HIV infects and depletes CD4+ T cells in Gut Associated Lymphoid Tissues (GALT) of the Gastrointestinal (GI) tract at a very early stage of infection. Furthermore, GALT are the major reservoirs of HIV-1 and may constantly shed virus and CD4+ T cells into the intestinal lumen throughout the entire course of infection. We hypothesize that the dynamic changes of HIV-1 and CD4+ T cell quantities in feces are linked to disease progression and can be used to predict disease prognosis. The aims of this study are to establish sensitive methods for detection and quantitation of HIV-1 and CD4 mRNA in feces, and to use the methods to monitor the amount of HIV-1 RNA/DNA and CD4 mRNA in feces samples of HIV infected patients and to correlate the findings with disease progression. In addition, since urine may potentially serve as a vehicle for HIV-1 transmission we have also measured HIV-1 RNA/DNA in the urine samples from the same population used for the feces study. Our results showed that using normal feces spiked with known copies of DNA and RNA, as low as 2.5 copies of HIV-1 DNA and 40 copies of HIV-1 RNA were detected per input in both nested PCR and RT-nested PCR reactions respectively. Human CD4 mRNA was also detected in feces. From HIV-1 infected volunteers of the Multicenter AIDS Cohort Study (MACS), HIV-1 DNA, RNA and human CD4 mRNA was detected in 8%, 19% and 31%, respectively, in the feces samples from patients with detectable viral load in blood. In the urine samples from the same study population, HIV-1 DNA was detected in 26% of HIV-1 infected donors and this detection is not always correlated with the presence of detectable viral load in blood. This study has major Public health significance as it demonstrates that HIV-1 RNA/DNA could be detected in feces and urine samples, which may lead to the development of a future non-invasive approach to evaluate disease progression and prognosis. In addition, our study demonstrated, for the first time, the presence of human CD4 mRNA in fecal specimens of infected donors, which could be used as a valuable tool in the future to assess the pathogenesis of Gut Associated Lymphoid Tissue over the course of HIV-1 infection.

CD4+T-cells have a central role in induction and homeostasis of the immune response, and are also the major target cells for HIV. HIV has devised mechanisms to subvert the immune system and further its cause of survival and dissemination. vpr is one of the accessory genes, which is essential for the virus survival in vivo. Being a soluble protein with an ability to transduce across cell membranes, Vpr can potentially affect bystander cells. We hypothesize that HIV-1 Vpr alters the functions of both infected and bystander T lymphocytes, utilizing direct and indirect mechanisms, and these Vpr-mediated effects contribute in part to the immune dysregulation, and aid in viral dissemination. The Specific Aims of this proposal are to: (1) Assess the immune modulatory effects of Vpr in infected and bystander T-lymphocytes; (2) Understand the role of Vpr in T lymphocytes, natural killer (NK) cells and dendritic cells (DC) interactions; and (3) Analyze the structure-function role of Vpr in immunopathogenesis. We utilized HIV-1wt and HIV-1ΔVpr viruses and compared the difference in the effects of these viruses under controlled simulated invitro conditions. The differences observed in the effect of these two viruses can be attributed to Vpr provided that the infections in both the experimental sets are similar. In some studies, to clearly identify infected cells, we employed EGFP reporter viruses. The effects in infected cells and bystander cells were evaluated. Results indicate that HIV-1 Vpr has a role in dysregulation of immune cells during HIV infection. Vpr differentially regulates the surface expression of T cell costimulatory molecules, CD28 and CTLA-4, and inhibits IFN-γ production in infected T cells. Vpr also inhibits NK cell function by augmenting TGF-β production and inducing altered expression of NK receptor ligands. Further, oligomerization of Vpr has a role in gag incorporation of Vpr and in viral pathogenesis. The findings presented in this study are significant for public health because mechanistic understanding of the pathogenesis will aid in development of novel anti-viral therapeutics.

Bacterial and viral meningitis are public health concerns as they are contagious and highly fatal without treatment. Newborn infants are at high risk for bacterial and viral meningitis. The onset of the infection is rapid and without quick diagnosis and treatment, many infants will die. Diagnosis requires the positive identification of the causative agent through culture or the use of polymerase chain reaction (PCR) from specimens of cerebrospinal fluid (CSF). Because these tests can take hours or days to perform, it is important identify children who have a higher likelihood of serious bacterial or viral infection so that empiric therapy can be initiated while awaiting further results. Previous studies have indicated that CSF characteristics can be accurate early predictors of viral and bacterial meningitis. Although CSF characteristics have been established for infection, normal values for infants less than 60 days of age are still not clear. To improve characterization of CSF values for infants, this study set to answer three questions: Is there a temporal relationship for CSF WBC, glucose, and protein? What are the means and confidence intervals for the means for each of these variables? What is the range of normal values that a physician could expect to find in infants less than two months of age? This study involved three independent retrospective chart reviews over a 15-year period to identify infants less than two months of age who presented to The Childrens Hospital of Pittsburgh emergency department with fever and had lumbar punctures performed but were not found to have bacterial or viral meningitis. For CSF WBC and protein, the data from the three cohorts were pooled and a single set of reference values was generated for infants less than two months of age. CSF glucose values were not pooled due to differences that existed between the cohorts and reference values were calculated for the cohorts individually. CSF white blood cell (WBC), glucose, and protein values were analyzed to answer our three study objectives. A temporal trend was found for CSF WBC and protein with values being highest during the first weeks of life. CSF glucose values did not change with time. These values will be potentially valuable reference tools in emergency departments for physicians who are faced with decisions regarding care and treatment of febrile infants.

The gut mucosa is an important portal for HIV-1 transmission and infection. Therefore, a vaccine which can prevent virus transmission at mucosal surfaces would be an ideal HIV-1 vaccine candidate. Clostridium perfringens has been used as a vehicle to deliver SIV proteins in large quantity to the terminal ileum. A mucosal immunization strategy using C. perfringens should be able to induce potent mucosal immune responses against HIV-1. A recombinant C. perfringens expressing large amount of HIV-1 Gag protein (Cp-Gag) was constructed. Under in vitro conditions, Cp-Gag was found to induce bone marrow derived dendrite cell (BMDC) to mature and stimulate HIV-1 Gag specific T cell responses. Then in vivo experiments were performed in mice to demonstrate orally delivered Cp-Gag ability to prime gut mucosal T cell responses. Since oral tolerance is a major obstacle for orally delivered immunization approaches, a combination of mutated heat-labile enterotoxin of E. coli (mLT) and CpG containing oligodeoxynucleotides (CpG-ODN) were used as adjuvants for oral administration with Cp-Gag. Orally delivered Cp-Gag was tested for induction of HIV-1 Gag specific T cell responses in a prime-boost model with intranasal inoculation of HIV-1 virus like particles (VLP). HIV-1 specific cellular immune responses in both the effector (Lamina propria) and inductive sites (Peyers patches) of the gastrointestinal (GI) tract were significantly higher in mice immunized using Cp-Gag and VLPs in prime-boost approaches compared to mice immunized with either Cp-Gag or VLPs alone. Such cellular immune response was found to be mediated by both CD8+ and CD4+ T cells. These groups of mice also seemed to have HIV-1 specific multifunctional T cells in PPs and LP of the GI tract. In summary, mucosal immunization of mice with a Cp-Gag and VLPs in a prime-boost mode led to strong HIV-1 Gag specific cellular immune responses in both mucosal and systemic immune compartments. Such strong mucosal immune response could be very important to control HIV-1 infection at mucosal surfaces. The proposed vaccine strategy has great public health significance for developing a practical vaccine against HIV due to its safety, low production cost and easy administration.

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.