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Viral appropriation of chemokine receptors is an effective way to prevent a host immune response against the invading virus. Many viruses, including poxviruses, subvert the host immune response by encoding several chemokine receptor homologues, capable of binding to and thereby precluding chemokines from activating their cognate cell surface receptors. All poxviruses employ strategies to modulate chemokine activity, including virus-encoded chemokine-binding proteins, receptor homologues and ligand mimics. The potential for the involvement of certain chemokine receptors in poxviral infection was suggested in studies utilizing the rabbit poxvirus, myxoma. Specifically, CCR5 was implicated in mediating cell target susceptibility to infection. Our data suggest virus-CCR5 interactions may lead to the selective activation of distinct signaling pathways that are advantageous for the virus. VACV, a member of the poxvirus family, produces two structurally distinct forms of virions, the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV), for which the immediate events following cell entry are ill-defined. Using confocal microscopy, we provided evidence that IMV and EEV enter both permissive and non-permissive cells, and that introduction of CCR5 into non-permissive cells – mouse fibroblasts and human PM1 T cells - renders them permissive for VACV replication. We showed that virus activation of CCR5 leads to the selective activation of distinct signaling pathways that are advantageous for the virus. We demonstrated that VACV infection in permissive cells is inhibited by siRNA knockdown of cell surface CCR5 expression and by the CCR5 antagonist, TAK-779. The importance of tyrosine phosphorylation of CCR5 was suggested by the observation that introduction of a CCR5 mutant, in which all the intracellular tyrosines are replaced by phenylalanines, effectively reduces VACV infection in permissive cells. Moreover, tyrosine-339 was implicated in CCR5 as the critical residue for mediating viral infection, since cells expressing CCR5.Y339F do not support viral replication. The cascade of events that leads to permissive phenotype of these cells includes phosphorylation activation of multiple signaling effectors: Jak-2, IRS-2, ERK1/ 2 and Grb2. These data were supported by findings that viral replication in permissive CCR5 expressing cells is blocked by Herbimycin A, and the Jak2 inhibitor, tyrophostin AG490, but not pertussis toxin. Viewed altogether, a critical role of post-entry events, specifically intracellular tyrosine phosphorylation events, was established in determining permissiveness of cells to VACV replication. Furthermore, evidence was provided that introduction of CCR5 in primary human T cells renders them permissive to VACV replication. Since permissive infection of T cells might represent a mechanism for VACV dissemination throughout the lymphatic system, we hypothesized that the absence of CCR5 may be protective against VACV infection in vivo. To test this hypothesis, wild-type and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies we identified aggressive viral replication in the lungs and spleens of CCR5+/+ mice, with no evidence of infection in the CCR5-/- mice. Moreover, associated with VACV infection, we provided evidence for CD4+ and CD8+ T as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5-/- mice, and showed that CCR5-expressing T cells harbor replicating virus. We showed that this CCR5-dependence is VACV-specific, since CCR5-/- mice were as susceptible to intranasal influenza (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments we provided evidence that adoptive transfer of CCR5+/+ bone marrow into CCR5-/- mice restored VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data showed a critical and novel role for CCR5 in VACV infection and dissemination in vivo. Moreover, our confocal studies suggested a possible physical interaction between cellular proteins and the VACV in cytosole. Using mass spectrometry-based proteomics, glomulin was identified as a host cell protein that interacts with VACV. Knockdown of glomulin expression in human PM1.CCR5 T cells reduced VACV infection. We demonstrate that treatment of PM1.CCR5 T cells with a c-Met phosphorylation inhibitor led to a significant reduction in VACV infectivity. The data indicated that inhibition of c-Met phosphorylation, reduces the cytosolic availability of activated glomulin, thus leading to a decrease in VACV infectivity. These data identify glomulin as a permissivity factor for VACV infection, and as a potential therapeutic target for VACV.

The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1 (NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1. Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-04-30 12:23:42.429

The notion that insulin binds to a specific site on the cell membrane was first proposed many years ago. However, experimental proof of a membrane bound insulin receptor did not come until the early 1970s when biologically active radiolabelled insulin was used in direct binding studies (Cuatrecasas, 1971). Recent advances in understanding the mechanism of insulin action are the result of studies on the structure and function of the insulin receptor. The membrane receptor would appear to have two functions: firstly, it must bind insulin and secondly, it must couple insulin binding to insulin action. Defects in either of these receptor functions will result in an impaired response to insulin, or insulin resistance (Taylor, 1985). Insulin resistance is a common disorder in a number of disease states in man. For example, non-insulin-dependent diabetes mellitus and obesity are associated with mild insulin resistance (Bar et aZ., 1976). There are also a number of relatively rare syndromes of extreme insulin resistance in which there is either impaired receptor function, or an immunological defect resulting in the development of auto-antibodies against the insulin receptor (Taylor et aZ., 1985).Studies on insulin receptor defects associated with these disease states have led to progress in understanding the molecular mechanisms of insulin action. Ideally when investigating these disease states one should study insulin action on classical target cells such as adipocytes, hepatocytes or muscle. However, it is now well established that the kinetics of insulin binding to its membrane receptor is similar in all human tissue whether or not it is a target for insulin action. This has led to a great deal of research on the more accessible human tissues such as monocytes, erythrocytes, cultured fibroblasts and Epstein-Barr virus (EBV) transformed B-Iymphocytes. The most convenient tissue to study is EBV transformed B-Iymphocytes, as these cells can be taken from individual patients and grown in culture in large quantities, which facilitates biochemical studies. Despite these advantages, it is important to establish that this virus-induced receptor is a true insulin receptor and not an artifact of viral transformation. Studies on B-Iymphocyte proliferation have shown that the insulin receptor appears on the cell membrane during the proliferative phase of B-cell activation. However , this is a transient event and once the cell reaches maturation the insulin receptor is no longer evident (Marchalonis & Galbraith, 1987). The insulin receptor has also been demonstrated in a number of cultured human lymphoblastoid cell lines (Gavin et aL, 1983; Maegawa et aL, 1983). It seems, therefore, that the insulin receptor is normally expressed by blast cells. The purpose of this study was to investigate insulin binding characteristics on a human lymphoblastoid cell line with B-cell characteristics which was originally derived from a patient with Burkitt's lymphoma. These cells, which are known as Raji cells, are unusual in that they carry multiple copies of the EBV genome in their DNA. For this reason they provide a useful model system for studying the insulin receptor in EBV transformed lymphocytes. In addition, studies on the mechanism of insulin action in these cells should give some insight into the function of the insulin receptor during B-cell proliferation. In this study four major characteristics of insulin binding to insulin receptors on Raji cells are described. Firstly, on the basis of kinetic studies a model for insulin-receptor interaction was established. Secondly, processing of insulin and the receptor was investigated to determine whether the receptor is functional. A third aspect was elucidation of the receptor structure and the insulin binding site. Finally, the cross-reaction between insulin and type I IGF receptors was studied, and the cellular response mediated by the insulin receptor and growth factor receptor was determined. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1988.

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No description available.

We used Experimental Autoimmune Uveoretinitis (EAU) as a model system to investigate the involvement of CCR2 and CX₃CR1 in regulating the trafficking and function of monocytes and microglia in an autoimmune context. METHODS: W.T. or CX₃CR1^GFP/GFPmonocytes were adoptively transferred into mice with EAU.  At 48 hours post transfer their phenotype was examined by flow cytometry and monocytes trafficking to the retina was imaged using Scanning Laser Ophthalmoscopy. An anti-CCR2 antibody (MC21) or antagonist (JE(9-76)) was used to examine the effect of CCR2 blockade on W.T. monocytes trafficking.  Infiltration of monocytes into the inflamed retina and activation of retinal microglia were examined by confocal microscopy on retinal flatmounts from W.T. and CX₃CR1^GFP/GFP mice and immunohistochemistry on cryosections from eyes. RESULTS: CCR2 increased on W.T. monocytes at 48 hours post transfer and at 24 hours on CX₃CR1^GFP/GFP monocytes.  However, blocking CCR2 by either method did not reduce the number of W.T. monocytes rolling along retinal vessels and infiltrating the retina.  Lack of CX₃CR1 did not alter microglial activation but infiltrating monocytes lacking the receptor could not migrate through the retina and clustered around vessels. CONCLSIONS: CCR2 may  not always be needed for recruitment into an inflammatory site.  CX₃CR1 has a role in neuroprotection in the retina by enhancing the migratory ability and distribution of infiltrating monocytes within inflamed tissue.  This work stresses the importance for careful dissection of the chemokine receptors’ mechanism of action before therapeutic possibilities are explored.

Here we demonstrate that both exogenous and endogenous cannabinoids affect different aspects of learning and memory in the rat.  For example, the potent CB₁ receptor agonist, WIN-2 was able to delay-dependently impair short-term memory (STM) sparing reference memory (RM).  This demonstrates that it is the STM but  not RM processes that are more sensitive to the effects of cannabinoids. In addition, given that cannabinoids were able to hinder the recruitment of hippocampal firing correlates that are crucial for correct performance of a STM task, suppress hippocampal principal cell firing during the encoding phase of a STM task, reduce spontaneous bursting and disrupt synchronous firing of hippocampal principal cells respectively, confirm that they do alter the neurophysiology of the hippocampus.  These cannabinoid induced alterations in hippocampal neuronal activity may well explain the observed deficits across numerous other working memory (WM) and STM tasks. The results also revealed that cannabinoid-induced deficits in learning and memory are brought about due to an interaction between cannabinoid and cholinergic systems.  Although endocannabinoids failed to produce impairments in STM under normal physiological conditions, STM deficits were observed when anadamide levels were pharmacologically elevated beyond normal physiological levels.  Moreover, results demonstrate that the endocannabinoid system is involved in behavioural flexibility (i.e. reversal learning) and modulation of acquisition and/or consolidation of certain spatial elements that are necessary to perform an operant conditioning risk. Overall, the results in this thesis show that cannabinoid induced deficits in learning and memory are produced as a result of their direct effects on hippocampal processing.  The exact mechanisms that mediate these cannabinoid-induced deficits in memory are yet unclear and remain to be determined.

Class of 2009 Abstract / OBJECTIVES: To develop a novel characterization system utilizing immunofluorescent FLAG tagged EP2 and EP4 receptors to assist in the explanation of their unique cell signaling properties for exploitation in future drug development design. METHODS: Plasmids were obtained and isolated that contained cDNAs encoding FLAG-tagged EP2 and EP4 receptors for transient expression in HEK-293 cells. The sequences of these plasmids were confirmed by restriction enzyme analysis and DNA sequencing. Transfected cells were treated with vehicle, PGE2 or forskolin to assess appropriate receptor functionality based on cAMP induction. RESULTS: The two PGE2 receptor subtypes, EP2 and EP4, are similar in their activation of adenylyl cyclase (AC) and subsequent up regulation of cAMP production. These receptors differ, however, in that EP2 more efficiently stimulates cAMP production and EP4 signaling involves the activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signal related kinases (ERKs). The PGE2- treated cells responded as predicted with intracellular production of cAMP, with the EP2 receptor responding more efficiently than the EP4 receptor. CONCLUSIONS: The intent is for these cells to be used as a novel assay system for the development of future selective EP2 and EP4 agonists. This research could potentially benefit in selectively targeting EP2 or EP4 pathways linked to prevalent ailments such as pain, fever, inflammation, possibly cancer or bone growth.

Dendritic cells play an essential role in activating immune responses upon recognition of pathogens. This results in maturation and migration to the lymph nodes, where T cells are stimulated by upregulated antigen presentation, co-stimulation and cytokine secretion. DCs are also considered important in inhibiting inappropriate immune responses against self-peptides which could lead to the development of autoimmunity. This has been attributed to DCs that demonstrate inhibited co-stimulation and cytokine secretion. It has been previously shown that the continuous ligation of an immunomodulatory receptor, LILRB1, during DC differentiation results in such a DC population that demonstrates an immature phenotype even after exposure to bacterial components and resulted in inhibiting primary T cell responses. The mechanisms by which LILRB1-DCs promote tolerance are, therefore, here investigated. Previous studies revealed significantly altered expression for a large number of gene targets which varied from immune to cytoskeletal and bone-related functions. One of these includes DcR3, a soluble protein with a poorly defined role in immune regulation. It is here demonstrated that DcR3 has a positive role in the induction of IL-17, a cytokine implicated in autoimmunity. However, DcR3 was not secreted by LILRB1-DCs, possibly accounting for some of their tolerogenic functions. In addition, the expression of several cytoskeletal proteins was significantly changed in response to LILRB1 ligation and was associated with decreased ability for phagocytosis and migration. Lastly, it has been recently identified that DCs are able to trans-differentiate into osteoclasts, the main cell type linked with inflammatory bone disorders, such as rheumatoid arthritis. It is here shown for the first time that ligation of LILRB1 inhibits this process and results in decreased bone resorption. Overall, these data provides evidence that ligation of LILRB1 on DCs affects normal inflammatory functions and suggests its potential for the development of new treatments against several autoimmune diseases.