Malaria is a major public health concern as greater than 40% of the worlds population is at risk. Globally and annually, there are approximately 300-500 million incident cases a year resulting in between 1 and 2 million deaths; the majority of these deaths occur in children under the age of 5 and in pregnant women. There are several disease complications that can arise from malaria, two of which are high density parasitemia (HDP) and severe malaria anemia (SMA). Not everyone who gets malaria gets HDP or SMA, and the underlying reason for this is unknown, however, research has shown that innate immune mediators, including Interleukin-12 (IL-12), play an important role. Currently there is no vaccine for malaria and drug resistance is a major issue. This study is of public health significance because it can give insight into the difference between those individuals who progress to severe disease complications and those that do not; potentially giving rise to novel drug and vaccine development. The severity and occurrences of these complications vary by age, region, and level of malaria endemicity. Previous studies have indicated a role for not only circulating levels of IL-12, but also for polymorphisms in the IL-12 and Interleukin-12 Receptor (IL-12R) genes. To gain a better understanding of the role that polymorphisms in the IL-12 and IL-12R genes may have we conducted a case-control study to compare phenotypic data to genotypic data. We investigated four Single Nucleotide Polymorphisms (SNPs) - rs2243113, rs2243140, rs383483 and rs429774 - in the IL-12 and IL-12R genes to determine if a correlation existed between disease status and genotype. We found that for all four SNPs, there was not a correlation between disease status and genotype. We also investigated the distribution of these four SNPs across populations with varying malaria endemicity. We also found that in all of the populations except for the Kenyan population there is a higher frequency of homozygous wildtype alleles for both rs2243113 and rs2243140 and a higher frequency of heterozygotes for rs383483.

CXCR3 is involved in numerous inflammatory disorders such as rheumatoid arthritis, multiple sclerosis, allograft rejection and inflammatory bowel disease. There is a strong and growing demand for novel and effective therapeutics that can mediate CXCR3 activity. In this study, a set of botanical compounds and a peptide mimetic of the second extracellular loop (ECL-2) of CXCR3 were examined for the ability to inhibit interactions between CXCR3 and its ligands in a murine model. EGCG, a green tea polyphenol, and gallotannin, derived from many plant sources, strongly inhibited the chemotaxis of stably transfected murine CXCR3-expressing L1.2 cells in response to murine CXCL9, CXCL10, and CXCL11. EGCG was also shown to bind directly to murine CXCR3 ligands with high affinities. Baicalin, a flavonoid found in the medicinal plant Scutellaria baicalensis, and ginkgolide A, from the Ginkgo biloba tree, did not significantly reduce cell migration towards murine CXCR3 ligands, nor did the peptide mimetic of the ECL-2 of murine CXCR3. Other green tea polyphenols similar in structure to EGCG were also analyzed and were less able to inhibit murine CXCR3 ligand-mediated chemotaxis than EGCG, with the following efficacies: ECG > EGC > EC. It was observed that the most effective test compounds contained more hydroxyl groups and hence were more negatively charged, similar to glycosaminoglycans, which are extracellular matrix components that bind many chemokines. It is possible that EGCG and gallotannin are able to bind the GAG-binding domains of murine CXCR3 ligands, which allows them to prevent receptor binding and inhibit their function. This possibility represents the public health relevance of this research, as EGCG and gallotannin may be attractive candidates as lead compounds for new therapeutics for CXCR3-mediated and other inflammatory diseases.

Our lab studies Epstein-Barr virus (EBV); therefore, we need a supply of cells that steadily produce virus for use in our experiments. Currently virus harvesting is unpredictable, as transfection of 293SL cells with a given Bacterial Artificial Chromosome (BAC) may produce cell lines that vary widely in the amount of infectious virus produced, with most lines producing no virus at all. Using quantitative real time PCR we quantified EBV mRNA expression pertaining to 16 specific targets including latent, lytic, and promoter transcripts. This was to determine if there was a correlation between the pattern of virus gene expression and the capacity of a cell line to produce virus. Such a correlation could be used to develop a screening assay predictive of a cell lines potential for virus production. We found that the genes most useful for creating a PCR-based screening assay were the genes belonging to the EBNA3 family. The public health significance of the steady production of Epstein-Barr virus would be that experiments could be conducted on quicker time tables, which in turn may increase the rate of knowledge obtained about EBV.

With an average HIV prevalence rate more than two times higher than the general population, the Cameroon military is in need of effective HIV/AIDS prevention intervention programs. The aim of this study is to examine socio-demographic factors associated to condom use among military personnel through an existing HIV prevention program and offer recommendations for HIV prevention interventions to the Cameroon military. Objectives: Analyze baseline condom use data collected from the 2005 HIV surveillance and behavioral study of the Armed Forces of Cameroon. Provide feedback to GVFI to effectively utilize this information for the 2009/2010 HIV/AIDS surveillance and intervention plan targeting the Armed Forces of Cameroon. Methods: The data included responses from a behavioral questionnaire and blood samples (n=2154) obtained from military personnel in Cameroon. Estimated population proportions of condom use data were compared for each of the following socio-demographic variables: military region, age, gender, marital status, military rank, and religion. Chi-square analyses were utilized to test for significance within each socio-demographic variable. Multivariate logistical regressions were executed based on the significant findings of the chi-square tests. Statistical analyses were completed using SYSTAT 13 and SAS 9.2. Results: Specific populations of military personnel demonstrated less condom use, including individuals from military Region 3, older personnel, women, married individuals, non-commissioned officers, and non-Christians. Discussion: This research has shown that there is a relationship between certain socio-demographic characteristics and lower reported rates of condom use. This information can be utilized for the new HIV/AIDS intervention prevention plan (2009/2010) targeting the Cameroon military. Conclusion: Training of trainers and peer educator programs targeting specific populations within the military can have an effect on decreasing the current STI/HIV prevalence rate. A multi-dimensional approach that focuses on intensive education at all levels of the military, outreach that includes condom distribution and counseling, and the availability of HIV testing is essential in creating the most effective HIV/AIDS prevention intervention program. Implications for public health: Consistent and proper condom use is a highly effective method for HIV/AIDS prevention. This research provides background data to inform the planning of an HIV intervention prevention program targeting military personnel in Cameroon. Such a program can be adapted for military programs around the world.

Background - The Martinson Lab is examining genetic characteristics related to cardiovascular disease (CVD) in men with HIV undergoing HAART therapy that are enrolled in the Multicenter Aids Cohort Study (MACS). CVD is a major side effect of HIV infection and HAART therapy. While the mechanism behind this remains unknown, the folic acid metabolic pathway may be involved. This study examines genes that encode enzymes involved in this pathway. Polymorphisms in these genes may lead to increased risk of CVD due to altered function of the enzymes they encode. The following polymorphisms in the MTHFR, MS, and MTRR genes have been found to affect enzymatic function of Methylenetetrahydrofolate reductase (MTHFR), Methionine Synthase (MS or MTR), and Methionine Synthase Reductase (MTRR) respectively: MTHFR C677T, MTHFR C1068T, MS A2756G and MTRR A66G. SNP genotypes for these loci were characterized for the MACS DNA samples. These results were compared with corresponding clinical data and statistics were used to determine how polymorphisms affect cardiovascular disease when influenced by HIV infection and HAART therapy. Methods MACS DNA samples were amplified using PCR, and each SNP was characterized using a Fluorescence Polarization Assay (FP). These data and clinical data for LDL, HDL, triglyceride, BMI, HIV status, HAART status, age, gender, and family history were analyzed using box-and-whisker diagrams, Kruskal-Wallis test, odds ratio calculations, and logistic regression analysis. Results Visual trends were seen between LDL levels and polymorphisms in MTHFR C1068T and MS A2756G. However, no significant associations were found statistically between SNP genotypes and LDL levels. A protective association may exist between the MS A2756G GG genotype and not-high LDL levels, but the small sample size of this genotype means that statistical significance was not reached. We intend to obtain data for more samples and repeat statistical calculations. This may lead to statistically significant outcomes. This thesis contributes to public health by furthering knowledge of how an individuals genetics influences CVD risk while being HIV+ and undergoing HAART therapy. This knowledge enables the patient to be given the best care possible with regards to their individual situations.

Introduction: Pseudomonas aeruginosa is a corneal pathogen and may cause corneal ulceration. The goal of this study was to determine the potential of PCR for detecting P. aeruginosa in corneal specimens from patients with keratitis. Study Aims: 1) To establish a specific real-time PCR assay to detect P. aeruginosa. 2) To determine a secondary target for P. aeruginosa that may provide a universal target for other bacterial pathogens. 3) To validate both assays for diagnostic testing with true positive and true negative clinical samples. Methods: 1) Analytical studies were conducted by testing P. aeruginosa and other bacteria isolated from patients with keratitis with a PCR assay designed to amplify the ecfX gene of P. aeruginosa. The outcome parameters were limit of detection, and amplification efficiency. 2) Similarly, P. aeruginosa isolates were tested for the 16S rRNA gene using the same parameters. 3) Validation of both assays was done by testing 20 cornea samples known to be positive for P. aeruginosa and 20 clinical samples known to be negative for P. aeruginosa DNA. Descriptive statistics were determined. PAGE analysis was performed to confirm the presence of amplified product. Results: 1) Amplification efficiency of the ecfX assay was 96.6%, with a limit of detection of 33.6 copies of target DNA/µl. All 21 P. aeruginosa isolates were detected, with no detection of the 35 non-P. aeruginosa isolates. 2) Amplification efficiency of the 16S rRNA assay was 103.4%, with a limit of detection of 8.12 copies /µl. All 21 P. aeruginosa isolates were detected. 3) The sensitivity, specificity, positive predictive value, negative predictive value, and efficiency for the ecfX and 16S rRNA assays were, [75%, 95%, 94%, 79%, and 85%], and [70%, 100%, 100%, 77%, and 85%], respectively. PAGE analysis supported specificity of the DNA amplified products. Conclusions: Both real-time PCR assays used in this study detected P. aeruginosa DNA from keratitis patient samples. These results indicate that aside from culture, PCR may be a useful adjunct method in the diagnosis of keratitis patients. Public Health Relevance: Real-time PCR can be used to detect P. aeruginosa from patients with keratitis to help preserve vision.

Lymphoblastoid cell lines (LCLs) are created by culturing lymphocytes from the peripheral blood and adding Epstein-Barr virus (EBV), a ubiquitous human herpesvirus which infects, activates, and transforms B cells. These cell lines are used for genotyping, as targets for cytotoxic cells, and as models for EBV immortalization of B cells, particularly post-transplant lymphoproliferative disease (PTLD) in which EBV-immortalized cells proliferate in the absence of a cytotoxic T-cell response. Studies have shown more diversity in LCLs than would be expected from cell lines that are often treated as interchangeable. It is not known how their diversity in factors like morphology, growth factor production, or cellular gene expression influences the EBV life cycle. In this study I investigated connections between LCLs cellular and viral phenotypes, categorizing them as either low in EBV copy number or fluctuating within a high range. As measured by lytic EBV replication and viral gene expression, LCLs showed high or low lytic permissivity, with permissivity defined as the likelihood that a cell will switch from stable latent infection into the lytic EBV life cycle. Permissivity was not affected by blocking the late events of the lytic cycle. I used flow cytometry to characterize 19 aspects of LCL surface phenotype, but found little association with lytic permissivity. Microarrays and PCR were used to identify genes expressed at higher levels in non-permissive LCLs, including transcription factors that maintain B cell lineage. Unfolded protein response (UPR) genes and the UPR protein Grp94 were expressed at higher levels in permissive LCLs. A drug was used to investigate effects of the UPR on permissive and non-permissive LCLs that had been maintained for short or long periods of time. The UPR enhanced permissivity, causing more cells to enter the lytic cycle, but this did not lead to lytic replication. This study enhances our knowledge about EBV life cycles by giving us new information about host factors that contribute to the lytic switch. This data about LCL diversity has public health relevance to the diversity of PTLD cases, since identifying risk factors for PTLD is a significant part of care for EBV-positive transplant recipients.

The chemokine CCL20 is thought to be an integral part of the communication between the innate and adaptive arms of the immune system, due to expression of the cognate receptor, CCR6, on immature dendritic cells and on memory T cells and B cells. Interest in this particular chemokine/chemokine receptor interaction has grown over time and more recently due to roles in SIV infection, mucosal immunology, and vaccinology. The need to further study the CCL20/CCR6 interactions is bolstered by our laboratorys previous findings of increased expression of CCL20 in acutely SIV infected lymph nodes and the increased expression of CCL20 in response to PAMPs in cells of lymphatic vessels. This thesis aims to develop and improve a system for studying the interaction between CCL20 and CCR6. I have found that the recombinant expression system utilized to obtain macaque chemokines provided highly pure fusion proteins. However, cleavage of the fusion protein into macaque CCL20 has been inefficient. Rhesus macaque CCL20 chemically synthesized using regioselective cyclization was highly biologically active using the chemotaxis assay and stable cell lines expressing CCR6. Chemotactic inhibition studies identified five compounds that inhibited CCL20 induced chemotaxis. The surfactant, GML, did not inhibit CCL20 induced migration. The anti-inflammatory botanicals, EGCG and gallotannin, both inhibited CCL20-driven migration at high concentrations. The three CCR6 extracellular loop mimetic peptides also partially inhibited CCL20 induced migration at high concentrations. In conclusion, I have utilized both a recombinant protein expression system and regioselective cyclization peptide synthesis to obtain bioactive, nonhuman primate chemokines. I have also successfully developed an in vitro system to study CCL20 induced migration, and have identified a number of botanical and biochemical elements that inhibit CCL20-induced migration. The public health significance of this study is related to the fact that vaccine efficacy may be affected by anti-inflammatory compounds that inhibit CCL20 mediated chemotaxis. Another way in which public health could be affected by this study is in using the anti-inflammatory compounds studied to treat chronic inflammatory conditions in which the pathology of the disease is related to up-regulation of CCL20 and CCR6.

Inhibition of reverse transcriptase (RT) is a vital tactic in the prevention of human immunodeficiency virus 1 (HIV-1). Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of compounds demonstrated to act as allosteric inhibitors of RT DNA polymerization. However, several lines of evidence suggest that polymerization may not be the main mechanism of inhibition of reverse transcription. It has been demonstrated that NNRTIs also have the ability to modulate RT ribonuclease (RNase) H cleavage. Additionally, recent evidence suggests that resistance to chain-terminating nucleoside reverse transcriptase inhibitors (NRTIs) is dependent on a balance between the polymerase and RNase H activities of the enzyme. In light of this, I hypothesize that NNRTIs block reverse transcription by exerting effects on both the DNA polymerase and RNase H active sites of the enzyme, significantly disrupting the equilibrium between these two enzymatic activities. Therefore, the ability for NNRTIs to be combined with other classes of RT inhibitors in antiretroviral therapies will depend on how these compounds respond to the NNRTI-induced shift in the polymerase/RNase H activity equilibrium. This study demonstrates that NNRTIs cause the accelerated appearance of secondary RNase H cleavage products that have decreased RNA/DNA hybrid structures. As a result, these template/primers(T/Ps) are not sufficient substrates for NRTI removal and therefore, excision is less efficient in the presence of NNRTIs. Additionally, fluorescent resonance energy transfer experiments demonstrate that NNRTIs cause a shift in the binding of RT and T/P such that the RNase H domain is moved away from the 5end of the primer. Finally, subunit-specific analysis shows that resistance to RTI combination therapy facilitated by the N348I mutation is a result of effects from the p51 subunit. I propose that the binding of NNRTIs cause RT to bind to T/P in a polymerase-incompetent mode, resulting in decreased polymerization and shorted RNase H cleavage products. Additionally, N348I can facilitate dual resistance by favoring the polymerase-competent binding mode. This work is of public health significance because it lays the foundation for the development of new reverse transcriptase inhibitors and highlights the importance of resistance in the connection domain of HIV-1 RT.

Severe anemia is the primary outcome of childhood malaria in holoendemic malaria transmission regions such as western Kenya. HIV-1 and bacteremia are equally important diseases in Kenyan children. Anemia is also the hallmark trait of pediatric HIV-1 infection, and despite our previous reports of exacerbated anemia in malaria/HIV-1 co-infected childrena, we also observed significantly lower parasitemias without worsening anemia in malaria/bacteremia co-infected children. Abundant production of pro-inflammatory cytokines is known to adversely affect erythropoiesis and is common to malaria, HIV-1, and bacteremia. As such, we performed a comprehensive bead-based 25-plex Multiplex assay to identify cytokines patterns and profiles associated with negative and positive hematolgic outcomes in co-infected children. Children in Aims 1 and 2 infected with P. falciparum malaria (Pf[+], aged 3-36 mos., n=542) were stratified into three groups: HIV-1 negative (HIV-1[-]/Pf[+]); HIV-1 exposed (HIV-1[exp]/Pf[+]); and HIV-1 infected (HIV-1[+]/Pf[+]). In Aim 3, malaria and bacteremia co-infected children (n=192) were divided into three categories: malaria alone, Pf[+]; Gram negative bacteremia/malaria co-infected, G[-]/Pf[+]; and Gram positive bacteremia/malaria co-infected, G[+]/Pf[+]. Univariate, correlational, and hierarchical regression analyses were used to determine differences among the groups and to define predictors of worsening anemia. Aim 1 analyses revealed HIV-1[+]/Pf[+] children had significantly more malarial pigment-containing neutrophils (PCN), monocytosis, increased severe anemia (Hb<6.0g/dL), and ~10-fold greater mortality. Hierarchical multiple regression revealed that worsening anemia was associated with elevated pigment-containing monocytes, younger age, and increasing HIV-1 status (HIV-1[-]→HIV-1[exp]→HIV-1[+]). Aim 2, addressing the inflammatory milieu, demonstrated that exacerbated anemia was associated with inflammatory mediator (IM) dysregulation, but not parasitemic or erythropoietic indices. A principal component analysis revealed that IL-12 was the most influential variable on Hb levels in HIV-1[+]/Pf[+] children, while the IL-1β:IL-10 ratio was most influenced by PCN. In Aim 3, both bacteremia co-infected groups had lower parasitemia compared to the Pf[+] group. A multiple mediation model examining IMs responsible for decreased parasitemia in the bacteremia co-infected groups identified IL-4, IL-10, IL-12, and IFN-γ as the key molecules in decreasing parasitemia. Thus, malaria/HIV-1 co-infection is defined by significantly enhanced anemia that is associated with unique IM profiles known to exacerbate anemia, while enhanced immune activation in malaria/bacteremia co-infected children appears to promote reduced parasitemia without adversely affecting anemia outcomes. By defining the inflammatory milieu associated with severe anemia, therapies can be developed to mitigate detrimental immune responses, thereby lessening the pediatric public health burden in western Kenya.