Molecular determinants of malaria pathogenesis are largely undefined. Chemokines and chemokine receptors, regulate immune responses, may thus determine malaria severity. Further, by regulating HIV pathogenesis, they may constitute a crucial link in malaria-HIV interaction. Understanding biologic mechanisms underlying malaria-HIV interaction has important public health utility in designing rational therapeutic and preventive strategies. Malaria could potentially modulate HIV-1 infection through alteration in expression of CD4 and chemokine receptors, required for cellular entry. This study has determined circulating levels and transcriptional profiles of β-chemokines (MIP-1α, MIP-1β, and RANTES) in ex vivo peripheral blood mononuclear cells (PBMCs) of children with varying degrees of malaria severity. Additional in vitro experiments assessed the effects of stimulation of PBMCs with crude hemozoin (Hz) or synthetic hemozoin (sHz) on CD4, β-chemokine and chemokine receptor (CCR5 and CXCR4) protein expression and transcript formation. Plasma MIP-1α and MIP-1β levels were significantly elevated in mild and severe malaria, while RANTES levels decreased with increasing disease severity. β-chemokine gene expression closely matched circulating β-chemokine profile, illustrating that PBMCs are a primary source for β-chemokine production during malaria. Healthy children with a history of severe malaria had lower baseline RANTES production than children with a history of mild malaria, suggesting inherent differences in RANTES production. In vitro experiments in PBMCs from healthy malaria-naïve donors showed that Hz and sHz promote a similar pattern of β-chemokine protein secretion and transcript expression. FACS analysis showed that Hz and sHz induced similar patterns of cellular surface expression of CD4, CCR5 and CXCR4 on PBMCs. Hz or sHz-exerted differential effects on CD14+ and CD3+ subsets, and this modulatory effect part to transcriptional regulation based on gene expression profiles obtained for respective antigens. Additional studies showed that HIV-1 replicates differently in monoctye-derived macrophages (MDMs) stimulated with either Hz or sHz. sHz enhanced HIV-1 replication while Hz had an inhibitory effect. Results presented here demonstrate a distinct profile of β-chemokine expression in children with severe malaria, which is promoted by P. falciparum derived hemozoin. Further, Hz modulates expression of surface antigens required for HIV-1 entry, defining a possible mechanism for HIV-malaria interaction.

HIV-1 Associated Dementia (HAD), the most severe neurological complication associated with HIV-1 infection, is commonly characterized by inflammation of the brain and neuronal degeneration, known as HIV Encephalitis (HIVE). HIVE develops in 20-30% of patients infected with HIV, which means that 9.5 million people are affected by HIVE throughout the world. While the introduction of highly active antiretroviral therapy (HAART) has decreased the incidence of severe late-stage HAD, the prevalence of its precursor HIVE is actually rising. Several HIV-1 viral proteins have been shown using in vitro models to have a role in the neurotoxic effects causing the neurodegeneration seen during HIVE. HIV-1 Viral Protein R (Vpr), a virion associated gene product which induces apoptosis in non-proliferating cells including neurons, is thought to contribute to the neuropathogenesis associated with HIVE. Previous studies have shown the presence of detectable levels of Vpr in the cerebrospinal fluid of HIV-1 infected patients. Extracellular Vpr released from HIV-1 infected macrophages has also been shown to be capable of transducing into cells not normally infected by HIV-1, causing death of these bystander cells. Additionally, Vpr has been shown in vitro to be able to induce apoptosis in human neurons. Although current research suggests that Vpr plays a significant role in neuropathogenesis, no work has been done yet in vivo to show the presence of Vpr in the brain tissue of HIVE patients. Using a panel of eight HIVE and four HIV seronegative patient brain tissue sections, I performed immunohistochemistry staining for Vpr, p24, and brain cell specific markers. Results indicate that Vpr was present in detectable amounts in both the basal ganglia and frontal cortex of all eight HIVE brain tissue samples tested. Double label immunohistochemistry was performed using antibodies specific for astrocytes macrophages and neurons. I detected the presence of Vpr in the macrophages and neurons, but not in the astrocytes, of HIVE patients. The results of this study strongly support the role of Vpr in the neuropathogenesis seen during HIVE. Further studies based on these findings could lead to the development of effective therapeutic treatments necessary to reduce, and possibly prevent, this public health epidemic.

A one-step real-time reverse transcription PCR (RT-PCR) assay was developed for the detection of human rhinovirus (RV) in nasal aspirate (NA) specimens. A set of primers was designed to amplify a 120-base target in the 5' non-coding region of RV RNA. The amplicon was detected using TaqMan minor groove binder (MGB) probes and ABI Prism sequence detectors. Three probes were evaluated using stock suspensions of 15 RV strains representing serotypes 1A, 2, 3, 7, 17, 21, 29, 37, 39, 40, 58, 62, 66, 72, and 87. The initial probe that was designed, Pic-4, detected 11 of the RV strains. Probe Pic-6 was designed with a degeneracy at the first 5' nucleotide to accommodate a target-probe mismatch. Although Pic-6 improved the detection limit for some strains, detection of RVs containing multiple mismatches was unsatisfactory. Probe Pic-5 was designed with nucleotide degeneracies at three positions to account for multiple mismatches. Pic-5 detected 14 of the RVs at a lower limit of detection of 0.00001 to 0.01 50% tissue culture infectious dose (TCID50) equivalents/PCR reaction. RV-87, recently classified as an enterovirus (EV), was not detected with any of the probes. The assay with Pic-5 detected 30 plus or minus 50 copies of a plasmid containing an RV-2 target sequence. The assay yielded negative results when nucleic acids from human cells, EVs, and a panel of bacteria and viruses found in the human nasopharynx were tested. The assay with Pic-4 yielded a detection limit of 1.0 TCID50 equivalents/PCR reaction in both neat and supernatant NA specimens seeded with RV-2. A total of 48 NA samples obtained from children with cold-like symptoms were tested by the RT-PCR assay with probe Pic-5; 8 (16.7%) of the samples were positive. In conclusion, the real-time TaqMan MGB RT-PCR assay with Pic-5 is rapid, sensitive, and specific and has the capability of detecting at least 14 serotypes of RV. The public health significance of this study is that the RT-PCR assay with Pic-5 may lead to improvements in the diagnosis and surveillance of RV infections, and to a better understanding of the ability of RV to cause serious infection in immunocompromised patients.

Most highly active anti-retroviral treatment (HAART) regimens eventually fail to provide complete and long-term suppression of virus replication due to the inability to fully clear virus from cellular reservoirs. The HIV-1 viral protein R, Vpr, increases virus replication in T cells and is necessary for the optimal infection of primary monocytes/macrophages and other non-dividing cells. In this essay, it is demonstrated that Vpr interacts with the cellular Glucocorticoid Receptor (GR) and transactivates the HIV-1 LTR through GRE and that this event can be blocked by the GR antagonist, mifepristone. Based on these observations, it is shown that targeting Vpr-mediated virus transcription with the glucocorticoid antagonist, mifepristone, can demonstrate a potent anti-retroviral therapy. Results demonstrated that Vpr-induced transactivation of both autologous and heterologous promoters was inhibited by mifepristone in a dose-dependent manner by >90% at a 1 µM concentration. Infectivity assays using T-tropic, dual-tropic, and macrophage-tropic viruses demonstrated antiviral effects on a dose-dependent regimen of mifepristone. The effects of mifepristone were also tested in HIV-1 latent cells that could be activated with extracellular viral protein and results exhibited a greater than 90% inhibition of re-activation in the presence of this antagonist. Cytotoxic effects of mifepristone demonstrated a CT50 from 10 to 100 µM in normal human primary cells, HeLa, HEK293, and CV-1 cells. Statement of Public Health Relevance: By utilizing the interaction between Vpr and the glucocortoicoid receptor, glucocorticoid antagonists such as mifepristone hold promise for anti-retroviral therapy by both preventing viral transactivation in currently-infected cell populations as well as preventing the reactivation of latent virus.

Childhood malarial anemia (MA) remains a global health burden with the vast morbidity and mortality occurring mostly in sub-Saharan Africa. Although design and testing of malaria vaccines is currently underway, the pattern of inflammatory mediator production that predicts a protective immune response against severe malaria, which would dramatically enhance vaccine testing, is largely unknown. Protective malarial immunity is regulated in part by cytokines, such as interleukin (IL)-12, IL-10, and tumor necrosis factor (TNF)-α, and effector molecules, such as prostaglandin E2 (PGE2) and nitric oxide (NO). Previous studies have illustrated that children with severe MA have lower levels of circulating IL-12p70 and PGE2, and increased plasma levels of IL-10, TNF-α, and NO relative to children with mild malaria, however, the mechanism(s) responsible for this pattern of immune production is unknown. Phagocytosis of parasitic products, such as hemozoin, by cultured peripheral blood mononuclear cell (PBMC) elicits dysregulation of inflammatory mediator production, therefore, the regulation and interactions of cytokines and effector molecules was investigated during acute childhood malaria and in cultured PBMC stimulated with Plasmodium falciparum-derived hemozoin. Children with high-density parasitemia had decreased IL-12p70 and increased levels of IL-10 and TNF-α. Experiments in cultured PBMC from malaria-naïve donors revealed that hemozoin suppressed IL-12p70 through induction of IL-10, but not over-expression of TNF-α transcripts and protein, which was independent of suppressor of cytokine signaling (SOCS)-3 induction. Hemozoin suppressed cyclooxygenase (COX)-2-dependent PGE2 production through reductions in COX-2 transcript and protein formation, and inhibition of COX-2 enzymatic activity. Suppression of PGE2, which was independent of hemozoin-induced IL-10, resulted in over-production of TNF-α. The ratio of plasma PGE2/TNF-α was decreased in children with severe disease. Cultured PBMC from children with severe malaria had elevated nitric oxide synthase (NOS)2 enzyme activity, which occurred at least in part through PBMC ingestion of hemozoin. Thus, ingestion of hemozoin by PBMC elicits a similar pattern of inflammatory mediator production to that observed in children with severe MA. Results presented here are of significant public health relevance in that understanding the regulation of cytokine and effector molecule production during severe malaria will vastly improve vaccine design and testing.

Due to shared routes of transmission, co-infection with Hepatitis C (HCV) and Human Immuno-deficiency Virus (HIV) has become prevalent worldwide, with approximately one-third of all HIV-infected individuals and about 10% of all HCV infected individuals in North America being co-infected with the other virus (1). Recent advances in HIV treatment have increased the life expectancy of HIV-infected patients, resulting in HCV-associated disease to develop into a major cause of morbidity and mortality among the co-infected population. HIV is consistently shown to have a negative effect on different aspects of HCV disease, from increased HCV RNA levels (2) to aggravated liver fibrosis and more rapid progression to cirrhosis and end-stage liver disease (3). The host immune responses play a major role in not only controlling HCV infection, but also in causing hepatic inflammation and damage. Despite major advances in the understanding of the pathogenesis of these two infections, the mechanisms underlying the role of immune responses in more rapid progression of HCV disease in HCV/HIV co-infection is not quite clear. This thesis is generated based on an investigation to understand why HIV infection worsens HCV pathogenesis. This question is addressed throughout this thesis from different immunological aspects, with a focus on the function of T-cells. I have demonstrated that in HCV/HIV co-infection, functional HIV-specific T-cells accumulate in the liver and produce an array of cytokines, including INF- and TNF-, which may represent a bystander role for HIV in the aggravation of HCV-induced liver damage. My data also demonstrate that co-expression of two defined T-cell exhaustion markers, Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, in the setting of HIV co-infection. Both total and HCV-specific peripheral T-cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared to HCV mono-infection. In co-infection, HCV-specific CD8+ T-cells showed greater frequencies of Tim-3/PD-1 dual-expression than those being HIV-specific, indicating a greater degree of exhaustion in the former. Additionally, I demonstrated that some HIV mono-infected individuals may contain CD8+ T-cells that cross-recognize two defined HLA-A2-restricted epitopes within the HIV and HCV proteome, the HIV-Gag: SLYNTVATL and HCV-NS5b: ALYDVVSKL. This T-cell cross-reactivity was further elaborated in the context of HCV/HIV co-infection, demonstrating that degeneracy of HIV-specific T-cells may play a role in the immuno-pathology of co-infection. Altogether, these data could be integrated into the foundation of potential mechanisms involved in the immunopathogenesis of HCV/HIV co-infection, and be applied to further investigation in basic science and clinical studies in this field.

NK cells are innate lymphocytes that are crucial for host immunity during infections. Discrimination between healthy and infected cells is facilitated through a sum of inhibitory and stimulatory signals. Host cells can modulate the expression of NK ligands in response to infection, transformation, and stress, while viruses can exploit these mechanisms to prevent the killing of the infected cells. This thesis focuses on the interaction between the NK receptor, NKR-P1B, and its ligand, Clr-b. First, we used a reporter cell-based protocol to establish hybridomas producing monoclonal antibodies against a putative viral immunoevasin, RCTL. We express CD3ζ-fusion proteins on the reporter cells, which we then use to immunize and screen hybridoma specificities. Our results demonstrate a rapid, efficient, and high-throughput method of monoclonal antibody screening, and provide the framework for our work on cytomegalovirus evasion of the NKR-P1B:Clr-b system. Next, we show that RCMV infection results in a notable downregulation of rClr-b at the protein and transcript levels. Conversely, RCTL is upregulated during infection, and binds to the same NK-inhibitory receptor as Clr-b, NKR-P1B. In the absence of RCTL, RCMV-mediated Clr-b loss leads to increased NK-killing of infected targets and an NK-dependent reduction of viral titers in vivo. Notably, NKR-P1B is highly polymorphic, and certain rat NKR-P1B alleles have lost binding to the viral RCTL but not to the host Clr-b molecule, suggesting co-evolution between the host and the virus. In the next chapter, we address some of the mechanisms responsible for CMV-mediated Clr-b downregulation, and show that Clr-b downregulation also occurs to mice in response to MCMV infection. Moreover, early gene expression (host or viral) appears to be required for Clr-b downregulation. Interestingly, engagement of any one conventional pattern recognition receptor is insufficient to mimic MCMV-mediated Clr-b downregulation. Similarly, fibroblasts lacking various intermediates for the interferon or inflammasome pathways still downregulate mClr-b, with the exception of the DNA sensor, Zbp-1. Lastly, a recently identified autocatalytic motif conserved in the rat and mouse Clr-b transcripts, the hammerhead ribozyme, appears to be involved in Clr-b regulation. Taken together, our results explore a novel and important role for NKR-P1B:Clr-b interactions in viral immunity.

Due to shared routes of transmission, co-infection with Hepatitis C (HCV) and Human Immuno-deficiency Virus (HIV) has become prevalent worldwide, with approximately one-third of all HIV-infected individuals and about 10% of all HCV infected individuals in North America being co-infected with the other virus (1). Recent advances in HIV treatment have increased the life expectancy of HIV-infected patients, resulting in HCV-associated disease to develop into a major cause of morbidity and mortality among the co-infected population. HIV is consistently shown to have a negative effect on different aspects of HCV disease, from increased HCV RNA levels (2) to aggravated liver fibrosis and more rapid progression to cirrhosis and end-stage liver disease (3). The host immune responses play a major role in not only controlling HCV infection, but also in causing hepatic inflammation and damage. Despite major advances in the understanding of the pathogenesis of these two infections, the mechanisms underlying the role of immune responses in more rapid progression of HCV disease in HCV/HIV co-infection is not quite clear. This thesis is generated based on an investigation to understand why HIV infection worsens HCV pathogenesis. This question is addressed throughout this thesis from different immunological aspects, with a focus on the function of T-cells. I have demonstrated that in HCV/HIV co-infection, functional HIV-specific T-cells accumulate in the liver and produce an array of cytokines, including INF- and TNF-, which may represent a bystander role for HIV in the aggravation of HCV-induced liver damage. My data also demonstrate that co-expression of two defined T-cell exhaustion markers, Tim-3 and PD-1 may play a significant role in HCV-specific T-cell dysfunction, in the setting of HIV co-infection. Both total and HCV-specific peripheral T-cells co-express Tim-3 and PD-1 in significantly higher frequencies, compared to HCV mono-infection. In co-infection, HCV-specific CD8+ T-cells showed greater frequencies of Tim-3/PD-1 dual-expression than those being HIV-specific, indicating a greater degree of exhaustion in the former. Additionally, I demonstrated that some HIV mono-infected individuals may contain CD8+ T-cells that cross-recognize two defined HLA-A2-restricted epitopes within the HIV and HCV proteome, the HIV-Gag: SLYNTVATL and HCV-NS5b: ALYDVVSKL. This T-cell cross-reactivity was further elaborated in the context of HCV/HIV co-infection, demonstrating that degeneracy of HIV-specific T-cells may play a role in the immuno-pathology of co-infection. Altogether, these data could be integrated into the foundation of potential mechanisms involved in the immunopathogenesis of HCV/HIV co-infection, and be applied to further investigation in basic science and clinical studies in this field.

NK cells are innate lymphocytes that are crucial for host immunity during infections. Discrimination between healthy and infected cells is facilitated through a sum of inhibitory and stimulatory signals. Host cells can modulate the expression of NK ligands in response to infection, transformation, and stress, while viruses can exploit these mechanisms to prevent the killing of the infected cells. This thesis focuses on the interaction between the NK receptor, NKR-P1B, and its ligand, Clr-b. First, we used a reporter cell-based protocol to establish hybridomas producing monoclonal antibodies against a putative viral immunoevasin, RCTL. We express CD3ζ-fusion proteins on the reporter cells, which we then use to immunize and screen hybridoma specificities. Our results demonstrate a rapid, efficient, and high-throughput method of monoclonal antibody screening, and provide the framework for our work on cytomegalovirus evasion of the NKR-P1B:Clr-b system. Next, we show that RCMV infection results in a notable downregulation of rClr-b at the protein and transcript levels. Conversely, RCTL is upregulated during infection, and binds to the same NK-inhibitory receptor as Clr-b, NKR-P1B. In the absence of RCTL, RCMV-mediated Clr-b loss leads to increased NK-killing of infected targets and an NK-dependent reduction of viral titers in vivo. Notably, NKR-P1B is highly polymorphic, and certain rat NKR-P1B alleles have lost binding to the viral RCTL but not to the host Clr-b molecule, suggesting co-evolution between the host and the virus. In the next chapter, we address some of the mechanisms responsible for CMV-mediated Clr-b downregulation, and show that Clr-b downregulation also occurs to mice in response to MCMV infection. Moreover, early gene expression (host or viral) appears to be required for Clr-b downregulation. Interestingly, engagement of any one conventional pattern recognition receptor is insufficient to mimic MCMV-mediated Clr-b downregulation. Similarly, fibroblasts lacking various intermediates for the interferon or inflammasome pathways still downregulate mClr-b, with the exception of the DNA sensor, Zbp-1. Lastly, a recently identified autocatalytic motif conserved in the rat and mouse Clr-b transcripts, the hammerhead ribozyme, appears to be involved in Clr-b regulation. Taken together, our results explore a novel and important role for NKR-P1B:Clr-b interactions in viral immunity.

The interaction of microbes with dendritic cells (DCs) is likely to have an enormous impact on the initiation of the immune response against a pathogen. In this study, we compared the interaction of Mycobacterium tuberculosis with murine bone marrow derived DCs and macrophages in vitro. M. tuberculosis grew equally well within non-activated DCs and MØ. Activation of DCs and MØ with IFN-g and LPS inhibited the growth of the intracellular bacteria in an NOS2-dependent fashion. However, while this activation enabled MØ to kill the intracellular bacteria, the M. tuberculosis bacilli within activated DCs were not killed. Thus, DC could restrict the growth of the intracellular mycobacteria, but were less efficient than macrophages at eliminating the infection. These results may have implications for priming immune responses to M. tuberculosis. In addition, they suggest that DCs may serve as a reservoir for M. tuberculosis in tissues, including lymph nodes and lungs. Dendritic cells (DC) possess anti-microbial mechanisms that may play a role in M. tuberculosis infection. Activated DC and MØ produce a comparable amount of reactive nitrogen intermediates, as measured by nitrite production but our data indicate that nitric oxide production is not directly responsible for the differences in the ability of DC and MØ to kill M. tuberculosis. Activated DC have the capacity to produce greater quantities of (reactive oxygen intermediate) ROI than activated MØ. The higher levels of ROI in infected DC may affect the formation of different (reactive nitrogen intermediate) RNI species, such as peroxynitrite, and alter the lethal effect on M. tuberculosis. In addition DC may not able to acidify their phagosomal compartment, where the bacilli reside, to the same degree as macrophages. This may contribute to the ineffectiveness of their anti-microbial compounds.