The population in the U.S. is aging. Data are sparse on cardiovascular disease in older adults, especially the oldest old, who are expected to number 18 million by 2050. The age at first myocardial infarction (MI) is increasing substantially, now to about age 71, and most of the cardiovascular disease (CVD) deaths in the U.S. occur in older individuals. CVD is the single most important cost of medical care in the US. In the early 1990s, the Cardiovascular Health Study (CHS), an NHLBI-supported cohort study of risk factors for coronary heart disease (CHD) and stroke in adults 65 years or older, recruited 5888 participants, who underwent extensive examinations at baseline and annually until 1999. Examinations included traditional risk factors as well as measures of subclinical disease. Since 1990, CHS has made major contributions to the understanding of CVD in older individuals, including the risks associated with subclinical disease, inflammation, diabetes, hypertension and renal disease. In 2008, CHS has the unique opportunity of evaluating clinical CHD in the oldest old-- MI, angina, heart failure (HF), atrial fibrillation-- and their contributions to both morbidity and mortality. In this competing continuation, the primary aim is to evaluate the incidence and determinants of cardiovascular disease and health in 1964 surviving participants aged 80 or older. The incidence of CVD in the oldest old will be related to demographic variables;measures of disability, physical functioning, and cognitive function;measures of subclinical disease;and traditional and novel clinical risk factors as well as their change over time. The data collection proposed in this application will increase the number of events in those 80 years and older by about 50% to 75% for stroke, CHD and HF. Power for assessing associations with stroke, CHD and HF is increased by 15 to 30%. The continued assessment of cardiovascular events is essential to identify determinants of successful cardiovascular aging among the oldest old. PUBLIC HEALTH RELEVANCE: Additional knowledge about the determinants of cardiovascular health and disease in older adults will help physicians and their patients make diagnostic, prognostic, and therapeutic decisions. PROJECT/


Non-insulin dependent diabetes mellitus (NIDDM) affects people of Latino origin more often and at an earlier age than whites. The Latino culture influences dietary behaviors which affect diabetes. Although nutrition intervention studies have demonstrated reductions in weight and glucose, culturally sensitive programs for a specific population of Latinos with diabetes have not been developed or validated. Boston, MA is home to a large population of Caribbean Latinos who originate predominantly from Puerto Rico and the Dominican Republic. An NIH planning grant has been used by the investigators to create a multidisciplinary team of practitioners and researchers and to identify and characterize a population of urban Caribbean Latinos with diabetes mellitus. Preliminary data show that these Latinos consume traditional Latino foods (especially rice and beans) primarily at lunch and dinner, use oil and other high fat foods extensively and rarely eat outside of the home. There is a strong need for programs that are culturally sensitive and target specific behaviors. We propose to design, implement, and evaluate a culturally-sensitive program that reduces dietary lipid intake. Using the information gained during the planning grant year, we will develop a program that is oriented towards Caribbean Latino culture, utilizes principles of behavior modification, and consists of proper nutritional guidelines. The intervention will focus on recipe modification, meal planning, informed shopping and group nutrition counseling. After pilot testing and modifying the program, diabetic Caribbean Latinos will be recruited from Boston City Hospital and the South End Community Health Center and randomized to either an intervention nutrition (IN) group or a usual care (UC) group. A separate UC group of Caribbean Latinos from Whittier St. Neighborhood Health Center will be followed to determine whether crossover occurs. The IN group will meet for twelve consecutive weeks, and then at weeks 14,16 18, 20 and 24. The UC group will be followed at 6, 12 and 24 weeks. Follow-up for both groups will be done at 9 and 12 months. Parameters to be measured in both groups include dietary intake, weight, lipid profile, HgbA1C, WHR, and blood pressure. Metabolic studies will include body fat distribution using dual energy x-ray absorptiometry (DEXA) and indices of insulin sensitivity and resistance as measured by IV glucose tolerance test. By designing culturally sensitive programs that target specific behaviors, we believe that dietary fat intake can be reduced in urban Caribbean Latinos with NIDDM.


Recent evidence suggests that injection drug use is increasing among suburban residents. Little research has focused on this trend, and the economic, social, and medical consequences have not been considered. To close this gap, we are proposing to identify, recruit, and study a valid sample of injectors who reside in Connecticut suburbs using respondent driven sampling (RDS). RDS was selected because of its strengths in identifying individuals from hidden populations and allowing statistical analysis of the sample. We are submitting this application in response to PAR-06-114, "Research on Pathways Linking Environments, Behaviors and HIV/AIDS". Not only will be focusing on an environment - the suburbs - that has been neglected, but we will further focus on suburban injectors who inject in two distinct environments. Preliminary studies revealed that there are those who inject predominantly in the suburban town in which they reside and those who inject in urban areas where they obtain their drugs. We are building our study around the hypothesis that the two environments result in differences in (1) injection drug use patterns; (2) knowledge of, risk for, and prevalence of HIV, hepatitis B, and hepatitis C infections; (3) sociodemographic stability; and (4) the use of and barriers to services that reduce the negative consequences of injection. Our study will recruit a sample of 600 active injection drug users who reside in the suburbs. The sample will be studied through structured interviews, serology, and in-depth life histories. The sample will be followed longitudinally to ascertain whether the pattern of suburban or urban injection is stable and to identify factors associated with changes in injection environments and in risk for syringe-borne viral infection. Our study is designed able to answer questions including (1) how representative the sample is of the underlying population, (2) whether two groups of suburban injectors who differ based on where they inject differ in terms of disease knowledge, risk, and prevalences, (3) how much of the prevalence of HIV, HBV, and HCV can be predicted by individual, group, and environmental level variables, (4) how suburban injectors are geographically clustered and whether this affects disease prevalence, (5) how interaction with the criminal justice system influences drug use and risky injection practices, (6) which factors are associated with arrest and incarceration and (7) how injectors use drug treatment and disease prevention programs and what they perceive as the barriers to larger use of these services. To meet the objectives of this study, we have assembled an experienced, multidisciplinary team that can collect and analyze the quantitative, qualitative, and serological data and disseminate research findings to the scientific community through scholarly and public health channels and to the service community through our Center for Interdisciplinary Research on AIDS. [unreadable] [unreadable] [unreadable]


Crohn's disease (CD) is a chronic and debilitating inflammatory bowel disease with unknown etiology;however, it is clear that there are both genetic components as well as environmental factors that are required for its development. Tremendous advances have been made recently in understanding the genetics of CD. Although several chromosomal loci have been associated with IBD susceptibility, until last year, only one gene had been conclusively identified as a CD susceptibility gene. This gene is NOD2 (nucleotide-binding, oligomerization domain 2), which encodes an intracellular sensor of bacteria in the innate immune system. Recent genome-wide association scans have identified multiple, novel CD susceptibility genes. Many of these new CD susceptibility genes also modulate immune responses, suggesting that dysregulation of an immune pathway rather than a single gene may be important in CD pathogenesis. One of these processes is autophagy. Autophagy (literally "self-eating") is best known as a cell survival mechanism in response to starvation, where organelles are broken down and recycled to provide nutrients. This mechanism also plays a crucial role in the clearance of intracellular bacteria by the innate immune system. Bacteria and bacterial components induce autophagy, linking innate immune microbial sensors to autophagy. One of these microbial sensors is Nod2, which detects a specific component of the bacterial cell wall called muramyl dipeptide (MDP). Our preliminary data demonstrates that stimulation of cells with MDP not only activates autophagy, but suggests autophagy may also be involved in activation of Nod2 by MDP. These results provide a link between two separate classes of molecules mutated in CD and lead us to propose the following central hypothesis: CD- associated mutations in the autophagy genes, ATG16L1 (autophagy related 16-like protein 1) and IRGM (immunity-related GTPase family, M), and NOD2 impair the same critical innate immune pathway and impairment of this pathway contributes to CD pathogenesis. We hypothesize autophagy and Nod2 function together in two non-exclusive ways: (1) MDP stimulation of Nod2 activates autophagy as an essential anti-microbial response, and (2) autophagy is involved in MDP internalization and Nod2 activation. This hypothesis will be tested by the following three specific aims using both primary cells and cell lines: 1) Determine the role of Nod2 in activation of autophagy - is autophagy a component of Nod2 stimulated anti- microbial responses?, 2) Characterize the mechanism of MDP internalization - is it autophagy?, 3) Analyze alterations in autophagy in phagocytic cells of CD patients. The results of our proposed studies may benefit individuals with CD by identifying a crucial processes altered in CD, leading to the design and use of more specific CD therapies targeting this pathway. PUBLIC HEALTH RELEVANCE: Recent advances in the genetics of Crohn's disease (CD) indicate that alterations in an immune pathway rather than a single gene may be important in CD pathogenesis. We propose to study the interactions of three genes associated with CD susceptibility and define if they are a part of the same innate immune system pathway. The results of our proposed studies may benefit individuals with CD by identifying a crucial process altered in the disease, leading to the design and use of more specific therapies targeting this pathway.


Estrogen is a fascinating hormone with seemingly paradoxical effects in the cardiovascular system. On the one hand, estrogen appears to be protective to the cardiovascular system of pre-menopausal women. Prior to the menopause have a lower incidence of cardiovascular disease than men. After the menopause, the incidence increases rapidly to reach and even exceed that of men. It has been widely assumed, form anecdotal evidence, as ell as several early studies, that replacement of estrogen would return the cardiovascular system of the post-menopausal woman to its pre-menopausal protected state. However, the early data from the Women's Health Initiative does not support the assumption; rather, these data suggest an increase in cardiovascular events in the first two years of hormone replacement therapy with estrogen. Estrogen remains the most likely cardiovascular protective factor for pre-menopausal women. Thus we propose that the failure to observe a protective effect of estrogen on cardiovascular function is the result of administering an estrogen that is an incomplete agonist in cardiovascular tissues, or that is administered in inappropriate mode. This project is designed to test the hypothesis that the dichotomous effects of estrogen on the cardiovascular system are due to differential Gene expression responses to different estrogen formulations and modes of administration. Two specific aims have been developed to test this hypothesis. Specific aim #1: To develop a profile of genes expressed in response to estradiol 17beta in vascular tissues and to examine the effect of estrogen formulation on the vascular reactivity of the mesenteric arteries.


A major goal of this laboratory is to understand the molecular mechanisms by which the nuclear hormone receptor peroxisome proliferator-activated receptor ? (PPAR?) controls adipogenesis and mediates antidiabetic effects of thiazolidinedione (TZD) drugs that improve insulin sensitivity but cause unwanted side effects. We hypothesize that target gene- and tissue- selective modulation of gene expression by PPAR? are dictated by different modes of PPAR? binding and synergy between PPAR? and cooperating transcription factors. Specific Aim 1 is to determine the direct transcriptional effect of TZDs in adipocytes and their relation to PPAR? binding on a genome-wide scale. We hypothesize that the direct effects of TZDs on transcription cannot be predicted from transcriptomic analysis. Preliminary Global Run-On sequencing (GRO-seq) results demonstrate the feasibility of measuring nascent transcripts in adipocytes on a genome-wide scale. Specific Aim 2 is to delineate the adipocyte epigenome and corepressor cistromes, and their relation to PPAR? binding and the effects of TZD treatment on a genome-wide scale. We hypothesize that TZD treatment of adipocytes alters coregulator recruitment to PPAR? at a subset of target genes, leading to epigenomic changes that alter gene transcription. This will be tested by ChIP-seq for coregulators and epigenomic marks in adipocytes treated with TZDs and non-TZD ligands with potentially fewer side effects. Specific Aim 3 is to understand the mechanisms of cell-type specific genomic binding and regulation by PPAR?. We hypothesize that PPAR? functions as a pioneer factor on some binding sites, particularly in adipocytes, whereas macrophage binding sites are more influenced by cell- specific transcription factors and epigenomic marks. This will be tested using cistromic approaches, and the ability of PPAR? and cell type-specific factors such as PU.1 to shape each other's genomic binding, as well as the adipocyte and macrophage epigenomes, will be explored using gain and loss of function studies. Our innovative approach will elucidate mechanisms underlying target gene- and tissue-specificity of PPAR?, which can be translated to the design of more selective insulin sensitizers to combat the epidemic of metabolic disorders.


Chronic administration of the beta-adrenergic agonist isoproterenol induces parotid gland hypertrophy and hyperplasia in the rat. This serves as a useful model for studying the physiological and biochemical alterations that occur in cell proliferation. An important event noted in the isoproterenol induced parotid acinar cell hyperplasia is the increase in cell surface 4 betagalactosyltransferase (Gal Tase). Numerous studies have shown that alterations in cell surface Gal Tase may be involved in cell-cell interaction during embryogenesis, differentiation and tumorigenesis. The elevated surface Gal Tase in parotid acinar cells then appears to interact with the EGF-R in a manner similar to growth factor associated 'receptor-ligand' interaction. It is believed that the EGF-R undergoes conformational changes in its carbohydrate moieties so as to serve as a substrate for cell surface-localized Gal Tase. However, it is not clear how the signal produced by such an interaction reaches the nucleus to modulate the processes such as DNA replication and gene expression. I therefore propose to examine the involvement of second messengers, and receptor crosstalk produced during the regulation of acinar cell growth. To accomplish this, phosphoinositol metabolism (along with their specific metabolic kinases), and adenylate cyclase G activating protein (GAP activity), will be studied in both the control and ISO stimulated parotid glands. Parallel studies with EGF and Gal Tase stimulation will be conducted to determine the extent of Gal Tase's ability to mimic EGF in regulating this specific receptor response. In addition, the characterization of the carbohydrate structure of EGF-R in the control and ISO-treated rat parotid gland will be made to confirm the unique Gal Tase - EGF-R interaction which appears to be a primary event in beta-agonist mediated signal transduction for proliferation. The injection of antibody to either Gal Tase or the EGF-R, along with ISO, have been shown to prevent acinar cell proliferation. The carbohydrate changes of the receptor may provide insights into the regulation and re-expression of fetal carbohydrate structures during parotid acinar cell hyperplasia. While not neoplastic, the expression of fetal carbohydrate antigens by these cells may reflect a common hallmark of transformed cells; namely a renewed synthesis of fetal developmental carbohydrate markers on the cell surface. These findings on the regulation of acinar cell signal transduction pathways in the isoproterenol induced parotid gland hyperplasia may be helpful in evaluating potential new treatments for the control of cell proliferation in human pathologies in general and those in particular of the oral cavity and soft tissue.


The broad, long-term objective of this research proposal is to improve the health and well being of the elderly via decreased risk for type 2 diabetes. The specific aim of the project is to examine the influence of an ad libitum 12- week low-fat/simple-carbohydrate diet, with and without aerobic exercise training, on body mass, energy balance, and insulin sensitivity in older subjects with impaired glucose tolerance. Preliminary data indicate that consumption of a low-fat/complex-carbohydrate diet, with no attempt at energy restriction, results in loss of body weight and improvement in insulin sensitivity. We hypothesize that replacement of dietary fat with simple rather than complex-carbohydrates will result in similar improvements in health. Thirty-six overweight men and women aged 55-80 years with impaired glucose tolerance will be randomized into one of three groups. Group 1 will receive a control diet (similar to baseline intake), group 2 will receive a low-fat/simple-carbohydrate diet (17% fat, 16% protein, 67% carbohydrate, 9g fiber/1000kcal), and group 3 will receive an identical low-fat diet and be placed in an aerobic exercise training program (4d/week, 80% VO2ma,). Body composition will be measured using air displacement plethysmography, energy and macronutrient metabolism will be measured using whole room calorimetry and de novo lipogenesis, and insulin sensitivity will be measured using a euglycemic-hyperinsulinemic clamp. Given the increasing prevalence of type 2 diabetes in the elderly U.S. population, completion of this project will more clearly define the specific dietary and exercise interventions that have the greatest potential positive impact on health in older individuals.


This competitive renewal proposal is to develop pulse saturation-recovery (SR) electron paramagnetic resonance (EPR) for application to spin labels in a context of site directed spin labeling (SDSL). The applicant's laboratory has recently made a breakthrough in SR detection based on a novel digital sub-sampling strategy, which has been termed time locked sub-sampling, and a new pulse spectrometer is under construction. Thorough engineering evaluation and design optimization including signal processing software are proposed for the next funding period. Feasibility for a new X-band bimodal loop gap sample resonator has also been demonstrated and further innovation is proposed. Engineering-based development follows naturally from the work of the previous funding period. SR can measure the rate of bimolecular collisions of spin labels with oxygen, and is a primary methodology for studying oxygen transport. Spin label oximetry is an important tool in identification of structural motifs such as alpha-helices or beta-sheets in proteins through SDSL methods. Three model systems are used here in each of three application-based specific aims. The model systems, in order of increasing biological relevance, are maleimide spin label in super-cooled sec-butylbenzene, hemoglobin labeled at beta93 in glycerol-water mixtures, and the ferric enterobactin receptor, Fep-A. The SR method is validated and compared with more conventional methodology. A novel SR method to separate overlapped spectra, termed discrimination by oxygen transport, is used to characterize multiple motional components of FepA. SR apparatus acquires intense free induction decay (FID) signals that are normally suppressed. Instead, this investigator proposes to use FID signals to measure site-to-site distances in double-labeled FepA. Frequency swept pulse electron-electron double resonance is introduced to characterize slow restricted angle motions in Fep-A in a situation where overall motion of the protein is negligible. The detection method, the bimodal resonator, use of FID signals for distance determination, use of SR to separate overlapped spectra, and measurement for the first time of restricted-angle motions are thought to be highly innovative. SDSL is a new and rapidly expanding methodology in structural biology. Addition of saturation recovery to SDSL methodology will, we hypothesize, revolutionize the field. The proposal lies at the foundation of modern biomedical research: development of tools that enhance one's ability to determine protein structure, dynamics and function. The investigator asserts that only on such a foundation can a rational approach to improved human health be based.


For risk assessment purposes it is important to establish the relationship between the applied or exposure dose and tissue specific concentrations and the relationship of this dose to toxicological or carcinogenic outcomes. It is therefore necessary to analyze tissue specific concentrations of 2,3,7,8-tetrachlorndibenzo-p-dioxin (TCDD) and other related chlorinated dibenzo-p-dioxins and furans in treated animals. We are therefore establishing a contract for analysis of TCDD and other related compounds in animal tissues. We will require high resolution gas chromatography/mass spectroscopy to quantitate these chemicals in these samples.


Fasting causes competition for nutrients between mother and fetus. The rate of maternal protein degradation may be lower in pregnancy in order to protect maternal stores while supplying the fetus. This rate will be measured by constant maternal infusion of I-13C leucine with application of steady state isotope dilution methods & will be compared in pregnancy and post-partum. Disturbance of the control of protein degradation may be responsible for altered fetal growth rate in gestational diabetes and preeclampsia.


Lysosomal storage diseases (LSDs) are a group of inherited metabolic conditions caused by defects in proteins critical for lysosomal function. While individually rare, their collective incidence is just over 1/7,000 live births, which makes the LSDs an important health problem. The majority of these diseases have a progressive neurodegenerative course, resulting in severe debilitation. Symptoms are not manifested unless there is more than 90% reduction in the residual activity of the deficient enzyme. Thus, an enzyme activity level above the critical threshold will prevent lysosomal storage and dysfunction, and can arrest, or reverse the disease process. Current therapies available for LSDs, such as enzyme replacement therapy (ERT) are only available to treatment of a few LSDs, and then only the non-neurological symptoms. Additionally, the approximate cost of over US$250,000/year/patient limits accessibility to ERT. For these reasons, therapies based on small molecules, which could cross the blood-brain barrier and be less expensive;appear to be an attractive approach. Small molecules can function as enzyme enhancement agents by binding, and stabilizing mutant misfolded proteins in the endoplasmic reticulum (ER), allowing them to evade the ER associated degradation (ERAD), and reaches the lysosome. Previously, we screened a small compound library of 1,040-FDA approved compounds and were able to identify and characterize two drugs that function as enzyme enhancement agents for two lysosomal enzymes: hexosaminidase A and glucocerebrosidase. My hypothesis is that small molecules may enhance residual activity found in patients with LSDs by direct interactions with the mutant enzyme, or by an indirect effect on different cellular pathways resulting in increased levels of the mutant and partially functional protein in the lysosomal compartment. I hypothesize that I can identify molecular probes that would restore arylsulfatase A (ASA), lysosomal enzyme deficient in metachromatic leukodystrophy (MLD), a neurodegenerative LSD for which a specific treatment is not available. To test this hypothesis, I aim to: (i) develop a robust and reproducible cell-based high throughput screening (HTS) assay to identify molecular probes that may function as enzyme enhancement agents for ASA;(ii) to configure a quantitative HTS assay for ASA, and carry out validation strategies for the "active" small molecules generated by the primary screening. I anticipate the submission of the screening project upon completion to the NIH Chemical Genomics Center for implementation. The candidate molecular probes generated by this screening will then be validated using different assays based on my experience previously acquired on the identification and characterization of enzyme enhancement agents for other lysosomal enzymes. The validated small molecules will be potential drug candidates to treat patients with MLD, and may also be used as tools to uncover novel biological pathways involved on the folding, maturation and trafficking of lysosomal proteins. PUBLIC HEALTH RELEVANCE: The development of high throughput screening assay (HTS) for arylsulfatase A (ASA) will allow the identification of molecular probes that, once characterized and validated, may be potential drug candidates to treat patients with metachromatic leukodystrophy, a lysosomal storage disease caused by deficiency of ASA. In addition, these compounds may be used as research tools to examine biological pathways involved on the folding, maturation and trafficking of lysosomal proteins.


This research project is aimed at developing a novel DNA sequencing, mapping, or diagnostic technique that requires no DNA labeling. The proposed innovative technique would even allow genomic DNA to be used for diagnostics, thus significantly reducing preparation cost (no polymerase chain reaction would be necessary). During Phase l we experimentally evaluated the most promising detection techniques and demonstrated the feasibility of the procedure. In Phase Il we plan to systematically determine the best parameters for probe immobilization and hybridization and determine the sensitivity of these procedures to variations in the parameters. We will also develop a less expensive prototype instrument that could be purchased by major facilities. Because of the potential cost effectiveness, accuracy and speed of this innovative technique, commercial benefits could be tremendous. PROPOSED COMMERCIAL APPLICATIONS: Commercialization could begin by supplying analytical services to researchers at national and private laboratories. Instrument sales would be first targeted to major research and clinical laboratories, but would grow to areas outside the biomedical field due to the wide applicability of the technique.


Candida albicans is both a ubiquitous part of the mammalian commensal flora and the most common systemic fungal pathogen of humans. Systemic disease is the fourth most common nosocomial infection and is associated with a ~40% mortality, due both to the severity of the infection and current inadequacies in diagnosis and treatment. Systemic, or disseminated, candidiasis mostly develops in patients whose innate immune system has been compromised by disease, chemotherapy, or medical intervention (surgery or implanted devices such as catheters). Thus, there is a fine line between Candida as a commensal and Candida as a pathogen and our long-term goal is to understand how this balance is maintained or disrupted to promote one state or the other. It is our premise that strengthening the immune system or weakening the fungus, even slightly, may tip this balance in favor of the patient, thus we have studied the interaction between C. albicans and cells of the innate immune system. From these studies, we have evidence that C. albicans secretes an immunomodulatory compound(s) that inhibits the release of nitric oxide (NO), a key antimicrobial and immunomodulatory compound, from macrophages. We have begun to characterize this inhibitor and have found that it is small, hydrophilic, heat-stable, and is not carbohydrate-based;one aim of this proposal is to identify this compound. In doing so, we have been aided by a series of genomic experiments that have defined the extensive and complex transcriptional response of C. albicans to phagocytosis by macrophages. One of the most surprising findings is an induction of the arginine biosynthesis pathway in its entirety. No other nucleotide or amino acid pathway is induced, making this a specific and unique response. In addition to being an essential amino acid, arginine is also the substrate for production of NO by the inducible Nitric Oxide Synthase (NOS2 or iNOS). Analogs of arginine inhibit iNOS;one such analogue has been shown to greatly reduce the anti-Candida activity of neutrophils. These arginine analogs fit our preliminary chemical characterization of the C. albicans-derived inhibitor. Thus, our central hypothesis is that C. albicans has co- opted the arginine biosynthesis pathway to produce an iNOS inhibitor, related to arginine, that promotes survival and pathogenesis of this organism. The experiments proposed here will test this hypothesis and identify the inhibitory compound. Serious fungal infections, caused mostly by Candida species, are increasingly common and severe. These affect mostly patients already debilitated by other medical treatments or illnesses and suggest that strengthening these patients'immune system would help fight these infections. We present data here that indicates that the fungus itself may be actively impairing the immune system and propose to characterize how this process occurs with the hope of eventually counteracting this ability.


1. It has been shown that the hypervariable region of flagellin of strains of Salmonella (potentially useful as typhoid vaccines) is a suitable locus for insertion of DNA coding for epitopes which are of interest as potentially effective immunogens for diseases other than salmonellosis. A nonapeptide which represents aa163-171 of Il-1 beta has been shown to be immunoenhancing. We shall test whether the expression of this nonapeptide enhances immune response to determinants which are juxtaposed to, near or spatially removed from this nonapeptide when it is inserted in the hypervariable region of vaccine strains of Salmonella. 2. Many polysaccharide antigens are considered to bc T independent in the sense that response to them does not require T cell help; anamnestic responses are not elicited and the immunoglobulin isotype response is restricted with such antigens. We propose to test a method to convert oligosaccharide determinants of such antigens to T dependence enabling both the elicitation of memory and unrestricted class switching. Furthermore, the process would make it possible to have such antigens expressed as peptide surrogates as piggy-back antigens in the hypervariable region of flagellin as in 1. A monoclonal anti Salmonella Vi will be used to screen a random peptide library for adventitious cross-reactive peptides which will be back-translated to DNA, and inserted for expression in the hypervariable region of flagellin. The essential hypothesis is that cross-reactivity as recognized in reaction will bc matched by cross-reactivity in induction of immune response. Specifically this might greatly enhance the utility of the typhoid vaccines being tested since they do not at present elicit anti-Vi.


Deficiencies in choroidal blood flow (ChBF) have long been suggested to underlie age-related retinal decline, and cause the outer retinal abnormalities that set the stage for AMD, given the necessary risk-conferring genes. Nonetheless, experimental evidence that this is the case has been lacking. We have been investigating the parasympathetic control of ChBF. We have found that the vasodilatory parasympathetic facial nerve input to choroid from the pterygopalatine ganglion appears to maintain high ChBF during low systemic blood pressure. We have found that this adaptive reflex, which we term ChBF baroregulation, is impaired by age, and when impaired by experimental manipulations in animal models leads to ChBF insufficiency and declines in retinal function. The studies proposed here will build on these findings and experimentally determine if impaired ChBF baroregulation leads to the types of outer retinal pathologies that characterize aging and/or early (i.e. dry) AMD. Three Aims will be pursued, all in rats. In the first Aim, we will determine the basic neural mechanisms by which ChBF baroregulation is mediated. These studies will provide basis for determining the defects that cause dysfunction of ChBF baroregulation, and identify points of intervention that might allow restoration of normal ChBF control. Our particular goal is to determine how blood pressure signals from the thoracic aorta via the aortic depressor nerve (ADN) regulate ChBF by their input to the nucleus of the solitary tract (NTS) and its output to the parasympathetic part of the facial nucleus (the superior salivatory nucleus, SSN), which project to the vasodilatory neurons of the pterygopalatine ganglion, which themselves innervate choroidal blood vessels. To this end, we will use imaging of circuit connectivity and pharmacological approaches to determine if SSN- projecting NTS neurons are excitatory and receive input from inhibitory ADN-receptive NTS neurons. In our second Aim, we will characterize by functional and morphological means the outer retinal abnormalities caused by long-term disturbance in ChBF baroregulation. We will be particularly interested in determining if the outer retina pathologies include those observed in aging, such as sub-RPE basal laminar deposits and photoreceptor loss, and/or those seen early in AMD, such as sub-RPE basal linear deposits, RPE and photoreceptor loss, and outer retinal waste accumulation (cholesterol, and proteins with advanced glycation end-product or peroxidized lipid adducts). Since light history is an AMD risk factor, we will also determine if impaired ChBF baroregulation exacerbates photoreceptor light damage. In the third Aim, we will determine if age-related choroidal baroregulatory impairments are associated with outer retinal dysfunction and pathology. Our studies may suggest choroidal baroregulation impairment as underlying age-related retinal decline, and as an AMD risk factor. This would suggest testing of choroidal baroregulation to assess AMD risk, and recommend drugs that boost ChBF as therapies. PUBLIC HEALTH RELEVANCE: Our studies will determine how the brain uses low blood pressure signals to maintain high blood flow in the choroidal layer of the eye, so the outer retina receives the nutrients it needs to remain healthy and function properly. We will also determine if impairment of this function underlies the retinal decline that occurs with aging, and explains why aging is the major risk factor for AMD. Our studies may lead to use testing of blood pressure regulation of choroidal blood flow as an AMD risk assessment, as well as lead to use of drugs that normalize neural control of choroidal blood flow to slow AMD onset and progression.


[unreadable] [unreadable] This proposal describes a 5 year training program for the development of an academic career in [unreadable] Cardiovascular Disease. The principal investigator (PI) has completed structured fellowship training in [unreadable] Cardiology at the University of Pittsburgh and will expand upon his scientific skills through interdepartmental collaboration. This plan emphasizes training in cardiac physiology, pathology, endocrinology, and biochemistry at the gross and molecular levels through investigation of the importance of leptin signaling in the heart. Dr. Charles McTiernan is an Associate Professor in the Cardiovascular Institute (CVI) and a recognized leader in the field of heart failure (HF) and cytokine research. Dr. Christopher O'Donnell, Associate Professor in the Pulmonary Division, is an expert on the study of leptin and murine physiology in heart failure. Together, both Drs. McTiernan and O'Donnell have trained numerous students and fellows in the past and will mentor the Pi's scientific development. To enhance training and provide scientific and career advise, an interdepartmental advisory committee will be formed. Dr. Michael Mathier, Assistant Professor in the CVI, will provide training on the functional assessment of heart failure in mice. Dr. Robert O'Doherty, Assistant Professor in Biochemistry, will provide guidance in the areas of leptin physiology, signal transduction, and cre-lox technology. Dr. Jorge Sepulveda, Assistant Professor of Pathology, is an expert in the field of cardiac pathology and will provide training in techniques related to leptin histopathology. The research proposed is based on preliminary data that shows benefit to leptin on cardiac function and survival after myocardial infarction (Ml). Therefore, we hypothesize that leptin is protective after Ml. Specific aims designed to test this over-riding hypothesis and provide mechanistic understanding of leptin's benefit [unreadable] after Ml include using mice to: 1) Examine of the effect of leptin-deficiency and systemic leptin replacement on survival and the development of HF after Ml. 2) Compare systemic versus central effects of leptin administration in ob/ob mice on survival and the development of HF after Ml. 3) Examine the effect of cardiac specific deletion of the leptin receptor on survival and the development of HF after Ml. Overall, the CVI at UPMC provides an ideal environment for training physician scientists by encouraging inter-disciplinary collaboration and sharing of resources necessary for productive scientific discovery. [unreadable] [unreadable] (End of Abstract) [unreadable] [unreadable] [unreadable]


Breast cancer is the second most prevalent cancer in women, with a mortality rate of 40,000 per year in the US. However, if detected and treated early, more than 95% of breast cancer patients will survive. Current method for breast cancer detection relies heavily on X-ray mammography, which produces many false positive findings. These false positive readings cause substantial mental anguish in patients and in many cases, costly and painful biopsies. A full 80% of all biopies uncover only benign masses and calcifications at an estimated annual cost of 2.5 billion dollars. To improve breast cancer detection, we propose a highly innovative design for a dedicated Positron Emission Mammography (PEM) camera utilizing long, linear, lead-walled straw (LWS) detectors that provide high spatial resolution and rigorously accurate depth of interaction determination. Thus detectors with ample field of view to contain the entire breast without translation can be employed and very close detector pair spacing can be used without resolution degradation. Our proposed camera offers markedly increased sensitivity and resolution with substantially reduced cost compared to crystal-based PEM cameras under development. Phase I of the project will achieve proof of concept by constructing and testing two substantial submodules of a full scale camera. In Phase II, a full scale high sensitivity prototype camera will be constructed and tested.


By utilizing various strains of rats, Rattus norvegicus, a paradigm of human culture is being explored. This paradigm is based on the following theoretical considerations: The evolution of species of mammals, including human beings, has resulted in optimum group sizes of 12 adults. Since each group occupies a fixed territory, eventual occupation of all available space by such groups culminates in a stable adult populations of species. However, if conditions permits elaboration of social roles (particularly cooperative ones) and development of complex communication networks, then more individuals can survive in a given area without a resulting stressful increase in rate of interaction among members of the population that would otherwise trigger limitation of population size. This process which has permitted successive doubling of the human population is being experimentally simulated by providing computer controlled contingencies for rats living in a complex physical environment that fosters development of cooperative social roles and more complex communication networks.


The Eighth Conference on the Neurobiology of Learning and Memory is the eighth in a series of conferences organized by the Center for the Neurobiology of Learning and Memory at the University of California, Irvine. The theme of the conference is: Memory and Brain: Basic Mechanisms and Clinical Implications. It will be held in Irvine, California on March 11-14, 2006. The overall goals of the conference are to communicate and discuss recent research findings in five currently important topics in the neurobiology of learning and memory, to stimulate discussion about how basic findings might be translated into therapeutic or other applications, and to educate and inform researchers in the field. The 2006 meeting will include five sessions: 1) Mechanisms for acquisition and maintenance of reinforcement related behavior: Relation to addiction/compulsion, 2) Sensory and perceptual learning, 3) Memory and normal and abnormal aging, 4) Modulation of neuroplasticity and long-term memory, and 5) Molecular and cellular mechanisms of memory. To date, fifteen speakers have agreed to make presentations; we are planning a total of twenty. These speakers are among the world's leading investigators in the topics listed. The conference format emphasizes discussion by all participants. Chairs will be chosen for each session, to lead the interchange of ideas. The general sessions will take place at the Hyatt Regency Hotel in Irvine; a poster session (approximately 100 posters) will be held in the Herklotz Research Facility on the DC Irvine campus. We anticipate an attendance of 350-400 people, including faculty, postdoctoral researchers and graduate students from the U.S. and many foreign countries.


Further studies on the subunit structure of triton X-100 and papain solubilized receptors will be undertaken to get an insight into the molecular weight of the anchor piece which inserts the receptor to the membrane. The subunits will also be characterized with respect to IF-cbl and Ca ion binding. Membrane lipids or synthetic lipids will be used to make lipososmes with the receptor to study the uptake of 125I IF-(57 CO)cbl by the liposomes. Using suckling rats the membrane bound and soluble alkaline phosphatases will be further characterized about their lipid binding abilities in order to make liposomes.


Exposure to stressful events during withdrawal from psychostimulants, such as cocaine or amphetamine, induces drug craving and drug relapse in both animal models and human individuals. The effects of stress on drug-seeking behavior persist for months following psychostimulant abstinence, and play a large part in chronic relapses associated with psychostimulant addiction. It is the chronic nature of psychostimulant addiction and relapse that produces significant social, health and economic impact. Identifying the neurological alterations that occur during psychostimulant withdrawal will have significant impact on the development of pharmacological treatments designed to prevent drug relapse. The studies outlined by this proposal will determine whether psychostimulant abuse results in increased sensitivity of stress-related neural systems. These studies will also establish the duration of these effects during psychostimulant withdrawal. This will be achieved by using laboratory rat models of psychostimulant withdrawal. Changes in stress behavior, and alterations to the activity of neurotransmitter systems that regulate stress behavior, will be determined during acute and protracted withdrawal from amphetamine. Furthermore, studies will determine whether alterations to neural stress systems and stress behavior following psychostimulant withdrawal can be reversed by chronic treatment of the antidepressant, fluoxetine. Together, the proposed experiments will elucidate the neural substrates of stress sensitivity during psychostimulant withdrawal, and will determine the feasibility of antidepressant treatment to reduce stress behaviors and thus psychostimulant relapse. [unreadable] [unreadable]


The objective of this proposal is to study the molecular basis for the decreased ability of the immune system in many elderly individuals, to produce high affinity antibodies and thus mount an effective immune response to vaccines or infections. To pursue this objective, the principal investigator will study the age-associated changes in the relationship between structure and function of immunoglobulin VH and VL genes which produce the natural anti-Gal antibody. Anti-Gal constitutes 1% of circulating IgG in humans and interacts specifically with the carbohydrate structure Gala1-3GalB1-4GlcNAc-R (termed alpha-galactosyl epitope). As many as 1% of circulating B lymphocytes can produce anti- Gal. The applicant has found that most of the sequenced VH genes in anti-Gal producing lymphoid clones, cluster within a distinct region of the VH3 immunoglobulin gene family, and that these genes undergo somatic mutations. Analysis of age-associated changes in activity of anti-Gal have demonstrated loss of affinity of this antibody in many elderly individuals. The principal investigator proposes to study two possible mechanisms which may cause the age-associated impairment in anti-Gal affinity in humans: 1) different utilization of VH and VL genes repertoire for anti-Gal synthesis; and 2) impaired affinity maturation in anti-Gal genes because of age-associated changes in the somatic mutations process. For this purpose he will construct combinatorial phage display libraries in young and in elderly individuals and isolate the anti-Gal displaying phage. Comparative analysis of the anti-Gal VH and VL gene repertoire in the young and elderly, and determination of the resulting antibody affinities, will enable the applicant to establish whether the age-associated decrease in antibody affinity correlates with the gene repertoire used for anti-Gal synthesis. Comparison of the V gene sequences with the corresponding germline genes will allow the investigators to establish whether the age-associated changes reflect the excess or paucity of somatic mutations in anti-Gal producing genes. The applicant will further determine whether elderly individuals with low affinity serum anti-Gal retain some high affinity clones, by selecting high affinity anti-Gal phage from the libraries of these individuals. Lastly, by correlating anti-Gal affinity with the immune response to an influenza virus vaccine, the principal investigator will determine whether anti-Gal can predict the competence of the immune system in elderly individuals to produce high affinity antibodies. Thus, by exploiting the anti-Gal system, the applicant aims to understand the molecular basis of the well-documented age-associated impairment of humoral immune responses in humans.


The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, or inappropriately recognize a normal component of the body (an auto-antigen). Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system cells and how recognition in infections or malignant cells is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. Our previous work has described the events within a cell that bring together the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). We are now conducting studies primarily at the cell and tissue level to relate the physiology of antigen recognition to the development of effector function, immune memory, or tolerance and to add to this picture the activities of other cell types such as non-hematopoietic cells like fibroblastic reticular cells (FRCs). By understanding these events, we will be able to determine how best to deliver antigenic substances to stimulate an effective immune response or to interfere with autoimmune reactions and to understand how innate and adaptive components of the immune system participate in host defense under normal and pathological conditions. We previously reported a new method for the direct confocal microscopic visualization in real time of the interactions of T cells and antigen presenting cells in intact lymphoid tissue. These studies showed that individual T cells bind stably to antigen-bearing dendritic cells for several hours before activation-associated events lead to dissociation of the cell pairs and rapid migration of the T cells in the lymphoid tissue. Immunological synapses between T cells and a critical antigen presenting cell (the dendritic cell) were visualized in vivo as was clonal T cell division. This experimental system has now been improved by moving to multiphoton rather than confocal imaging and true intravital observation methods have been developed. We have worked out methods for imaging the bowel, which have allowed us to directly image the interaction of microorganisms with gut epithelium and dendritic cells in the mucosal immune system. Our studies in the small bowel have revealed the dynamic extension of lamina propria dendritic cell processes across the villus epithelium and the capture of lumenal bacteria by these processes. Other studies revealed the movements of B cells in lymphoid tissues and have provided evidence that dendritic cells outside of the follicle are involved in presentation of antigen not only to T cells but also to B cells. Using these new imaging approaches in concert with classical cellular immunological tools such a flow cytometry, we have recently shown that the small adapter protein SAP is essential for sustained interaction of antigen-specific T and B-cells that is needed to produce robust B cell clonal expansion and the development of germinal centers. Surprisingly, the same molecule is unnecessary for effective T cell interaction with dendritic cells, revealing an unexpected dichotomy in the molecular control of antigen-dependent cell-cell interactions involving T cells. New transgenic mice with cells ubiquitously expressing fluorescent proteins are being generated to provide new tools for long-term tracking of cells and genetically engineered strains of mice that have fluorescent reporters under the control of cytokine gene regulatory elements are being employed to relate the physical behavior of immune cells to their function.


We propose to hold an international conference involving approximately 350 scientists and 50 students from the U.S. and abroad. A major objective for the conference, Genetic Engineering of Animals: An Agricultural Perspective, is to bring together scientists working in the area of molecular genetics to discuss methodologies, philosophy, and potential applications as it relates to improvement of domestic animal agriculture and domestic animal research. To address the topic, we envision a tentative program that will be presented by approximately 24 of the leading scientists in genetic engineering of animals. The program will open with a conceptual session which will serve to meet a major objective of educating people who may have potential application of genetic engineering to increase the efficiency of their research efforts or to aid them in addressing important new research problems related to their research interests and the improvement of domestic animal production. A section on applications in laboratory and domestic animals will include discussions on sources of genetic material and examples of applications, i.e. influencing physiological, immunological, and developmental processes. A final section will address philosophical issues such as future application, moral, and legal implications, and where does it lead to. Proceedings of the conference will be published in an edited book within six months of the conference and the book should serve as a resource for the current state of our knowledge about genetic engineering as it relates to domestic animal agriculture.


The long-term goal of this Challenge Grant project is develop on a fast-track several existing new drugs which are already known to have activity in animal models of tuberculosis (TB), but which will not be developed by the respective companies in which they were discovered. Sequella, Inc. is in licensing negotiations for the rights to at least two, and possibly a third promising drug lead, selected with an emphasis on the following criteria: 1. Evidence of efficacy in a mouse model of tuberculosis 2. Simple chemical composition and synthesis, for ease of production and pricing 3. Representation of different drug classes 4. Evidence that they do not share cross-resistance with existing TB drugs. Sequella, Inc. will cost share with the NIAID to support the necessary remaining preclinical studies for each drug lead, according to the compound's present state of development. Preclinical studies include medicinal chemistry to enhance solubility and bioavailability, pharmacodynamic studies in animals, toxicological tests in animals and/or in vitro systems, and purity/stability testing in bulk drug prepared under GMP guidelines. Based upon the data obtained from the preclinical studies, Sequella, Inc. will apply for Investigational New Drug (IND) approvals for all compounds that show a duration of pharmacodynamic effects consistent with dosing intervals of no greater than once a day, and that appear likely to have an acceptable therapeutic ratio. The decision to proceed with Phase I/II trials of individual drug leads will be made cooperatively with NIAID staff. For each drug to be tested further, Phase I trials to determine tolerability will be carried out in volunteers at the NIH Clinical Research Center (if approved) or under contract. Phase II studies of each individual drug to test for efficacy in human TB patients will be carried out using a clinical trial design intended to reduce the risk of drug-resistant strains arising or spreading. The aims of the two-month Phase I time period would be to complete licensing agreements on targeted drugs (PA-824), identify promising TB drug leads in addition to those of which we are already aware, to meet with investigators who have worked with these compounds, to begin the negotiating process with Universities or companies to secure right-to-license agreements, and to outline clinical development plans for each TB drug candidate.


This application is in response to a Letter of Invitation (LOI-HD-05-111) to conduct community-linked studies to investigate the role of prenatal alcohol exposure in the risk for SIDS, stillbirth and FAS, and to determine how these different outcomes are inter-related. The proposed research will be conducted by the investigators the Prenatal Alcohol, SIDS, and Stillbirth (PASS) Research Network in a cooperative agreement with NICHD and NIAAA. This research involves the collaboration of: 1) two comprehensive clinical sites serving populations that are high risk for prenatal alcohol exposure, SIDS, and stillbirth, i.e. the American Indians in the Northern Plains and the Cape Coloured in Cape Town, South Africa;2) a central Developmental Biology and Pathology Center (DBPC);3) a central Data Coordinating and Analysis Center (DCAC);4) a central Physiology Assessment Center (PAC);and 5) program scientists and officers at the NICHD and NIAAA. This particular application pertains to the Tygerberg Comprehensive Clinical Site (CCS). The experimental design involves a prospective study of 7,000 pregnancies, and two retrospective, autopsybased studies of SIDS and stillbirth. The long-term goals of the SAFE PASSAGE STUDY are to decrease fetal and infant mortality and improve child health in communities at high risk for prenatal maternal alcohol consumption. The Specific Aims of the Network are as follows: 1) to determine the association between prenatal alcohol exposure and the risk for SIDS and stillbirth;2) to determine the role of the timing, pattern, and amount of prenatal alcohol exposure and other environmental factors in the risk for morbidity and mortality in early human life;3) to determine the role of specific genes in modifying the risk for morbidity and mortality in early life that is associated with prenatal alcohol exposure;4) to determine the role of alcohol exposure during pregnancy, and interactions among alcohol exposure and environmental and genetic modifiers, in altering profiles of autonomic activity of the fetus and infant, and neurobehavioral outcomes in the infant;5) to determine the role of maternal alcohol exposure, as influenced by specific environmental and genetic factors, in the impairment of placental function, and thereby the increased risk for fetal and/or infant morbidity and mortality;and 6. To determine abnormalities in key neurotransmitter systems in the brains of fetuses and/or infants that convey risk for sudden death, and to determine the role of prenatal alcohol exposure, as influenced by specific environmental and genetic factors, in their pathogenesis. The mission of the CCS is to ensure the proper identification of the perinatal risks of the use of alcohol during pregnancy, early detection of these risks and to perform basic research related to alcohol-induced injury in the human brain and placenta. This multidiscipline international study will determine whether the exposure of the fetus to alcohol during pregnancy leads to SIDS stillbirths and other complications. Knowledge gained from the study will help health care workers to provide better care for pregnant women.


The purpose of this study is to assess the efficacy and safety of 3,4-diaminopyridine in patients with myasthenia gravis and the Lambert-Eaton myasthenic syndrome.


The effect of prolonged space flight on the size and function of the heart is unknown. Using ultrasound, we measured the wall thicknesses, internal dimensions and the performance during blood ejection of the hearts of the Skylab 4 astronauts. Studies immediately following splashdown and subsequently over 68 days post-flight revealed no impairment of heart performance after 85 days in space.


Specific Aims 1. To facilitate the implementation of patient oriented HIV/AIDS related research at UCLA 2. To assist investigators with the regulatory aspects of biomedical and behavioral patient-oriented research 3. To establish and maintain a clinical database that will interface with the CFAR/CNICS program. The primary responsibility of the Clinical Research Facilitation Core is to facilitate the process of IRB submissions for faculty, fellows, new researchers and translational researchers who do not have a great deal of experience with human subjects research. While funding to facilities and equipment are crucial to the conduct of research, regulatory delays in the conduct of research are often overlooked. This core in itself is innovative as it provides investigators with resources not previously available to reduce delays. By its very nature, this core will support new initiatives. It will encourage research among new faculty, young faculty and fellows. The core is fully linked into the CFAR seed grant process and allows recipients to become productive in their research far earlier than if they did not have the Core to assist them. The Core has created both a website and a blog to share the resources learned while submitting applications. Additionally, the Core provides seminars to investigators and staff to increase knowledge of regulatory pitfalls and procedures. In reducing regulatory delays in. the conduct of patient-oriented research, this Core helps to maintain UCLA investigators on the cutting edge of research in HIV. PERFORMANCE SITE(S) (organization, city, state) University of California, Los Angeles Center for Clinical AIDS Research and Education 9911 W. Pico Blvd. Suite 980 Los Angeles, CA 90035 PHS 398 (Rev. 04/06) 614


The principal objective of this multi-center study is to evaluate and compare safety and growth velocity effectiveness of graded Norditropin dose levels after administration to growth hormone deficient children for 2 years. The secondary objective is to determine safety and efficacy with chronic Norditropin administration and follow-up of patients until they reach adult height. There are 100 children enrolled nationally, randomly assigned at 3 dosage levels, 0.025, 0.050, and 0.1 mg/kg/day. The study is divided into 2 sequential segments: Segment I 24 months, Segment II lasts from end of Segment I until they reahc adult height. We have enrolled 6 children on this protocol 4 males, 2 females. Doses are as follows: 1 male on 0.1 mg/kg/day, 1 male on 0.05 mg/kg/day, 2 males and 2 females on 0.025 mg/kg/day. All children are growing well. We have lost one child for compliance reasons. No child has yet reached final height.


The melanoma stem cell can be identified by a few markers such as TIE-1, CD133, nestin and ABCG2. Recently, a novel marker, ABCB5 was identified by Schatton et al. This protein is a transporter protein responsible for the efflux of chemotherapeutic drugs (doxorubicin specifically) from melanoma cells. Schatton et al demonstrated that ABCB5 was expressed in CD133 + cells, and that ABCB5+ cells were capable of self-renewal, and could be serially transplanted in animal xenograft models. ABCB5 was expressed in both primary and metastatic melanomas, and constituted about 2-20% of the tumor cell population. Given recent data that suggested that the Wnt protein, Wnt5A, can affect the proliferation and maintenance of hemaopoietic stem cells, we are trying to determine what the role, if any, of Wnt5A in the maintenance and profileration of the melanoma stem cell might be, using ABCB5+ as a marker of stemness. We have recently shown that Wnt5A can cause the loss of expression of melanoma differentiation antigens (MDAs), while promoting metastasis. Melanoma stem cells give rise to differentiated tumor cells, and so we are currently exploring the link between ABCB5, MDAs and Wnt5A. We have found thus far that cells which express high levels of MDAs tenc to also express high levels of ABCB5. A very interesting finding is that PCR anlaysis indicates, in our less differentiated, more metastatic melanoma cells, ABCB5 is alternatively spliced. We are currently sequencing this gene to determine if this is due to mutation. If it is, we propose to analyze a series of patient tumors for the ABCB5 mutation, and then perform a functional anlaysis to uncover the significance of this mutation, using both in vitro and in vivo methodology.[unreadable] Another family of proteins involved in melanoma stem cell development, and melanoma metastasis is the Notch family of proteins. Current data from our laboratory indicates that Wnt5A and Notch proteins may interact to promote wound-healing, and melanoma metastasis. We are exploring this link further, and trying to better understand the interactions between these two families of proteins not just in a cancer setting, but also in the context of normal skin, and skin aging.


The goal of our proposed research is to improve diagnosis of patients presenting to the Emergency Department [ED] with dizziness, some of whom are misdiagnosed with potentiallygrave medical consequences. The prevailing diagnostic paradigm for the evaluation of the dizzy patient is based upon a 'pathophysiologic' approach. This approach begins a search for etiology with the assumption that the quality of symptoms (vertigo, presyncope, imbalance, or non-specific dizziness) reflects the underlying pathophysiologic mechanism (vertigo = vestibular, presyncope = cardiovascular, imbalance = neurologic, and non-specific = psychiatric). Although this assumption often holds true, the 'pathophysiologic' approach mandates a thorough etiologic search in each organ system, not only the one suggested by symptom quality. This strategy is well suited to the referral clinic setting where it was developed, but poorly suited to the time-pressured environment of the ED, where the high index of illness severity demands effective triage rather than diagnostic certainty. We hypothesize that (a) potentially serious misdiagnoses of dizzy patients are uncommon but not rare events in the ED and may result from an over-reliance on the diagnostic importance on symptom quality; (b) a novel 'triage' approach to diagnosis would reduce misdiagnoses and improve outcomes in an 'in vitro' computer model of the diagnostic approach to dizziness; and (c) a clinical decision-support system based on this approach would reduce misdiagnoses under simulated patient conditions. To test our hypotheses, we have designed three specific aims to: (1) measure the frequency, potential severity, and possible cause of misdiagnosis of dizzy patients in the ED (by gathering extensive case data on each ED dizzy patient and referencing ED physician [EP] diagnoses against those of a multidisciplinary expert panel); (2) design a computerized decision model to test a new 'triage' approach to diagnosis (by comparing 'in vitro' simulations of the two diagnostic approaches using hypothetical case scenarios); and (3) 'pilot' a web-based decision support system to reduce misdiagnosis of simulated ED dizzy patients (by comparing EP performance on a video-case-based examination with or without the use of the decision support system, using a randomized trial design). Results of this study will form the foundation for subsequent research into the effectiveness of error-reduction interventions among dizzy patients. The research career award candidate has devoted himself to acquiring the clinical and research skills required to complete this project and launch a successful career as an independent investigator. He has garnered the support and enthusiasm of both his clinical department and a large, multidisciplinary team that will enable him to complete the stated objectives. This research project and the research paradigms derived from it will form the nucleus of a career devoted to research in medical decision-making, causes of diagnostic errors, and methods to preventthem.


Accumulating evidence from many different types of scientific studies indicates that inadequate intake of the B vitamin, folate, increases the risk of colorectal cancer. Among the observations supporting this concept are those which indicate that a common polymorphism in the folate-dependent enzyme, methylenetetrahydrofolate reductase (MTHFR), is associated with modulation of colorectal cancer risk. The biochemical and molecular mechanisms by which the effect of folate is mediated remain ill-defined, although the prevailing concept focuses on folate's central role in DNA synthesis and repair, and biological methylation. Although less compelling, there exists both scientific rationale and empiric evidence that other B-vitamins involved in one-carbon metabolism can interact with folate status in determining the risk of neoplastic transformation in the colorectum. An understanding of the pathways by which folate modulates colorectal carcinogenesis would be greatly facilitated by defining the mechanisms by which vitamins B2, B6, B12, as well the common MTHFR polymorphism, interact with folate in determining cancer risk. These factors can therefore serve as mechanistic probes to further our understanding of the processes involved. The proposed experiments will be conducted in established mouse models of B-vitamin depletion and intestinal carcinogenesis, as well as in some novel mouse models that are under development. Experiments are planned to examine the means by which interactions of the abovementioned dietary and genetic factors alter the biochemical, molecular and cell cycle mileu of the intestinal epithelium and thereby modulate the risk of carcinogenesis. Although several genetic pathways will be examined proactively, identification of novel, previously unsuspected, pathways towards cancer will be elucidated by use of DNA microarray technology. Elucidating the mechanisms responsible for the effect of folate on carcinogenesis will greatly enhance our ability to translate this field of scientific investigation into public health initiatives that effectively reduce the risk of cancer.


Description (adapted from applicant): The goal of the COEP is to provide environmental health information and developing educational programs in response to identified community needs. Reflecting the Center?s research theme on environmental agents as modulators of disease processes, Center faculty are actively involved in COEP delivering presentations at community forums and teacher workshops, assisting in curriculum development, or participating in the Community Advisory Board. Outreach efforts span the entire range of the community, from children to older adults, from rural to urban residents. In addition to the Community Advisory Board, the COEP Community Outreach Initiatives include a Lead Poisoning Prevention Program, Occupational and Health Care Provider Outreach, Community Lectures, Newsletters, and Web Site. The COEP Science Education Outreach Initiatives include three main programs: ?My Environment, My Health, My Choices?, an Environmental Health Curriculum for middle and high school students; Project Begin which involves the ethical, legal, and social implications of genetic research in environmental health sciences; and the Life Sciences Learning Center, itself comprised of five programs?Laboratory Investigations, Lab Skills Development Program, Saturday Morning Science, Summer Science Camps, and Teacher Workshops. Many of these COEP programs are new initiatives within the Center, developed since the 1999 NIEHS Center grant renewal.


Toxic oxygen species are important mediators of lung injury. An array of antioxidant defenses have evolved to protect cells against reactive oxygen species. An important component of this defensive system is glutathione. The specific aims in this study are to identify and characterize genetic factors which can influence the intracellular concentration of glutathione in mouse embryonic stem cells, and to generate transgenic mice from these cells. We hypothesize that selected mouse embryonic stems cells with alterations in glutathione content will display either increased or decreased resistance to reactive oxygen metabolites. Transgenic mice formed from such stem cells should also show altered sensitivity to free-radical damage. During the study period we will 1) isolate retrovirus-induced mutants of mouse embryonic stem cells with altered GSH content, by using a fluorescent dye specific for GSH; 2) isolate and characterize the mutated DNA that resulted in altered GSH metabolism; 3) analyse the effects of the altered GSH on cell sensitivity to free-radical damage; and 4) establish colonies of transgenic mice bearing the GSH mutations of interest. Creation of transgenic mice with genetically altered GSH content will be especially useful in future studies of free radical-induced lung injury.


Summary: Infections due to Neisseria gonorrhoeae represent a significant cause of morbidity and mortality in women and children throughout the world. The pathogenesis of gonococcal infections is not well understood, particularly with respect to the effect of patient hormonal status on the infectious disease process. Currently, there are no licensed vaccines for prevention of gonorrhea. This project is investigating the effect of reproductive hormones, estradiol and progesterone, and oral contraceptives, on the expression of gonococcal outer membrane antigens, and whether or not these hormones affect attachment and/or invasion to human cervical and endometrial tissue culture cells. The goal is to identify potential new antigens that may be particularly relevant in gonococcal infections in women and to determine if hormonal status may be a factor to be considered in the evaluation of models of infection. The specific aims of this project are: 1. to assess the effect of estradiol and progesterone on expression of gonococcal outer membrane antigens; and 2. to determine whether estradiol and progesterone alter gonococcal adherence and/or invasion to human cells. Progress to date on specific aims: 1. We have determined the effects of reproductive hormones on the cultured cell line Hec1B: estradiol was not inhibitory to the growth of Hec1B cells in culture over a range of concentration from 1x 10-6 to 1 x 10 -12 M/L, and the cell line grew optimally at concentrations of 1x 10 -8 and -9 M/L. These concentrations are similar to the physiologic levels measured in serum taken from women during the early proliferative stage of the menstrual cycle. Progesterone and combined progesterone and estradiol are slightly inhibitory to cell proliferation. Phenol red has a weak estrogenic-like activity and we have identified this common component of cell culture media as a possible confounder in gonococcal adherence assays. 2. Outer membrane proteins from HEC1B cells grown with and with out estradiol were analyzed and at least two proteins at 80kDa and 180 kDa demonstrated increased expression when exposed to estradiol. These proteins will be excised from gels and sequenced using Liquid chromatography mass spectroscopy/mass spectroscopy. 3. Gonococcal adherence assays were conducted and an increase in gonococcal adherence in the presence of 1 x 10-9 M/L estradiol was observed. Our laboratory has recently developed an assay that will more accurately quantitate the number of bacteria bound to cultured cells. 4. Outer membrane proteins isolated from gonococci bound to Hec1B cells in the presence or absence of estradiol revealed an altered protein profile by SDS-PAGE. Several proteins were down-regulated and one protein was up-regulated. These proteins were excised from gels and sequenced using Liquid chromatography mass spectroscopy/mass spectroscopy. Sequenced proteins were used to search the FA1090 Neisseria genome at the University of Oklahoma and the NCBI-Blast database. The down regulated genes were identified as groEL, dnaK, L23, and a gene encoding a putative lipoprotein. The up regulated protein was identified as neisserial catalase. The gene for each identified protein has been cloned from MS11mkC genomic DNA. Promoterless b-galactosidase (lacZ) and GFP reporters were constructed which will be used to evaluate differences in gene transcription of identified genes in the presence and absence of hormones. Our laboratory is in the process of identifying other proteins that differed in their expression when in the presence of estradiol. High precentage SDS-PAGE is being used to isolate lower molecular weight proteins.


Project 3: Dissecting p53 Action in a Mouse Lymphoma Model Scott Lowe, Ph.D. p53 is a potent tumor suppressor that limits cancer development and contributes to the anti-tumor effects of certain cytotoxic agents. Accordingly, p53 mutations are associated with aggressive cancers, poor patient prognosis and, in some cases, therapy resistance. Our project studies the p53 network using the Ep-myc transgenic mouse, a well characterized model of B cell lymphoma in which p53 profoundly impacts tumorigenesis and treatment outcome. We previously used the E?mu?-myc system to study how p53 loss promotes tumor development and drug resistance, and to explore the relationship between cancer genotype, tumor progression, tumor maintenance, and treatment sensitivity. These studies produced new insights into p53 regulation and effector mechanisms in diverse contexts where it limits proliferation. They also established that p53 loss is required for tumor maintenance and that translational control of cell survival is a druggable mediator of oncogenesis. To facilitate our goals, we developed methods to genetically manipulate E?mu?-myc lymphomas, enabling the rapid and cost-effective production of mice harboring tumors with diverse genotypes. We also incorporated small animal imaging methods to track lymphoma progression and response, and stable and inducible RNAi technology to fine tune and study loss of function phenotypes. Moving forward, we seek to further explore the interplay between cancer genetics and cancer therapy in the E?mu?-myc system in order to produce a more complete picture of the p53 network and its action in vivo. We also wish to understand in detail how p53 mutations impact tumor behavior, and identify new therapeutic targets based on the vulnerabilities they create. We will perform focused RNAi screens to identify new modulators of p53 action relevant to suppressing tumorigenesis and/or modulating therapy response, test the hypothesis that the deletions on chromosome 17p influence cancer biology beyond simply inactivating p53, and identify and characterize drug targets that interact with the p53 tumor suppressor network. Our approach uses genomic data from human lymphoma to inform function studies in mice, and implements a suite of new animal modeling and genetic tools that are time and cost effective.


The purpose of this project will be to evaluate a simple blood test and an interview process proposed as methods to screen for the presence and control of diabetes. If accurate these tests may be clinically useful for dentists interested in treating geriatric patients and needing to asssess the presence and degree of control of diabetes in this population prior to initiating dental treatment. Methods: 100 subjects of which at least 30 will be diabetic, will be recruited from the patient population of two extramural geriatric dentistry clinics run by the U.S.C. School of Dentistry. A medical history and relevant symptom report will be taken from each subject using an interview procedure. In addition, the diabetic subjects will be asked to respond to questions regarding the history and control of their diabetes. All subjects will be asked to fast from midnight, and will then have three tests run: a venous blood glucose, a hemoglobin A 1c, and a capillary blood glucose test. Those subjects who have never been diagnosed as being diabetic will then be given a 75 gram carbonated drink for glucose testing. Two hours later venous and capillary blood will be drawn. The data received will be tabulated and coded and subjected to descriptive statistical analysis. Significance: It has been well-reported that the prevalence of diabetes increases with age. With the increasing numbers of elderly patients seeking dental care, and the significant complications that can occur in treating an undiagnosed or uncontrolled diabetic, and in-office technique to uncover undiagnosed diabetics or to assess the level of control of diagnosed diabetes is needed. Proper understanding of the role of diabetes in the production and maintenance of oral disease will not only benefit the patient community, but it can be of great significance to dental clinicians who often treat oral diseases as isolated phenomenon with little or no knowledge of the interrelationship between unsuccessfully managed or undiagnosed diabetes, and the oral problem they may be seeing.


Mucosal organs such as the intestine are highly vascular organs with extensive metabolic demands. Epithelial cells which line the intestine function to orchestrate a multitude of mucosal responses, and given their anatomic location, are primary targets for diminished blood flow and resultant tissue hypoxia. Our previous studies have explored the response of intestinal epithelial cells to hypoxia and these studies defined a transcriptional signaling pathway mediated by hypoxia-inducible factor (HIF). Activation of HIF results in the coordinated induction of a cluster of apically-localized, barrier protective gene products. Such induction parallels the accumulation of polymorphonuclear leukocytes (PMN, neutrophils). In this proposal, we will test the hypothesis that HIF coordinates protective epithelial responses to hypoxia. Three specific aims are proposed to test this hypothesis. First, we will elucidate the role of PMN to inflammatory hypoxia using in vitro and in vivo models of intestinal inflammation. Second, we will build on recent findings to further explore the role of HIF signaling to intestinal inflammation. In particular, we will define the contribution of HIF-1 and HIF-2 to protection afforded by inflammatory hypoxia. Third, we will extend our recent findings with pharmacological approaches that activate HIF (prolyl-hydroxylase inhibitors) to define specific targets and mechanisms of protection in both chemically- and genetically-induced murine models of intestinal inflammation. The overall aim of this proposal is to elucidate the how hypoxia and inflammation coordinately influence disease outcomes at the mucosal interface. PUBLIC HEALTH RELEVANCE: These studies are proposed to better understand basic mechanisms of inflammation in the intestine. Specifically, these studies will define how metabolic shifts present during episodes of inflammation might be harnessed to develop novel therapies for mucosal diseases. It is our hope that extensions of this work in human patients might impact inflammatory disease outcomes.


The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signaling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behavior that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signaling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signaling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents. Protein kinase C, the best studied downstream target for diacylglycerol, represents the classic system for tumor promotion and is a therapeutic target for cancer chemotherapy. The vanilloid receptor represents a promising therapeutic target for cancer pain, among other indications, and thus represents an important direction in palliative care for cancer patients. DAG-lactones represent a synthetically accessible platform for probing the structure activity relations of protein kinase C and the other targets downstream of the second messenger diacylglycerol. We have continued to evaluate DAG-lactones designed to have selectivity for the RasGRP subclass of diacylglycerol targets. This class is of particular importance because it functions as an activator of Ras, which is many tumors shows enhanced activity without being mutated. The atypical protein kinase C isoforms zeta and iota play important role in cellular polarity and cellular invasion. We have previously identified the specific structural differences between their DAG recognition domain and that of typical DAG responsive signaling proteins such as the classical and novel PKC isoforms. We have described a first generation of DAG lactones with selectivity for the specific differences in the recognition domains of PKC zeta and iota. We have evaluated the role of aliphatic side chains on the DAG lactone for providing selectivity for protein kinase C epsilon, a pro-tumorigenic protein kinase C isoform. Bryostatin is an agent in clinical trials with a unique mechanism of action. It binds to protein kinase C with high affinity and activates the enzyme but paradoxically antagonizes many protein kinase C mediated responses. We find, using microarray analysis of gene expression along with detailed examination of the time and dose dependence of genes representative of the differences in expression revealed by the microarray analysis, that transient duration is the predominant difference in the mode of action of bryostatin as compared to typical protein kinase C activators such as the phorbol esters. In collaboration with the groups of Gary Keck and Michael Krische, we seek to define the critical structural elements in bryostatin conferring its unique pattern of activity, with the goal of developing the next generation of bryostatin analogues. One recent insight is our identification of the B ring structure as critical for the bryostatin like activity. Protein kinase C is subject to post-translational modification, in particular phosphorylation. In collaboration with the CCR Collaborative Protein Technology Resource, we have shown that the extent of protein kinase C modification is much more extensive than had been recognized. Moreover, the pattern of modification was different for different ligands such as phorbol ester or bryostatin. We suggest that such modification signatures may be of particular value for the structure activity evaluation of ligands with complex effects where, as in the case of protein kinase C, ligand binding causes both activation and change in subcellular localization. For protein kinase C or other targets of diacylglycerol, ligand binding drives membrane association. We show that the charge on the DAG recognition domain plays a critical role in the selectivity for membrane phospholipids. Further, this selectivity also depends on the specific ligand, with more hydrophilic ligands showing greater sensitivity to membrane composition. On our TRPV1 project, we have made considerable progress in defining the optimal structures for binding of antagonists to human TRPV1, as part of our effort which seeks to advance the development of drug candidates for this target. We further are continuing our efforts to use photoaffinity labeling to confirm directly the ligand binding site on TRPV1 as well as to use mutational analysis to refine computer modeling of the putative binding site.


Centralized Omics REsource (CORE) Technologies is a multiple award Indefinite Delivery Indefinite Quantity (IDIQ) begun in 2015 and provides IDIQ contract awards for whole genome sequencing and other omics measures, including DNA methylation, RNA expression patterns, and metabolic profiling. CORE, in conjunction with NHLBI's overarching TOPMed Program, seeks to discover factors and variants that influence disease risk, identify subtypes of disease, and develop more targeted and personalized treatments. Studies and proposed study designs and omics are selected via peer review and are approved samples supplied to one of eleven selected omics centers; the resulting raw and processed data is processed using TOPMed approved and designed pipelines and shared with originating studies, TOPMed PIs working in TOPMed domain working groups, and are also made available to NHLBI directed data repositories for sharing with approved investigators. TOPMed studies include multiple non-European (underrepresented populations) as well as studies that include or are entirely female.


Icosahedral capsid assembly is a highly coordinated process involving sequential addition of multiple proteins, ultimately leading to an infectious virion of proper size and morphology. The long-term goal for this project is to achieve a mechanistic understanding of the protein: protein interactions involved in capsid assembly. The development of new anti-viral drugs is impeded by a lack of understanding of how viral capsid proteins are programmed to adopt the correct conformations to produce the correct assembly product. Capsid assembly will be investigated using bacteriophage P22, a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the capsid is formed from a coat protein having the ubiquitous HK97 fold. The initial assembly product is a procapsid (PC). Scaffolding protein (SP) directs proper assembly of coat protein (CP) to form PCs. SP also directs the incorporation of the portal protein complex, which is essential for genome encapsidation. Phage P22 provides an excellent model assembly system because complex in vivo processes are easily mimicked in vitro. The simple genetics and well-established biochemistry of phage P22 offers significant advantages as an assembly model over complex mammalian dsDNA viruses. Our central hypothesis is viral capsid assembly is driven by specific weak protein: protein interactions, which control nucleation and elongation to form the proper assembly products. In this granting period we will test our central hypothesis with the following aims. Aim 1. Define how communication between domains of coat protein affects capsid morphology. Our data suggest that proper capsid morphology is controlled by communication between domains of CP, thereby affecting the curvature of the subunit. We will test this hypothesis by generating and characterizing site- directed mutants in different domains of P22 CP. Aim 2. Determine the structure and function of the I-domain from distantly related phages. We defined important roles for an inserted domain in the folding and assembly of P22 coat protein. Phages Sf6 and CUS-3 have low coat protein sequence identity but have an identifiable inserted domain. We will determine the NMR solution structure, and the function of the inserted domain in the CP folding and assembly from these related phages. Aim 3. Elucidate the protein conformational changes occurring during assembly. Protein conformational changes regulate proper capsid assembly. We will use single molecule FRET experiments to understand how individual capsid proteins in a population change conformations between monomeric and assembled states. Aim 4. Investigate the assembly of the portal protein complex during PC assembly. Portal protein is essential for genome packaging, yet how this complex is assembled into procapsids at a single vertex is not understood. Incorporation of portal protein will be characterized using RNA atpamers identified by SELEX.


Studies of the effects of environmental agents on human health are a vital part of the mission of the institute. As such studies are almost always observational rather than experimental, interpretation of the data is complex. Good statistical collaboration in all phases, including conception, design, analysis, and interpretation, is essential to the success of this work. During the current year, we were active collaborators on many projects. Included were studies evaluating biomarkers, examining health effects of various environmental or occupational exposures and drugs, and describing biology of several conditions.


Cervical spine injuries, degenertion (spondylosis), nerve root compression (radiculopathy) and spinal cord compression (myelopathy) are the major causes of pain, deformity and neurological disfunction in the cervical region. Studies of the spinal column alone, while important, provide limited information about the cord and nerve roots. The purpose of the present proposal is to study in a comprehensive manner the mechanical basis of local, radicular and myelopathic symptomatology in the cervical spine. Such information is not available presently. This comprehensive study has three basic parts. Firstly, to determine ligamentous strains and intervertebral foramenal changes due to physiological movements of the cervical spine. Secondly, to measure physiological movements of, and strains in,, the nerve roots and spinal cord. Finally, to quantify 3-dimensional anatomy of the cervical spine in various physiological postures using cryomicrotome sectioning, X-rays and CT-scans. Significance of this work will be in several different areas. The comprehensive set of biomechanical data about the functioning relationships between the various components of the cervical spine system, namely, vertebrae, ligaments, spinal cord and nerve roots will be available for the first time. It will provide the basis for understanding the functioning of the cervical spine system. Thereby making it possible to correlate precisely the anatomic derangements with clinical presentation of symptoms in a patient. It will also be helpful in the design of new and more efficient diagonostic and treatment modalities for the various problems of the cervical spine.


The objective of this project is to develop and implement sophisticated point-of-care EHR-based clinical decision support that (a) identifies and (b) prioritizes all available evidence-based treatment options to reduce a given patient's cardiovascular risk (CVR). After developing the EHR-based decision support intervention, we will test its impact on CVR, the components of CVR, in a group randomized trial that includes 18 primary care clinics, 60 primary care physicians, and 18,000 adults with moderate or high CVR. This approach, if successful, will (a) improve chronic disease outcomes and reduce CVR for about 35% of the U.S. adult population, (b) maximize the clinical return on the massive investments that are increasingly being made in sophisticated outpatient EHR systems, and (c) provide a model for how to use EHR technology support to deliver "personalized medicine" in primary care settings. PUBLIC HEALTH RELEVANCE: The objective of this project is to develop and implement sophisticated point-of-care EHR-based clinical decision support that (a) identifies and (b) prioritizes all available evidence-based treatment options to reduce a given patient's cardiovascular risk (CVR). The prioritized list of treatment options is provided in different formats to both the primary care physician (PCP) and patient at the time of each office visit made by a patient with moderate to high CVR and sub-optimally controlled and potentially reversible CVR factors. Available evidence-based treatment options are prioritized based on the magnitude of potential CVR reduction of each treatment option. This intervention strategy, referred to as Prioritized Clinical Decision Support (PCS), is specifically designed for widespread use in primary care settings and has the potential to substantially augment current efforts to control CVR in the 35% of American adults with 10-year Framingham CVR of 10% or higher. To assess the ability of the PCS intervention to reduce CVR in adults, we will randomize 18 primary care clinics with 60 primary care physicians (PCPs) and approximately 18,000 eligible adults with baseline Framingham 10-year risk of a major CV event (either heart attack or stroke) of 10% or more into one of two experimental conditions: Group 1 includes 9 clinics (with 30 PCPs and 9,000 patients) that will receive prioritized clinical decision support (PCS) to reduce CVR at the time of each clinical encounter made by an eligible adult. Group 2 includes 9 clinics (with 30 PCPs and 9,000 patients) that receive no study intervention and constitute a usual care control group. The study will formally test the hypothesis that after control for baseline CVR, post- intervention 10-year Framingham CVR will be better in Group 1 than Group 2 at 12 and 24 months after start of the intervention. In addition, impact of the intervention on specific components of CVR (BP, lipids, glucose, aspirin use, and smoking) will be assessed, and the cost-effectiveness of the intervention will be quantified. This innovative project builds upon 10 years of prior work by our research team, and extends prior successful EHR clinical decision support interventions by introducing prioritization, by providing decision support to both patients and PCPs at the time of the office visit, and by extending the decision support across the broad and critically important clinical terrain of CVR reduction. The results of this project, whether positive or negative, will extend our understanding of how to maximize the clinical return on massive public and private sector investments now being made in sophisticated outpatient EHR systems. If successful, this decision support tool could be broadly used to both standardize and personalize care delivered by case managers, pharmacists, and other providers in a wide range of care delivery configurations.