IL-10, an immunoregulatory cytokine with biological effects primarily on inhibition of inflammatory responses, has also been shown to stimulate a variety of functions including CD14 expression on human monocytic cells. CD14, a receptor for lipopolysaccharide (LPS), plays a critical role in the synthesis of proinflammatory cytokines by LPS-stimulated monocytic cells. Herein, I show that LPS-induced CD14 expression on monocytic cells may be mediated by endogenously produced IL-10. In this study, I have investigated the molecular mechanisms by which IL-10 enhances CD14 expression in normal human monocytes and a promyelocytic HL-60 cells as a model system. IL-10 induced the phosphorylation of PI3K and p42/44 extracellular signal-regulated kinase (ERK) MAPKs. By employing specific inhibitors for PI3K (LY294002) and ERK MAPKs (PD98059), I provide evidence that LY294002 either alone or in conjunction with PD98059 inhibited IL-10-induced phosphorylation of STAT1 and consequently CD14 expression. However; IL-10-induced STAT3 activation remained unaffected under these conditions. Furthermore, LY294002 and PD98059 inhibited the binding of STAT1 transcription factor to its binding site in the CD14 promoter. Finally, STAT1 siRNA inhibited IL-10-induced CD14 expression. Taken together, results show for the first time that IL-10-mediated CD14 upregulation may be mediated by STATI activation independently of STAT3. Furthermore, IL-10-activated STAT1 may be regulated through PI3K either alone or in concert with the ERK MAPKs.

In this study we sought to demonstrate the effect of endostatin on endothelial cell adhesion, proliferation, survival and migration in the presence of various ECM proteins. We found that endostatin was able to decrease VEGF-induced adhesion of endothelial cells to a variety of matrices including laminin, collagen IV, fibronectin, collagen I, tenascin-c and vitronectin. Endostatin was also able to inhibit the proliferation of endothelial cells however, this function was more dependent on the ECM proteins upon which the endothelial cells were cultured with the strongest inhibitory effects noted following growth on plastic and the weakest inhibitory effects observed following growth on collagen I or tenascin C which are both tumor associated ECM proteins. We also showed that endostatin did not induce apoptosis of VEGF-treated endothelial cells and in parallel showed that endostatin did not reduce the VEGF-induced activation of the survival protein Akt. (Abstract shortened by UMI.)

Apoptosis is characterized by an intracellular proteolytic cascade consisting of a family of proteases called Caspases, and activation of this cascade by extracellular or intracellular stress signals results in the characteristic morphological and biochemical features of apoptotic cell death. The Inhibitors of Apoptosis Proteins (IAPs) are a family of proteins that bind and inhibit Caspases and have been demonstrated to be the only known endogenous inhibitors of the terminal caspase cascade. Defects in apoptosis allow cancer cells to survive under conditions of enormous stress and increased expression of several members of the IAP family has been observed in tumor biopsy samples and cancer cell lines. XIAP Associated Factor 1 (XAF1) was identified as a nuclear protein that binds to the X-linked Inhibitor of Apoptosis protein (XIAP) and antagonizes the anti-caspase activity of XIAP, thereby reversing the protective effect of XIAP overexpression in cancer cell lines. The expression of XAF1 is significantly reduced in cancer cell lines and several primary malignancies as a result of promoter hypermethylation, and re-activation of xaf1 by DNA methylation inhibitors sensitizes cancer cells to apoptosis-inducing agents. Xaf1 has recently been identified as an interferon (IFN)-stimulated gene that sensitizes tumor cells to apoptosis by TRAIL (TNF-related Apoptosis Inducing Ligand) following treatment with IFN-beta. This thesis documents the role of XAF1 in cell cycle and its potential as a negative regulator of Survivin, an unusual IAP that possesses poor anti-Caspase activity, and is known to be involved in the cell cycle as a member of the chromosomal passenger complex. In HEL 299 and wild-type mouse embryonic fibroblast (MEF) cells, FACS profiling of DNA content suggests that XAF1 overexpression triggers a G1 cell cycle arrest by upregulating p21. This XAF1-mediated upregulation of p21 is not seen in p53-null MEFs and is attenuated by a dominant-negative p53-mutant in HEL 299 cells, suggesting that XAF1 functions primarily upstream of p53 in the cell cycle arrest pathway and results in activation or repression of p53-target genes. Survivin is negatively regulated by p53 and these results suggest that XAF1 may regulate Survivin expression in a p53-dependent manner. Furthermore, overexpression of XAF1 in p53-deficient cancer cells results in significant downregulation of Survivin expression, indicating the existence of a p53-independent mechanism for the observed effect. Immunoprecipitation experiments indicate that XAF1 binds to all the IAPs tested except Survivin. Interestingly, it was observed that XAF1complexes with Survivin only in the presence of XIAP. Furthermore, such an association was associated with a decrease in expression levels of Survivin. XIAP is a RING-bearing protein which functions as an E3 ligase and is involved in the ubiquitin-proteasome pathway. Treatment of transfected cells with proteasomal inhibitor or the use of a XIAP RING-mutant lacking E3 ligase activity was shown to restore Survivin protein expression indicating that XAF1 mediates Survivin downregulation by promoting the E3 ligase activity of XIAP. Taken together, these results suggest that XAF1negatively regulates Survivin expression by both p53-dependent and -independent mechanisms.

Salmonella typhimurium (ST) causes gasteroenteritis in humans and typhoid-like disease in mice. Since CD8 T cells facilitate acquired immunity, we evaluated the development and function of the CD8 T cell response against ST. Responses were compared to the acute intracellular pathogen, Listeria monocytogenes (LM). Because ST replicates within phagosomes and causes chronic infection, it was hypothesized that CD8 T cell priming may be muted and dysfunctional. While LM-induced CD8 T cells differentiated rapidly and displayed a mainly central-memory phenotyope in the long-term, CD8 T cells failed to become activated rapidly during ST infection and differentiated mainly into an effector/effector-memory phenotype. While the CD8 T cells induced against ST were functional, owing to the delay in CD8 T cell activation during ST infection, even conventional memory CD8 T cells failed to respond rapidly. Thus, the phagosomal lifestyle may allow escape from CD8+ T cells, conferring a survival advantage to the pathogen.

Interleukin 12 plays a central role in NK cell activation and CD4 +T cell differentiation. IL-12 acts via a receptor composed of IL-12Rbeta1 and IL-12Rbeta2 subunits. IL-12Rbeta1 plays a primary role in ligand binding whereas IL-12Rbeta2 is responsible for signaling. IL-18 shares some functional activities of IL-12. However, there is a considerable amount of contradictory data in the literature regarding the requirements for IL-12 and IL-18 responsiveness. This work examined the expression of IL-12Rbeta2 on murine NK and T cells and the requirements for acquisition of IL-12/IL-18 responsiveness. NK cells stimulated with IL-2+IL-12 or IL-2+IL-18 exhibited rapid upregulation of IFN-gamma expression followed by upregulation of IL-10 or IL-13 mRNA, respectively. NK cells from IL-12Rbeta2-deficient mice responded to IL-2+IL-18 independently of IL-12. Furthermore, flow cytometric analyses revealed that NK cells activated in vitro with IL-2 differentiate into two distinct cell subsets expressing different levels of IL-12Rbeta2. Like NK cells, NK-T cells also responded to IL-2+IL-12 or IL-2+IL-18 without stimulation of the antigen-specific T cell receptor (TCR). Previously, it was thought that all T cells require activation via the TCR in order to respond to IL-12 or IL-18. Thus, responsiveness of conventional T cells to IL-2+IL-12 or IL-2+IL-18 in the absence of TCR ligation was assessed. Naive CD4+T cells from wild type or DO11.10/Rag2-/- OVA-specific TCR transgenic mice responded to IL-2+IL-12 or IL-2+IL-18 by expressing IFN-gamma and cells grown in IL-2+IL-12 exhibited signs of polarization towards a TH1 phenotype. Transgenic BALB/c mice constitutively expressing the IL-12Rbeta2 chain were generated and the role of the IL-12Rbeta2 in TH2 phenotype development was analyzed using the TH1-dependent murine leishmaniasis model. Despite constitutive expression of the IL-12Rbeta2 chain, transgenic TH2 cells did not revert to TH1 when restimulated with IL-12 and mice remained susceptible to Leishmania. The role of IL-12Rbeta2 in TH1 cell development was also analyzed using the same model. Resistant C57BL/6 mice defective in IL-12Rbeta2 gene exhibited susceptibility to L. major infection and expressed a T H2 phenotype, consistent with a critical role for IL-12 in this model. Thus understanding the expression of IL-12Rbeta2 in NK and CD4 +T cells and its regulation by cytokines may constitute an important element in studies in cancer and autoimmune disease therapy.

Apoptosis signal pathways converge on the Caspases, a family of proteases that form a self-amplifying cascade, the activation of which generates the characteristic morphological and biochemical features of apoptotic cell death. The Inhibitors of Apoptosis (IAPs) are a family of proteins that bind and inactivate Caspases at both the initiation and terminal effector stages of this cascade. Over-expression of IAPs, as observed in cancer cell lines and tumour biopsy samples, results in a cellular phenotype of resistance to a variety of apoptotic stresses including chemotherapeutic drugs and irradiation. One negative regulator of IAP function, Smac (second mitochondrial activator of Caspases a.k.a.Diablo) has been identified as a mitochondrial protein that is processed and released concomitantly with cytochrome c following an apoptotic stress. Proteolytic processing of the mitochondrial signal peptide sequence generates a novel amino terminus that interacts with XIAP and disrupts XIAP-meditated Caspase-9 inhibition. By disrupting the interaction of XIAP with Caspase-9, Smac is believed to promote apoptotis. Furthermore, the structure determinations of XIAP, in conjunction with either Caspases or Smac, have led to the identification of a critical pocket and groove on the surface of each BIR domain ( Baculovirus IAP Repeat). Within XIAP BIR3, the binding pocket interacts with Caspase-9 and can be disrupted by the presence of Smac, which competes for the same site. In this thesis work, the utility of Smac as a chemo-sensitizing agent for the treatment of cancer was enhanced by three different strategies. The first approach involved the development and characterization of an ubiquitin-Smac fusion system that circumvents the targeting of Smac to the mitochondria and results in the expression of a fully mature, biologically active Smac protein. The second approach entailed developing a strategy for increasing the affinity of Smac binding to the IAPs. A random peptide phage display screen was used to identify novel, high affinity peptide ligands that bind to the IAP-BIR3 motifs. These peptide sequences were similar in structure to the amino terminus of Smac and Caspase-9. The data generated from these two approaches showed that both over-expressed, mature Smac and Smac-like peptide sequences bind to the IAPs in vitro and in vivo and sensitize cells to chemotherapeutic drugs. The third and final approach involved developing a Smac system that was a more potent apoptosis-inducer than wild-type Smac, which would be predicted to have greater therapeutic potential. It has been shown that Smac interactions with the IAP BIR domains may accelerate IAP auto-ubiquitination and destruction via the IAP carboxy terminal RING (Really Interesting New Gene) domain. Adding an IAP-RING domain to the carboxy terminus of Smac was predicted to enhance IAP degradation while maintaining the IAPs in a non-functional protein complex. Indeed, it was observed that generating a Smac protein with RING-conferred E3 ligase activity significantly decreased IAP levels, with a concomitant increase in the sensitivity to apoptosis in the presence of chemotherapeutic agents.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis affecting over 170 million people worldwide. The mechanisms behind the pathogenesis of chronic HCV infection are not well understood. The aim of this thesis is to elucidate how the HCV structural proteins affect the dynamic structural and functional properties of hepatocytes and measure the extra-hepatic manifestations induced by these viral proteins. To pursue this goal, a transgenic mouse model was established by expressing Core, E1 and E2 proteins downstream of a CMV promoter. HCV RNA was detected in transgenic mouse model tissues, such as liver, kidney, spleen, and heart. Immunofluorescence analysis revealed the expression of core, E1 and E2 proteins predominantly in hepatocytes. Histological analysis of liver cells demonstrated steatosis in transgenic mice older than 3 months, which progressed with mouse age. Electron microscopy analysis revealed alterations in mitochondria, and in the endoplasmic reticulum. The animals became more prone to liver and lymphoid tumor development and hepatocellular carcinoma. It is likely that the HCV structural proteins mediate some of the histological alterations in hepatocytes by interfering with liver biological processes. To study that, we compare differential gene expression patterns in the liver of HCV transgenic and non-transgenic mice using complementary DNA (cDNA) microarrays and confirmed the expression of altered genes by real-time RT-PCR. 15 600 genes were analyzed and 394 genes were identified to be differentially expressed at a statistically significant level, while 196 genes were identified as up-regulated and 198 genes were identified as down-regulated. The expression of HCV structural proteins in transgenic mouse livers induces alterations in the expression of genes involved in many important biological processes such as lipid metabolism and transport, antigen processing and presentation, protein biosynthesis, carcinogenesis, etc. Also, some of the differentially expressed genes were associated with important cell signaling pathways such as Mitogen activated protein kinase (MAPK) pathway and Writ oncogenic pathway. The third study aims to elucidate the role of immune-mediated cell damage in hepatitis C infection using HCV Transgenic mice. Adoptive transfer of carboxyfluorescein diacetate suuccinimidyl ester (CFSE) labelled splenocytes from HCV immunized mice into HCV transgenic mice was performed. After the adoptive transfer, we observed that there was a significant decrease in the percentage of CFSE-labeled CD4+ and CD8+ T cells in the blood of transgenic mice receiving transfers from immunized donors. Moreover, the percentage of CFSE-labeled CD4+ and CD8+ T cells were significantly higher in the spleen of transgenic and non-transgenic mice when they received transfer from non-immunized mice. Interestingly, transgenic livers of mice received transfers from immunized mice had significantly high percentage of CFSE-labeled T cells than non-transgenic livers receiving non-immunized transfers. This suggests that the T cells from HCV immunized mice recognized HCV antigens in the liver. Taken together, the research results indicate that our transgenic mouse model is a suitable model to study hepatitis C pathogenesis.

Apoptosis of CD4+ T cells and their eventual depletion constitute a hallmark of HIV infection and disease progression. However, monocytes/macrophages unlike CD4+ T cells survive HIV replication and hence represent a major reservoir of virus. The mechanism underlying this resistance to HIV-mediated apoptosis in monocytic cells is not clear. It is believed that TNF-alpha which is produced during HIV infection may play a major role in inducing resistance in monocytes. In this study I investigated the molecular mechanisms involved in regulation of resistance to HIV-Vpr induced apoptosis in monocytic cells. LPS and LPS induced TNF-alpha are known to induce resistance to monocyte apoptosis. However, the molecular mechanism regulating this process is not known. cIAP2, one of the anti-apoptotic genes is known to be induced in monocytes in response to LPS. First of all, I demonstrated that LPS and TNF-alpha induced survival of human monocytic cells through the induction of the c-IAP2 gene. Further, I studied the involvement of MAPKs and PI3K signaling pathways in c-IAP2 induction by using specific pharmacological inhibitors. My results suggest that neither MAPKs nor PI3K is involved in c-IAP2 induction. Since calcium signaling pathway is considered to be one of the upstream signaling molecules in signaling cascade, I investigated its involvement in c-IAP2 induction in monocytic cell line. I showed that binding of LPS/TNF-alpha to their respective receptors induces calcium influx. Calcium is also known to activate calmodulin (CaM) which in turn activates various kinases and phosphatases. My results revealed that activation of CaM and CaM kinase II (CAMKII) is involved in c-IAP2 induction, whereas calcineurin, the protein phosphatase activated by CaM is not involved in this process. I have shown for the first time that LPS/TNF-alpha induced c-IAP2 and associated anti-apoptotic activity is regulated by CaM/CAMKII through the activation of NFkappaB. Since TNF-alpha is believed to be involved in inducing resistance to HIV induced apoptosis in monocytes, further I used Vpr, one of the HIV proteins to induce apoptosis in monocytes. My results showed that HIV-Vpr induces apoptosis in monocytes and pretreatment of cells with either LPS/TNF-alpha induces resistance to Vpr mediated apoptosis. To determine the molecular mechanisms involved in Vpr induced apoptosis, I used the C-terminal synthetic peptide (Vpr52-96) which mimics the whole Vpr protein for induction of apoptosis. I also used the N-terminal (Vpr1-45 aa) peptide as control. I demonstrated that both the C-terminal and N-terminal peptides phosphorylate all the three MAPKs such as p38, ERK, and JNK MAPK; however, only C-terminal (Vpr52-96) peptide induced apoptosis is regulated selectively by JNK MAPK. To determine the involvement of pro- and anti-apoptotic genes in Vpr induced apoptosis, RNAse protection assay was performed. My results revealed that in response to Vpr52-96, there was downregulation of Bc12, whereas other pro and anti-apoptotic genes of Bc12 family remained unchanged. I also demonstrated the involvement of c-IAP1, one of the inhibitor of apoptotic proteins (IAPs) in Vpr induced apoptosis. My results show that Vpr peptide-induced apoptosis is mediated by down regulation of anti-apoptosic Bc12 and c-IAP1 genes through JNK MAPK activation. Deregulation of the JNK pathway has been implicated in cancer and other diseases. Therefore, investigation of the molecular mechanisms that govern the role of the JNK pathway in apoptosis should provide insight into its biological functions and strategies to target this pathway for prevention and treatment of human diseases and cancer. Finally, I demonstrated that pretreatment of cells with LPS or TNF-alpha induced resistance to Vpr52-96 mediated apoptosis. My results suggest that LPS induced resistance is mediated by endogenous production of TNF-alpha in monocytes. I also demonstrated that LPS/TNF-alpha mediated resistance is regulated by induction of c-IAP2 through NFkappaB by activation of CAMKII. However, pretreatment with Vpr followed by LPS or TNF-alpha stimulation no longer gave protection to Vpr induced apoptosis. Vpr peptide also inhibited LPS/TNF-alpha-induced calcium influx, activation of CAMKII, and c-IAP2 induction. Taken together, my results suggest that the c-IAP2 gene plays a critical role in LPS and TNF-alpha-induced resistance to HIV-Vpr-mediated apoptosis in human monocytic cells. Since the calcium/CAMKII pathway is involved in LPS/TNF-alpha induced resistance to Vpr mediated apoptosis, strategies based on manipulation of these molecules, which would suppress c-IAP2 induction may be useful in clearing virus reservoirs in monocytes.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease dependent on gene-environment interaction. Food components are considered as environmental agents that influence the incidence of islet cell autoimmunity and T1D. Wheat proteins are harmless in healthy individuals, but in genetically susceptible individuals, wheat may be immunopathogenic. There are several reports that link wheat with increased risk of autoimmune diabetes. The major aim of this study was to determine whether there is an abnormal immune response to a mixture of wheat proteins (WP) or a purified wheat storage globulin homologue, Glb1 in T1D patients. If so, the question arises regarding the nature of the immune response and whether such reactivity is genetically controlled. A sensitive CFSE-based proliferation assay and a capture ELISA technique were established to evaluate T cell immune response and Glb1 antibody level. Studies of young adults with T1D indicated that a subset of patients displayed abnormal humoral and cellular immune response to Glb1. T cell responses to WP and Glb1 were predominantly Th1 type and were associated with the diabetes risk gene HLA-DR4, and not HLA-DQ2, the main coeliac disease (CD) risk gene, suggesting the effect was diabetes-specific. Additional studies in children at high risk for diabetes with islet autoimmunity revealed that Glb1 antibody and zonulin were highly correlated, suggesting the presence of gut abnormalities related to tight junctions. In addition, increased immune response to Glb1 was present in children at high risk for diabetes who developed antibodies to the CD autoantigen, tTG. Interestingly, Glbl antibodies appeared before tTG antibodies, suggesting that Glb1 antibody could have predictive value in people at risk of disease. The data from this study clearly demonstrate for the first time the presence of abnormal humoral and cellular responses to wheat related dietary antigens in patients with T1D. Further, these data strongly suggest that the gut is not functioning normally and raises the possibility of a defect in the gut barrier, an immune imbalance and/or impaired oral tolerance.

Signature-tagged mutagenesis (STM) is an in vivo mutation-based genetic screen that allows for the identification of virulence genes. A working model of STM in the temperature-dependent rabbit model (TDRM) of Haemophilus ducreyi has been established. A unique signature-tag (ST) was inserted upstream of the aphA-3 gene in the transposon Tn1545-Delta3. Eleven different ST were used, each consisting of 21 unique nucleotides that were designed for PCR. A library of ST transposon H. ducreyi 35000 mutants was generated. A STM pool consisted of 10 unique ST-mutants that were inoculated intraepithelially into the TDRM. Ulcer development and histopathology of the lesions produced by the STM mutants inoculated at 105 CFU showed the same morphology as those produced by wild-type inoculated at 105 CFU. Ulcers that developed by day 4 were excised and recovered mutants in the output pool were screened by PCR. A previously characterized attenuated hemoglobin (HgbA) mutant was ST to serve as a positive control and was not recovered in the output pool. A total of 176 STM mutants were screened and 26 attenuated H. ducreyi mutants were recovered. The 26 candidate genes were grouped according to their functional categories: classical virulence factors, cell surface components, stress response, DNA recombination and repair, transport, metabolism, regulation and hypothetical genes. This STM study resulted in the identification of known virulence genes such as the cdtA, hgbA and lspAl which validated the STM approach. The identification of a heat shock protein and genes involved in DNA repair, emphasize the importance of stress response genes in vivo. Many novel genes were discovered that had not been previously identified as having a role in pathogenesis. Genes involved in metabolism were identified which highlight the fitness contribution these factors play within a hast. Two of these genes (frdA and sucA) may facilitate the ability of H. ducreyi to grow anaerobically. As in other STM studies, genes with hypothetical functions were identified. One gene, hicB, is suggested to encode an RNA toxin-antitoxin cassette. This study provides proof-of-principle for the STM approach for the recovery of genetic determinants in H. ducreyi responsible for the pathogenesis of chancroid.