Browsing by Subject "chemokines"
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Item Characterization of the effects of Interferon gamma protein-10 (CXCL10) against the protozoan parasite Leishmania amazonesis(2007-06-26) Rene Ernesto Vasquez; Lynn Soong; Thomas K. Hughes; Randall Goldblum; Johnny W. Peterson; David CorryLeishmania amazonensis causes progressive disease in most inbred strains of mice. We have previously shown that L. amazonensis-infected C57BL/6 mice have profound impairments in expression of pro-inflammatory cytokines, chemokines and in activation of antigen-specific CD4+ T cells. These impairments are independent of IL-4. The precise mechanism of pathogenesis associated with L. amazonensis infection remains largely unresolved. Since chemokines are essential mediators of leukocyte recruitment and effector cell function, we hypothesized that these molecules are important for the initiation of early responses locally and the eventual control of the infection. In this study, we found that CXCL10-treated bone marrow-derived macrophages from both BALB/c and C57BL/6 mice showed decreased numbers of L. amazonensis parasites, which was partially due to increased nitric oxide production, as well as elevated production of pro-inflammatory chemokines. When susceptible C57BL/6 mice were locally injected with CXCL10 following L. amazonensis infection, there was a significant delay in lesion development and reduction in parasite burdens, accompanied by a 7- and 3.5-fold increase in IFN-γ and IL-12 secretion, respectively, in re-stimulated lymph node cells. This study confirms that CXCL10 assists in the reduction of intracellular parasites. To address the mechanism underlying this enhanced immunity we utilized stationary promastigotes to infect bone marrow-derived DCs of C57BL/6 mice and assessed the activation of DC subsets and the capacity of these DC subsets in priming CD4+ T cells in vitro. We found that CXCL10 induced IL-12p40, but reduced IL-10 production in DCs. Yet, L. amazonensis-infected DCs produced elevated levels of IL-10, despite CXCL10 treatment. Elimination of endogenous IL-10 led to increased responsiveness to CXCL10 treatment, as judged by increased IL-12 production in DCs, as well as increased proliferation and IFN-γ production by CD4+ T cells. In addition, CXCL10-treated CD4+ T cells became more responsive to IL-12 via increased expression of the IL-12Rβ2 chain and produced elevated IFN-γ. This study indicates the interplay between CXCL10 and IL-10 in the generation of Th1-favored, pro-inflammatory responses and further highlights the utility of CXCL10 as a potential therapeutic for the control of non-healing cutaneous leishmaniasis.Item Role of interleukin-8 monomer-dimer equilibrium and glycosaminoglycan interactions in neutrophil recruitment(2008-08-08) Pavani Kumari Gangavarapu; Dr. Sarita Sastry; Dr. Roberto Garofalo; Dr. Krishna RajarathnamChemokines are small, soluble secreted proteins that induce cell migration through activation of G Protein Coupled Receptors (GPCR), and also bind extracellular matrix\r\nglycosaminoglycans (GAG) for recruiting target cells. Interleukin-8 (IL-8) is a proinflammatory chemokine, recruits neutrophils as a host response to infection and tissue injury.. An imbalance in the recruitment process has been attributed to a variety of autoimmune and inflammatory diseases. Knowledge of in vivo IL-8„ŸGAG interactions is one of the first steps towards understanding the in vivo physiology and molecular mechanisms regulating neutrophil recruitment. All chemokines including IL-8 exist as both monomers and dimers and in vitro studies have shown that both monomers and dimers of IL-8 can bind to GPCRs and GAGs, though with different affinities and specificities. However, the role of monomers, and dimers and of monomer-dimer equilibrium in regulating in vivo IL-8 function is not known. We hypothesize that the dynamic equilibrium between monomers and dimers in solution and in GAG-bound form is essential for regulation of in vivo neutrophil recruitment. Studying the role of monomers and dimers in isolation is experimentally challenging, as the monomers and dimers exist in equilibrium. This equilibrium prevents studying one species without interference from the other. We used an innovative approach, where we have used obligate monomers and obligate dimers to overcome this hurdle, and have studied the role of monomer-dimer equilibrium and GAG-binding in neutrophil recruitment. The knowledge from this study could provide valuable information for the inhibition of chemokine function, and be used for designing drugs for inflammatory and autoimmune diseases.\r\nItem 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.Item The role of CCL5 (RANTES) in the immune response against Mycobacterium tuberculosis in the guinea pig(Texas A&M University, 2005-02-17) Skwor, Troy ArthurTuberculosis is the second leading cause of morbidity and mortality worldwide due to an infectious disease. Development of a new tuberculosis (TB) vaccine would be facilitated by a better understanding of the mechanisms of protection induced by the current TB vaccine, Mycobacterium bovis BCG. Recombinant guinea pig (rgp)CCL5 and anti-rgpCCL5 were developed and characterized. The biological activity of rgpCCL5 was determined in a chemotaxis assay using T lymphocytes and pleural exudate cells. The specificity of rabbit anti-rgpCCL5 polyclonal antibody was confirmed by Western blot. RgpCCL5 was used to stimulate alveolar and peritoneal macrophages in vitro. and cytokine/chemokine gene expression was evaluated using real-time PCR. RgpCCL5 stimulated TNFα, IL-1β, CCL2, and CXCL8 mRNA expression and TNFα protein production (as assessed in the L929 cell bioassay) in macrophages. The effect of BCG-vaccination on CCL5 expression and production in leukocytes infected with M. tuberculosis was examined in vitro and in vivo. Polyclonal anti-rgpCCL5 was used to develop an ELISA assay to quantify gpCCL5 protein levels, and real-time PCR was used to detect CCL5 mRNA. Leukocytes isolated from BCG-vaccinated guinea pigs and infected in vitro with virulent M. tuberculosis demonstrated significantly elevated gpCCL5 mRNA and protein compared to cells from naive animals. The response of gpCCL5 to M. tuberculosis in vivo was studied in tuberculous pleural effusions, where peak levels of CCL5 mRNA and protein were reached at day 4 post-induction. Disease severity, cellular differentiation, histology, and cytokine/chemokine mRNA levels in pleural cells and granulomas were analyzed on day 4 in guinea pigs induced with tuberculous pleurisy and treated with either rgpCCL5 or anti-rgpCCL5 by direct intra-pleural injection. In these studies, neutralizing CCL5 resulted in reduced macrophage accumulation, diminished levels of IFNγ, TNFα, and CCL5 mRNA in pleural effusion cells, and reduced spontaneous lymphocyte proliferation. Together these studies suggest an important role for gpCCL5 in activating leukocytes during M. tuberculosis infection.