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Delta Omega

2013 Poster Competition Winners

Applied Science:

1st Place - Andrea L. Wirtz, MHS

Title: Mixed methods research to understand burden of HIV infection and transmission among men who have sex with men (MSM) in Malawi: understanding risks among a criminalized population to inform HIV prevention interventions

Background: Recent years have witnessed an increased knowledge of the high burden of HIV among men who have sex with men (MSM) across the globe and the increased transmission efficiency of HIV through receptive anal intercourse -18 times higher than that of vaginal sex. The term 'MSM' is used in recognition of the sexual behavior, rather than identity: many men who have sex with men do not identify as homosexual or bisexual. Malawi has a generalized HIV epidemic with approximately 8% of adult men living with HIV, yet a pioneer study of HIV risks among men who have sex with men (MSM) documented HIV prevalence as high as 21%. Yet, Malawi is one of several countries in which homosexuality is criminalized and highly stigmatized, creating a situation in which MSM do not have equal access to HIV prevention programs. In collaboration with a community based organization, The Center for Development of People (CEDEP) that provides HIV prevention services to MSM, and the Malawi College of Medicine we designed a mixed methods research study to understand the baseline prevalence, risks, and followed with a combination HIV prevention program (CHPI) to be tested among a cohort of MSM for one year.

Methods: From May to July 2011, we conducted qualitative research which included in-depth interviews among purposely sampled MSM and health service providers working in Blantyre, Malawi. Using peer and key informant recruitment, a total of eight in-depth interviews were conducted with MSM participants representing a range of ages, social and behavioral characteristics, sexual orientations, and marital patterns (with a woman). A total of five participants were recruited from the district hospital, local public health clinics, STI research clinics, as well as from a HIV prevention service organization. Quantitative baseline research included enrollment of a total of 339 MSM who were recruited via respondent-driven-sampling (RDS) from August 2011-March 2012 in Blantyre, Malawi. Study activities included a structured survey instrument and biological assessment of HIV and syphilis and those testing preliminarily positive were referred for confirmatory testing and treatment as indicated. From this baseline population, 100 MSM with negative HIV diagnosis were recruited and enrolled in the 12 month CHPI study cohort. Qualitative research served to provide inputs to the design and methods for implementation of the CHPI. Participants of the CHPI are followed prospectively, with quantitative and qualitative follow-up assessments.

Results: Preliminary analysis of the baseline data provided demographic characteristics: mean age of 25.1yrs. (range:18-49) and 46.6% unemployed. Only slightly more than half were gay-identified (61.9%). A high proportion reported some kind of relationship with women: 10.3% currently married to a woman; 17% divorced/separated; 3% cohabitating; and 32% reporting a female sexual partner in the last year. Sexual risks were high: participants reported a mean of 3 male sex partners in the last 12 months; 49 - 54% reported consistent condom use with regular or casual male partners, respectively, and fewer (33%) with female partners. The prevalence of HIV prevalence was high: 15.8% (unadjusted) and, among those with HIV infection, 91% were unaware of their HIV status and 39.9% had never been tested for HIV. Nearly 60% reported that vaginal sex was the highest risk form of sex, indicating low knowledge of transmission risk related to anal intercourse. Qualitative research explained this low awareness of transmission risks among MSM: most HIV prevention information is targeted to heterosexual, married couples and MSM are not informed or knowledgeable of risk of transmission and acquisition related to anal intercourse. As a result, some believe that transmission happens only between wife and husband. Participants also described fears related to disclosure during health visits, including concerns about unintentional disclosure to others by health professionals and fear that homosexual behavior would be reported to authorities. Many avoided HIV prevention and STI treatment as a result of these fears. Providers likewise voiced concern that they would be seen condoning illegal behavior. With these findings the CHPI was designed to include several components: 1) HIV/STI counseling and testing and referrals to trained health facilities, 2) HIV and health education and prevention materials (condoms and lubricants) provided through peer educators, and 3) health sector trainings to inform providers of HIV risks and provide general information on competent healthcare for MSM. As of January 2013, the cohort has maintained 99% retention and 7 HIV seroconversions reported. The study will conclude in April 2013.

Conclusions: This study reinforces that MSM are an important population in Malawi's HIV epidemic and deserve targeted HIV prevention services. As of May, 2012, the changing government in Malawi publicly announced intention to decriminalize homosexuality and Parliament has requested report briefs from this study to consider decriminalization of homosexuality as a public health priority. The data here highlight the need to take advantage of this opportunity to provide services to MSM, given the limited HIV-related knowledge and high-risk practices and the use of epidemiologic data for national advocacy. Preliminary results were presented to UNAIDS, USAID, and CDC in January 2013 who have agreed to support expansion of the baseline study to six other sites in Malawi and will include the results and recommendations during the redrafting of the National HIV Strategy (February 2013). The intervention may be generalizable to other settings where homosexuality is criminalized or stigmatized.

2nd Place - Julie K. Bower, PhD, MPH

Title: The Association of Lifestyle Factors With Circulating Levels of the Soluble Receptor for Advanced Glycation End Products (sRAGE): The Atherosclerosis Risk in Communities (ARIC) Study

Background: Accumulation of advanced glycation end products (AGEs) is hypothesized to contribute to oxidative stress, vascular inflammation, and atherosclerosis. The soluble form of the receptor for AGEs (sRAGE) may act as a "sponge" for AGEs and is lower in persons with vascular damage (e.g., individuals with CVD). Influenced by endogenous factors, low sRAGE may also be determined by modifiable factors including tobacco use and lifestyle behaviors. Previous literature also suggests that intake of foods with high AGE content might be important contributors to AGE accumulation in the body.

Objectives: This study aimed to evaluate the association of major lifestyle factors with serum sRAGE levels in the Atherosclerosis Risk in Communities (ARIC) Study.

Methods: We conducted a cross-sectional analysis of 1,214 participants aged 47-68 years with measured sRAGE at ARIC Visit 2 (1990-1992); participants were selected based on estimated GFR values (>60 mL/min/1.73m2) and individuals with missing data for the variables of interest were excluded. Multivariable logistic regression was used to examine associations with low levels of sRAGE (lowest quartile: 119.4-723.9 pg/mL). Lifestyle factors included adiposity, smoking, alcohol consumption, high AGE-content food intake, and physical activity.

Results: Smoking, alcohol use, and body mass index were strongly associated with low sRAGE. Consumption of high AGE-content foods and physical activity were weakly associated with low sRAGE and became non-significant after multivariable adjustment.

Conclusions: Modifiable behaviors, such as smoking and alcohol use, were associated with sRAGE levels. Additionally, higher body mass index may contribute to low sRAGE levels. Further studies that examine the effects of changes in these factors would provide information about whether sRAGE levels are modified by changes in lifestyle factors over time.

3rd Place - Kirsten S. Vannice, PhD

Title: Active surveillance of adverse events following immunization with meningococcal A conjugate vaccine MenAfriVac™ in Mali

Background: New vaccines are being developed exclusively for use in developing countries, making post-licensure safety surveillance infrastructure in these settings a priority. The new meningococcal A conjugate vaccine MenAfriVac™ was introduced in Mali over three 10-day campaigns in 2010 and 2011. We conducted a post-marketing active vaccine safety surveillance study in Mali using novel methods that have been only used in industrialized countries previously.

Objective: The objective of the study was to assess the safety of the meningococcal A conjugate vaccine MenAfriVac™ in Mali.

Methods: We used Mali's existing national health care system to test a new model for active vaccine safety surveillance in a developing country. Thirty-seven community health centers and three reference health centers contained within three health districts participated for a total study population under surveillance of approximately 400,000. Data from patient visits were extracted from handwritten external consultations, general/pediatric hospitalizations, and obstetric/gynecologic hospitalizations registers. MenAfriVac status was based on self-reporting. To determine if there were increased rates of clinic visits for adverse health outcomes following immunization, we conducted both population and individual analyses and calculated rates of events using the conditional exact test, the vaccine cohort risk interval method, and self-controlled case series method for pre-specified events.

Results: Many pre-specified adverse events of interest were infrequently reported. The data suggest a possible increased rate of reporting for fever with eight of nine analyses showing statistically significantly elevated incidence rate ratios (IRR) greater than 1.0. For convulsions two of nine statistically significant IRRs greater than 1 were found for associations with receipt of MenAfriVac™. These associations are hard to disassociate from the malaria burden, as the vaccination campaign occurred during the decline of the malaria season. Some of the associations may have been due to declining rates of malaria in control time windows used for self-controlled analyses. Rates of other pre-specified adverse events did not suggest an association with MenAfriVac™ across campaigns and methods.

Conclusions: Results from our study are consistent with other studies indicating a strong safety profile for MenAfriVac™. Active pharmacovigilance is needed in developing countries as new products are developed exclusively for these markets. Our study provides useful data for MenAfriVac™ safety and serves as a model for future vaccine safety monitoring in low-income countries.

Basic/Lab Science:

1st Place - Meredith C. Foster

Title: APOL1 and Risk of Incident and Progressive Chronic Kidney Disease in African Americans: the Atherosclerosis Risk in Communities (ARIC) Study

Background: Although African Americans with compared to those without common coding variants in the APOL1 gene are 5-29 times more likely to have focal segmental glomerulosclerosis, HIV-associated nephropathy, and end-stage renal disease in case-control studies, the impact of these variants on chronic kidney disease in a prospective, community-based sample of middle-aged African Americans has never been examined.

Objective: To determine whether the APOL1 G1 and G2 risk alleles are associated with the development of chronic kidney disease and progression to end-stage renal disease events among African Americans in the Atherosclerosis Risk in Communities Study (ARIC) Study.

Methods: We analyzed data from 3,067 African Americans free of chronic kidney disease at ARIC baseline to evaluate the association of APOL1 G1 and G2 risk alleles (2 vs. 0 or 1 allele) with incident chronic kidney disease (eGFR<60mL/min/1.73m2), end-stage renal disease, and the subsequent progression to end-stage renal disease amongst the group with chronic kidney disease using time-to-event analyses. Cox models were adjusted for age, sex, study center, and percent European ancestry based on 1350 ancestry informative markers.

Results: Overall, 13.2% of participants (n=404) carried 2 APOL1 risk alleles, 6.2% (n=190) developed chronic kidney disease, and 3.7% (n=114) had an ESRD event. Carrying 2 risk alleles was associated with a 1.49-fold increased risk of chronic kidney disease (95% confidence interval [CI] 1.02-2.17, p=0.04) and a 1.88-fold increased risk of end-stage renal disease (95% CI 1.20-2.93, p=0.005) when compared to 0 or 1 risk allele; associations persisted with adjustment for percent European ancestry. Among participants who developed chronic kidney disease, those with 2 risk alleles were more likely to progress to ESRD than their counterparts with 0 or 1 risk allele (hazard ratio 2.22; 95% confidence interval 1.01-4.84).

Conclusion: APOL1 risk variants are novel risk factors for the development of chronic kidney disease and strong risk factors for progression from chronic kidney disease to end-stage renal disease in the general African American population.

2nd Place - Frances R. Belmonte, PhD

Title: Over-expression of epidermal growth factor receptor 2 (ErbB2) in the heart alters redox-sensitive molecules that modulate reactive oxygen species (ROS)

Background: One signaling pathway that is shared by the heart and cancer is epidermal growth factor receptor 2 (ErbB2), a receptor tyrosine kinase and member of the epidermal growth factor receptor (EGFR) family. The link between ErbB2 and heart function is based on the important discovery that when anti-ErbB2 (Trastuzumab/Herceptin) and doxorubicin are jointly administered to treat ErbB2-overexpressing breast cancer, a synergistic cardiac toxicity ensues in a subgroup of patients. Previous studies have shown that doxorubicin induces ErbB2 in rat hearts, thereby predisposing the heart to off-target toxicity to anti-ErbB2. To further understand the role of cardiac ErbB2 in vivo, we developed a transgenic murine model that over expresses ErbB2 in the cardiomyocyte. The resultant phenotypes from the cardiac ErbB2 over-expression are cardiac hypertrophy that does not progress to heart failure and activation of pro-survival pathways.

The rationale for a possible link between ErbB2 signaling and oxidative stress is from a study where neuregulin (ligand for the ErbB family of receptor tyrosine kinases) upregulated the mRNA transcripts and induced expression of oxidative stress defense proteins in cardiomyocytes. Additional evidence for a potential role of ErbB2 signaling on antioxidant mechanisms is from a study in which neuregulin upregulated glutathione reductase mRNA and reduced oxidative stress in adult rat ventricular cardiomyocytes. The same study found that the PI3K/Akt pathway was involved in the neuregulin-induced reduction of oxidative stress through ErbB2 signaling. Thus, these studies highlight the importance of ErbB2 signaling in promoting pro-survival pathways in the heart that will affect the antioxidant capacity of the cardiomyocyte. However, whether ErbB2 affects in vivo systems is unknown.

Objective: In our present study, we will use the ErbB2 transgenic murine model to investigate the underlying mechanism for ErbB2 over-expression on redox signaling and ROS formation.

Methods: Total RNA was extracted from the hearts of the wild type and ErbB2 transgenic mice (12-weeks-old, 3 mice per genotype) for whole genome expression analysis using an Illumina BeadArray. The oxidative stress genes were analyzed by cluster analysis to determine the patterns of expression between ErbB2 transgenic mice and wild type mice. Total heart lystates or mitochondria from the hearts of the transgenic and wild type mice were probed for the presence of Gpx1, Gpx3, Gpx4, c-Abl, p-c-Abl (Y245), Arg, prohibitin, lamin b, alpha actin, and Akt proteins. The Amplex Red assay was used to measure the production of hydrogen peroxide (H2O2) produced from isolated heart mitochondria. Electron Paramagnetic Resonance (EPR) was used to measure oxidative stress in total lysates from wild type and ErbB2 transgenic left ventricular tissue. A cyclic hydroxylamine (CMH) spin probe that detects superoxide was added to the total lysates. Left ventricular tissue was homogenized in 5% metaphosphoric acid with or without M2VP, a scavenger of reduced GSH. Supernatant with the M2VP was used to measure GSSG while the supernatant without the M2VP was used to measure GSH. Glutathione peroxidase activity was measured with a NADPH-linked enzymatic assay that monitors the decrease in NADPH absorbance at 340 nm. Glutathione reductase activity was measured by the rate of NADPH oxidation at an absorbance of 340 nm. Thioredoxin reductase 2 activity was measured by the reduction of 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) by TrxR2 and NADPH at an absorbance of 412 nm.

Results: Microarray data that compare the transgenic mice to wild type mice suggest that ErbB2 alters various oxidative stress genes. We found that ErbB2 over-expression in the heart upregulates protein levels of glutathione peroxidases 1, 3, and 4. Glutathione peroxidase activity is higher in the transgenic mice compared to the wild type group. We measured ROS production and found that the transgenic hearts produce less ROS than the wild type hearts. However, the GSH/GSSG ratio is reduced in the transgenic hearts. Since NADPH oxidase has been implicated in cardiac hypertrophy, our findings suggest that the reduced GSH/GSSG may be due to increased Gpx activity and possibly NADPH oxidase activity. Our data suggest that the heightened antioxidant capacity in the ErbB2 transgenic mouse hearts may be play an important role in preventing oxidative stress and heart failure from occurring in this murine model of hypertrophy.

Separate studies have shown that glutathione peroxidase 1, the primary glutathione peroxidase isoform, is regulated by the c-Abl and Arg non-receptor tyrosine kinases. Additionally, it has been shown that c-Abl and Arg share similar activation pathways. We found that c-Abl and Arg and the phosphorylated forms of c-Abl are upregulated in the hearts of the transgenic animals.

Conclusions: Our data suggest that ErbB2 over-expression upregulates gluthatione peroxidases through c-Abl and Arg to modulate ROS production by increasing glutathione peroxidase activity. The increased Gpx activity results in elevated GSSG and a reduction in the GSH/GSSG ratio. These findings are similar to those found in studies that use a cardiac hypertrophy model. We have shown through two different methods (Amplex Red and EPR) that the ErbB2 transgenic mice have less mitochondrial- and cellular-derived oxidative stress, respectively. Our studies may be the first to show that ErbB2 regulates glutathione peroxidase through the c-Abl and Arg non-receptor tyrosine kinases in the heart. The heightened antioxidant capacity in the transgenic mouse hearts may be play an important role in preventing oxidative stress and heart failure from occurring in this murine model of hypertrophy.