The studies to be carried out will concern the pathobiology of bone marrow suppression in AIDS or AIDS-related complex. The applicant intends to approach this problem in three ways. The first will utilize fresh marrow from HIV-infected individuals to determine whether injury to the megakaryocyte (MK) and the myeloid lineages, is due to immune complexes, inhibitory factors related to infection or the virus itself. Inhibitory substances will be tested in vitro on colonies from normal marrows. A direct effect of HIV will be explored by subcellular localization of HIV components immunohistochemically and by in situ hybridization with nucleic acid probes on the EM level. If cells prove to be infected, the route of entry, e.g. whether via CD4, Fc receptors or emperipolesis will be studied by analysis of normal marrow incubated with HIV for varying time periods. The second approach, to be executed in collaboration with Drl Malcolm Moore, will establish whether HIv can infect pluripotent stem cells. These will be selected with My-10 MoAB after ablation of proliferating progenitors with 4-hybroperoxycyclophosphamide, by FACS sorting or in SCID mice. Such cells are recognized by their requirement of SCF plus IL-1 for proliferation. IF pluripotent cells cannot be infected with HIV this precursor compartment could be amplified in vivo in the hope of outpacing the suppressive effect of HIV or chemotherapeutic agents such as AZT or ddI. Moreover, an argument for the clinical use of IL-1, in addition to CSF, would be provided. SImilar studies will be conducted on marrow stromal cells which elaborate the growth factors. Whether AIDS stromal cells are functionally intact will concern the structure of the virus itself and its interaction with plasma membranes. These studies will employ freeze-fracture to determine which structural components (e.g. """"""""knobs"""""""", envelopes) are responsible for various antigenic epitopes and whether insertion of HIV polypeptides affects the number and/or distribution of intrinsic membrane proteins represented by intramembranous particles (IMP). Cells cultured with HIV for 2,4,8 and 24 hrs. will also be subjected to """"""""label fracture"""""""" which permits identification of antigens on the external membrane leaflet coincident with the IMP. Redistribution of integral membrane proteins could elucidate the lytic and fusogenic phenomena exhibited by infected cells and perhaps lead to rational intervention at this level.
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