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2008 POSTER COMPETITION WINNER ABSTRACTS

Overall winner  1^st Place Basic/Lab Science 

Project Title: Silencing of an immune pathway negative regulator renders mosquitoes resistant to the human malaria parasite

2^nd Place Basic/Lab Science  April Neal

Project Title: Effects of Long Term Lead Exposure on Pre- and Post-Synaptic Protein Markers

Project Summary:

Lead (Pb2+) exposure can result in cognitive deficits in humans and in animal models.  The developing neuron is particularly sensitive to Pb2+, although the exact cause of Pb2+-mediated cognitive deficits is unknown. A large body of evidence indicates that the N-methyl-d-aspartate receptor (NMDAR) may mediate Pb2+ effects on synaptic plasticity and cognition. Other evidence suggests changes in pre-synaptic elements by Pb2+. Therefore, synaptic glutamatergic transmission may play a critical role in Pb2+-induced neurotoxicity. In this study we investigated the composition of synapses in developing neurons at 7 days in vitro (DIV7) using western blotting and immunocytochemistry.  DIV7 hippocampal neurons were exposed to 0, 0.01, 0.1 and 1.0 uM Pb2+ for 5 days, at which point cells were harvested for protein analysis. Using western blots, we found that synaptophysin protein levels decreased in response to Pb2+, with significant changes occurring at 0.1 uM and 1.0 uM Pb2+. In contrast, the protein levels of MAP2 and NR2B remained the same. Analysis showed that the amount of NR1 and NR2A co-localized with synaptophysin decreased significantly at 0.1 and 1.0 mM Pb2+ while the amount of NR2B co-localized with synaptophysin did not change. Additionally, the area of PSD-95 puncta increased significantly at 0.01 uM, while the overall number of puncta per mm did not change significantly at any dose. Taken together, these data indicate that Pb2+ causes changes at both pre-synaptic active zones and post-synaptic spines. We have shown a specific decrease in the pre-synaptic protein synaptophysin which may represent a direct effect of Pb2+ on protein levels and not a change in pre-synaptic terminals since MAP2 protein levels and PSD-95 immunopostive spine density were unaffected by Pb2+.   However, PSD-95 puncta area significantly increased at 10 nM Pb2+ exposure, possibly indicating an increase either in the total spine area or an increase in the levels of splitting synapses at very low levels of Pb2+ exposure. Reduced co-localization of synaptophysin with the NMDAR subunits NR1 and NR2A but not NR2B suggests that glutamatergic synapses in dendritic spines expressing NR2A-containing NMDAR are significantly decreased by Pb2+ exposure. Since the age-dependent switch of NR2B-containing synapses to NR2A-containing synapses is an important milestone of developing neurons, our results suggest that Pb2+ may interfere with processes affecting maturation of signaling pathways by NR2A-containing NMDAR. [Grant #ES06189 to TRG]

3^rd Place Basic/Lab Science  David Sintasath

Project Title: Identification of a Novel Simian T-lymphotropic virus (STLV) Lineage in Two Monkey Species from Cameroon: High STLV Diversity at the   Hunter-Primate Interface

Project Summary:

The recent discovery of novel HTLV-3 and HTLV-4 viruses in persons hunting and butchering  nonhuman primates (NHPs) in Cameroon demonstrates that the genetic diversity and natural history of primate T-lymphotropic viruses (PTLVs) are far from being understood. To better understand the origin, genetic relatedness, and epidemiology of these novel HTLVs we investigated the prevalence and diversity of STLV in NHPs hunted in the forests of Cameroon. DNA was extracted from dried blood spots from 170 NHPs representing 12 different simian and prosimian species. PCR analysis using generic tax primers detected an overall STLV prevalence of 7% (12/170) in four simian species. Analysis of conserved tax sequences revealed a high PTLV diversity, including STLV-1 (n=7) and STLV-3 (n=3). Samples from a Cercopithecus mona and C. nictitans each had highly divergent STLV-3-related sequences. Phylogenetic analysis of larger tax sequences from these twomonkeys inferred a novel lineage outside the diversity of PTLV-3 suggesting a long, independent evolution of these viruses. STLV-3 LTR sequences from three different monkey species were all distinct including those from Lophocebus albigena, an NHP not known to be infected with STLV. STLV-1LTR sequences obtained from two monkey species (n=5) all clustered in the HTLV-1 D and F subgroups supporting a primate origin for these clades. The discovery of a novel STLV lineage and several unique STLV-3s significantly expands the range of PTLV diversity. The propensity of STLV to transmit to humans highlights the need for more active surveillance at the primate-human interface for cross-species transmission of deltaretroviruses.

3^rd Place Basic/Lab Science  Allison Brown

Project Title: In utero exposure to maternal Schistosoma mansoni infection in mice modulates immune responses to vaccines in their offspring

Project Summary:

Observational studies in humans indicate that in utero exposure to maternal helminth infection modulates the immune responses to vaccines in children; however, the mechanisms responsible are not known. Helminth antigens have been shown to cross the placenta but transfer of cytokines, antibodies and maternal lymphocytes also may play a role. To investigate 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 live Schistosoma mansoni cercariae and mated at 6 weeks post-infection. Offspring of infected and uninfected dams received either an alphavirus replicon expressing measles virus (MV) H protein (VCR-H), adsorbed tetanus toxoid (TT) or a PBS control vaccine at 4 weeks of age and were sacrificed 2 weeks later. Splenocyte production of IFN-³ and IL-4 was measured by ELISPOT in response to soluble adult worm antigen (SWAP), a T cell mitogen (ConA) and vaccine-specific antigens (MV H peptides and TT). VCR-H vaccinated offspring of infected dams displayed increased IFN-³ and IL-4 production upon stimulation with SWAP, mitogen and measles virus H peptides compared with offspring of uninfected dams (P = 0.03). Furthermore, there was a direct relationship between maternal egg burden and offspring cytokine production. VCR-H vaccinated offspring of dams with high hepatic S. mansoni egg counts produced significantly more IFN-³ and IL-4 than offspring born to dams with a lower egg burden (P = 0.05). Evidence of a dose response effect also was observed among the TT vaccinated offspring, with high maternal egg burden resulting in higher IFN-³ and IL-4 production upon SWAP, mitogen and TT stimulation (P = 0.06).  A separate study aimed to determine whether host immune responses to repeated schistosomal infection differed from those after single exposure.  Balb/c females were either singularly exposed to 30 live cercariae or exposed three times 3 weeks apart to 10 live cercariae.  Mice repeatedly exposed to schistosomes experienced significantly increased production of IFN-³ in response to both ConA and SWAP (0.02 and 0.03, respectively) and increased IL-4 in response to ConA (0.05).  Thus, both maternal schistosomiasis and repeated schistosome exposure result in increased IFN-³ and IL-4 production in response to schistosomal and non-schistosomal antigens.  These studies demonstrate that prior immune sensitization to schistosome antigens enhances immune activation and may contribute to a variety of unforeseen consequences, including increased susceptibility to HIV transmission.

1^st Place Applied Science  Stephen Sozio

Project Title: Statins Do Not Increase Stroke Risk in Patients Initiating Dialysis: The Choices for Healthy Outcomes in Caring for End Stage Renal Disease (CHOICE) Study

Project Summary:

INTRODUCTION: Kidney disease has reached epidemic proportions with a predicted prevalence in 2030 of more than two million patients   with end-stage renal disease.  The 5-year survival of these patients is less than 50%, with stroke being the third leading cause of death.  Contrary to the general population, there was an unexpected higher rate of stroke among those treated with HMG-CoA reductase inhibitors (statins) than placebo among prevalent diabetic hemodialysis patients in a prior randomized trial. It is not known if this was a type I error or a true association.

METHODS: We investigated the association of statin use at baseline and cerebrovascular events (CVE) among a national, incident dialysis cohort of 1,041 patients enrolled from 10/95 to 7/98 in the CHOICE study. Incident CVE were defined as both non-fatal (hospitalized stroke,

carotid endarterectomy) and fatal (stroke death) events after dialysis initiation. Participants were censored for transplant, non-stroke death, or 12/31/04. With Cox proportional hazards regression analysis, we assessed the independent risk of CVE associated with statin use after adjustment in separate models for a propensity to use statins score and for age, race, sex, history of CVE, smoking status, diabetes, hypertension, albumin, and cholesterol.

RESULTS: Mean age was 58 years with 54% male, 67% White, 74% on hemodialysis, and 16% taking statins. A total of 164 patients experienced CVE; incidence rate was 5.2/100 person-years (95% confidence interval (CI)[3.3-7.8]) for those taking statins and 4.9/100 person-years (95%CI[4.1-5.7]) for non-statin users. Statin use was not associated with increased hazard of CVE in univariate (HR 1.07, 95%CI[0.72-1.60]), propensity-adjusted (HR 0.96, 95%CI[0.62-1.48]), or traditional multivariate analyses (HR 0.91, 95%CI[0.57-1.46]).

CONCLUSIONS: We conclude that statin use among incident dialysis patients neither increases nor decreases the risk of CVE. Further studies are needed to understand the pathophysiology and prevention of stroke in patients with ESRD.

2^nd Place Applied Science  Bruce Swihart

Project Title: The Lasagna Plot: A saucy new alternative to the Spaghetti Plot

Project Summary:  In public health, data are often collected at the subject level longitudinally.  The gold standard for graphically displaying and exploring such data is the spaghetti plot, which involves plotting a subject's outcome measures (vertical axis) versus time (horizontal axis) and connecting the dots.  Although useful for fewer subjects, trends and patterns tend to become obscured for large numbers of subjects.  This is because trajectories can overlap in a spaghetti plot, for the magnitude of the outcome measure is the vertical dimension. Enter the lasagna plot, which uses color or shading to depict the magnitude of the outcome measurement and fixes the vertical dimension per subject. Thus each subject forms a "layer" in the lasagna plot.  Two advantages of the lasagna plot over the spaghetti plot are 1) group level and individual level information are preserved regardless of the number of subjects or time points (no "overplotting"); 2) dynamic sorting of data to better ascertain group level behavior over time is possible

3^rd Place Applied Science Stephanie Richard

Project Title: The 1918 Influenza Pandemic Experience in Japan: Age and Geographic Mortality Patterns

Project Summary:

A better understanding of past influenza pandemics is essential to prepare for future pandemics. Based on excess mortality models applied to historical vital statistics, we quantify the age and geographic mortality patterns of the 1918 pandemic in Japan, and compare with those in the US and the UK. During the 1918-20 pandemic period, all three countries experienced unusually high mortality in young adults. However, in contrast to the US and UK, Japanese elderly were not entirely spared and excess mortality in Japan was balanced between two seasons, 1918-19 and 1919-20. Surprisingly, Okinawa and prefectures in the densely populated region around Tokyo, partially escaped the first season, only to be more severely hit the following season. These geographical differences were not related to differences in population demographics or density. Our study and others suggest that influenza pandemic mortality patterns can vary substantially between countries
and regions, for reasons yet unclear.

2007 POSTER COMPETITION WINNER ABSTRACTS

Overall winner  1^st Place Applied Research Sheng Luo

Title: Analysis of Smoking Cessation Patterns Using a Stochastic Mixed Effects Model with a Latent Cured State

Summary:

Background: Smoking is the leading preventable cause of death in the U.S. In the U.S. alone, 44.5 million adults, or 20.9% of the adult population were smokers in 2004; 440,000 annual premature deaths are attributable to smoking. Smoking is a major cause of a large number of diseases, e.g. cancers of the lung, larynx, and pharynx, etc. The slow reduction of adult smoking prevalence is partly due to high rates of relapse following quit attempts among smokers. A major problem when studying addiction behavior is that participants typically make several quit attempts before they successfully quit. Thus, for efficient development, targeting and evaluation of interventions, it is necessary to distinguish transient quitting (temporarily smoking-free but relapse later) from permanent quitting (lifelong smoking-free) and identify the risk factors associated with permanent quitting.

Objective: To identify and quantify baseline factors associated with success of permanent quitting and describe the full stochastic nature of the smoking addiction pattern using the Alpha-Tocopherol, Beta-Carotene (ATBC) Lung Cancer Prevention study data set.

Method: The ATBC study is a large (29,133 participants) longitudinal cohort study and it contains unique information about smoking and health status of each participant during every 4-month interval throughout the follow-up period. We used the ATBC data set to model smoking cessation patterns using the proposed discrete-time stochastic mixed-effect model with three states: smoking, transient quitting and permanent quitting (absorbent state). Random participant specific transition probabilities among these states were used to account for participant-to-participant heterogeneity. Another important innovation in our research was to design computationally practical methods for dealing with the size of the data set and complexity of the models. This was achieved by integrating over the Beta distribution of random effects and obtaining the marginal likelihood as an explicit function of the model parameters. We thus avoided complicated model fitting techniques, e.g. numerical integration or Markov Chain Monte Carlo (MCMC). This provided large computational advantages over the more popular model using logistic multivariate normal random effects.

Results: Baseline age was positively associated with probability of making quit attempts (p<0.001). However, years of smoking, cigarette and alcohol consumption had inverse association with probability of making quit attempt (p<0.001). If the quit attempt was made, more cigarette consumption per day was associated with lower probability of relapsing (p=0.003), while more alcohol consumption per day was associated with higher probability of relapsing (p=0.004). Moreover, 5.1 years in age increased the odds of permanent quitting by 10.4% (95% CI: -0.40%~22.4%; p=0.06). Individuals with psychological symptoms were significantly less likely to be successful permanent quitters (p=0.03).

Conclusion: An important, previously unknown, but often debated, finding was that baseline risk factors have different effects on different transition probabilities. We provided mathematical quantification of the participant-to-participant variation in transition probabilities. Our modeling strategy enriched the statistical arsenal of cure modeling and provided new and valuable scientific insight into the smoking addiction behavior.

2^nd Place Applied Research  Honghong Zhu

Title: A Validation Study on Smoking Questionnaire in the Shanghai Women's Health Study

Summary:

In order to determine the potential exposure levels of secondhand smoke (SHS) at a population level and validate the self-reported smoking questionnaire in the Shanghai Women’s Health Study, SWHS, we designed a cross-sectional validation study of urinary cotinine at baseline. A total of 571 urine samples were randomly selected by strata of smoking levels of neither active smoking nor SHS exposure (never SHS), former smokers, ever only exposure to SHS (only SHS), and current smokers. The geometric means (95% confidence intervals) for urinary cotinine were 2.8 (2.2-3.5), 3.4 (2.6-4.4), 5.2 (4.7-5.8) and 933.2 (551.4-1579.4) in ng/ml urine among women who reported never SHS, former smokers, only SHS, and current smokers, respectively. All women, including never SHS, had cotinine detectable in their urine samples. In the linear regression analysis, all the self-reported smoking measures at baseline were significantly correlated with urinary cotinine after adjusting for creatinine and other covariates. Urinary cotinine significantly increased by 0.370 ng/ml urine per one additional cigarette smoked per day by women and by 0.035 ng/ml urine per one additional cigarette smoked per day by their husbands. Spearman correlation coefficient, r_s, with creatinine-adjusted urinary cotinine was 0.94 (p< 10^-5) for cigarettes smoked per day by current-smoking women at baseline with a great variation of cotinine levels across individuals and 0.51 (p< 10^-5) for cigarettes smoked per day by their husbands. The weighted Kappa coefficient was 0.95 (p< 10^-5), indicating almost perfect agreement between self-reported smoking status and the urinary cotinine-indicated smoking status. Sensitivity for self-reported current smokers was 88% and specificity was 100%, when using the urinary cotinine-indicated smoking status as a gold standard. In conclusion, the smoking questionnaire of both active smoking and SHS in the SWHS is valid. However, the validity decreases slightly in quantitative measures of smoking due to the large variation of cotinine across individuals. The universal presence of cotinine in the urine samples suggests SHS is a severe public health issue in China.

3^rd Place Applied Research  Valerie Harder

Title:  Applying alternative propensity score techniques to test whether marijuana problems cause depression

Summary:

Increased marijuana use among adolescents and reports linking marijuana use to other psychiatric disorders has prompted research on the relationship between adolescent marijuana use and later depression. Using propensity score (PS) adjustment techniques we test the potential causal link between adolescent marijuana problems (abuse or dependence) and young adult depression (past-year major depression diagnosis). A cohort of 2,311 first-graders was interviewed over a 15-year period with 75% retention. We used both parametric and non-parametric PS estimation techniques and five alternative applications of the PS, including matching, subclassification, and weighting. The PS adjusted the final logistic regression of depression on marijuana problems by balancing all observed confounding covariates. Unadjusted, 24% of individuals with marijuana problems reported depression whereas only 13% of individuals without marijuana problems reported depression. The PS-adjusted analyses all resulted in increased odds of depression among those with marijuana problems [Odds Ratio range 1.12 (p>0.05) – 1.88 (p<0.001)]. The variation in the magnitude of the effect and statistical significance does not directly suggest one of the PS techniques to be superior. We use statistical simulations and comparisons of the baseline covariate balance to determine if one PS method is most effective. This work suggests both methodological and substantive conclusions: non-parametric estimation of the PS may be an improvement over parametric estimation for these data, and adolescents with marijuana problems are at increased odds for young adult major depression, but the causal link is modest.

1^st Place Lab Research  Lindsey Garver

Title: The immunoglobulin superfamily in Anopheles gambiae: potential immune molecules in the malaria vector

Summary:

Although falciparum malaria is one of the most devastating infectious diseases in the world, proper control of the parasite and its vector, the Anopheles gambiae mosquito, remain elusive.  Because the mosquito vector is required for transmission and because vector control can be cost-effective and is unaffected by compliance of individuals, we are focused on finding a way to control malaria though molecular manipulation of the mosquito.  The mosquito’s immune system is active against the malaria parasite yet determination of the molecules and mechanisms involved is still in its infancy.  Because immunoglobulin superfamily members are among the most important and best characterized molecules of the immune system and this family has not been well-studied in the context of insect immunity, we initiated a screen of mosquito proteins containing immunoglobulin domains to identify candidate immune molecules.  First, a bioinformatics-based analysis was used to define the IgSF in Anopheles gambiae and select and test candidates for implication in immune functions. This screen identified 145 genes that have at least one immunoglobulin domain.  From here, a gene expression-based analysis was performed in order to determine the infection responsiveness of individual exons representing each of the 145 IgSF genes.  Expression profiles of adult female mosquitoes and mosquito cell lines challenged with Plasmodium parasites, Gram negative bacteria, Gram positive bacteria and fungi show that 237 exons from 84 genes are significantly regulated in response to at least one challenge, many responding to multiple challenges.  We think that some of this regulation is attributable to immune defense. Data from both the computational and microarray screens were used to choose six genes as candidates for further functional analyses to establish possible immune relevance.  Candidates were named Infection Responsive with Immunoglobulin Domain 1-6 (IRID1-6), and chosen for testing based on domain composition suggestive of immune relevance and significant regulation of at least two exons upon challenge.  Each of the six IRID genes were silenced in adult female mosquitoes using RNAi and subsequently challenged with Gram positive bacteria, Gram negative bacteria or Plasmodium.  Survival rates and oocyst loads from these assays suggest several of these candidates are involved in defense against these pathogens.  One candidate, IRID3, seems to have a drastic effect on a mosquito’s ability to survive after bacterial challenge and another, IRID6, has a modulatory effect on Plasmodium falciparum infection in the mosquito.  From here, we can begin our characterization of IRID3 and IRID6 which may not only represent a novel class of innate immune molecules.  IRID6 is of particular interest as a potential target for malaria control at the vector level.

2^nd Place Lab Research  Judith Easterbrook

Title: Regulatory T Cells Mediate Seoul Virus Persistence and Pathology in Norway Rats

Summary:

Hantaviruses cause one of two diseases in humans, hantavirus pulmonary syndrome (HPS) or hemorrhagic fever with renal syndrome (HFRS).  Human pathology is hypothesized to be caused by sustained, elevated levels of proinflammatory cytokines.  In rodents, hantaviruses are transmitted horizontally and cause persistent infection in the absence of disease.  The mechanisms mediating hantavirus persistence in rodents are unknown.  Regulatory T cells contribute to persistence of several pathogens and act locally at sites of pathogen replication by suppressing proinflammatory responses.

Following inoculation with Seoul virus, regulatory T cells (CD4+CD25+FoxP3+) are elevated in the lungs during the persistent phase of infection (i.e. at Day 30 p.i.).  Additionally, expression of foxp3, a transcription factor that is unique to regulatory T cells, and TGF-?, a cytokine produced by regulatory T cells, is elevated in the lungs of male Norway rats 30 days post-inoculation (p.i.), but remains unchanged in the spleen during infection.  To test the hypothesis that regulatory T cells affect persistence of Seoul virus, male Norway rats were administered anti-CD25 mAb, to deplete regulatory T cells, or saline and were inoculated with Seoul virus or vehicle.  Following depletion of regulatory T cells, Seoul virus RNA copies in the lungs and proportion of rats shedding virus in saliva are reduced during infection.  Additionally, depletion of regulatory T cells significantly reduces pathology, including development of hemorrhage and edema, in the lungs during Seoul virus infection. Taken together, these data suggest that regulatory T cell responses are elevated during infection and may contribute to Seoul virus persistence and pathology in male Norway rats.

3^rd Place Lab Research  JInchun Yan

Title: Increased androgenic sensitivity during aging contributes to the prostate proliferative potential

Summary:

Benign prostate hyperplasia (BPH) is a prevalent condition in aging men. Spontaneous epithelial cell hyperplasia in the dorsal and lateral lobes of old Brown Norway rats is analogous to BPH in humans. Therefore, the Brown Norway (B-N) rat represents an excellent model to investigate the mechanisms that regulate cell proliferation and prostate hyperplasia. The majority of cells in the adult prostate are quiescent in the G0 phase and in order for hyperplasia to occur, cells must escape from cell cycle arrest and enter S phase through G1/S restriction. In the androgen withdrawn-replenished Brown Norway rats, proliferation rate quantified by BrdU labeling index of all three lobes in response to androgens is time and dose dependent, and peak at day 3. Under the similar serum testosterone levels monitored by radioimmunoassay, aging dorsal and lateral prostates have higher proliferation rate than the young animals, while there is no age dependent difference in proliferation rate of ventral lobes. Immunoblots demonstrated the abundance of cyclinD1, cdk6, cyclin E, cdk2 was significantly upregulated, and P27Kip1 abundance was significantly downregulated, affected by androgens. Interestingly, aging dorsal and lateral prostates have higher cyclin D1 and cyclin E abundance than the young ones, no matter the intact control animals, or the 3days androgen replenished young and aging animals under the similar serum testosterone levels. Androgens can also affect Rb phosphorylation, shown by immunoprecipitation and immunoblots.  Immunostaining demonstrated changing patterns of cdk4 subcelluar localization and probability of cyclinD1 nuclei localization corresponded to the time frame of cell proliferation affected by androgens. These data suggested increased androgenic sensitivity and its effect on the key cell cycle regulators (especially those related to G1/S transitions) may be one of the multiple mechanisms leading to BPH, which develops during aging while the serum testosterone levels decrease.

2006 POSTER COMPETITION WINNER ABSTRACTS

OVERALL Xiaoyan Song   Association of Serum Ferritin and Mortality in Incident Hemodialysis Patients

Summary:
Background:  Each year, about 300,000 patients in U.S. receive hemodialysis (HD) to treat end stage renal disease. Given that almost every HD patient suffers from anemia and thus requires recombinant human erythropoietin (EPO) treatment, intravenous (IV) iron therapy has become one of the most important adjunctive treatments in anemic HD patients. Although IV iron is effective in correcting anemia, excessive iron in human body increases patient’s risk of developing infections and cardiovascular diseases. Epidemiological studies addressing iron and mortality in HD patients provided controversial evidences, partially because these studies only followed patients for less than 2 years which were insufficient to assess the causal effect of iron on mortality.  
Objective:  To examine the relationship between serum ferritin (SF), a marker of body iron storage, and all-cause mortality in incident HD patients.
Method:   A prospective longitudinal cohort study.  Eligible patients were incident HD patients who were enrolled in the Choices for Healthy Outcomes in Caring for ESRD (CHOICE) study, an ongoing prospective study that had followed 1,041 incident dialysis patients recruited from 81 clinics nationwide since 1995, and had baseline SF values that were measured within 3 months of dialysis onset.  Baseline patient characteristics (age, gender, race, smoking, employment and marital status) and co-morbidity were collected through a self-administered questionnaire.  Clinical laboratory values including SF were obtained from an electronic database. Death was prospectively identified from medical records, national Death Certificate Index database, and U.S. Renal Disease System. Statistical analyses included descriptive analysis to examine associations between SF (grouped into <100 mg/dL, 100~500 mg/dL, 500~800 mg/dL, and >800 mg/dL for the ease of clinical interpretation), death and covariates. Unadjusted and adjusted Cox regression model was used to determine the independent effect of SF on mortality. Laboratory values including inflammatory marker C-reactive protein (CRP) were adjusted as time-dependent covariates. To further illustrate a potential causal effect of iron on mortality, we analyzed a group of patients who survived for at least 6 months from dialysis onset and excluded SF values that were measured within 6 months before the events (death or censoring) occurred. 
Results:  Of 767 HD patients enrolled in the CHOICE study, 687 (89.6%) had baseline SF available and 614 (80.1%) survived for at least 6 months after dialysis onset.  Patients in the four SF groups were similar in baseline age, race, smoking, marital and employment status, and education levels.  Baseline SF was positively associated with CRP (p=0.04), but negatively associated with albumin (p<0.001). No statistical association was found between baseline SF and mortality in the analysis using either whole- or sub- group data. Cox regression model using whole-cohort data found no association between follow-up SF and mortality. Comparing to the reference group 100~500 mg/dL, Hazard Ratio (HR) was 0.74 (95% Confidence Interval (CI): 0.45~1.23), 1.02 (95% CI: 0.75~1.39), and 1.16 (95%CI: 0.93~1.45) for SF<100, 500~800, and >800 mg/dL groups, respectively. Cox regression using sub-cohort data and excluding SF measured in the last 6 months before events occurred revealed that SF <100 mg/dL was associated with elevated mortality (HR: 1.26, 95%CI: 1.01~1.57).  The association remained statistically significant after adjusting for CRP and albumin.
Conclusion:  We found that low, rather than high, SF was associated with increased mortality.  Our results suggested a reverse causal effect when using SF as a marker of body iron to assess its relationship with mortality in HD patients.  Further study was warranted to confirm this finding.   

Laboratory Research
1st Place  Caren Chancey Combination of antibodies to CD18 and ICAM-1 reduces transmission of cell-associated HIV-1

Summary:
Background:   A better understanding of the interactions between HIV-1 positive cells and the cervical epithelium is essential for development of microbicides to prevent sexual transmission of HIV-1 to women.  Our lab has previously demonstrated that cell-associated transmission of HIV-1 by monocytes is the most efficient route of transmission across a model cervical epithelial monolayer and in a hu-PBL-SCID model of vaginal HIV-1 transmission.  In addition, antibody to ICAM-1 has been shown to block transmission of cell-associated HIV-1 in both of these models.  Use of lactobacilli to produce antibody-like single-chain Fvs (scFv) offers many advantages over traditional microbicides, but the available concentration of antibody is limited by the amount of scFv that can be secreted in vivo.  In order to determine whether a significant reduction in the amount of antibody required to block HIV-1 transmission could be achieved, we evaluated the blocking capabilities of antibodies to CD18, the ?-chain of the ICAM-1 ligands LFA-1 and Mac-1, both separately and in combination with anti-ICAM-1.

Methods:  Peripheral blood mononuclear cells (PBMC) infected with HIV-1 BaL were added to the apical side of confluent monolayers of HT-3 cervical epithelial cells grown on permeable transwell supports.  After 24 hours, PBMC in the basal compartment were counted, and HIV transmission was measured by p24 ELISA on the basal supernatant.   Data were analyzed using one-way ANOVA with Bonferroni correction by the STATA statistical package.

Results:  Two different anti-CD18 antibodies, H52 and 7E4, were able to reduce transmission of cell-associated HIV-1 by 90±1% and 67±6%, respectively.  Anti-CD18 and anti-ICAM-1 used in 50:50 combination at a total concentration of 5 ug/ml reduced migration of PBMC from HIV-1 infected cultures significantly better (p<0.05) than 20 ug/ml total of either antibody alone.
 
Conclusions:  These findings indicate that a combination of antibodies to the adhesion receptor pair ICAM-1 and CD18 offers better protection against cell-associated HIV-1 transmission than either antibody alone, and that this combination can protect at a concentration which is feasible for use in a lactobacillus-based microbicide delivery system.

2nd Place John Pisciotta Hemozoin Formation and Detection in P. falciparum

Summary:
The intraerythrocytic malaria parasite forms intracellular heme crystals, called hemozoin, as a means of detoxifying free heme released through hemoglobin catabolism. This process is targeted by the quinoline antimalarials. The intracellular mechanism of heme crystallization initiation has not been fully substantiated; however, histidine rich proteins, polar lipid and/or neutral lipids have all been purported to play a role. The current study employs the lipid fixative malachite green to observe neutral lipid nanospheres enveloping nascent hemozoin within P. falciparum digestive vacuoles (DV). Mass spectrometry lipidomics identifies significant saturated mono- and diacylglycerol neutral lipids and a paucity of residual polar lipids associated with purified hemozoin. Under conditions consistent with the DV, the neutral lipid mixture rapidly initiates heme crystallization with reversible, pH dependent quinoline inhibition. Formation of a heme-drug complex is required for drug entry into neutral lipid nanospheres and inhibition of heme crystallization. This work identifies neutral lipid nanospheres as both the microenvironment of heme crystallization and the nonaqueous, nonpolar site of quinoline inhibition.

3rd Place Judy Easterbrook Potential Mechanisms Mediating Persistence of Seoul Virus in Male Norway Rats

Summary:
Hantaviruses cause severe disease in humans and pathology is hypothesized to be mediated by sustained, elevated levels of proinflammatory cytokines.  Rodents are the natural hosts for hantaviruses; unlike humans, rodents display no pathology during infection, yet remain persistently infected.  This study examined whether Seoul virus suppresses rodent host immune responses that would allow persistent infection.  Male rats were inoculated with Seoul virus and proinflammatory (interleukin (IL)-1?, IL-6, and TNF) and anti-inflammatory (IL-10, IL-1 receptor antagonist (ra), and corticosterone) responses were assessed.  Copies of Seoul virus were significantly elevated in the lung 15 and 30 days post inoculation (p.i.).  Expression of splenic IL-1? was cyclical, with transcription elevated 3 and 60 days p.i., but reduced to baseline 15-30 days p.i.  Interleukin-1? protein production was significantly below baseline 15-60 days p.i.  Similar, but less dramatic, trends were observed for transcription of IL-6 and TNF.  Expression of IL-10 and IL-1ra remained at baseline 3-30 days p.i., but was significantly elevated 60 days p.i. Synthesis of IL-10, however, was significantly suppressed 3-60 days p.i.  Corticosterone concentrations remained low 3-30 days p.i., but were elevated at 60 days p.i.  These data indicate that Seoul virus infection suppresses proinflammatory cytokine production.  The reduction of proinflammatory immune responses does not correlate with elevation of anti-inflammatory factors produced by the host, including IL-10, IL-1ra, and corticosterone, suggesting that virus-induced immunosuppression may be involved.

Applied Research
1st Place Xiaoyan Song Association of Serum Ferritin and Mortality in Incident Hemodialysis Patients

2nd Place Joachim Bleys Vitamin / Mineral Supplementation And The Progression Of Atherosclerosis. A Meta-Analysis Of Randomized Controlled Trials

Summary:
Joachim Bleys, MD, MPH; Edgar R. Miller III, MD, PhD; Roberto Pastor-Barriuso, PhD; Lawrence J. Appel, MD, MPH; Eliseo Guallar, MD, DrPH
From the Departments of Epidemiology and Medicine, and the Welch Center for Prevention, Epidemiology and Clinical Research, the Johns Hopkins Medical Institutions, Baltimore, MD (J.B., L.J.A., and E.G.), the National Institute on Aging / National Institutes of Health, Baltimore, MD (E.R.M.), and the Division of Biostatistics, National Center for Epidemiology, Instituto de Salud Carlos III, Madrid, Spain (RPB).

ABSTRACT
Background.  Laboratory and observational studies suggested that antioxidant and B-vitamin supplementation may prevent atherosclerosis.  Although trials have not shown a benefit of these supplements on clinical cardiovascular events, it is unknown whether they affect the progression of atherosclerosis as measured by imaging techniques.
Methods and results.  We performed a meta-analysis of randomized controlled trials assessing the effect of vitamin / mineral supplementation on atherosclerosis progression.  We searched MEDLINE, EMBASE, and CENTRAL for relevant studies.  No language restrictions were applied.  We separately analyzed trials using antioxidant compounds (vitamins E, C, beta-carotene or selenium) from trials using B-vitamins (folate, vitamin B6 or vitamin B12).  The progression of atherosclerosis was evaluated by B-mode ultrasound, intravascular ultrasound, or angiography.  Effect sizes were calculated for the difference in slope of atherosclerosis progression between participants assigned to supplements and those assigned to the control group.  In trials not involving percutaneous transluminal coronary angioplasty (PTCA), the pooled effect size was -0.06 (95% CI, -0.20 to 0.09; 7 trials) for antioxidants, and -0.93 (95% CI, -2.11 to 0.26; 4 trials) for B-vitamins.  In post-PTCA trials, the pooled relative risk for restenosis was 0.82 (95% CI, 0.54 to1.26; 3 trials) for antioxidants and 0.84 (95% CI, 0.34 to 2.07; 2 trials) for B-vitamins.
Conclusions.  Our meta-analysis showed no evidence for a protective effect of antioxidant vitamin / mineral and B-vitamin supplementation on the progression of atherosclerosis, thus providing a mechanistic explanation for the lack of effect of these supplements on clinical cardiovascular events.

Key words: Meta-analysis, Antioxidants, Folate, Atherosclerosis, Prevention

3rd Place  Jeffrey Willis Perceived Neonatal Morbidities in Rural Uttar Pradesh, India: Effects of a Community Mobilization and Behavior-Change Communication Intervention

Summary:
Objective: To test the effects of a community mobilization and behavior-change communication (BCC) intervention on perceived neonatal illnesses in rural Uttar Pradesh, India. 

Background: Poor neonatal health is a barrier towards improving child survival. One mechanism of improving newborn health in low-resource, high mortality areas such as rural Uttar Pradesh, India, is mobilizing households to engage in community-based essential newborn health practices.  Johns Hopkins University and King George Medical University in Lucknow, India, have implemented a community mobilization and BCC intervention that delivers a package of preventive essential newborn care messages to families and communities in rural Uttar Pradesh.  It is hypothesized that this intervention will reduce the incidence of newborn illnesses. 

Methods: The study was nested in a cluster-randomized trial of the impact of a package of essential newborn care in Shivgarh, a rural block of Uttar Pradesh.  The study prospectively enrolled 802 mothers between February and August, 2005, and administered a structured questionnaire to recently delivered women in Hindi at the end of the neonatal period.  Multiple logistic regression analysis was used to evaluate the statistical significance of the interventions on perceived neonatal illnesses.   

Results: Newborns in the intervention area had significantly lower odds of perceived diarrhea [Adjusted Odds Ratio (OR) 0.66, 95% C.I. 0.46-0.95] and perceived skin-related complications [Adjusted OR 0.62, 95% C.I. 0.42-0.89)] compared to newborns in the comparison area.  On average, the day in which the initial newborn illness was recognized was earlier among households in the comparison arm (10.2 ± 8.0 days after birth) than households in the intervention arm (12.3 ± 8.7 days) (p=0.01). 

Conclusions: An intervention program focusing on essential preventive newborn health practices significantly reduced the perceived odds of diarrhea and skin-related complications in newborns during the neonatal period.  Onset of neonatal illnesses may also have been delayed. Community mobilization and BCC interventions are important tools to reduce neonatal morbidities, although future research should consider using health professionals to more accurately document the changes in neonatal morbidity rates.

SCIENTIFIC POSTERS SUBMITTED TO THE 2006 DELTA OMEGA COMPETITION