Chemokines and their receptors have been recently implicated as important factors in the progress of HIV infection. A systematic characterization of their distribution within the brain is essential to understand changes in their expression during HIV encephalitis (HIVE). In S.A. number 1 we will compare abundance and distribution of chemokines and their receptors in the brain of AIDS patients who died with various degrees of HIVE and determine the relationship with viral burden. Levels of chemokine and receptor gene expression and localization will be assessed by quantitative reverse-transcription-PCR (QRT-PCR). Cellular localization will be determined by double immunofluorescent laser confocal microscopy (IFLCM). Brain viral burden will be measured by new quantitative HIV mRNA assays and PCR for proviral genes. In vitro, HIV infection of human macrophages is chronic and productive. Using fetal and postnatal microglial cultures and various viral stains, in SA number 2 we will study mechanisms of viral spread within the brain macrophage cell population and try to clarify the inconsistencies reported in previous studies regarding the role played by the newly described co-receptors. Gene expression and cellular distribution of chemokine receptors will be analyzed by RT-PCR, immunostaining and receptor binding studies using fluorescent labeled ligands. HIV infection of microglia will be compared to monocyte derived macrophage (MDM) cultures using IFLCM, ELISA and immuno-EM for HIV proteins and RT- PCR for HIV genes. To identify other genes potentially involved in the spread of HIV infection within brain macrophages we propose to use a recently developed technique called mRNA differential display analysis. In SA number 3 we will try to block infection using chemokine receptor ligands and treatments with new antiretroviral drugs. In addition to natural and synthetic chemokine molecules, we will test combinations of nucleoside analogs and protease inhibitors in MDM and microglial cultures infected with HIV. The neurotoxicity (measured by decreased neuronal survival and differentiation) of various treatments will be tested in vitro in HIV infected microglia or MDM co-cultured with uninfected neuroglial (mixed neuronal and astrocytic) fetal cells. The relevance of the results from autopsy studies regarding the role played by various new co-receptors will be tested in an in vitro model based on primary cultures of human neuroglia and macrophages. Comparison of autopsy and in vitro data will identify important mechanisms of disease that can be subjected to therapeutic interventions. We believe that the recently proposed HIV co-receptors are important but they are not unique or sufficient to explain the HIV spread in the brain macrophage population. We also hypothesize that chemokine receptors expressed on human (non-macrophage) neuroglia may mediate neurotoxicity in HIVE and, consequently, the proposed treatments with ligands may have to be carefully assessed.
| Harrold, S M; Dragic, J M; Brown, S L et al. (2001) Neurotrophic factor regulation of human immunodeficiency virus type 1 replication in human blood-derived macrophages through modulation of coreceptor expression. Adv Exp Med Biol 493:41-7 |
