The lymphatic endothelium is involved in the drainage of interstitial fluid and in the migration of immune cells like dendritic cells (DCs) from the periphery to draining lymph nodes (LNs). Tuberculosis has been declared a pandemic infectious disease accounting for more than 2 million deaths annually and is caused by the intracellular bacteria, Mycobacterium tuberculosis. The chronic inflammatory response to M. tuberculosis infection is characterized by the formation of granulomatous structures in the pulmonary compartments of infected individuals. These structures contain excess interstitial fluid and are enriched with immune cells including DCs. Therefore, the lymphatic vessels might play important roles in regulating drainage of fluid and migration of immune cells from granulomas to the draining LNs. My hypothesis was that there is an increased concentration of lymphatic vessels in these granulomatous structures and that the inflammatory environment including mycobacterial components present in granulomas and at other sites of infection elicit an inflammatory response from these lymphatic vessels which contribute to the overall immune response to M. tuberculosis infection. To address this hypothesis I have examined the distribution of lymphatic vessels in granulomatous and LN tissues obtained from nonhuman primates infected with M. tuberculosis and analyzed their expression of multiple chemokines and lymphatic markers. In addition, I evaluated the response of LECs to inflammatory mediators that included multiple TLR ligands, M. tuberculosis components and cytokines. I observed an association of lymphatic vessels with granulomas, and found that there was heterogeneity in the expression of chemokines and lymphatic markers by LECs in tissues. I also found that primary human LECs expressed multiple TLR molecules and responded to TLR ligands, cytokines and M. tuberculosis components by increasing expression of inflammatory chemokines, cytokines and adhesion molecules. These LECs also demonstrated phenotypic similarities with DCs. Overall my findings support the involvement of the lymphatic endothelium in the inflammatory immune response to pathogens like M. tuberculosis. From the perspective of public health relevance, these studies provide direction in the development of new therapeutic targets against M. tuberculosis infections and aid in the development of better adjuvants for vaccines for infectious diseases and cancers.

More than 40 million people are living with human immunodeficiency virus-1 (HIV-1). A prophylactic vaccine inducing a 'sterilizing immunity' is desired to prevent further infections, but will require many years to develop. Moreover, prophylactic vaccines will not help the millions of people who are already infected with the virus, and who face life-long treatment with expensive and toxic antiretroviral therapy (ART). This dissertation is based on the proposal that the best strategy for these individuals is a therapeutic vaccine that will attack residual viral reservoirs by expanding HIV-1 specific, primary T cell responses to the persons's own, autologous virus. Previously, this laboratory demonstrated that mature dendritic cells (DC) loaded with immunodominant HIV-1 peptides or HIV-1 infected apoptotic bodies can activate residual HIV-1 specific memory T cell responses. However, such memory T cells are only partially restored during ART. I hypothesized that targeting naive CD8⁺ T cells through a DC-based immunotherapy could elicit a robust and broad T cell response to HIV-1. Furthermore, most immunotherapy studies have used consensus strains of HIV-1 antigens that I believe inadequately represent the host's diverse pool of HIV-1 quasispecies. The current study has provided initial data that support that CD8⁺ T cells can be primed by in vitro engineered DC, even against autologous HIV-1 peptides representing immune escape variants. This study therefore supports the concept of using autologous virus as an antigen in immunotherapy and demonstrates that the use of autologous viral sequences expands both memory and primary T cell responses in vitro. Thus, a potential advantage is that future immunotherapies could use autologous virus representing a large repertoire of the host's diverse HIV-1 antigen pool. This could elicit primary immune responses specific for each patient's quasispecies of HIV-1, as well as activation of residual HIV-1 specific memory T cells, giving the broadest immune control of HIV-1 infection during ART. Such an approach has important public health implications by having a strong positive impact on, and improve the control of, HIV-1 infection in persons on ART. It also serves as an in vitro priming model for development of prophylactic vaccines against HIV-1 and other infectious agents.

Plasmodium falciparum malaria is one of the predominant causes of morbidity and mortality in children under five years of age in sub-Saharan Africa. In malaria endemic regions, the intensity of transmission and the age at which malaria is first acquired are important in conditioning disease outcomes. In addition, inter-individual variability in disease severity among age-matched children (aged <3 yrs) with similar levels of parasite exposure is largely determined by genetic variability. Historical exposure to malaria in endemic populations has exerted tremendous selective pressure on the human genome, particularly in the host-immune response genes that mediate susceptibility and clinical outcomes. Hemoglobinopathies, such as the alpha thalassemia 3.7 kb deletion and sickle-cell trait (HbAS) also confer protection against severe malaria through a mechanism(s) that are yet to be fully elucidated. As such, this study examined the role of alpha thalassemia 3.7 kb deletion and HbAS in protection against severe malaria anemia (SMA) in children (n=468; aged 3-36 months) residing in a holoendemic P. falciparum transmission region of western Kenya. These investigations demonstrated that successful genotyping of the deletion required high-quality genomic DNA from large volumes of whole blood that was unavailable for most of the small, underweight-for-age, severely anemic children in which DNA was isolated from dried blood spots. Results presented here further demonstrated that the HbAS genotype was significantly associated with a reduced burden of both low (<10%; P=0.03) and high (greater than or equal to 10%; P<0.001) pigment-containing monocytes (PCM). In addition, hemoglobin (Hb; P=0.05) and red blood cell (RBC; P=0.04) counts were significantly higher in the HbAS group relative to children with the HbAA genotype. The HbAS genotype was also significantly associated with protection against SMA using both the World Health Organization (i.e., <5.0 g/dL; P=0.04) and modified definitions of SMA (i.e., <6.0 g/dL; P=0.02). Taken together, results presented here suggest that the HbAS genotype confers protection against SMA by reducing the natural acquisition of malarial pigment (hemozoin) in monocytes. This study has significant public health importance by demonstrating that one of the mechanisms by which HbAS provides protection against SMA is through reducing the overall burden of hemozoin in monocytes.

In India, HIV-1 subtype C has been the predominant subtype throughout the course of the HIV-1 epidemic, regardless of geographic region in the country. We hypothesize that the dominance of HIV-1 subtype C compared to other subtypes in India is due to enhanced replication fitness and/or enhanced transmission efficiency of this subtype across the mucosal surface over other subtypes present in India. The specific aims of this project are: (1) to compare the replication fitness between Indian HIV-1 subtype A and subtype C; (2) to evaluate the transmission efficiency of Indian HIV-1 subtype A and subtype C across the mucosa of cervical tissue; and (3) to determine the role of the LTR and env gene in replication fitness and transmission efficiency. Replication fitness was assessed using a dual infection growth competition assay. We observed that primary HIV-1 subtype C isolates had higher overall relative fitness and transmission efficiency than primary subtype A isolates in PBMC and in an ex vivo cervical tissue derived organ culture, respectively. Furthermore, a comparison of replicative fitness between a subtype A/subtype C half genome chimeric virus and parental subtype A virus indicates that the higher replication fitness and transmission efficiency of subtype C virus over subtype A virus from India is not due to the env gene alone. We have also characterized the genetic structure and functional characteristics of subtype A and subtype C LTRs from India. Despite their apparent variability, no significant difference was observed in the transcriptional activity between the LTRs of subtype A and subtype C. Therefore, the LTR region alone is not responsible for higher replication fitness of subtype C over subtype A. The findings presented in this study are significant for public health because an understanding of the mechanism of the asymmetric distribution of HIV-1 subtypes in India is an important component in the development of strategies to control HIV-1 infection in this country.

Introduction: Acanthamoeba keratitis (AK) infection needs to be diagnosed definitively to optimize therapy in order to avoid possible visual impairment. Aims: 1) To optimize two noted Real-time PCR (RT-PCR) TaqMan methods (Rivière and Qvarnstrom) using the Cepheid SmartCycler® II system. 2) To identify potential inhibitory effects from topical drugs on RT-PCR. 3) To validate and compare the two assays using ocular clinical samples. Methods: 1) Primers and probes were optimized for both assays to detect genus-specific Acanthamoeba 18S rDNA. 2) Thirteen topical ophthalmic drugs were diluted to determine the level of inhibitory effect present. The lowest non-inhibitory concentrations were then used to determine RT-PCR amplification efficiency. 3) Excess clinical samples (139) were processed for culture and assayed by both assays on the SmartCycler® II and the results were compared. Results: 1) The Rivière RT-PCR plasmid DNA, cyst and trophozoite limits of detection and amplification efficiency were 10.13 copies/10μl, 0.7/300µl, 2.3/300µl, 94% respectively. The Qvarnstrom RT-PCR plasmid DNA, cyst and trophozoite limits of detection and amplification efficiency were 43.8 copies/10μl, 0.7/300µl, 2.3/300µl, 92% respectively. 2) Out of the thirteen topical drugs, the most noteworthy result was that of Polyhexamethylene biguanide (PHMB). The non-inhibitory dilution and RT-PCR efficiency were 1/2560 and 72.7%. 3) The results of the clinical validation indicated that 134/139 (96.4%) results correlated between the two assays of which 4/134 samples were culture negative but RT-PCR positive. Conclusions: The two RT-PCR assays were optimized successfully on the SmartCycler® II system with comparable results in detecting genus - specific Acanthamoeba DNA. In examining the effects of thirteen topical drugs on RT-PCR, PHMB was demonstrated to both inhibit the reaction at a high dilution and reduce amplification efficiency substantially. Ocular samples (139) were tested using both assays and results thus far indicate that both could be used to diagnose AK in the laboratory. Public health relevance: RT-PCR can be used to rapidly diagnose AK. Commencement of AK specific therapy earlier will substantially reduce the patients the pain and suffering. Also by examining the effects of topical ophthalmic drugs on RT-PCR, the potential for false negative results and result delays could be minimized.

Retroviral Gag polyproteins are necessary and sufficient for virus budding, but little is known about how thousands of Gag polyproteins are transported to the budding sites. The actin cytoskeleton has long been speculated to take a role in retrovirus assembly and recent studies suggest that HIV-1 assembly is regulated as early as viral RNA nuclear export, however specific mechanisms for these regulations are unknown. In contrast to numerous studies of HIV-1 Gag assembly and budding, relatively little is reported for these fundamental pathways among animal lentiviruses. In this project, we used bimolecular fluorescence complementation (BiFC) (1) to reveal intimate (<15nm) and specific associations between EIAV Gag and actin, but not tubulin; (2) to characterize and compare assembly sites and budding efficiencies of EIAV and HIV-1 Gag in both human and rodent cells when the mRNA nuclear export context is altered to be Rev-dependent or Rev-independent; (3) to reveal co-assembly of Rev-dependent and Rev-independent HIV-1 Gag and rescued assembly of Rev-independent HIV-1 Gag in human cells by in cis provided membrane targeting signals. The results of these studies showed that (1) multimerization of EIAV Gag was required for association with filamentous actin and this association correlated with Gag budding efficiency, suggesting that association of Gag multimers with filamentous actin is important for efficient virion production; (2) HIV-1 and EIAV Gag assembled in different cellular at sites, and HIV-1 but not EIAV Gag assembly was affected by mRNA nuclear export pathways, suggesting that alternative cellular pathways can be adapted for lentiviral Gag assembly and budding; (3) Rev-independent HIV-1 Gag was deficient in lipid raft targeting and its assembly and budding could be restored by membrane targeting signals provided in trans or in cis, suggesting that raft association is critical for HIV-1 assembly and budding and is regulated as early as nuclear export of Gag-encoding mRNA. The findings presented in these studies are significant for public health because a better understanding of the mechanism of retrovirus assembly and budding increase the potential to develop novel antiviral therapies targeting this critical step in the viral life cycle.

The need for a therapeutic human immunodeficiency virus (HIV) vaccine is urgent for the control of the acquired immunodeficiency syndrome (AIDS) epidemic. The variability of the virus as well as its ability to undergo escape mutations in T cell epitopes are important obstacles facing the development of an AIDS vaccine. Consequently, a powerful strategy would be the induction of robust antigen specific T cell responses targeting patient-specific virus sequences expressed during the course of infection. An attractive vaccine approach to achieve this is the use of dendritic cells (DC) transfected with in vitro transcribed mRNA encoding autologous virus sequences. The rhesus macaque model provides an ideal preclinical setting to test the therapeutic potential of DC-based vaccination. We hypothesize that optimal antigen presentation and stimulation of potent T cell responses could be achieved by loading DC from SIV-infected macaques with mRNA encoding virus-derived sequences isolated during the course of infection. This represents a powerful strategy for the generation of a potential therapeutic AIDS vaccine. In support of our hypothesis, we generated the following evidence: (1) nucleofection is a superior method for efficient transfection of human and monkey monocyte-derived DC with DNA and mRNA to conventional electroporation and lipofection; (2) nucleofection of DC with mRNA led to better protein expression and DC maturation as compared to DNA transfection; (3) mRNA nucleofection of DC resulted in rapid and sustained gene expression, a critical factor in DC-based immunotherapy for durable antigen presentation; (4) nucleofection of monkey monocyte-derived DC with wild-type non codon-optimized gag mRNA was efficiently expressed and induced strong antigen-specific T cell responses whereas DNA transfection led to non-specific T cell stimulation; (5) enhanced CD4+ T cell responses were observed when Gag was redirected to the lysosomal pathway via the targeting signal of the lysosome-associated membrane protein (LAMP-1) following nucelofection of DC with mRNA; (6) rhesus DC transfected with lysosome-targeted gag mRNA encoding an escape mutation in an immunodominant CTL epitope stimulated CD4+ and CD8+ T cell responses of almost equivalent magnitude directed towards undefined epitopes outside of the mutated region; (7) gag or env mRNA transfected-DC from SIV-infected macaques stimulated significant antigen-specific T cell responses in an entirely autologous system; (8) DC cotransfected with gag mRNA as well as mRNA encoding CD70 or OX40L did not result in enhanced immunostimulatory functions. HIV/AIDS is a significant public health problem demanding action. This work demonstrates that mRNA-transfected DC expressing SIV antigen from infected monkeys stimulate broad and relevant T cell responses, thus supporting this approach for the generation of a therapeutic HIV vaccine to decrease the burden associated with the infection.

Methicillin-resistant Staphylococcus aureus (MRSA) is an infectious disease that has been a cause of nosocomial infections since the 1960s, but has more recently become an emergent disease in community settings. MRSA infections that develop outside hospitals have been associated with risk factors such as young age, recent antibiotic use, recent contact with health care, and dermatological conditions. To provide descriptive epidemiological data and evaluate potential risk factors, we undertook a case-control study of Allegheny County residents with laboratory-confirmed MRSA and methicillin-sensitive S. aureus (MSSA) cultures from January through August of 2007. A random sample of each group was contacted and interviewed using a standardized questionnaire. Comparing 54 MRSA culture-positive residents to 50 MSSA culture-positive residents, we found that having a reported self history of MRSA (p<.001), having a household member or self recently having been in the hospital (p=.041), and having a household member or self recently having been in a community living setting (p=.032) were significant risks of having a positive MRSA culture. These findings have public health significance because as a greater number of people become infected or colonized by MRSA, the reservoir in the community will continue to grow, resulting in a greater number of infections and increased morbidity and mortality from the disease.

Epstein Barr virus persists in the human host by establishing a latent infection following primary infection. The virus periodically reactivates; producing virus that can infect new cells or be shed in saliva to infect new hosts. EBV is also implicated in malignant B cell proliferation in the immunocompromised and a variety of haemopoetic cancers, indicating that it is of public health significance. The LMP2 gene of EBV encodes 2 protein isoforms: a 497aa protein (LMP2a) and a 378aa protein (LMP2b). These isoforms are identical, with the exception of an N-terminal cytoplasmic signaling domain of 119aa encoded in the LMP2a exon 1. The remaining residues (including the entirety of LMP2b) encode an integral membrane protein consisting of 12 transmembrane spanning regions with short alternating intracellular and extracellular connection loops. Most research on the LMP2 isoforms has focused on the LMP2a protein and its role in blocking B-cell receptor mediated signaling, degradation of associated proteins, and transformation. LMP2b, lacking the obvious signaling domain, has been largely ignored. Recently studies have suggested that LMP2b is a negative regulator of LMP2a. In the following studies, we have evaluated the contribution of LMP2b to the block in BCR signaling using LMP2b expressing BJAB cell line. Our results demonstrate that LMP2b has the ability to singularly block BCR signaling. LMP2 proteins have been described at both the plasma membrane as well as in the intracellular membranes of cells. Our studies indicate that the intact 12-TM region of the LMP2 proteins is necessary for intracellular localization. Through progressive deletions of 2TM segments from both the N- and C-terminal ends of the protein, we find that an intact 12-TM domain is necessary for localization, and there are at least 2 domains required for multimerization. The role of LMP2 in immortalization is also contested, with groups reporting that LMP2a is both necessary and dispensable for immortalization. We utilized an established system of recombinant EBV construction to demonstrate that LMP2a, but not LMP2b plays a role in establishment and maintenance of viral latency. Taken together, these results indicate a function for LMP2b in signaling and immortalization separate from LMP2a.

We have previously demonstrated a significant association between human herpesvirus 8 (HHV-8) seroprevalence among men with prostate cancer in the Caribbean island of Tobago compared to cancer free men. HHV-8 DNA has been detected in semen and prostatic tissues in some, but not all reports. I have performed immunohistochemistry (IHC) on prostate tissues from HHV-8 seropositive men from the United States and Tobago with prostate cancer for the expression of viral proteins and determined if expression of these proteins are associated with increased inflammation. My results demonstrate the presence of viral proteins in prostates from seropositive men and among tissues expressing these viral proteins, there is increased inflammation as measured by macrophage infiltrate. I have also looked for the presence of polymorphisms in several genes previously associated with increased risk of prostate cancer to determine if there was a genetic link with the greater risk for prostate cancer in Tobago. We have found single nucleotide polymorphism in the IL-6 gp130 signaling receptor that is associated with increased prostate cancer risk among HHV-8 seropositive men. The public health relevance observed by these results suggests an interaction between HHV-8 infection, increased inflammation and genetic polymorphisms resulting in increased prostate cancer risk. I hypothesize that HHV-8 is a cofactor for prostate cancer in Tobago by establishing a chronic infection which leads to chronic inflammation and ultimately prostate cancer.