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2013-14 Award Recipients

Javed Butler, MD
Medicine
Epigenetic Control of Inflammatory Pathways in Heart Failure

HF is the leading cause of morbidity and mortality in the world, with a prevalence that is projected to greatly increase over time. The economic burden of outpatient and inpatient HF care is increasing in-step with the increasing prevalence, heightening the need for effective HF therapies. Examining molecular targets implicated in the pathological disease processes of worsening HF are vital to our understanding of HF. Inflammatory pathways and inflammasome modulation have been proposed as novel therapeutic targets in HF, but new treatments or interventions targeting a modifiable inflammatory pathway are urgently needed in HF. Epigenetic regulation of the inflammatory response has been identified as an important therapeutic approach, but trials in persons with HF have not been reported to date. This study examines a method of epigenetic regulation of the inflammasome, a key mediator of sterile inflammation in HF. Stored samples from The Atlanta Myocardial Consortium will be used for this study. The objectives of this study are to determine the relationships between ASC methylation and inflammatory cytokines, IL and IL-18, in persons with heart failure, examine the differences in methylation of ASC among persons with heart failure with reduced ejection fraction and persons with heart failure with preserved ejection fraction, examine the relationships between patient reported outcomes and the proposed inflammatory pathway, and determine the relationships between aerobic capacity and ASC methylation and inflammatory cytokines in persons with heart failure.

Ann Chahroudi, MD, PhD
Pediatrics
Analysis of SIV Reservoirs in Infant Rhesus Macaques

Worldwide, over 30 million people are infected with HIV, including 2.5 million children. While combination antiretroviral therapy (cART) suppresses viral replication and greatly reduces the mortaility and morbility of HIV infection, viral rebound quickly ensues if drugs are stopped. As such, a sterilizing or functional cure remains a key priority in HIV/AIDS research. A critical obstacle to curing HIV infection is the persistent reservoir of latently infected cells that are not eliminated by cART. However, we lack detailed knowledge of the cellular and anatomic reservoirs that are responsible for HIV persistence in the settling of suppressive cART, particularly in pediatric patients. The objective of this proposal is to interrogate the latent viral reservoir using a novel model of SIV infection and cART treatment n infnt rhesus macaques (RM). Based on our preliminary data identifyinf SIV target cells in lymphoid and gastrointestinal tissues of infant RM, we hypnotice that latently SIV-infected CD4+ memory T cells in multiple anatomic locations represent a source of persistent viral infection in RM infants. We aim to (1) demonstrate sustained suppression of viremia in SIV-infected, cART-treated infant RM; and (2) identify cellular and anatomic SIV reservoirs in these infant RM using state-of-the-art virologic and immunologic techniques. With a better understanding of persistent viral reservoirs, novel reservoir-directed therapeutic strategies can be designed and tested in nonhuman primates, then scaled up to clinical trials in HIV-infected patients.

Kristen Criado, PhD
Pediatrics
Assesment of Feeding Problems in Autism: Initial development of a Standardized measure based on FDA Guidance

Children with autisum s[ectrum disoders (ASD) have a fivefold increase in the odds of a feeding problem compared with peers (1). With ASD affecting an estimated 1.14% of children (6), high prevalence of feeding concerns in this population poses a significant public health concern. Estimates of food selectivity (i.e., only eating a narrow variety) in ASD reach as high as 95% (5), suggesting feeding problems may occur near epidemic levels. Evidence also suggests poor dietary diversity in ASD is associated with detrimental nutritional and medical consequences, including vitamin and mineral deficiences, poor bone growth, and constipation (7,8,9). Chronic feeding difficulties also represent a significant source of stress for parents (10), and many lead to greater social isolation for families (1). Despite such negative outcomes, the etiology, topography, and remediation of feeding problems in ASD remain poorly understood. A major barrier to progess in this area is the absence of reliable and valid assessment (13). Thus, there is a pressing need for a gold-standard instrument to support clinical and research efforts. Using the methods described in the FDA Guidance (14), we propose to develop a parent-report insturment to asses feeding problems in ASD. The purpose of this proposal is to develop a parent-reported outcome (PRO) instrument to: 1) characterize feeding problems in ASD, 2) serve as an end point for clinical trials, and 3) increae early detection in order to support best standards of care.

Methods

The project will result in the initial draft of a PRO for feeding problems in children with ASD to support both clinical and research activities with the potential for rapid and broad application in the ASD community. The findings have immediate and important implications for the work of practitioners serving children and familites with autism, who, in the absence of such reliable and valid assessment and screening tools, may struggle to identify children at risk for significant feeding problems. Without appropriate assessment and evidence-based interventions, parents may fill the void with alternative treatments that may be ill-conceived or even harmful to children and families

Charles A. Downs
School of Nursing
Cigarette Smoke Activates RAGE to Affect Lung Fluid Balance

Cigarette smoking causes chronic obstructive pulmonary disease (COPD). Increased expression of the receptor for advanced gycation end-products (RAGE) and increased activity of the epithelial sodium channel (ENaC) occurs in the distal lung with cigarette smoke exposure. The central hypothesis is that cigarette smoke activates RAGE-dependent, Nadph oxidase (Nox)-generated ROS signaling to regulate ENaC activity and ultimately lung fluid balance. The first aim is to investigate the signaling pathway through which cigarette smoke activates RAGE signaling. Lung tissue and isolated alvelor epithelial cells from RAGE-/- and WT mice will be used for single channel patch clamp analysis to determine changes in ENaC activity before and after exposure to cigarette smoke extract (CSE). Pharmacological inhibitors of Nox subunits will be used to clearly articulate the mechanism through which RAGE regulates ENaC within the setting of CSE. FACS-sorted alveolar epithelial type 1 and type 2 cells (from RAGE-/- and WT mice) will be subjected to CSE and ROS measured. The second aim is to demonstrate that inhibition of RAGE signaling ameliorates the effects of cigarette smoke on inappropriate lung fluid clearance. RAGE-/- and WT mice will receive a tracheal instillation of CSE and then changes in lung fluid clearance measured over time. Nox inhibitors will be given concurrently to demonstrate the role of Nox-generated ROS. The overall hypothesis of this study is that cigarette smoke activates ENaC in the pathogensis od COPD, thus providing a potential therapeutic target for the treatment and prevention of COPD.

Erica Duncan
Psychiatry and Behavioral Sciences
Genetics of acoustic startle latency in schizophrenia

Many studies have shown a large heritability (up to 85%) of schizophrenia (SCZ), but finding vulnerability genes for this disease has proven very difficult. The findings from genome-wide association studies (GWAS) have been difficult to replicate without tens of thousands of subjects and do not fully explain the inheritance pattern of this disease (Mulle 2012; Hamshere et al. 2013). The current thinking in the field is that SCZ is not inherited in a Mendelian manner, but rather is associated with rare mutations, common mutations, and copy number variants of varying effect sizes (Stefansson et al. 2008; International Schizophrenia Consortium 2008; Purcell et al. 2009; Ripke et al. 2011; Mulle 2012; Hamshere et al. 2013). A strategy to unravel the bewildering genetic heterogeneity of SCZ is to use an endophenotype or intermediate phenotype approach (a measurable trait associated with a disease that can be discovered by a biologic test). SCZ subjects are sub-grouped based on endophenotypes in order to discover underlying genetics and etiopathophysiology specific to that subgroup that is thought likely to be less heterogeneous that all patients with SCZ. The hope in our field is that this approach will lead to specific and more effective treatments for SCZ patients subtyped in this manner. The acoustic startle response (ASR) is a reflex contraction of the muscles in response to a sudden acoustic stimulus. It is seen in all mammals and is thought to prepare the animal for movement in response to threatening stimuli. Startle latency is the time required between the startling stimulus and neural transmission through a 3-synapse subcortical circuit to the effector muscles of the response and is an index of neural processing speed. Latency is prolonged in SCZ compared to control subjects (CON) and is very highly heritable (up to 90%) in a recently published study from our lab. Furthermore, latency is not affected by medication status in SCZ patients. Thus startle latency is a putative endophenotype for SCZ. Prolonged latency identifies those young subjects at clinical risk for SCZ even before they develop SCZ symptoms. Other less heritable endophenotypes have begun to yield positive findings in genetic association studies (imaging, neurocognitive, prepulse inhibition of startle; Greenwood et al. 2007; Greenwood et al. 2011; Greenwood et al. 2012). Therefore we conducted a preliminary candidate gene analysis of latency in a predominantly non-SCZ population and found a modest association between latency and several single nucleotide polymorphisms (SNPs). We now propose a candidate gene association study of our collected population of SCZ and CON subjects in whom we have already established that latency is highly heritable. Objective 1. Using 370 well-characterized SCZ and CON subjects already collected at the Atlanta VA, we will evaluate the association between startle latency and SNPs on genes with association to SCZ or SCZ endophenotypes in humans or to latency in the animal literature. Objective 2. We will leverage publically available post mortem expression data from subcortical brain regions (pons, region that mediates startle) to investigate the relationship between the startle-associated candidate SNPs and gene expression.

Michael J Haber, PhD
Rollins School of Public Health
Estimation of the number of influenza cases prevented by vaccination

Estimation of the number and fraction of influenza cases prevented by vaccination is challenging because it involves the comparison of the number of cases observed in a given population to the expected number of cases in the absence of vaccination, which is usually unknown. We have recently completed writing and testing a detailed agent-based stochastic simulation program generating an outbreak of influenza in a structured population. In this project we plan to program a complex computational algorithm to facilitate estimation of the input parameters to the simulation program, using observed data from recent influenza seasons. We will then run stochastic simulations of influenza outbreaks, using the estimated input parameters, while the entire population remained unvaccinated. This will allow us estimate and compare the incidence of influenza illness with and without vaccination.

Estimation of the number of influenza cases prevented by vaccination is very important as it will enable health officials promote vaccination programs. It will also lead to better estimates of the costs associated with influenza outbreaks and facilitate the comparison of different vaccination strategies and selection of the most effective strategy under given conditions. The proposed research will also help understand and quantify the important role of indirect (herd immunity) effects of vaccination programs.

The software to be developed in this project will be made available to the community of scientists who are interested in influenza-related research. We believe that the outcomes of this project will help us secure additional funding for similar research projects focusing on other vaccine-preventable diseases.

Adam Pomerleau, MD
Emergency Medicine
Describing Emergency Physician Ppioid Prescribing Behaviors and Attitudes: A Multicenter Survey

The United States (US) is in the midst of a prescription drug overdose epidemic. In 2008, poisoning deaths became the leading cause of accidental death, surpassing motor vehicle deaths. Drug overdose account for greater than 90% of annual poisoning deaths and opioid medications specifically are implicated in 40% of these deaths, more than any other class of prescription medication. Emergency medicine physicians are among the top fice specialties in terms of the number of prescriptions for opioid medications dispensed to patients under 40 years old. Little is performed to date reveal significant variability in prescribing decisions among physicians. Therefore, I propose a multicenter survey study of emergency physicians' self-reported behaviors and attitudes around prescribing opioid medications in the emergency department. My proposal intends to utilize a national consortium of emergency departments recently created explicitly for the purpose of investigating issues associated with the administration and prescription of opioid medications and other controlled substances from the emergency department. My preliminary work in this area will guide development of the proposed survey instrument. The results of the proposed study will help identify factors that correlate with differences in prescribing behaviors that occur at the hospital, state, or regional levels. In addition, the results may suggest areas to target interventions that promote safer opioid prescribing practices among emergency physicians. ultimately, I intend to use the proposed study as the next step in my efforts to obtain extramural and ongoing support from sources such as the National Institute on Drug Abuse and the National Center for Injury Prevention and Control to continue to study this new public health crisis.

M. Alice Shillingsburg
Pediatrics
Increasing Vocal Language in Minimally Verbal Children Diagnosed with ASD

Language deficits are one of the most common presenting complaints of parents of children with Autism Spectrum Disorder (ASD; De Giacomo & Fombonne, 1998). It has been estimated that 20-50% of those diagnosed do not develop functional vocal language (Graziano, 2002; Lord, Risi, & Pickles, 2004). Evaluating techniques that foster the development of vocalizatons is an essential line of research in the efforts to provide effective intervention to children diagnosed with ASD. Although it is widely accepted that intensive behavioral intervention leads to significant progress in communication, individuals without existing vocal abilities often present treatment challenges that require specialized attention. Although decades of research support the use of operant reinforcement and behavioral shaping procedures for increasing vocalizations (Harris, 1975), for some children, these procedures are ineffective. One limitation of existing treatment procedures for language deficits is th lack of procedures for producing vocalizations in children considered minimally verbal who do not respond to shaping and differential reinforcement. Thus, a critical gap in the literature includes a technology for producing vocalizations in these individuals. Research investigating an alternative procedure for producing vocalizations in individuals characterized as minimally verbal ASD has emerged but with variable results. Stimulus-stimulus pairing (SSP) is a procedure based on the principles of respondent conditioning that involves intensive and systematic pairing of adult produced vocalizations with preferred items. Though some studies have shown positive results in terms of vocal development, a more systematic analysis of the intervention is needed. A recent review of the literature revealed only 11 studies with 34 total participants. Additionally, these studies all involved distinct differences among the procedures used, as well as a wide variety of participants in terms of age, diagnosis, and vocal language ability limiting the conclusions that many be drawn regarding the efficacy of SSP. Further, previous research studies only reported change in vocalizations during treatment sessions. Therefore, the longterm impact of the intervention following the intervention and outside of teh intervention setting is unknown. Identifying a more specific profil for which the SSP procedure is effective and obtaining vocalization data in the participant's natural environment (e.g., home or school) is warranted. The purpose of the present study is to investigate the feasibility and preliminary efficacy of the SSP procedure with a well characterized sample during treatment sessions and during follow-up observations in the natural environment. Deploying technology, specifically the LENA vocal recorder, will provide the opportunity to gather valuable data (e.g., spontaneous vocal language in the natural environment) in a non-clinical setting, as well as, a data collection technique that does not require a skilled clinician to collect eliminating observer error. As the science on interventions for children with autism continues to progress, it is critical that studies examining methods to produce vocal language in minimally verbal children with ASD be conducted to ensure this particularly vulnerable population does not get overlooked.

Rabindra Tirouvanziam, PhD, Engineer
Pediatrics
T-cell suppression by mature neutrophils: a novel mechanism in chronic human airway disease

Severe chronic airway inflammation as seen in cystic fibrosis (CF) and non-CF bronchiectasis (NCFB) involves the continuous and massive recruitment of polymorphonuclear neutrophils (PMNs) from blood into the airways. In prior analyses of airway samples from CF patients, we identified a viable PMN fraction with marked exocytosis of neutrophil elastase and myeloperoxidase, two enzymes endowed, among other properties, with T-cell suppressive abilities. Following up on this discovery, we show in preliminary data presented here that CF airway PMNs express the bona fide T-cell suppressor molecules programmed death-ligand 1 and arginase 1 in vivo, and that incubation in CF airway fluid in vitro upregulates arginase 1 expression on PMNs and suppresses T-cell function.

In this pilot study, we propose to test the hypothesis that mature airway PMNs drive the disease process in CF and NCFB, in part, by undergoing adaptive steps in the lumen that lead them to exert strong suppressive effects on T-cells. Our goals are to:

Goal 1: Determine T-cell suppressive functions in airway PMNs and the status of cognate T-cells, in vivo. We will perform extensive comparisons of sputum (and matched blood) from CF and NCFB patients. These include cellular analyses by high-content flow cytometry, mRNA profiling by multiplexed qPCR and molecular mediator profiling by Luminex and photometry.

Goal 2: Determine mechanisms inducing T-cell suppressive functions in airway PMNs and how they impact T-cells, in vitro. PMNs will be exposed to airway fluid from CF and NCFB patients by direct or indirect incubation (the latter using a novel air-interface airway / PMN transmigration model that recapitulates the process of pathological PMN conditioning). To assess potential suppressive effects of airway fluid and airway PMNs on T-cells, blood T-cells will be incubated with airway fluid and / or airway-like PMNs (produced in vitro) and analyzed for proliferation potential. In vitro-conditioned PMNs and T-cells will be analyzed analyses by high-content flow cytometry, their mRNAs profiled, and molecular mediators studies as above.

This pilot study will help solidify our discovery of T-cell suppressor function by mature human airway PMNs. This discovery helps explain a long-standing paradox of CF airway disease (mirrored in other pathologies), i.e., the compartmentalization of T cells in the lamina propria and their exclusion from the lumen where pathogens thrive. Indeed, mature airway PMNs may promote T-cell tolerance in the face of chronic infection. We expect to identify novel targets in PMNs or T-cells that will be modulated pharmacologically in a follow-up R01 study, with the goals of overcoming the refractory nature of CF and NCFB airway disease to existing therapies and improving quality of life for patients. Finally, the demonstration of T-cell regulation by airway PMNs as a disease mechanism will open the way for fruitful basic and clinical research well beyond CF and NCFB.

Hongjie Yuan
Pharmacology
Molecular Mechanisms of Human NMDA Receptor GRIN2B Subunit Mutations in Neurodevelopmental Disorders

Neurodevelopmental disorders are associated with disabilities in brain function that affect a child's behavior, memory or ability to learn. Such disabilities carry devastating mental, emotional, and economic consequences for the individuals, their families, as well as society. The molecular basis for a subset of disabilities involve disease-causing mutations in various ion channel families, including NMDA receptors (NMDARs). The cation-selective NMDAR channels formed from assembly of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits mediate a slow, Ca2+-permeable component of excitatory synaptic currents that can trigger changes in synaptic strength, a cellular correlate of learning. NMDARs also play an important role in normal brain development. A large number of mutations (>140) have been reported in just the last three years, leading to the view that these mutations are present in a subset of patients with various neurological disorders. Surprisingly, the incidence of NMDAR mutations found in pediatric patients presenting with neurological problems is 5.7% (202/3549). Mutations in NMDAR GRIN2B subunits have been identified in children with a broad range of neurodevelopmental problems, including attention deficit hyperactivity disorder, autism spectrum disorders, developmental delay, mental retardation, schizophrenia, and intellectual disability, as well as seizures. Unfortunately, virtually no functional analysis of these mutations exists, making it impossible to evaluate effects of mutations in the context of clinical phenotype.

We proposed 3 lines of experimentation addressing the molecular mechanism underlying development disorders suggested to arise from 21 NMDAR GRIN2B subunit mutations. All experiments will utilize our recently developed approach to expressing receptors that contain one mutant and one wild type NMDAR GRIN2B subunit, enabling an accurate assessment of receptor function in heterozygous patients.

Aim 1. How do human NMDAR GRIN2B mutations alter pharmacological properties? We will evaluate all mutations in human NMDAR GRIN2B subunit using automated two-electrode voltage clamp recordings (TEVC) in Xenopus oocytes.

Aim 2. How do human NMDAR GRIN2B subunit mutations alter channel function ? We will analyze the synaptic response time course and single channel properties of a subset of mutations to understand the mechanisms underlying receptor dysfunction in patients.

Aim 3. How do human NMDAR GRIN2B subunit mutations alter receptor trafficking? We will evaluate receptor trafficking of a subset of mutations with loss-of-function using Western blot and fluorescence-based imaging studies.

Linsheng Zhang
Pathology
Genetic Profiling of Myelodysplastic Syndrome with RNA-Seq

Recently revealed gene mutation profile of MDS demonstrate that majority of mutations present in MDS are in genes involving RNA splicing, chromosome remodeling, transcriptional regulation, and epigenetic regulations. The array based RNA expression profile of hematopoietic cells in MDS has been associated with clinical outcome. Demethylation therapy is one of the most effective treatment options for low to intermediate MDS patients who are transfusion dependent; however, study on the baseline methylation status of a small group of gense did not seem to predict treatment response to demethylation therapies. We postulate that all the pathobiologic process of MDS will eventually reflected in the change of transcript in the hematopoietic cells. The aim of this study is to optimize a clinically applicable RNA-Seq approach to investigate the RNA expression profile, as well as coding region mutations that may predict treatment response to epigenetic therapies. We propose to perform RNA-Seq on 12 bone marrow samples from low and intermediate risk MDS patients who need demethylation treatment in addition to supportive care. The analysis will be focused on identifyingg differential gene expression profile, fusion transcripts and coding region mutations associated with treatment response. The genetic profile will be confirmed by RT-PCR, and information generated from RNA-Seq will be used to improve the RNA based targeted gene panel deep sequencing for myeloid neoplasms. The study will also provide critical preliminary data for further funding application to support a larger and more complete genetic profiling research to screen for biomarkers that will help diagnosis and treatment decision of MDS patients.