2008 Poster Competition Winners
Project Title: Silencing of an immune pathway negative regulator renders mosquitoes resistant to the human malaria parasite
Malaria is one of the mostly costly infectious diseases in the world both in terms of human life and economic loss. In areas of developed nations, malaria eradication has been made possible by control of the Anopheles mosquito that transmits Plasmodium, the malaria parasite. We and others hypothesize that manipulating the molecular immune response of the mosquito to render the insect unable to support infection would offer a novel way to control malaria. Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd pathways, which control the nuclear translocation of two NF-kappaB-like transcription factors, Rel1 and Rel2, respectively. Activation of Rel1 and Rel2 by these pathways is controlled by the negative regulators AgCactus and AgCaspar. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito_s ability to manage infection with the rodent malaria parasite (Meister et al. 2005, Frolet et al. 2006). Using RNA interference to deplete the negative regulators, we found that Rel2 activation resulting from AgCaspar depletion rendered the mosquitoes resistant to the human malaria parasite Plasmodium falciparum at the pre-mature oocyst stage. Mosquitoes devoid of the Toll pathway regulator Cactus were, on average, 66% more resistant than control mosquitoes. High-density microarray-based, genome-wide expression analyses identified a plethora of genes that were regulated by the activation of the two Rel factors via AgCactu and AgCaspar depletion. The A. gambiae Toll pathway displayed a significantly more diverse role in mosquito biology than did the Imd pathway, which was more immunity-specific. Gene silencing of AgCactus induced 471 and repressed 117 genes, while AgCaspar silencing induced 61 and repressed 55 genes. Depletion of AgCactus and AgCaspar affected the expression of 31 and 17 immune genes, respectively, including anti-Plasmodium factors. The fitness cost of AgCaspar depletion, as measured by mosquito longevity, blood-feeding propensity and oviposition, was insignificant. These findings suggest that this broad-spectrum immune activation in A. gambiae offers an interesting approach to developing novel malaria control strategies
Project Title: Effects of Long Term Lead Exposure on Pre- and Post-Synaptic Protein Markers
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]
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
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.
Project Title: In utero exposure to maternal Schistosoma mansoni infection in mice modulates immune responses to vaccines in their offspring
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- 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- 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- 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- 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- 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- 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.
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
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.
Project Title: The Lasagna Plot: A saucy new alternative to the Spaghetti Plot
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
Project Title: The 1918 Influenza Pandemic Experience in Japan: Age and Geographic Mortality Patterns
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.