Most of our experimental knowledge of microbial pathogenesis comes from experiments with a microbe infecting cultured isolated cells otherwise sterile. In vivo, however, critical events of this pathogenesis occur in tissues where diverse cells are immersed in a heterogeneous microenvironment populated by pathogenic and commensal microbes affecting each other. HIV interactions with other microbes, such as human herpesviruses (HHV), may determine the clinical course of HIV disease. In particular, they may differentially affect CCR5-using HIV-1 (R5), which selectively transmits infection and dominates its early stages, and CXCR4-using HIV-1 (X4), which often evolves at the late stages of the disease. To explain the selective transmission of R5 over X4 HIV-1 a ?gatekeeping? mechanism was suggested, however its actual location and mode of action remain to be understood and are thought to be associated with the properties of gut-associated lymphoid tissue (GALT), which is an early HIV target. The study of human microbial pathogenesis requires adequate an experimental tissue model. Below, we propose a new hypothesis on the ?gatekeeping? mechanism and study the role of GALT in selective transmission of R5 HIV-1 (Project 1), identify the mechanisms of the differential interactions of HHV-7 with R5 and X4 HIV-1 in coinfected human lymphoid tissue ex vivo (Project 2), and report on the development of new tissue-like models based on experimental primate embryonic stem cell (ESC) differentiation (Project 3). ? ? 1. Differential infection of human tissue ex vivo by CCR5- and CXCR4-using HIV-1. ? R5 virus is almost exclusively associated with acute infection, irrespective of the route of transmission. However, body fluids that transmit infection contain both R5 and X4 HIV-1. The ?gatekeeping? mechanism that restricts the transmission of X4 more efficiently than of R5 has not been identified. We hypothesize that there is not a single ?gatekeeper? mechanism but that instead, at various stages of HIV transmission, a range of mechanisms restrict the establishment of infection by X4, although no stage, individually, seems to provide a perfect barrier. Nevertheless, the superimposition of several of these leaky ?gatekeepers? provides an almost perfect protection against X4 transmission in a more reliable manner than a single efficient firewall could do. We are testing this hypothesis by studying GALT that provides a strong barrier against X4 HIV-1 infection. Towards this goal, we developed a system of intestinal explants derived from both endoscopy and surgical resection specimens. The observed higher frequency of R5 target cells in rectal tissue suggests that this may be a mechanism for preferential R5 HIV-1 transmission. This system is now used to compare GALT infectivity by X4 and R5 HIV-1 variants. Also, it can be used for screening and evaluating candidate microbicides.? ? 2. Effect of herpesviruses 7 on HIV-1 in coinfected human lymphoid tissue. ? HHV-7 is a common HIV copathogen. To understand the complex interactions of HHV-7 and HIV-1 we investigated them in ex vivo human lymphoid tissue. Blocks of human lymphoid tissue inoculated with HHV-7 become productively infected without exogenous activation. Like HHV-6, which we studied earlier, HHV-7 suppresses the replication of R5 but only mildly inhibits the replication of X4 HIV-1. In spite of the fact that HHV-7 and HHV-6 are closely related and have a high degree of genetic homology, and despite the striking similarities in the pattern of their modulation of HIV-1 infection, we found that their molecular mechanisms of HIV-1 suppression are dramatically different. In contrast to HHV-6, HHV-7 does not enhance chemokine production. Instead, HHV-7 infection abrogates CD4 expression in T lymphocytes in both productively infected and in uninfected (bystander) cells. The downregulation of CD4, which leads to a generalized decrease in the number of potential HIV targets, reduces the susceptibility of these tissues to HIV-l infection. HHV-7 exerts its inhibitory effect predominantly on R5 HIV-1, as in tissues coinfected with X4 HIV-1 replication of HHV-7 is severely inhibited. These ex vivo results suggest that HHV-7 may interfere with HIV-1 in lymphoid tissue in vivo, thus affecting disease progression, in particular facilitating a switch of dominance from R5 to X4 HIV-1, which in turn may affect HHV-7 replication. ? ? 3. Differentiation of rhesus embryonic stem cells in tissue-like three-dimensional structures. ? The lack of human tissues and the variability of tissues between humans have hampered the deciphering the mechanism of human microbial pathogenesis. Therefore, the development of new highly reproducible human tissue models is required. Here, we aimed to design various tissue-like structures by differentiating ESCs into particular lineages in three-dimensional structures that would become targets for various pathogens. We have used several extracellular matrixes to differentiate non-human primate ESCs in multicellular structures. We compared embryonic stem cell differentiation, adhesion, and spreading on three types of ECM: three-dimensional bioactive collagen I, Matrigel and a biologically inert agarose. ESC readily attach to and spread on type I collagen gels. On Matrigel, ESCs formed aggregates with round cells while, in the periphery of these aggregates, cells spread. ESCs did not attach to agarose remaining in suspension-like cultures that facilitated cell-cell adhesion and the formation of large often hollow aggregates. Differentiation was low on collagen gels, while Matrigel promoted strong expression of endoderm genes, and ESC grown on agarose expressed liver-specific genes and proteins, and differentiated into cardiomiocytes capable of beating. In general, the extent of cell-cell interaction vs. cell-substrate adhesion is closely related to the pattern of ESC differentiation. Understanding the effects of ECM on the differentiation of ESCs in various types of cultures may lead to the design of tissue-like structures consisting of aggregates of ESC differentiated into particular lineages, especially into endoderm and mesoderm; This will be a new experimental system for studying microbial pathogenesis.
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