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.

Despite the significant need for prevention education and updated disease curricula in elementary schools, there is a deficit of model units, lesson plans, and activities at the fourth grade level. An infectious disease and prevention teaching unit has been developed, following guidelines specified by the Centers for Disease Control and Prevention and a format consistent with proven pedagogical methods. This curriculum was tested in five classrooms with a total of 94 students. Prior to implementation, an assessment of all fourth grade teachers in the district examined their perceived knowledge of infectious diseases and their perceived self-efficacy in teaching such content. Evaluation of student progress included student pre and post-tests to assess changes in knowledge. Upon completion of the unit, teachers evaluated the unit to determine its relevance, effectiveness, and ease of implementation, and completed a post-test on their own knowledge and efficacy. Results indicate that the unit was effective in increasing student comprehension and interest in infectious disease prevention, and teacher efficacy in delivery of the material. This model curriculum can serve as a foundation to increase school health education in critical public health areas such as infectious diseases and preparedness, and provide an early introduction to public health careers.

Previous studies have demonstrated that the increased incidence of invasive disease caused by serogroup C Neisseria meningitidis in the United States during the 1990s was attributed primarily to strains belonging to the ST11 clonal complex. Subcapsular genotyping of a subset of isolates from Maryland identified distinct early and late clones defined by antigenic shift at the FetA outer membrane protein. Representational difference analysis (RDA) was used to identify additional genetic differences that may have contributed to the emergence of the late clone. A collection of serogroup C isolates representative of the early and late clone was subjected to pulsed field gel electrophoresis (PFGE) to determine genetic relatedness among the isolates and to identify a candidate tester/driver pair for RDA. RsaI-digested tester genomic DNA (late clone) was ligated to specific adaptors followed by two rounds of subtractive hybridization with RsaI-digested driver genomic DNA (early clone). PCR amplification of subtracted tester DNA with adaptor specific primers generated at least three late clone-specific bands that were absent from the early clone. These products were cloned and sequenced and confirmed by Southern blotting with tester and driver digoxigenin-labeled genomic DNA probes to be tester specific. A BLAST search of late clone-specific sequences identified homology to either IS1301 or pJS-B plasmid N. meningitidis sequences. PCR with primers specific to either IS1301 or pJS-B plasmid sequences amplified these elements from late clone isolates but not from early clone isolates. Thus, RDA successfully identified two unique genetic elements present in an emergent N. meningitidis serogroup C ST-11 clone that had undergone antigenic shift at FetA. Further investigation is required to determine the potential role of these elements in clonal emergence and N. meningitidis pathogenesis. The public health significance of this project stems from increased incidence of meningococcal disease being a major concern: morbidity and mortality increase, outbreaks produce panic and disruption in communities, public health agencies must respond for control and prevention, and mass immunization and antibiotic prophylaxis are often required.

Human immunodeficiency virus (HIV), which progresses into the disease commonly referred to as Acquired Immunodeficiency syndrome (AIDS), has become one of the world’s most destructive epidemics since its discovery in the early 80's. To date, the virus has killed more than 25 million people, with an average of 5 million newly infected cases last year alone. The HIV-1 genome is comprised of structural and enzymatic polyproteins as well as regulatory/accessory, which are essential for viral replication. Viral protein R (Vpr), which is identified as one of the regulatory/accessory genes, is responsible for carrying out several of the virus life functions, including virus replication, cell cycle regulation, apoptosis, and immune dysregulation. Through research of the virus, the disease has been divided into two very distinctive categories: Rapid Progressors (RPs) and Long Term Non-Progressors (LTNPs). The differences between these categories are due to the varying quasispecies, which infect the population, and ultimately disease progression. Several well-known mutations that occur within vpr have been associated with disease progression, linking them to one of the category types. Using a population from the Multicenter AIDS Cohort Study (MACS), patient vpr genotypes were analyzed and compared with current findings in research. Several of the patients deduced amino acid sequences revealed different gene variants, truncations, as well as a number of point mutations. Functional analysis revealed a decrease in cell apoptosis, which could have been caused by the observed point mutations. Further analysis is needed in order to determine if any other functions of the virus are disrupted due to the observed mutations. Because the virus has the ability to make changes within, as of right now the only hope in counteracting the effects of HIV is through the use of antiviral medication, such as HAART. But studies have shown that not everyone has the same positive effect when these drugs are administered. By understanding the virus and its pathogenesis, researchers will be able to develop new targets for therapeutic interventions. The public health significance of this project is to provide the valuable research that will lead towards such viable HIV-1 therapeutic interventions.

Abstract HIV/AIDS among students at Historically Black Colleges and Universities (HBCU) in the rural South is a growing public health concern. Lack of basic HIV/AIDS knowledge, underestimating risky behaviors, and lack of discussions relating to sexuality are some factors that contribute to the spread of HIV/AIDS within the HBCU population. Objectives of the study included the following: 1) To examine how the issue of HIV/AIDS is viewed by student leaders and organizations on campus; 2) To examine what student organizations and administrators are doing to educate the student body on HIV/AIDS; 3) To identify barriers that student organizations and administrators face when providing education to students; and 4) To examine how student leaders rate their leadership influence when interacting with their peers. This study utilized a qualitative research design in which student leaders and administrators were interviewed and asked a series of questions related to HIV/AIDS education on their campus. Student leader participants were recruited from a university site located in the rural South. Interviews were collected through a 30 minute tape-recorded session on campus. Interview data were analyzed using principles of grounded theory. The findings of the study suggest that student organizations could be a useful vehicle for HIV/AIDS peer-led interventions if their members are well trained and first address underlying issues such as cultural homophobia, sexuality, and stigma relating to HIV/AIDS. Administrators of the university should encourage students to be creative when addressing their peers about issues surrounding HIV/AIDS. Researchers and public health officials must create appropriate interventions to address issues surrounding HIV/AIDS before effective education of HIV/AIDS can take place. Public health significance: Improving HIV education among HBCU students presents a potentially effective strategy that addresses the larger issue of HIV/AIDS among African Americans by focusing their efforts and targeting a smaller sub-population first. The public health relevance of improving education among HBCU campuses is evident when considered in light of this promising possibility. This sub-population is particularly important because many of these individuals will become leaders of the African American community, and influence community behavior and attitudes towards HIV/AIDS.