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.

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.

One-third of the worlds population is infected with Mycobacterium tuberculosis, which causes over 2 million deaths annually. M. tuberculosis typically infects humans through the inhalation of aerosolized microorganisms and the hosts immune system controls the infection by developing a granuloma, which consists predominantly of macrophages and lymphocytes. The factors initiating the formation and maintenance of these granulomas are not well understood, but immune cells are likely recruited to the site of inflammation to maintain immune control of the infection. Chemokines and cytokines play important roles in cell trafficking and migration of immune cells, and DC initiate an adaptive immune response. My hypothesis is that DC (in conjunction with macrophages) recruit immune cells to the granulomatous site by the expression of IFN-g-inducible chemokines, which are expressed due to mycobacterial antigen stimulation. To determine local cytokine- and chemokine-specific and DC-associated mRNA expression patterns in granulomatous lesions, I performed in situ hybridization (ISH) on paraformaldehyde-fixed, cryopreserved lung tissue sections obtained from cynomolgus macaques (Macaca fascicularis) infected with a low dose of virulent M. tuberculosis. In addition, we evaluated the presence of mycobacterial 16S rRNA to determine the distribution of the mycobacteria and the mycobacterial burden within the granulomas. To model the immune environment in the pulmonary granulomas, I infected human monocyte-derived DC with M. tuberculosis in the presence of IFN-g. Although I found an abundant expression of the IFN-g-inducible chemokines and numerous DC-associated genes within the granulomas, the IFN-g-inducible chemokine expression was predominantly produced by macrophages. The presence of DC in the granuloma may serve to skew the immune response to a type I environment, but our data do not suggest a direct role in the production of IFN-g-inducible chemokines. These studies provide further information on the potential roles for chemokines and DC in granuloma formation and maintenance as well as the composition of local DC populations. These studies further illustrate the complex microenvironment of granulomas, which are important in the control of tuberculosis infection. Further understanding of granuloma formation and maintenance could lead to the development of therapeutic treatments needed to reduce this public health epidemic.

Escherichia coli O157:H7 is an important food-borne pathogen and public health risk that infects thousands of people a year in the United States alone. While many infections may remain undetected, some develop into hemorrhagic colitis and/or hemolytic uremic syndrome especially in young children and the elderly. The development of the molecular subtyping technique pulsed-field gel electrophoresis (PFGE) greatly enhanced the detection of outbreaks caused by this organism, but its technical limitations had researchers searching for alternative techniques. The use of variable-number tandem repeats (VNTRs) for human forensics and subtyping of extremely clonal bacterial species such as Bacillus anthracis provided a potential new technique for examining E. coli O157:H7. This new technique, multi-locus VNTR analysis (MLVA), examines multiple VNTR loci, which are some of the most rapidly evolving genetic elements in the genome. We demonstrated the utility and superiority of MLVA over PFGE as a molecular subtyping technique for E. coli O157:H7. With the establishment of the MLVA protocol, the need arose to understand how often the MLVA loci mutate to help characterize which isolates are highly related. Using an experimental protocol of 10 serial subcultures, one of the 7 MLVA loci was found to be hypervariable with a tendency of single, addition TR mutation. Two other loci were found to be slightly variable while the remaining 4 loci had no mutation events during the experiments. The establishment of a protocol based on VNTRs and the initial understanding of mutational dynamics only touched upon the genotypic roles of VNTRs but not the functional roles. A preliminary examination of the functional roles of a few selected VNTRs was undertaken by performing a variety of tests. A detailed description of all this projects results is presented in the following work.

Abstract Description of problem: APIs in the US have experienced an increase of AIDS cases at a rate greater than other ethnic communities due to relatively low levels of knowledge regarding HIV and mechanisms of transmission, the high prevalence of risky sexual practices, and the absence or lack of educational interventions designed to reach API communities. Findings from this study further public health in terms of HIV prevention by providing information about the ideas APIs have about HIV/AIDS. Objectives: 1) To describe API knowledge level with regards to HIV/AIDS. 2) To identify HIV risk-related behavioral characteristics of APIs. 3) To identify types of prevention/intervention activities accessible to the API community. Methods: This study utilized a descriptive research design. Key informant participants recruited from the University of Pittsburgh Community were interviewed with questions about HIV/AIDS using a snowball sampling method, that is, request peoples reference to other key informant participants. Subjects were paid $20 for their participation. The data was collected through a single 60-minute tape-recorded interview session in a common setting where the participant would not feel labeled in any manner and could openly participate. Confidentiality was guaranteed to encourage honest responses because the participants had to reveal personal information such as HIV status and HIV risk-related behaviors. Results: The participants demonstrated good levels of knowledge with regards to HIV/AIDS; the relationship between HIV and AIDS, and also about ways their community can be at risk. Participants mentioned using condoms as a safe activity that places people less at risk for HIV/AIDS and not using condoms as an unsafe activity that puts their ethnic community more at risk of HIV/AIDS. Discussion: The myth that American APIs are the model minority is contradicted as APIs are often underserved in healthcare due to cultural, linguistic and lack of peer and community support for sexual and racial diversity hindering self-esteem and positive self-identity. Therefore, such issues are identified within the API community that are important to consider when developing HIV prevention and education programs, benefiting the target community, health professionals and researchers reaching a diverse population for future studies and interventions being promoted.

Non-human primates are often used in the development and testing of vaccines and therapeutics for malaria. Infection of rhesus macaques (Macaca mulatta) with Plasmodium coatneyi causes cerebral malaria (CM) at high levels of parasitemia. To prevent mortality, parasitemia is frequently treated prior to this point when cerebral signs are largely absent. The public health significance was to determine the validity of this as a model for studying malarial anemia (MA) by examining hematological profiles, cytokines, and effector molecules shown to be important in childhood MA, including tumor necrosis factor (TNF)-α, interleukin (IL)-10, (IL)-12p40, and interferon (IFN)-γ, and nitric oxide (NO) at eight different time points during the acute infection. Peripheral parasitemia was monitored daily throughout infection. Venepuncture was performed for hematology panels on days 0, 2, 5, 7, 8, (peak parasitemia), 10, 15, 22, 34, 42, 56, and 150 post-infection (PI) for monitoring acute and chronic malaria infection. Complete blood counts (CBC) revealed that monocytes (MO) were highest during the initial rise in parasitemia, neutrophils (NEU) were highest at peak parasitemia, and lymphocytes (LY) remained constant throughout infection, except at peak parasitemia where levels were dramatically reduced. Platelets (PLT) declined shortly after infection and decreased until day 15, where levels sharply increased to higher than baseline values on day 19 PI. Hemoglobin (Hb) was lowest at parasite clearance(day 15 PI). Elevated peripheral blood mononuclear cell (PBMC) transcripts (determined by real time RT-PCR in circulating blood mononuclear cells) and plasma levels (determined by ELISA) were associated with enhanced disease severity (peak parasitemia, thrombocytopenia and anemia). Plasma cytokine levels were not correlated with PBMC transcript levels, suggesting that the plasma cytokine levels may be from multiple cellular sources in addition to PBMC. Low IL-10 relative to TNF-α (IL-10/TNF-α plasma ratio) coincided with the lowest Hb concentration, a finding we have shown in children with MA. Taken together, these results demonstrate similar patterns of cytokine and effector molecule dysregulation in rhesus macaques infected P. coatneyi as that observed in humans with P. falciparum-induced malarial anemia.

Approximately ¼ of AIDS patients develop HIVE, the pathologic entity associated with cognitive, motor, and behavioral deficits attributed to synaptic damage and neuronal loss. It still remains unclear why only a subset of HIV-infected individuals develops abundant central nervous system (CNS) macrophage/microglia infection that characterizes HIVE. The overarching hypothesis of this body of work is that simian immunodeficiency virus (SIV) encephalitis (SIVE) is the CNS manifestation of a systemic increase in SIV infection and activation of monocyte/macrophage elements. Specifically, we examined the relationship of infected and activated monocyte/macrophage elements outside of the CNS during the evolution of lentiviral encephalitis to the presence of infected macrophages in the CNS. We studied three models of SIV infection: SIV-infection of rhesus and pigtailed macaques and SIV-infection of CD8+ T cell depleted macaques. Antibody-mediated CD8+ T cell depletion did not increase the incidence of SIVE in infected rhesus macaques. In SIV-infected rhesus macaques, we examined whether presence of activated macrophages or SIV-infected macrophages is associated with the presence of neuronal damage. The presence of abundant infected macrophages in the CNS is related to postsynaptic neuronal damage in macaques with SIVE. At the same time cerebrospinal fluid viral load increased in SIV-infected CD8-depleted rhesus and non-depleted pigtailed macaques that developed encephalitis, monocyte-derived macrophages produced more virus ex vivo than macaques that did not develop encephalitis. Compared to pigtailed macaques that did not develop SIVE, the monocyte associated SIV-DNA load of monocytes was elevated in macaques that developed SIVE. Pigtailed macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes, than macaques without SIVE. Longitudinal analysis of phenotypic markers of monocyte activation show that increases in proportion of CD14+/CD16+ monocytes is associated with chronic disease. Brains with SIVE have greater numbers of T cells with cytotoxic potential. In conclusion, these findings suggest that inherent differences in host macrophage viral production or immune response to macrophage infection are associated with development of encephalitis. Further understanding of the differential role monocyte/macrophages have in the development of lentiviral encephalitis will identify therapeutic targets to halt this public health epidemic.

The HIV/AIDS epidemic is one of the most important public health problems facing this generation. The failure of recent vaccine trials and growing resistance to anti-retroviral drugs underscores the need for novel therapeutic strategies. Design of such therapies will depend on a detailed understanding of the mechanism of action of the HIV-1 gene products. To further that goal, we have undertaken a detailed investigation of the HIV-1 viral protein R (Vpr). We employed cDNA microarray and antibody array analyses using isogenic virus with or without Vpr to determine the effect on host cellular gene expression. Vpr induced differential regulation of 109 cellular genes representing diverse families of signaling molecules. Two gene products, NHE1 and TNF alpha, were further studied for their potential roles in Vpr-mediated apoptosis. NHE1 expression was decreased by 50% at both the protein and mRNA levels in the presence of Vpr. Vpr-mediated NHE1 downregulation correlated with a dose dependent decrease in intracellular pH as well as a decrease in the active form of the pro-survival kinase Akt. The loss of these anti-apoptotic functions of NHE1 is proposed to contribute to the apoptotic role of Vpr. The pro-inflammatory cytokine TNF alpha may also play a part in Vpr-mediated apoptosis. Macrophages infected with vpr-expressing virus secreted 1.1-8.5 fold more soluble TNF alpha in response to LPS stimulation than their counterparts infected with isogenic virus lacking Vpr expression. Fold upregulation of TNF alpha directly correlated with induction of apoptosis in uninfected lymphocytes, implicating TNF alpha regulation by Vpr in bystander cell death. Two polymorphisms in the TNF alpha promoter, positions -238 and -963, were found at a higher prevalence in donors showing the lowest and highest effect of Vpr on the TNF alpha response. These results suggest that host genetic determinants may affect bystander cell death and thus the course of HIV pathogenesis. Together, the results of this study present a molecular basis for changes induced in the host cell by HIV-1 Vpr and elucidate two potential pathways for the design of anti-retroviral therapeutics targeting HIV-1 Vpr.