Browsing by Subject "CCR5"
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Item Immunopathogenic mechanisms of non-healing cutaneous leishmaniasis caused by Leishmania amazonensis infection(2007-04-10) Nanchaya Wanasen; Lynn Soong; Victor E Reyes; Richard G Titus; Gary R Klimpel; Allan R BrasierThe objective of my dissertation project is to understand mechanisms employed by Leishmania amazonensis to evade host immune components, particularly B cells and interferon-gamma. Our data have shown that L. amazonensis infection induces B cell activation, leading to enhanced lesion development. In the absence of B cells, L. amazonensis-infected mice developed a delayed onset of disease, correlating with an impaired activation of antigen-specific pathogenic CD4+ T cells. Flow cytometry and immunohistochemical studies suggest that B cells may provide secondary signals for activating T helper cells and recruiting leukocytes to the site of infection. The pathogenic role of B cells was suggested to be partly due to the presence of antibodies, which induced dendritic cell activation and promoted efficient priming of parasite-specific T cells to produce high levels of IL-10. The investigation of the role of IFN-g in L. amazonensis infection revealed that IFN-g alone could promote parasite growth within macrophages (MFs) by inducing the expression of mouse cationic amino acid transporter-2B (mCAT-2B), a key transporter for L-arginine. Although a clear upregulation of mCAT-2B and L-arginine transport was detected, no measurable iNOS or arginase activities were observed in IFN-g-treated, infected MFs. These data suggest an involvement of a novel L-arginine usage independent of iNOS and arginase activities during IFN-g-mediated parasite growth enhancement. The ability to scavenge available L-arginine directly by the parasites is proposed. In addition to inducing pathogenic B cells and interfering with L-arginine pathways, these parasites were found to express molecules similar to the mammalian chemokine receptor, CCR5. Although the function of a parasite-derived CCR5-like molecule remains unclear, it may help the parasites to find immature “safe target” cells. Together, this project provides new insights into the complex interactions between the parasite and host immune system and indicates the challenges we will face in the control of non-healing cutaneous leishmaniasis.Item Structural and Functional Studies of the Receptor-binding and Glycosaminogly-canbinding Mechanisms of a Viral Chemokine Analog vMIP-II and Rational Design of Chemokine-based Highly Potent HIV-1 Entry Inhibitors(2012-07-16) Zhao, BoChemokines are small immune system proteins mediating leukocyte migration and activation, and are important in many aspects of health and diseases. Some chemokines also have the ability to block HIV-1 infection by binding to the HIV-1 co-receptors CCR5 (CC chemokine receptor 5) and CXCR4 (CXC chemokine receptor 4). The first part of this work is to determine the mechanism of action of a human herpesvirus-8 encoded viral chemokine analog vMIP-II (viral macrophage inflammatory protein-II) by characterizing its interactions with endothelial surface glycosaminoglycans (GAGs) and cell surface receptors. Nuclear magnetic resonance (NMR), mutagenesis and molecular-docking were conducted and results show that vMIP-II tightly binds glycosaminoglycans using residues distributed along one face of the protein, such as R18, R46 and R48, and that there is a shift in the GAG binding site between the monomer and dimer form of vMIP-II where the N-terminus is involved in GAG binding for the dimer. This study, for the first time, provides a model that explains the mechanism of how quaternary structure affects chemokine-GAG binding. Mutagenesis and competition binding assays were conducted to study the receptor-binding mechanism of vMIP-II. Preliminary results suggest that vMIP-II uses the same positively charged binding surface comprising R18, K45, R46 and R48 to interact with the negatively charged N-termini of CCR5 and CXCR4. NMR studies on how vMIP-II interacts with N-terminal peptides of CCR5 and CXCR4 is on-going. The second part of this work was to rationally design HIV-1 entry inhibitors based on our knowledge of the mechanisms of chemokine-receptor binding and HIV-1 cell entry. We successfully designed two chimeric HIV entry inhibitors composed of CCR5-targeting RANTES variants (5P12-RANTES and 5P14-RANTES) linked to a gp41 targeting C-peptide, C37. In in vitro assays, chimeric inhibitors 5P12-linker-C37 and 5P14-linker-C37 showed the highest anti-viral potency yet published with IC50 values as low as 0.001 nM against certain virus strains. On human peripheral blood mononuclear cells, the chimeric inhibitors also exhibited very strong inhibition against R5-tropic and X4-tropic viruses, with IC50 values as low as 0.015 nM and 0.44 nM, respectively. A clear delivery mechanism was observed and characterized. These fully recombinant inhibitors can be easily produced at low cost and are excellent candidates for HIV microbicides.