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Delta Omega

2009 Poster Competition Winners

Introduction: Geographic disparities in HIV-related mortality highlight areas that may require more resources. Published US HIV-related death rates by state have used general population denominators. We used HIV-infected population denominators to control for prevalence differences among states, better reflecting the quality of care.

Methods: We calculated HIV-related death rates per 1,000 HIV-infected person-years among persons age 15+ for 2001 through 2006 by state, age-adjusted to the 2000 US Standard Population. Numerators were HIV-related deaths (defined by ICD-10 codes B20-B24) by state of residence at death from the National Vital Statistics System, which records the underlying cause of all US deaths. Denominators were person-years based on yearly differences between cumulative HIV infection diagnoses and cumulative deaths among persons with those diagnoses, adjusted for reporting delays, by state of residence at diagnosis. Denominator data came from the national HIV/AIDS Reporting System, which documents all persons diagnosed with HIV infection and their deaths, as reported to state health departments. Negative binomial regression determined rate ratios (RRs) among states, adjusted for age, sex, race/ethnicity, and year. Analysis was limited to 34 states with confidential name-based HIV reporting for at least four years.

Results: Based on 2,437,274 HIV-infected person-years, the overall death rate due to HIV was 22.8/1,000 person-years (95% confidence interval [CI], 22.5-23.2). Rates by state ranged from 10.5 (95% CI 8.8-12.3) to 35.2 (95% CI 31.8-38.7), showing significant heterogeneity across states even after adjusting for race/ethnicity (p<0.0001). Rates increased by age (RR 1.36 per decade, 95% CI 1.35-1.38) and decreased by calendar year (RR 0.91, 95% CI 0.90-0.91). States with the 11 highest rates were all in the southern region.

Conclusions: Our findings suggest that, among US states, the need for more resources to prevent HIV-related mortality is concentrated disproportionately in the South. Policymakers should consider state or regional differences within a country when analyzing death rates.

1st Place- Basic/Lab Science: Jose Ramirez, PhD Candidate

Title: The Toll pathway is a conserved immune defense active against different dengue serotypes and present in multiple Aedes aegypti strains

The dengue virus has become one of the most important arboviral pathogens given the recent increase in incidence in the tropics and subtropics. It is transmitted among humans, primarily by the mosquito Aedes aegypti. Dengue transmission and disease dynamics are exacerbated by the existence of four closely related dengue serotypes. Mosquito vectors are able to limit infection with certain pathogens by mounting a range of immune responses. Although great advances have been made in understanding the mosquito responses to other pathogens such as Plasmodium, little is known about the mosquito antiviral immune responses.

Our previous studies have demonstrated the implication of the Toll Pathway as part of the anti-dengue defense repertoire at 7days post-infection. In this study we have assessed the anti-dengue effectiveness of this pathway at the early stage of infection and against different dengue virus serotypes and in field-derived mosquitoes. We observed that the Toll pathway is active at the early stages of infection (72h post-infection), at the time when new virions are released from the midgut for widespread dissemination. Furthermore, this immune defense is effective against other dengue virus serotypes and present in different strains of field-derived Aedes aegypti mosquitoes. This work adds important information to our understanding of mosquito-dengue virus interactions at the early stages of infection and key factors that modulate dengue transmission. The implication of the mosquito’s innate immune system in the defense against infection was investigated through reverse genetic RNAi methodology and viral plaque assays.

2nd Place- Basic/Lab Science: Shuzhen Sim

Title: Dengue virus inhibits immune signaling in Aedes aegypti cells

The ability of many viruses to manipulate the host antiviral immune response often results in complex host-pathogen interactions. In order to study the interaction of dengue virus (DENV) with the Aedes aegypti immune response, we have characterized the DENV infection-responsive transcriptome of the immune-competent A. aegypti cell line Aag2. As in mosquitoes, DENV infection transcriptionally activated the cell line Toll pathway and a variety of cellular physiological systems. Most notably, however, DENV infection down-regulated the expression levels of numerous immune signaling molecules and antimicrobial peptides (AMPs). Functional assays showed that transcriptional induction of AMPs from the Toll and IMD pathways in response to bacterial challenge is impaired in DENV-infected cells. In addition, Escherichia coli, a Gram-negative bacterial species, grew better when co-cultured with DENV-infected cells than with uninfected cells, suggesting a decreased production of AMPs from the IMD pathway in virus-infected cells. Pre-stimulation of the cell line with Gram-positive bacteria prior to DENV infection had no effect on DENV titers, while pre-stimulation with Gram-negative bacteria resulted in an increase in DENV titers. These results indicate that DENV is capable of actively suppressing immune responses in the cells it infects, a phenomenon that may have important consequences for virus transmission and insect physiology.

3rd Place- Basic/Lab Science: Talibah Metcalf, PhD Candidate

Title: Characteristics of trafficking and long-term maintenance of B-cells in the central nervous system in response to Sindbis virus infection

Alphaviruses are mosquito-borne message-sense RNA viruses that can cause encephalitis in a wide range of vertebrates including humans. Previous studies with Sindbis virus (SINV), the prototype alphavirus, have shown that infectious virus is cleared within 7-9 days, but that viral RNA persists. IFN-gamma plays a role in noncytolytic clearance of virus from neurons and anti-viral antibodies are important for both viral clearance and suppression of viral reactivation. After recovery, SINV-specific antibody secreting cells are present in the central nervous system (CNS) for the life of the animal. However, little is known about the changing functional characteristics of B-cells and important determinants of B-cell trafficking and long-term maintenance in the CNS.  To characterize the B-cell subset populations in the periphery and CNS tissue, as well as to understand the role of chemokines, C57BL/6 mice were infected intracerebrally with SINV and tissue was assessed at various times after infection by flow cytometry and qRT-PCR.

Plasmablasts and memory B-cells (CD19+CD38+CD138-IgM-IgD-) were 70% of the B-cell population for at least 6 months, while plasma cells (CD19+/-CD38-CD138+) were less than 5% of the population. Staining for intracellular and surface IgG at day 60 identified 40% of the B cells as plasmablasts and 50% as memory cells.  A small population of CD19+CD38-CD138- cells, characteristic of germinal center B-cells, was detected and this was confirmed by staining for the germinal center marker GL7. Levels of CXCL9/CXCL10/CCL3/CCL5 (leukocyte trafficking), CCL19/CXCL13 (follicle formation), and BAFF (B-cell survival) mRNAs peaked between days 5-7 after infection, followed by a gradual decreased and return close to baseline by 6 months, except for BAFF that was maintained at a low level. The expression of CXCR3 (receptor for CXCL9/10), CCR5 (receptor for CCL3/5), CCR7 (receptor for CCL19), and BAFF receptor was detected on CNS B-cells.

These results show that plasmablasts and memory B-cells are present for months after infectious virus has been cleared and could play a role in the long-term suppression of SINV replication. The detection of germinal center B-cells along with the early upregulation of chemokines involved in follicle formation suggest the formation of germinal centers in the brain in response to SINV infection. The low levels of BAFF mRNA coincide with the low number of B-cells present after 2 months suggesting the brain provides a microenvironment for differentiation and survival of these cells.

1st Place-Applied Science and Overall Winner: Brandon Brown, PhD Candidate

Title: Peruvian FSWs: understanding HPV and barriers to vaccination

Objectives: To determine the level of awareness of human papillomavirus (HPV), and investigate the potential acceptability of HPV vaccine among female sex workers (FSWs) in Peru.

Methods: Behavioral and vaccine related knowledge data were collected from FSWs aged 18-29 in Lima, Peru. These questionnaires were administered individually by trained interviewers at Patrucco Clinic and surrounding sex venues using convenience sampling.

Results: The average age of the 319 women in our study was 24 years, and the mean age of first sex work is 20.51 years. Less than half (44%) of FSWs had heard of HPV and 47% correctly reported HPV as the cause of cervical cancer. 65% of women reported that condoms prevent HPV infection, while only 7% knew of a vaccine to prevent cervical cancer. Overall, clinic attendees were more knowledgeable about HPV than women approached at sex venues. Nearly 100% of participants would like to receive HPV vaccine, but many listed potential barriers to vaccination. Only eight women said they would not pay for an HPV vaccine, while the average amount women were willing to pay is 27.7 dollars (range 1.8-357 dollars). 99% of women reported being able to complete all 3 vaccine doses.

Conclusions: FSWs are a population at high risk of HPV infection and subsequent cervical cancer, thus they should have access to HPV vaccine. Due to low knowledge of HPV and cervical cancer, FSWs should be targeted for HPV education campaigns. Barriers to vaccination of FSWs can be overcome.

2nd Place-Applied Science: Miranda Jones, MHS Candidate

Title: Secondhand Tobacco Smoke Exposure in Motor Vehicles

Context: Increasing research has given rise to more smoke-free environments in an effort to protect non-smokers from the adverse health outcomes associated with secondhand tobacco smoke. However, many non-smokers still remain vulnerable to secondhand smoke exposure when traveling within a motor vehicle with a smoker. Due to the confined space within a motor vehicle, tobacco smoke concentrations may increase rapidly and intensify the hazards of secondhand smoke exposure.

Objective: To assess exposure to secondhand tobacco smoke in motor vehicles using passive airborne nicotine samplers.

Methods: Seventeen smokers and five non-smokers who commute to and from work in their own vehicle participated. Two passive airborne nicotine samplers were placed in each vehicle for a 24-hour period, one at the front passenger seat headrest and the other in the backseat behind the driver. At the end of the sampling period, airborne nicotine was analyzed by gas chromatography.

Results: Median (IQR) air nicotine concentrations in smokers vehicles were 9.6 g/m3 (5.3-25.5) compared to non-detectable concentrations in non-smokers vehicles. After adjustment for vehicle size, window opening, air conditioning, and sampling time, there was a 1.96-fold increase (95% CI 1.43, 2.67) in air nicotine concentrations per cigarette smoked.

Conclusion: Air nicotine concentrations in motor vehicles were much higher than air nicotine concentrations generally measured in public or private indoor places, and even higher than concentrations measured in restaurants and bars. These high levels of exposure to SHS support the need for education measures and legislation that regulate smoking in motor vehicles when passengers, especially children, are present.

3rd Place- Applied Science: Prabu Selvam, MHS Candidate

Title: Determining Potential Infectiousness using Cough Plates in Tuberculosis Endemic Settings in Peru

Background: This study aims to evaluate a novel cough aerosol capture technique that utilizes a selective solid culture medium and a direct patient cough on to a Petri dish containing a selective growth medium. Using a cough sample to isolate Mycobacterium tuberculosis may give a direct measure of infectivity that can guide control measures in endemic settings.

Methods: Suspected tuberculosis patients were asked to cough on a cough plate for direct culture. Each aerosol sample was accompanied by a sputum sample for comparison with MODS sputum liquid culture and acid-fast sputum smear microscopy. A total of 395 subjects were recruited from a shantytown community near Lima, Peru. Of all subjects, 45.6% (180/395) were MODS sputum culture positive.

Results: The cough plate returned a positive result for 14% (23/164, 95%CI: 9.1 20.3) of all MODS culture positive patients, while 31.0% (9/29, 95%CI: 15.3 50.8) of culture positives were cough plate positive among subjects with sputum smears indicating high bacterial load (BK=3). Specificity was 100% (204/204) when comparing cough plate to MODS. There were five subjects who were positive under MODS and cough plate but negative with sputum smear.

Conclusion: Cough Plate positivity perfectly predicts MODS positivity. Since higher bacterial load increases the chances of expelling M. tuberculosis in cough aerosols, the results of the cough plate confirm a generally understood relationship between high bacterial load and potential for infectiousness. Only a minority of patients were identified by the cough plate technique, and this outcome is in line with prior work regarding the identification of infectious patients

1st Place-Basic/Lab Science: Christopher Harvey, PhD Candidate

Title: Nrf2- A novel target to improve host antibacterial defenses in high risk populations

Rationale: Immunocompromised populations, including COPD patients, are at high risk for bacterial and viral infections that exacerbate preexisting disorders. While ninety percent of COPD is associated with cigarette smoking, smoke exposure itself has been associated with impaired phagocytosis in alveolar macrophages. Several studies have also indicated that chronic intake of corticosteroid drugs, which are commonly administered to treat the symptoms of COPD, leads to immunosuppression and enhances the risk of bacterial pneumonia. Recently, our laboratory has indicated that Nrf2 is the primary transcription factor that regulates innate antibacterial defenses, including antimicrobial peptides and the scavenger receptor MARCO. The present study was designed to investigate if Nrf2 activation improves innate immune antibacterial defenses using a smoke exposed mouse model and human COPD macrophages.

Methods: Wild type (Nrf2+/+) and Nrf2 deficient (Nrf2-/-) mice were exposed to cigarette smoke or filtered air for 1 day. Subsequently, mice were administered sulforaphane, a phytochemical activator of Nrf2, or vehicle via nebulizer followed by intranasal instillation of P. aeruginosa. After infection, BAL was analyzed for inflammation and bacterial colonization. Human macrophages were derived from COPD patients and treated with either vehicle or sulforaphane followed by inoculation with P. aeruginosa. Bacterial clearance was measured 4hr after addition of P. aeruginosa.

Results: Cigarette smoke impairs bacterial clearance in lungs of mice. Nrf2 deficiency further impairs bacterial clearance and killing following smoke exposure. Pharmacological activation of Nrf2 by sulforaphane restores the phagocytic activity of only Nrf2+/+ macrophages and significantly decreases bacterial colonization. Culture media obtained from sulforaphane treated Nrf2+/+ macrophages also showed significant bactericidal activity. Macrophages derived from COPD patients show impaired bacterial killing. However, pharmacological activation of Nrf2 restored bactericidal activity (3-fold increase in bacterial clearance). Conversely, P. aeruginosa continued to multiply in the media of vehicle treated human macrophages.

Conclusion: Modulation of innate immune activity through consumption of a phytochemical activator of Nrf2 may be a viable prophylactic/therapeutic means of preventing severe bacterial infections in immunocompromised individuals.

2nd Place-Basic/Lab Science: Talia Chalew, PhD Candidate

Title: Development of Assay to Assess Environmental Impacts of Engineered Nanoparticles on Chesapeake Bay Oysters

Over 500 consumer products are currently on the market that include and utilize engineered nanoparticles. Every day usage of these products is associated with the release of nanoparticles to the aquatic environment. As sessile filter feeding organisms, oysters are ecologically relevant indicator species for water contamination and economically important to the Chesapeake Bay region. However, little is known about the effects of engineered nanoparticles on these aquatic organisms, as methods for assessing the toxicological effects of engineered nanoparticle exposure are under development. Native Chesapeake Bay oysters have been devastated by Dermo (Perkinsus marinus) disease, which infect oysters through phagocytosis. In our study, titanium dioxide (TiO2) was utilized as a test particle due to high consumer usage and expected high abundance in the environment. Our experiments were focused on in vitro studies exploring the effects of nanoparticles on oyster hemocytes, which are the first line of oyster's immune responses against pathogens, including P. marinus. Our data show that waterborne engineered nanoparticles alter the dynamic of hemocyte phagocytosis, qhich might contribute to the decrease in Chesapeake Bay-native oyster populations. This research project impacts the health of the Chesapeake Bay oyster populations and public health, as oysters harvested from the Bay are consumed raw.

3rd Place-Basic/Lab Science: Allison Brown, PhD Candidate

Title: In utero exposure to maternal schistosomiasis modulates both acute and memory cellular and humoral immune responses of their offspring

Observational studies in humans indicate that in utero exposure to maternal helminth infection modulates immune responses to vaccines in their children, although the immunological mechanisms have not been identified. Helminth antigens cross the placenta and transplacental transfer of cytokines, antibodies or maternal lymphocytes may play a role in immune modulation. To investigate the mechanisms by which maternal helminth infection modulates immune responses to vaccines in their offspring, we developed a murine model of in utero exposure to maternal schistosomiasis. Balb/c dams were exposed to ~20 live Schistosoma mansoni cercariae and mated with Balb/c males 6 weeks after infection. Offspring of infected and uninfected dams received an alphavirus replicon expressing measles virus H protein (VCR-H) or a PBS control vaccine at 4 weeks of age, and were sacrificed at 2 or 6 weeks after vaccination. Using ELISPOT assays to measure splenocyte responses to bystander and vaccine-specific antigens, a dose response effect was observed between maternal egg burden and IL-4 and IFN- cytokine production at 2 and 6 weeks post-vaccination. Acute responses at 2 weeks post-vaccination were directly correlated with maternal egg burden. In contrast, memory responses at 6 weeks were inversely correlated with maternal egg burden, suggesting that in utero exposure results in short-term activation but long-term suppression of T cell responses after vaccination. CD4+ T cells were the primary producers of IL-4 by intracellular cytokine staining, and CD8+ T cells were the primary producers of IFN- . Increased egg burden and longer duration of infection in dams were associated with CD4+ T cell populations with regulatory T cell profiles (expressing CD25, Foxp3 or both) in their offspring, a potential mechanism of long-term immune suppression following in utero exposure to maternal schistosomiais. Among VCR-H vaccinated offspring, the IgG2a to IgG1 ratio increased with maternal egg exposure, consistent with enhanced Th1 skewing, but total vaccine-specific IgG titers decreased with increasing maternal egg exposure. These observations demonstrate that in utero exposure to maternal infection modulates both the acute and memory cellular and humoral immune responses of their offspring, and may decrease vaccine efficacy in offspring