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.

HIV-1 Vpr is known to regulate both viral and host cellular promoters resulting in transcriptional regulation of various cellular factors in host immune cells, such as T cells, macrophages and dendritic cells. It has been shown that Vpr has a role in the upregulation of proinflammatory cytokine TNF-á, which affects immune regulation during infection. However, the mechanisms by which TNF-á is regulated by HIV-1 Vpr are not well understood. A goal of this project is to determine the effects of Vpr in its biologically relevant forms and identify the domains of Vpr involved in TNF-á production. Additionally, we also sought to determine whether TNF-á is up-regulated in infected/exposed cells and/or bystander cells. From our experiments, we conclude that HIV-1 Vpr increases TNF-a production in the context of infection as well as exposure in the absence of other viral proteins. Furthermore, HIV-1 Vpr has multiple domains capable of inducing TNF-á production. However, the increase in TNF-alpha production in DC is dependent on LPS stimulation. We were unable to conclusively determine the cell type that is responsible for this observed phenotype however the results from our studies indicate that infected/exposed cells could be the dominant producers. Due to the association of Vpr with transcriptional regulation of various cellular factors, we investigated the domains of the TNF-á promoter involved in Vpr-mediated TNF-á regulation. Using the HeLa T4 cell line, TNF-á promoter mediated transactivation was increased by two fold when exposed to HIV-1 Vpr(+) as opposed to HIV-1 Vpr(-) as detected by luciferase reporter assay. A six fold increase was observed in the transactivation of full length and mutant TNF-á promoter in macrophage-derived microglia cell line in the presence of Vpr expression. Results from mapping studies indicate that HIV-1 Vpr can regulate TNF-á production via multiple domains of the TNF-á promoter, however for maximum transactivation, the full-length promoter is required. Statement of Public Health Significance: By determining the details of HIV-1 Vpr and TNF-á interaction and the mechanisms for which they interact could reveal novel targets for the development of HIV-1 therapeutics in the fight against this epidemic.

Neisseria meningitidis is a leading cause of invasive meningococcal disease and humans are the only known host. The administration of meningococcal vaccines has reduced the number of meningococcal cases and carriage rates in humans. Current vaccine strategies target important immunological determinants. Insertion sequence 1301 (IS1301) has been shown to facilitate evasion of the host immune response by disrupting antigen expression. The public health importance of this study is in the design of future vaccines against N. meningitidis and in understanding the emergence of new clones. In the 1990s there was an increase in serogroup C meningococcal disease in Maryland that was associated with antigenic shift at the fetA gene. The isolates were characterized as either an early clone or late clone based on the outer membrane protein sequence profiles. The 2:P1.5,2:F.1-30 sequence profile is classified as an early clone while the 2:P1.5,2:F.3-6 sequence profile is classified as a late clone. Previous studies determined that the late clone contained IS1301, while the early clone did not. The goal of this present study is to characterize the IS1301 insertion sites in the late clone to determine if this genetic element contributed to clonal emergence. Early and late clone isolates were characterized by DNA sequence analysis of the housekeeping gene, fumC. A single nucleotide polymorphism characteristic of the hypervirulent ET-15 clone was identified in the late clone isolates. Southern blot analysis using an IS1301 probe revealed a heterogeneous population with multiple insertion sites, ranging from five to ten insertions, within the serogroup C late clone genomes. Of note was a high molecular weight triplet banding pattern common to the majority of isolates. Several different IS1301 specific, PCR-based strategies were performed in an attempt to clone the IS1301 elements corresponding to the bands of the IS1301 triplet. In addition, whole genome sequence analysis was performed on one of the late clone isolates. Initial whole genome analysis demonstrates IS1301 integration within an opacity-associated protein (Opa), which promotes adherence to host cells. Further investigations are necessary to determine the effect of IS1301 insertion on antigenic variation.

Studies of viral accessory genes have progressed in order to understand pathogenesis and develop effective therapeutics and vaccines. For human immunodeficiency virus type-1 (HIV-1), one such gene receiving special focus is vpr. Vpr has been implicated in dysregulation of host cellular events (including cell cycle arrest and apoptosis), infection of non-dividing cells, and increased viral replication in infected T cells. In simian immunodeficiency virus (SIV), a similar gene is seen in a slightly different form, including vpr as well as a duplicate, vpx. In SIV, these two genes have been shown to split the functions of HIV-1 vpr. In order to use SIV as a model for HIV-1, it must be determined which SIV gene is responsible for mediation of different functional effects. HIV-1 vpr has been shown to downmodulate surface markers on dendritic cells and alter cytokine environments in vivo. Studies have shown that HIV-1 vpr pushes natural killer cells into anergy, rendering them non-functional. Results presented in this study indicate SIV infection also results in these effects, but responsibility for these effects is split between SIV vpr and vpx. The vpx gene appears to play a role in downmodulation of surface receptors on dendritic cells and changes the cytokine environment within the dendritic cells. The vpr gene, however, appears to be responsible for decreased functionality of NK cells, leading to a non-functional anergic state. These findings suggest SIV vpx and vpr cause similar effects compared to HIV-1 vpr and, as expected, the SIV genes split the functions of their HIV-1 homolog. Statement of Public Health Relevance: HIV infection and disease is a growing epidemic and it has become increasingly apparent that in vitro studies are not sufficient to provide the data needed to create an effective vaccine. Because vaccine research cannot be performed on human subjects, the best mode for transition would be a shift to in vivo studies on non-human primates using SIV as a model for HIV-1 infection and disease. Before this can be adopted, it will be necessary to show HIV-1 and SIV have similar effects in vitro on immune cells and can be used interchangeably.

Dendritic cells (DCs) are a heterogeneous population of immune cells that influence a wide variety of immune responses, including immunity to infectious diseases and malignant tumors, and in the generation of tolerance. In their immature state, DCs are highly specialized at capturing and internalizing exogenous antigens. Cell-associated antigens are of special interest because they play a role in both the induction of immunity and tolerance. This study aimed to add to the field of DC biology by further describing how DCs handle cell-associated proteins from both live and apoptotic cells. We hypothesized that the DCs ability to capture, internalize, and process integral membrane proteins would vary based on the target cells viability and that the DCs ability to capture cell-associated protein would vary based on the proteins intracellular localization. To quantitatively and qualitatively characterize uptake, we created a biologically relevant system using the Epstein Barr virus latent membrane protein 2 and the melanoma protein gp100, each fused to the enhanced green fluorescent protein (EGFP) and expressed at the outer plasma membrane of a tumor cell line, along with a cell line expressing EGFP in the cytoplasm. We found (1) DCs captured integral membrane proteins but not cytoplasmic protein from live cells; (2) DCs captured membrane and cytoplasmic proteins from apoptotic cells more efficiently and at a faster rate than from live cells; (3) during direct physical interactions DCs transiently surveyed live cells capturing small quantities of membrane, but stayed in prolonged contact with apoptotic cells while continuously internalizing membrane fragments; (4) DC internalization of membrane protein from live cells was clathrin-dependent while uptake from apoptotic cells was clathrin- and caveolae-dependent; and (5) internalized membrane protein from both live and apoptotic cells was found in early endosomes, late endosomes, and lysosomes. This work has potential broad public health implications as it is important to understand all aspects of DC biology when developing vaccines for both chronic and acute diseases. We hope that by uncovering the intricacies of DC handling of cell-associated proteins we will gain a better understanding of how to possibly manipulate DCs in order to influence the immune response.