Critical events of normal and pathologic processes, including those caused by microbes, occur in tissues where cells contact neighboring cells and are immersed in a rich heterogeneous signal-rich microenvironment. In our Section, we study pathogenesis of microbes, in particular HIV-1 variants that use either CCR5 (R5) or CXCR4 (X4) for cell entry in human lymphoid tissues ex vivo. The former in vivo transmit infection and dominate early stages of HIV disease, whereas later, in approximately 50% of cases there is a switch toX4 dominance, and this switch is followed by a rapid progression to AIDS. The cause of the R5-to-X4 switch remains to be understood and possibly is a function of both viral and host factors. In our first project, we studied the possible role of viral factors, namely whether R5 variants dominating early stages of HIV disease are different in patients later undergoing an R5-to-X4 switch and in those progressing without a switch. In the second project we addressed potential host factors, namely whether infection of hosts by other microbes differentially affects R5 and X4 HIV. In the third project, we applied the developed ex vivo tissue system to study in pre-clinical trials the effects of potential antivirals on R5 and X4 HIV variants 1. Differential Pathogenesis of Primary CCR5-using HIV-1 isolates in human lymphoid In the course of HIV disease, R5 HIV variants, which typically transmit infection and dominate its early stages, persist in approximately half of infected individuals (nonswitch virus patients), while in the other half (switch virus patients) viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. We compared the pathogenesis of sequential primary HIV-1 isolates all of which were of the R5 phenotype, from switch and nonswitch virus patients, in ex vivo lymphoid tissue. We found that viral factors are major determinants of the relative replication ability of these isolates. In contrast, the absolute replicative capacity of HIV-1 isolates is controlled by host (tissue) factors. R5 HIV-1 infection results in depletion of their natural targets, i.e., CCR5+CD4+ T cells. We found that depletion of these cells was accompanied not only by downregulation of CD4 but also by downregulation of CCR5. Depletion of CCR5+CD4+ T cells seems to be related to the modes of disease progression in the patients that harbored them. Infection of tissues from fourteen donors with six R5 isolates from three nonswitch virus patients resulted in the loss of 59.6 ? 2.7% of CD3+CD8?CCR5+ cells, whereas infection of tissues from nine donors with five R5 isolates from the two switch virus patients resulted in a significantly smaller loss of CD3+CD8?CCR5+cells. Thus, R5 HIV-1 isolates from nonswitch virus patients are more cytopathic than R5 variants from switch virus patients, and this difference may explain the steady decline of CD4+ T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value 2. Measles virus inhibits HIV-1 replication in human lymphoid tissue ex vivo In vivo, humans are continuously exposed to microbes that interact with each other and with HIV-1 in the context of human tissues. Observations in vivo have recently revealed several microbes that can inhibit HIV-1 replication. In particular, acute infection with measles virus (MV) has been shown to suppress HIV-1 replication in coinfected children. The mechanism of this phenomenon remains largely unknown. We addressed these in our study. Inoculation of human lymphoid tissue with a laboratory-passaged wild-type isolate (Chicago-1) or an attenuated MV (Edmonston) strain resulted in productive MV infection. Both strains readily infected B and T lymphocytes in lymphoid tissues, including the CD4+ and CD8+ subsets. These cells were infected in proportion to their relative abundance in lymphoid tissue. MV dramatically inhibits replication of R5SF162. Replication of X4LAI.04 was also inhibited in MV-coinfected tissues, but to a lesser extent. We analyzed, in infected tissues, levels of key chemokines and cytokines that may be induced by MV and inhibit HIV-1 infection. Analysis of nineteen cytokines/chemokines revealed that the ones significantly upregulated in MV-infected tissues were RANTES, MIP-1alpha, and MIP-1beta the well-known CCR5 ligands and inhibitors of R5 entry. MV and R5 HIV-1 coinfection significantly increased RANTES upregulation to a level about fourfold higher than that in uninfected tissue. Deciphering the molecular mechanisms by which MV and other pathogens alter local cytokine/chemokine networks and cause tissue microenvironments to become detrimental to HIV-1 may significantly contribute to the development of effective anti-HIV therapies. 3. Immunostimulatory and HIV inhibitory properties of pertussis toxin B oligomer We found that pertussis toxin B oligomer PTX-B and its genetically modified variant used for vaccination, PT-9K/129G, inhibited HIV-1 replication in ex vivo-infected human lymphoid tissue. Also, these compounds increased the number of cells emigrated from the tissue blocks into the surrounding medium and stimulated the proliferation of emigrated cells, most of which were CD4+ T lymphocytes. Furthermore, cells emigrated from PTX-B-stimulated tissues upregulated the expression of typical activation antigens, such as CD25, HLA-DR, and CD69. X4 HIV-1 inhibited PTX-B-induced CD4+ T-cell activation while R5 HIV variants had no effect on cell activation. Cells emigrated from the tissue did not replicate HIV for at least as long as 3 weeks in culture, nor did the treatment of these cells with mitogenic concentrations of PTX-B induce HIV replication, as evaluated from RT activity. Therefore, we investigated whether these cells did indeed include latently infected ones or simply were not infected at all. For this purpose, emigrated cells were stimulated with either IL-2 or PHA. High levels of HIV replication, comparable with those in acutely infected mitogen-activated PBMC, were readily induced, indicating that a fraction of these cells were latently infected with a replication-competent virus. Moreover, HIV DNA were found in cells emigrated from control and from PTX-B-treated tissue. Thus, in this ex vivo system PTX-B maintains viral latency despite triggering lymphocyte activation and proliferation, and thus represents a new experimental model of latent inducible R5 and X4 HIV-1 infection of CD4+ T lymphocytes. PTX-B promotes activation and proliferation of cells emigrated from lymphoid tissue but, unlike PHA or IL-2, does not induce viral replication, thus uncoupling T-cell activation from HIV replication. PTX-B and the clinically approved PT-9K/129G are potential antiretroviral agents endowed with immunostimulatory capacity. Ex vivo-infected blocks of human lymphoid tissue can be used to evaluate antiviral potentials of new drugs prior to animal and clinical trials.
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