 使用EMSA分析，星形胶质细胞产生一种结合RRE-rev的因子 很复杂。该因子对细胞因子和PMA刺激也有反应。 核因子-kB和rev结合因子似乎都受限于 与对HIV-1感染高度敏感的细胞相比，这种药物的浓度更高。 这些数据指出了HIV-1在大脑中潜伏感染的机制 涉及生产所需的细胞因子水平的降低 HIV-1增殖。星形胶质细胞作为HIV-1储存库的作用 正在使用体外感染模型进行研究。

We have established an in vitro model of latent HIV-1 infection in human fetal astrocytes. Several weeks following infection or transfection, cocultivation with uninfected lymphocytes or stimulation with the cytokines TNF-alpha and IL1-beta will increase viral production from this cell type. We have demonstrated that phorbol 12-myristate 13-acetate (PMA) also increases HIV-1 p24 production from the primary human astrocyte. Using electrophoretic mobility shift assay (EMSA) in combination with supershift studies using specific antibodies, we demonstrated that PMA, like TNF-alpha increases the p50/p65 form of NF- kB. Furthermore, we also showed that the protein kinase inhibitor H7 inhibits PMA and TNF-alpha associated increases in HIV-1 expression at a time when it has little to no inhibitory effect on the associated increases in p50/p65 NF-kB. Thus, unless p50/p65 NF-kB or its binding is affected by H7 in a manner that cannot be resolved by EMSA, an increase in this form of NF-kB is not always sufficient to increase HIV-1 expression from the astrocyte. The laboratory is also investigating the ability of specific RNases to inhibit the multiplication of HIV-1 in lymphocyte cell lines. Onconase and bovine seminal RNase were shown to block infection of HIV-1 in productivity infected cell lines. This block appears from an intracellular mechanism of RNase activity. In addition to the regulation of NF-kB in infected astrocyte cultures, the viral rev protein also seems to be a target for cellular control. Using EMSA assays, astrocytes produce a factor which binds the RRE-rev complex. This factor also responds to cytokine and PMA stimulation. Both the NF-kB and the rev binding factor appear to be limited in concentration compared with cells highly susceptible to HIV-1 infection. These data point to a mechanism of HIV-1 latent infection in brain involving reduced levels of cellular factors necessary for productive HIV-1 multiplication. The role of the astrocyte as a reservoir of HIV-1 in the brain is being investigated using the in vitro model of infection.