Datasets:

haritzpuerto

/

the_pile_00_NIH_ExPorter

like 0

Applicant's Abstract This grant will renew a 25-year training program in cardiovascular disease epidemiology and prevention. The overall purpose of the program is to produce behavioral and medical scientists who can conduct inter-disciplinary research aimed at the prevention of cardiovascular disease in communities. The training will be derived principally from direct research experience in an existing interdisciplinary research resource, the Stanford Center for Research in Disease Prevention (SCRDP). The SCRDP and its closely affiliated programs offer community, policy, behavioral, clinical, and laboratory studies for participation by trainees. Current SCRDP research totals about $10 million annually from NIH (66 percent), state, foundation, and industry sources. Current research programs include community-based interventions to reduce sales to and social exchange of tobacco among adolescents; evaluation of the California statewide tobacco control program; studies of ways to limit marketing and promotion of tobacco use; clinical trials of psychopharmacologic interventions for smoking and chew tobacco use; a study of advocacy training for youth at high risk for tobacco and drug use; a school-based study of the health effects of an intervention to reduce television watching; other school-based studies of nutrition and exercise interventions; studies of the effects of various dietary changes and supplements on cardiovascular and cancer risk factors; a study of exercise for preventing obesity and diabetes in poor Hispanic women; a study of exercise effects on health and well-being in elderly women care-givers; a study of exercise effects on sleep in older adults; and a study contrasting interventions based on two behavioral theories for achieving change. A program of directed study and data analysis and a core seminar program, required of each trainee, enhance the research training, as do selected opportunities for other coursework, limited patient care, and teaching. Postdoctoral trainees are closely supervised by the faculty and encouraged to publish three to four articles during their 1-3 years of training (average 2 years); predoctoral trainees are all enrolled in Ph.D. programs at Stanford University. Trainee selection is based on interest in cardiovascular disease prevention, potential for academic research career, and demonstrated excellence. Two predoctoral trainees will be selected from graduate students in the School of Education and the Departments of Epidemiology and Communication. Six postdoctoral trainees will hold either the M.D. or Ph.D. degree. Physician trainees will generally enter with three years of clinical experience in medicine, psychiatry, pediatrics, or preventive medicine. Two postdoctoral trainees will be able to enroll in a fourwquarter, 45-unit M.S. degree in epidemiology with an emphasis on clinical investigation methods.

Cyclin-dependent kinase 5 (Cdk5) is a neuronal kinase that has been implicated in many diverse processes in the central nervous system. Cdk5 has been shownto regulate dopamine signaling in the neostriatum and is a molecular target of chronic cocaine exposure. We intend to investigate the role Cdk5 plays in glutamtatergic neurotransmission in the ventral striatum (nucleus accumbens). Of particular interest is the role of Cdk5 in dopamine signaling, the main target of drugs of abuse. Using Cre/loxP technology, a conditional cdk5 knockout mouse has been generated to allow temporal control of tissue-specific knockout of Cdk5. The effect of striatal loss of Cdk5 on dopamine signaling pathwaywill be assessed using a pharmacological approach combined with quantitative immunoblotting. The electrpphysiological properties of striatal neurons will be defined following loss of Cdk5. These studies will be conducted following chronic cocaine exposure, in order to better define the role of Cdk5 in the neostriatum.

Abstract ? Project 1 African-American (AA) men experience the highest prostate cancer (PCa) incidence and mortality rates of all U.S. racial/ethnic groups. They are also known to present with more aggressive high-risk disease, especially of higher Gleason score and PSA levels. Factors contributing to the high burden of PCa among AA men are not known. AAs are exposed to considerably higher levels of social stressors such as institutional and interpersonal discrimination, crime, low socioeconomic status, social isolation, and resource-poor environments. These social stressors exist at multiple levels, from individual to neighborhood to institutional, and across the lifecourse, leading to chronic stress. Social stressors experienced among AA men may thus be a contributor to the development of aggressive PCa and high mortality. We will apply recently developed multilevel frameworks that emphasize the consideration and evaluation of exposures from ?cells to society? to understand how ?stress gets under the skin? to cause biological vulnerability, specifically the high burden of PCa among AA men. Our specific aims are: 1) Examine the associations between exposures to neighborhood social stressors and risk of aggressive PCa and mortality among AA and non-Hispanic White (WH) men. Among population-based samples of all AA (N=149,000) and WH (N=668,000) men diagnosed with PCa in the RESPOND catchment areas, we will link geospatial neighborhood data on segregation, racial composition, socioeconomic deprivation, and other social and built environment attributes to cancer registry data and examine the associations between these neighborhood factors and aggressive PCa and risk of mortality; 2) Examine the associations between exposures to multilevel social stressors across the lifecourse and risk of aggressive PCa among 10,000 AA men in RESPOND. Each stressor will be examined individually and combined, and for selected time points (early, mid, adult life) and cumulatively over time; 3) Examine the associations between exposures to multilevel social stressors across the lifecourse and genetic factors, as well as their combined effects in association with aggressive PCa. More specifically, we will assess the association between the multilevel social stressors and: a) proportion of African genetic ancestry, b) frequency and type of somatic profiles, and c) whether social stressors, germline genetics (including PCa aggressive loci), and somatic profiles are jointly associated with risk of aggressive disease. To address these aims, we have designed a multilevel study involving cross-sectional, prospective, and retrospective designs that integrates multilevel data from multiple sources including cancer registry, patient survey, geospatial (to characterize neighborhood-level stressors) and public record data (to construct adult residential history), germline genetics from Project 2 and somatic tumor profile data from Project 3. Covering 6 states and 1 metropolitan region, and representing 38% of all AA men with PCa in the U.S., RESPOND will represent the single largest coordinated research effort to study aggressive PCa in AA men, with an innovative focus in Project 1 on social stressor exposures that are most relevant to this population.

The goal of the proposed study is to study the neural basis of aspects of self and social cognition - negative self appraisals and elevated attention to negative emotional social signals - that are highly relevant to understanding the development of adolescent depressive disorders. Persistently negative self appraisal and elevated attention to negative emotional social signals, (e.g. negative facial expressions) are key processes that denote risk for depressive disorders across the lifespan. These processes are particularly relevant to understanding risk for depression in adolescence, because this is a period during which there is rapid transformation in self appraisals and interpersonal social functioning as part of the key developmental task of forming a positive and coherent self representation. Suboptimal resolution of this developmental task is linked to onset and recurrence of depressive disorders and risks for suicide in adolescence. Therefore, understanding the neural basis of negative self appraisals and attention to negative facial expressions in adolescent depression will provide valuable insights into specific neural mechanisms of depression during this vulnerable developmental period to guide intervention strategies. Furthermore, this research will also help identify objective, neurobiological markers of adolescent depressive disorders that can be used in the future to detect those adolescents who may be most at risk of future depression or who are on a trajectory to a recurrent course of the illness.

This project will continue a 30 year surveillance effort assessing the levels and patterns of substance use among American Indian adolescents attending reservation schools. Each year of the five year project a representative sample of 1500 Indian youth living on reservations will be given a comprehensive drug use survey in their school classroom. In addition, the survey will contain questions regarding violence, victimization and delinquent behaviors; which will continue surveillance of these behaviors over a 10-year period. The purposes of the surveillance work are to observe changes over time, to accurately describe these domains, to provide insight into the nature of drug use, violence and victimization and to inform the efforts of those designing and evaluating intervention efforts. Due to the continuing high rates of marijuana and drunkenness among American Indian youth in many reservation communities, reaching levels that are normative or near normative, extended survey data will be obtained on the attitudes and perceived norms for these behaviors. Substance-specific variables for marijuana, getting drunk (normative behaviors) and inhalant use (non-normative) will be obtained from reservation schools and from a random sample of non-Indian youth living in rural areas. New items regarding attitudes toward marijuana will be included in the survey and the Theory of Normative Social Behavior will be used to compare the attitudes between Indian and non-Indian youth. Hierarchical linear models will be used to assess both the individual and school-level effects of the perceived normative environment for these three types of substance use (alcohol, inhalants and marijuana) which represent varying levels of normative and non-normative use. In addition to the effects of the perceived normative environment, the role of cultural identification will also be examined as a moderating or mediating variable. A final goal of the project is to develop a series of recommendations, based on project findings, for the design of drug, alcohol and violence prevention programs that will be effective specifically for American Indian youth. Schools will be provided with extensive feedback on their survey results which is intended to raise community awareness of drug use levels and patterns, to obtain funding for prevention activities and to evaluate the effectiveness of prevention interventions that have been implemented in the community. PUBLIC HEALTH RELEVANCE: This project will continue 30 years of surveillance of drug use among American Indian youth. These data have been used by various agencies to create policies for reducing substance use for these youth. Importantly, the tribes involved have used the data to raise awareness of the levels of drug use in their communities, to evaluate prevention interventions and as a statement of need when applying for prevention funding.

In this proposal, we combine chemical and biochemical approaches to examine fundamental questions relating to the mechanism of action and toxicity of bleomycin. We recently isolated a series of nucleoside base-propenals produced by the action of bleomycin on DNA. These compounds proved highly cytotoxic and resemble bleomycin in their effects on tumor cells in culture. These observations lead to the development of a novel family of structurally-related site-directed inhibitors and suggest the possible involvement of base propenals in the toxic actions of bleomycin. We propose to explore the molecular basis by which bleomycin produces strand-scission in DNA and to define the mechanisms involved in the production of base propenals and free bases which are released during this process. Experiments have been designed to precisely identify products of reactions in which oligonucleotides of defined base sequence are cleaved by bleomycin. The action of "activated" bleomycin on organic molecules will be utilized to establish chemical mechanisms involved. The reaction of ionizing radiation with DNA, a process which resembles the action of bleomycin, will also be examined. Finally, we propose a series of studies to test the hypothesis that base propenals produced by the action of bleomycin on nuclear DNA account for the cytostatic and cytotoxic properties of the drug. Results of these experiments should contribute to an understanding of radical mechanisms involved in the degradation of DNA by drugs, chemical and gamma-irradiation. They hold potential significance for the design and development of new analogs of bleomycin.

Our long-term objective is to understand the molecular and anatomical basis of cortical plasticity. Closely allied to this aim is to understand whether remote memory is based on cortical plasticity processes and more specifically whether remote memory depends on structurally based cortical plasticity. To these ends we will test two main hypotheses: that [unreadable] Animals showing remote but not short-term memory deficits will show experience-dependent cortical plasticity deficits [unreadable] Remote memory deficits will be associated with specific deficits in experience-dependent dendritic and spine plasticity By testing these hypotheses on mutants with deficits in both remote memory and cortical plasticity we will simultaneously begin to reveal the molecular basis of structural cortical plasticity. To test these hypotheses we plan to study four main properties of barrel cortex in animals generated by the remote memory screen: (1) the ability of barrel cortex to undergo experience-dependent plasticity (EDP), (2) normal anatomical and receptive field development of the cortex, (3) spine and bouton stability/turnover in whisker deprived and undeprived animals, (4) excitatory synaptic transmission and the ability to undergo spike-timing dependent plasticity (STOP). By identifying the mechanisms involved in remote memory and cortical plasticity, we may acheive several objectives: the capactiy for modification could be extended in cases of impaired development;insight could be gained into memory and learning deficits in adults;new approaches could be envisioned for restoring cortical function after brain damage. These objectives are therefore directly related to the agency's mission to improve public health because they are aimed at understanding processes that go wrong in disease conditions such as Alzheimer's and restoring function in trauma conditions such as stroke.

The brush border (BB) Na/H exchanger NHE3 is rapidly up and down regulated as part of digestion, with[unreadable] prolonged inhibition contributing to the pathophysiology of diarrhea. NHE3 exists in the BB in large,[unreadable] multiprotein complexes of varying size. In these complexes, NHE3 associates with 4 BB PDZ domain[unreadable] containing proteins, NHERF1, NHERF2, PDZK1 and IKEPP. The BB PDZ proteins are in somewhat different[unreadable] locations in Na absorptive cells, although each has a BB component. This project will test the hypotheses[unreadable] that acute NHE3 regulation in intestinal BB requires its presence in large multiprotein complexes scaffolded[unreadable] by these BB PDZ proteins; and that these complexes are dynamic and change in their location and makeup[unreadable] as part of NHE3 regulation. Proposed studies will knock down (shRNAi in Caco-2 cells) and out (gene[unreadable] targeting in mice) each of these BB PDZ proteins alone and in combinations. Physiologic and[unreadable] pathophysiologic regulation of NHE3 in mouse ileum and Caco-2 cells will be evaluated using the Ussing[unreadable] chamber/voltage clamp approach and two-photon microscopy with SNARF-4F to measure intracellular pH.[unreadable] Basal and acutely stimulated and inhibited NHE3 activity will be examined, with determination whether[unreadable] trafficking of NHE3 is affected; what size complexes in the BB NHE3 and the BB PDZ proteins are in, using[unreadable] sucrose density gradient centrifugation, under these conditions; and whether NHE3/NHERF proteins change[unreadable] their direct association as part of NHE3 regulation as assessed by FRET. Knockout/down models of the 4[unreadable] BB PDZ proteins (NHERF1, NHERF2 already on hand and PDZK1 is available) will be evaluated to[unreadable] understand their contribution to NHE3 regulation. NHE3 associates with the cytoskeleton directly by ezrin[unreadable] binding and indirectly by binding to ezrin via binding NHERF1, NHERF2 and PDZK1. Point mutations of[unreadable] NHE3 have been identified which separately eliminate this direct and indirect ezrin binding. These mutants[unreadable] will be expressed in Caco-2 cells and effects determined on NHE3 BB localization and on NHE3 basal and[unreadable] regulated activity, NHE3 complex formation and associating proteins, as well as on NHE3 mobility in the BB[unreadable] as assessed by FRAP. These studies will provide insight into how salt is absorbed normally in the intestine[unreadable] and becomes abnormal in diarrheal diseases.

The goals of our work are to determine the extent to which mRNA populations of adult, immature, regenerating and neoplastic rat liver may differ quantitatively and qualitatively from each other and to identify messenger RNAs (mRNA) and sets of active genes which might be specific for a certain growth pattern. We have established that differences between mRNA populations of normal and regenerating liver are quantitative rather than qualitative and that the transcription of at least two oncogenes increases in parallel with DNA synthesis during liver regeneration. Cells of the normal liver appear to contain most, if not all, of the cellular sequences required for neoplastic transformation; during liver carcinogenesis restriction of genomic expression occurs.

The Clinical Services Core of the CNMD represents a unique resource that is organized around the recruitment and characterization of unmedicated schizophrenic subject at both early and later phases of the illness. Such subjects represent an extremely limited and important patient population. Specifically, the Clinical Services Core: 1) screens, recruits and assesses subjects using standardized assessment methods found in the Core Assessment Battery in order to generate a uniform data base shared by all investigators; 2) provides reliable and valid diagnostic evaluation using a well-developed longitudinal methodology and initial clinical follow-up and care; 3) utilizes well developed policies and procedures to coordinate a range of protocols and avoid duplication of efforts; and 4) Provides clinical and diagnostic expertise for postmortem diagnostic conference for the Human Brain Bank Core-B. Because the Clinical Services Core integrates and coordinates the recruitment and assessment of subjects, Projects 7- Cohen and 8-Sweeney in the CNMD and other funded collaborating investigators shares these subjects efficiently and productively. The Clinical Services Core also relies on the Statistics and Data Management Core-D to provide data management for the Core Assessment Battery.

The research can be divided into three parts. The first is the analysis of the arrangement of the different classes of base-pair sequences in the chromomeres of Drosophila chromosomes through the isolation of homogeneous populations of hybrid DNA molecules between fragments of Drosophila chromosomal DNA and the lambda bacteriophage or plasmid DNAs. E. coli are infected with hybrid molecules which are constructed by enzymatic methods in vitro; clones of infected cells are then isolated which contain specific hybrids as autonomously replicating plasmids or as prophage; and the hybrids isolated from the clones by standard methods. The particular chromomere(s) associated with a specific hybrid can be determined by in situ hybridization to polytene chromosomes, and the sequential arrangements in the chromomeric DNA determined by techniques worked out for viral DNAs. The second project derives from our ability to isolate Thomas circles formed fragments of chromosomal DNA after exonuclease treatment. This is a powerful tool for the topographical analysis of proximate repetitive sequences in animal chromosomes. We are presently using this tool to determine the arrangement of moderately repetitive sequences in the Drosophila chromosomes. The last project is concerned with the regulation of replication origins in Drosophila chromosomal DNA. In the first phase we have determined the fork rates and the distribution of origins in the rapidly replicating clevage nuclei (S phase equals 3-4 min) and in cell cultures (S equals 600 min). A model derived from these results is presently under test.

Lactoperoxidase, peroxide, and thiocyanate ion form an antibacterial system in saliva. The proposed research is designed to determine how this system kills or inhibits growth of bacteria. Our goal is to find ways to increase the effectiveness and selectivity of this natural defense that will be relevant to prevention of oral disease. Also, it will be determined whether the mechanism of action of the salivary system is different from that of the leukocyte antimicrobial system, myeloperoxidase, peroxide, and halide ion. Such studies may help to obtain more effective prevention of infection in other parts of the body. The chemistry of antibacterial reactions will be studied using radioactive forms of halide ions or thiocyanate. The chemical nature and cellular location of chemical modifications will be determined. These results will be correlated with studies on killing or growth inhibition, to determine which chemical reactions are essential to antibacterial action. To make more effective use of antibacterial action in the oral environment, the optimum conditions for action will be determined, and the role of bacterial cell structure and metabolism in determining susceptibility will be studied. Also, the combined action of the peroxidase systems and other agents, such as salivary components, will be studied to attempt to make antibacterial action selective against oral pathogens. BIBLIOGRAPHIC REFERENCE: Aune, T.M. and Thomas, E.L. (1976) Modification of Protein Sulfhydryls by Products of Peroxidase-Catalyzed Oxidation of Thiocyanate Ion. Fed. Proc., 35, 1630.

DESCRIPTION: How Plasmodium sporozoites invade hepatocytes, and develop within them, remains largely unknown. Our Preliminary Studies indicate that sporozoites can rapidly enter and leave host cells by disrupting the host cell plasma membrane. After few cycles of non-productive entry and exit, sporozoites invade host cells, and develop into the exo-erythrocytic stages. We propose that the passage of sporozoites through several cells is a required maturation step in the life cycle, and that it occurs during the parasite's journey from the site of entry in the skin to the liver. This hypothesis will be tested using cell and molecular biology approaches involving in vitro and in vivo models for hepatocyte infection. Our Preliminary Studies suggest that Plasmodium sporozoites ensure their complete development into the exo-erythrocytic stages by inhibiting apoptosis of hepatocytes. In contrast, irradiation of sporozoites prior to infection results in hepatocyte apoptosis. We propose to determine the genetic pathways involved in apoptosis inhibition of hepatocytes, and to identify the Plasmodium genes that contribute to this process, by examining gene expression in the host and pathogen in the presence and absence of irradiation. A protective immune response to malaria is induced by immunization with irradiated sporozoites. We propose that the apoptotic bodies derived from hepatocytes infected with irradiated sporozoites mediate the initiation of a protective anti-malaria immune response. Immunological and cell biological approaches will be used to further examine this hypothesis in vitro and in vivo.

The proposed Flint Center for Health Equity Solutions will establish an NIMHD Transdisciplinary Collaborative Center (TCC) for health disparities research on chronic disease prevention to be based in Flint, Michigan. This proposal was developed in collaboration with community members from its inception, and evolved from a conversation between Flint community members and the Flint-based Michigan State University researchers that they helped to hire. The TCC will strengthen community-engaged health disparities research in the Flint area, serving as regional focal point for organizing and nurturing productive working relationships with across a broad cross-section of stakeholders with an interest in eliminating health disparities. The TCC has 4 cores and 2 intervention research projects. Research Project 1 will examine the effectiveness of a community-designed, community-based, multilevel physical activity and healthy food intervention (the Church Challenge) relative to enhanced treatment as usual in primarily African-American Flint-area churches. The intervention targets individual, church, and church as driver of community policy-level changes (e.g., improving healthy food and physical activity opportunities in Flint). Research Project 2 will evaluate a multi-tiered intervention program for men and women in recovery from substance abuse. Tier 1 consists of a peer coaching and advocacy recovery support service. Tier 2 is an evidence-based Strengthening Families Program to support family reunification and support. Each of the proposed projects represent an area of unmet need to address health inequities, including access via affordable health care; structural inequality and social isolation and stratification. Each project has high potential for translation. The TCC's Administrative Core will provide leadership for the Center's overall strategic planning, including scientific leadership and oversight. This Core will manage, coordinate and supervise the entire range of proposed TCC activities, monitor progress and ensure that component plans are carried out. The Consortium Core will organize and nurture productive working relationships with a broad cross-section of academic partners, community organizations, minority and health disparity populations, health care provider organizations, for-profit or non-profit organizations and foundations, governmental agencies, and other stakeholders to advance TCC-related health equity efforts. The Methodology Core will conduct a community needs assessment and provide the TCC with statistical, mapping, and cost-effectiveness analysis services. The Dissemination and Implementation Science Core will develop and manage a highly effective translation program (i.e., conduct research dissemination) and also produce generalizable knowledge about how best to do so (i.e., conduct dissemination and implementation research that is synergistic with other Center efforts). Our initiatives will reduce health inequities in Flint, Michigan and provide promising insights to promote translational approaches in Region 5 and more broadly across the nation.

DNA mismatch repair (MMR) exemplified by the E. coli methyl-directed MMR pathway, targets base pair mismatches that arise through DNA replication errors, homologous recombination and DNA damage. Inactivation of MMR results in a large increase in the rate of spontaneous mutation and is associated with both sporadic and hereditary cancers. In addition to its role in post-replication repair, components of the MMR pathway also influence the expansion of trinucleotide repeats associated with syndromes such as Huntingtons disease, play an essential role in assuring the proper pairing of chromosomes during meiosis, modulate homologous recombination involving closely related sequences, and participate in the generation of antibody diversity at immunoglobulin gene loci.[unreadable] [unreadable] A key step in mismatch repair is the recognition of DNA mispairs by MMR proteins and the licensing of excision repair. This is a critical problem in cells because the gapped DNA intermediate of MMR is easily converted into lethal double-strand breaks if not efficiently repaired. In collaboration with Dr. Dorothy Erie, we are using atomic force microscopy (AFM) to examine the conformations of individual protein-DNA complexes that are formed during MMR. Measurements of fractional occupancies at specific locations on a mismatched DNA allow us to determine the binding affinity, specificity, and stoichiometry of MutS bound to mismatched DNAs. Single molecule studies of MMR proteins harboring single amino acid changes in the mismatch binding site are being exploited to define key aspects of mismatch recognition. We are also examining how human MMR proteins MutSalpha (hMSH2-MSH6), MutSbeta (hMSH2-hMSH3), and MutLalpha (hMLH1-hPMS2) interact with each other at the sites of DNA mismatches and how nucleotide cofactors modulate such interactions and license downstream excision steps. These studies have important implications for understanding how this mismatch repair pathway contributes to genome stability and cancer avoidance.

The objective of this mechanism-based research is to devise safe and efficient protein particulate nanocarriers (PPC) for small RNA delivery. To accomplish this, we plan to exploit the intracellular trafficking machinery to direct the delivery f small RNAs to their site of action while maximizing small RNA delivery. Small RNAs are used in a range of health applications. However, their potential has yet to be fully realized. This is largely due to inefficient delivery: only 1-2% of small RNAs reach the RNA-induced silencing complex (RISC): the site of action using conventional techniques. Small RNA delivery is a multistep process in which inefficiencies at any stage can compromise the efficacy of gene silencing. In particular, the intracellular fate of synthetic carriers is not well understood and tus poorly controlled. Recent studies indicate that active RISCs are functionally and physically coupled to MVBs and are not free in the cytoplasm as previously thought. Thus, we hypothesize that the effect of small RNA delivery can be significantly increased by actively targeting MVBs. We propose the design of PPCs containing signaling moieties that are recognized by the cell sorting machinery and that serve as molecular zip codes for the directed transport of small RNAs to the RISC. This could ultimately lead to enhanced gene silencing. This strategy could improve RNA-based therapy by allowing for the administration of lower doses of therapeutics, thereby reducing side effects and improving safety profiles.

During the past 40 years there has been a remarkable decline in the prevalence of dental caries in children and adults due to the widespread use of fluoride in public health programs, such as communal water fluoridation, dental practice and in oral hygiene products. In spite of this dramatic improvement, dental caries continues to be the most prevalent dental disease. The early detection and monitoring of tooth surface demineralization is a critical step in reducing the prevalence of dental caries. The identification and validation and then use of new technologies should increase the efficiency of caries clinical trials and reduce their cost. One of the major goals of this grant is to validate new and refined technologies for the clinical assessment of tooth surface demineralization. This proposal will concentrate on using Quantitative light-induced fluorescence (QLF) alone, in combination with Red Fluorescence, and with the incorporation of a new hand piece and drying procedure. These technologies are designed to detect early signs of demineralization and should be able to monitor lesion progression and/or regression. This grant will also use the recently developed International Caries Detection and Assessment System (ICDAS) for the visual exam and record both cavitated and non-cavitated lesions. This grant will take advantage of the working relationship developed between three established teams of Cariologists during their previous program project grant and the experiences gained from that endeavor. As such the aims of this grant will include both in vitro studies (Iowa) to monitor demin/remin as well as in vivo studies (Indian and Texas) to validate monitoring of demin/remin clinically. The data generated will then be used in the fourth specific aim to develop a guide for clinical decision making that include the QLF measurements and the ICDAS visual exam. The clinical validation studies will use an Orthodontic population for permanent caries (SA 3) and a child population for deciduous caries (SA 2). The "exfoliated tooth model histopathology" will be used with the studies on the deciduous dentition as the gold standard for validation. The clinical data will be paired with the PLM for validity measurements and analyzed by the biostatisticians.

The specific role of epinephrine and cortisol in modulating the inflammatory response elicited during endotoxemia is not well defined. The proposed study seeks to assess in normal subjects, the effect of elevated levels of cortisol, catecholamines or both during five days, upon the ex vivo cytokine production by whole blood in response to endotoxin.

The study is currently in progress. 11/12 subjects have completed both interaction portions of the study.

The project on the effects of retinoic acids on brain, behavior and drug interactions investigates the pathophysiology of retinoic acid action in vivo. Rats are the current model but expansion into mouse and guinea pig models is anticipated. The primary focus of the rat model is the assay of the pharmacokinetics of the interaction of 13-cis-retinoic acid with neuroleptics. Early results have shown statistically significant changes in the blood levels of haloperidol and one of its metabolites after the concurrent administration of 13-cis-retinoic acid to rats.

We developed methods to build confidence interval and bands to detect time-varying effects of treatments in Cox's proportional hazards regression model. Dr. Sundaram and her collaborators have addressed in the need for statistical inference which in confidence intervals and confidence bands which give more tighter intervals than the standard Wald-type (normal approximation based) confidence intervals/band. This work extended the results of Tian, Zucker and Wei (2005, JASA) and shows that the proposed intervals/bands are tighter than those proposed by Tian, Zucker and Wei. This was achieved by developing empirical likelihood (EL) point-wise confidence regions for the time-dependent regression coefficients via local partial likelihood smoothing. Asymptotic properties were established for the proposed methods. Extensive numerical studies conducted indicated that the EL point-wise/simultaneous confidence regions/bands have better performance than the Wald-type estimators. The proposed methods illustrated on two real examples: the gastric cancer data and the Mayo Clinic primary biliary cirrhosis data showed similar findings of more precise (narrower) confidence intervals (bands). [unreadable] [unreadable] [unreadable] Another project developed method for randomly truncated data which are frequently encountered when the study design is retrospective and/or due to inability of experimental design to be able to capture the study participant before the initiation of the event. For example, pregnant women get selected based on their first visit to the gynecologist for confirming their pregnancy, resulting in loss to follow up of women who had early pregnancy loss. Estimation based on randomly truncated data becomes very challenging as the risk set over time is non-monotonic, making it very different from random right censoring. One of the problems addressed by Dr. Sundaram is developing robust inference for two sample accelerated failure time data for this type of data. Dr. Sundaram has also developed robust methods for analyzing proportional odds model for randomly truncated data. Proportional odds model provides a useful alternative to proportional hazards when the hazards converge over time (e.g., for modeling treatments that are successful). The large sample properties like asymptotic normality and strong consistency of the proposed estimators were established and the finite sample properties investigated through extensive simulations indicating good performance. The proposed methods are easy to compute, which is not the case with likelihood based estimators as it is not possible to profile out the non-parametric (baseline odds function) as is done with proportional hazards and right censored data.

One of the main problems in patients with carcinoma of the prostate is the pain suffered from skeletal metastases. Several radionuclides such as Sr-89 which concentrate in such metastases are used to alleviate the pain and its effect on the quality of life. The aim of this project is to ablate the skeletal secondary deposits. To do this we give greatly increased activities (so far up to 4000 MBq compared with the more usual approximately 2000 MBq) of Re-l86 HEDP with peripheral blood stem cell support (PBSCS) to overcome the effects of bone marrow irradiation by uptake of the radiopharmaceutical in normal bone. So far it seems that the upper levels of activity we have given are more effective in ablating metastases not visible on the bone scintigram rather than ablating those already visible. In a Phase I activity escalation protocol we are treating patients with skeletal metastases which have escaped hormonal control. On finishing the Phase I study at the end of April 2000 we will have determined the maximum activity which can be administered safely and will use this level of activity in the Phase II study which is the subject of this grant application. In this Phase II study we will measure the number of new metastases at three to twelve months and analyze the behavior of the metastases originally visible. Based on the results of this study we will then design a new study comparing the effect of high dose unsealed source therapy with peripheral blood stem cell support in patients with rising prostate specific serum antigen but no visible metastases with those receiving standard therapy alone. We will analyze the data already acquired from the Phase I study to attempt to determine a metastatic dose response relationship. From this we will be able to evaluate the possibility of giving even higher activities of a shorter half-life bone seeker such as Sm-153. We would then be able to give higher doses/dose rates yet have lower residual radiation at 12 days post treatment when the peripheral blood stem cells are re-infused.

This Program Project is based in the Linus Pauling Institute, an emerging international leader in research and education on micronutrients and antioxidants, and one of a few centers in the US to focus entirely on health promotion and disease prevention by dietary and CAM approaches. The Center of Excellence for Research on Complementary and Alternative Medicine (CAM) Antioxidant Therapies (CERCAT) will investigate two specific categories of CAM antioxidants: (i) Antioxidants that modulate the cellular redox environment and, thus, cell signaling and transcriptional activation, e.g. by affecting critical thiols with a low pKa or upregulating endogenous antioxidant systems. The CAM antioxidants to be investigated from this category are dithiol compounds (e.g. alpha-Iipoic acid) and metal chelators (e.g. EDTA and desferrioxamine). (ii) Highly conjugated or aromatic compounds that inhibit tyrosine nitration by peroxynitrite and other reactive nitrogen species. The principal antioxidant to be examined in this category is uric acid. Using cell culture studies and relevant animal models, CERCAT will determine the molecular and cellular mechanisms of action of these CAM antioxidants, and their safety and efficacy in treating amyotrophic lateral sclerosis (ALS) and cardiovascular diseases (CVD) and reversing the loss of cellular resistance to stress that occurs with aging. These goals of CERCAT are buttressed by NCCAM's "increased emphasis on studies of the mechanism underlying CAM approaches" and its "FY 2003 Research Priorities" of "studies of the biology of EDTA chelation therapy in animal models of CVD" and "neurodegenerative disorders using in vitro studies and animal models." CERCAT's research goals will be accomplished through three highly interactiveprojects: 1) "Metal chelators and thiols in endothelial function, and CVD" (Balz Frei); 2) "Lower vulnerability to toxins in aging by treatment with lipoic acid" (Tory Hagen); and 3) "CAM antioxidants and ALS" (Joseph Beckman). Center Investigators will be aided by an Administrative Core, which handles budgetary, reporting, and external advisory needs. In summary, CERCAT will investigate the efficacy of CAM antioxidants in ALS, CVD and aging, and provide the essential knowledge about the underlying mechanisms, dose-response effects, and relevant biological targets to advance these CAM therapies to human trials; equally important, the studies will test for untoward effects that might discourage CAM antioxidant therapies from proceeding to human studies.

The long-term goal of our research is to understand the mechanisms that generate neuronal networks during development and then apply this knowledge to regenerating the neural circuitry lost after injury or neurodegenerative diseases. An important step towards this goal is to identify the molecular cues that permit axons to navigate towards their synaptic targets. However, although many of the extrinsic factors that orient axons to project in a particular direction are well-described, the mechanism(s) that control the rate of axon outgrowth remain unresolved. Our recent studies have shed light on this issue; we showed that cofilin, and its negative regulator Lim kinase 1 (Limk1), control the speed of growth for a population of dorsal commissural interneurons in the spinal cord. Thus, axons are also instructed by extrinsic signals to grow at a particular rate. Such temporal cues have the potential to control the rate and/or time at which directional information is interpreted and are a important mechanism that ensures that axonal circuits develop in concert with the rest of the developing embryo. Moreover, this finding raises the possibility that the signaling pathways that control the rate of axon growth during development could be manipulated to accelerate axonal outgrowth in a regenerative or neuroprotective context and thereby speed up the lengthy process of regrowing neural circuits in a human patient. To work towards this goal, we will determine whether the signals that regulate the rate of axon growth are important for the establishment of spinal motor circuits during development and in an embryonic model that tests the functionality of stem-cell derived MNs. In Aim 1 of this proposal we will test the hypothesis that the balance between the activation states of cofilin and Limk1 controls the rate of endogenous motor axon extension during development. We will utilize in ovo electroporation of chicken embryos and mouse loss-of-function genetics to increase the activity levels of cofilin in developing embryos and assess the effect of elevated cofilin activity on the rate and trajectory of spinal motor axon extension. In Aim 2 of this proposal we will test the hypothesis that elevating levels of cofilin in embryonic stem cell (ESC)-derived motor neurons (MN) results in their generating functional motor circuits more rapidly. We will use lentivirus transfection methods to intrinsically increase cofilin activity in ESC-derived MNs and then will assess the rate of motor axon extension as well as their ability to form functional neural circuits in culture. ESC-derived MNs are a promising candidate to replacing MNs that are damaged or lost after injury or disease. The ability to intrinsically accelerate axon extension from ESC-derived MNs may lead to more efficient recovery times when paired with other axon regeneration therapies.

In the previous grant, 'After-effects of entrainment of the human circadian pacemaker', we were able to demonstrate that non-photic time cues elicited a phase-shifting effect on circadian rhythms in totally blind subjects that, while unable to entrain non-24-hour circadian rhythms in society, was able to modify phase to a greater extent than previously thought. We also discovered that the prevalence of intact circadian photoreception (i.e., maintenance of the melatonin suppression response following ocular exposure to white light) in the absence of light perception was greater than prior studies suggested. In the current renewal application, we propose to further investigate these initial findings. Following screening to test for the presence or absence of an intact circadian photoreception response, subjects will enter one of two studies. [unreadable] Study 1 will study those with an intact circadian response to light and examine the spectral sensitivity of circadian photoreception in the absence of photopic and scotopic photoreception. These results will be compared with similar studies that have already been completed in sighted subjects who maintain both circadian and photopic/scotopic photoreception to assess the relative contributions of these photoreceptor systems to circadian responses. Study 2 will study those who are not affected by light and will determine the limits of entrainment for non-photic time cues (sleep-wake, rest-activity, meals, showers, social contact) in order that the strength of such time cues can be determined for therapeutic use. Specifically we will test the hypotheses that: Study 1) exposure to monochromatic light of 460 nm for 6.5 h in the early biological night will cause a 3 h delay in circadian phase and a 85 percent suppression in pineal melatonin production whereas exposure to the same photon density of 555 nm will have no effect on circadian phase or melatonin suppression (compared to 1.7 h delay and 37 percent suppression in sighted subjects); Study 2) Exposure to a non-photic schedule advanced by 0.4 h relative to baseline intrinsic period will cause a phase advance of period of 0.4 h. This work has significant implications for understanding the novel photoreceptor system(s) underlying circadian and non-image-forming effects of light in humans and for further understanding the impact of non-photic time cues on the human circadian pacemaker by defining the limits of entrainment for non-photic synchronizers in order to potentially evaluate the efficacy of such time cues for treatment of circadian rhythm sleep-wake disorders in both the blind and sighted populations [unreadable] [unreadable]

Cutaneous malignant melanoma (CMM) incidence is rising, accounting for approximately 4% of cancer diagnoses in U.S. Recently, a novel CMM susceptibility locus was identified on chromosome 1 (1p22). To identify this gene Aim 1, we will use a multidisciplinary approach to comprehensively identify, prioritize, and screen genes within the critical region. We will construct a custom oligonucleotide microarray with probes representing all evolutionarily conserved sequence at 1p22 and assay expression in melanocytes and melanoma cell lines to identify all expressed genes in the region. We will use these arrays for CGH to screen for deletions in familial CMM patients and sporadic melanoma cell lines. We will SNP-genotype our families and perform a linkage disequilibrium based association study to narrow the 1p22 critical region. Based on these experiments, we will prioritize 1p22 candidates for high-throughput mutation screening. Once the CMM susceptibility gene is identified, we will look for genotype-genotype correlation, testing the hypothesis that Iow-penetrance genetic CMM risk factors act as modifiers to the penetrance of 1p22 mutations (Aim 2). Finally, we will determine the prevalence of 1p22 mutations in sporadic melanoma cell lines and tumors (Aim 3).

This proposal concerns conceptual combination, i.e., the process by which people combine existent simple concepts (e,g., brown and apple) into novel combinations (e.g., brown-apple). Conceptual combination may be the fundamental means by which we enlarge our stock of concepts, and hence is criticl to the psychology of learning and reasoning. But little prior work on this topic has been done with natural concepts. What has been done suggests that the extent to which an object is judged to be a member of a conjunctionis always a simple logical function of the extent to which that object is judged to be a member of each constituent of the conjunction (e.g., the typicality of a particular object as a brown-apple is the minimum of that object's typicality as a brown thing and as an apple). We show that this suggestion is incorrect. We propose that there are different kinds of conjunctions, and we develop a taxonomy of conjunctions whose major dimensions include the semantic relation between the adjective and noun concepts and the extent to which conjunction provides a true description of the object to be categorized. Several proposed experiments will evaluate this taxonomy. Other studies will determine performance differences among objects that vary in how typical they are of conjunctions, and investigate how learned conjunctive concepts are represented in memory. We will also focus on a particular model, which assumes that categorization is often based on similarity to known exemplars, and develop detailed predictions in the model for the relation between a conjunction and its constituent concepts. Our prior research on simple concepts has already turned up important implications for medical diagonisis in general and psychiatric diagnosis in particular (see Cantor et al, 1980), and we expect the current work to do so as well.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication. Cristea IM, Rozjabek H, Molloy KR, Karki S, White LL, Rice CM, Rout MP, Chait BT, Macdonald MR.Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: a role for G3BP1 and G3BP2 in virus replication, Journal of Virology. 84(2010)6720-32

The overall goal of this program is to train fellows in areas of basic research relevant to Pediatric Infectious Diseases, especially those in the immunocompromised host. The fellowship is designed as an intensive laboratory-based research training program with 2 years of protected laboratory time and 1 year of clinical training. Research can focus on issues related to the pathogen or the host. Faculty with successful basic research programs will serve as laboratory mentors. These faculty, selected from the Departments of Pediatrics, Medicine, Biochemistry, and Microbiology and Immunology, have a documented history of productive collaborative research. In addition, these laboratory mentors have all been actively involved in training pediatric fellows in projects relevant to Pediatric Infectious Diseases. [unreadable] [unreadable] The training program consists of: 1) a formalized research project in the area of basic microbiology, immunology, molecular biology, or virology under the direction of one of the laboratory mentors 2) an organized series of graduate level courses, lectures, seminars, journal clubs, and other conferences; 3) regular presentation of research in progress for constructive criticism; and 4) involvement in ongoing clinical activities within the Division of Pediatric Infectious Diseases (qualifying the fellow to sit for Board certification in Pediatric Infectious Diseases). The Department of Pediatrics will support successful fellows wishing additional years of research training. This training program has as its goal producing well-trained physician-scientists who are capable of developing their own independent research programs in areas relevant to infections in the immunocompromised host. [unreadable] [unreadable]

PROJECT SUMMARY Cognitive Behavioral Therapy (CBT) is typically considered the current gold standard and first line treatment for disorders characterized by recurrent binge eating such as bulimia nervosa (BN) and binge eating disorder (BED). Outcomes, while clinically significant, leave substantial room for improvement with recent systematic reviews and meta-analyses finding that 40-50% of patients with BED and nearly 70% of patients with BN remain at least partially symptomatic after a full course of CBT. In an effort to improve outcomes, a growing number of researchers have begun to evaluate the use of mindfulness and acceptance-based behavioral treatments (MABTs) for BN and BED and preliminary evidence suggests such treatments can be effective for this population. Although the research on MABTs for binge eating remains nascent, MABTs are frequently used in clinical practice for BN and BED, with one recent study showing that patients are more likely to report that their therapist has used mindfulness-based techniques than CBT-specific techniques. Given the widespread clinical interest in MABTs and the growing body of research supporting the preliminary efficacy of these treatments, a fully powered tests of the efficacy, mechanisms of action, and moderators of outcome for MABT appears warranted. In particular, rather than testing any specific MABT treatment package (e.g. acceptance and commitment therapy, dialectical behavioral therapy, mindfulness-based eating interventions) in a large clinical trial, we believe that a study that can isolate and evaluate the independent and synergistic efficacy of the most commonly used MABT components has the highest potential for impact. A review of MABTs for BN and BED suggests that there are four commonly used MABT components: (1) Mindful Awareness, (2) Distress Tolerance, (3) Emotion Modulation, and (4) Values-Based Decision Making. MABT treatment packages have vary widely in which of these components they incorporated and which they emphasized. By identifying which components of MABTs are most effective (and for whom they are effective), we can emphasize the powerful elements of MABTs and deemphasize or eliminate inert components. A traditional RCT would not provide the power necessary to evaluate the independent and synergistic efficacies of four distinct treatment components compared to an active treatment approach such as CBT. Instead, the proposed study will use a Multiphasic Optimization Strategy (MOST) approach (including a full factorial design) in which 256 individuals with transdiagnostic binge eating pathology are assigned to one of sixteen behavioral treatments, i.e., representing each permutation of the MABT component described above being included or excluded from the base treatment (a version of CBT that emphasizes the key behavioral ingredients of this treatment approach). Results of the component analysis set up future work to evaluate an optimized treatment containing only effective components (which can be expected to have superior efficacy, efficiency and disseminability) against current gold-standard CBT.

This proposal for a Mentored Clinical Scientist Development Award focuses on elucidating the role membrane trafficking mechanisms play in the regulation of D1 and D2 dopamine receptor cell-surface number and how antipsychotics influence this process. These studies are of critical clinical significance since dopamine receptor number is dysregulated in various neuropsychiatric disorders including schizophrenia and secondary to treatment with antipsychotics. Membrane trafficking is fundamental for achieving the ordered placement of receptors and for the processes of up- and down-regulation of the number of receptors present in specific membrane domains. Disturbances in membrane trafficking processes have been implicated in the pathophysiology of several different disorders such as retinitis pigrnentosa, diabetes insipidus, and schizophrenia. The proposed studies examine the hypothesis that regulated membrane trafficking mechanisms, in particular post-endocytic mechanisms which target receptors to either recycling or degradation, play an important role in 1) regulating the cell surface receptor number of D1 receptors and 2) in mediating antipsychotic-induced upregulation of specific D2-like dopamine receptor subtypes. The proposal will examine the subtype-specific endocytic trafficking of dopamine receptors, examine how antipsychotics affect the endocytic trafficking of D2- like receptors and determine the mechanisms of D 1 receptor membrane insertion. Studies will utilize the well-established HEK-293 cell heterologous model system which is amenable to biochemical studies, and cultured striatal neurons which are more physiologically relevant. These studies are expected to provide insight into the dysregnlation of dopamine receptors observed in a variety of psychiatric disorders. [unreadable] [unreadable]

Commercialization of silver sheath HTS wires/tapes for applications in all-HTS superconducting magnets require significant improvements in their reliability, production yield, economy, and mechanical properties. Superconducting magnets are key components in Magnetic Resonance instruments. Their manufacturing and maintenance are major cost items. Fabrication of economical HTS conductors can lead to development of all-HTS magnet systems that can operate at temperatures of higher 30K, which are more user friendly and inexpensive to maintain. This project offers a materials processing solution that will lead to fabrication of HTS wires/tapes that: l) Use significantly less silver. 2) Have more uniform HTS cores. 3) Produce longer length wires/tapes leading to more production yield. 4) Have, significantly improved mechanical properties. The Phase I work will: A) Demonstrate reinforcement of silver matrix by metal-oxide fibers on prototype samples. B) Fabricate prototype reinforced HTS wires/tapes. C) Characterize mechanical and superconducting properties of prototype samples. PROPOSED COMMERCIAL APPLICATIONS: Availability of reliable and economical HTS wires/tapes will have a profound impact on many US scientific and commercial applications including: medical, biology, physics, electric power, energy, military, space, and transportation.

In response to Request of Proposal (RFP), NHLBI-HV-1 0-11, SRI International (SRI, formerly Stanford Research Institute) proposes to act as a flexible and responsive resource contractor as the Pharmacology and Toxicology Center (PTC) to support the Science Moving towArds Research Translation and Therapy (SMARTT) program of the National Heart, Lung, and Blood Institute (NHLBI). The purpose of the SMARTT program is to provide resources, such as consulting, manufacturing, pharmacology and toxicology testing, preclinical and early-phase clinical study design support, and administrative and regulatory expertise, to assist translation to the clinic of novel synthetic, natural, or biologic therapeutic interventions arising in the scientific community for the treatment of heart, lung, and blood diseases.

The goal of the proposal is to determine the effects of ear acupuncture on children with ADHD. Subjects will be recruited from student populations in schools in the Richmond, Virginia metropolitan area who receive their medication management through the ADHD Follow-up Clinic of the Childrens Medical Center of the Medical College of Virginia Hospitals. The study will recruit 53 children from grades 1-6 who have a primary diagnosis of ADHD. A small preliminary study involving five subjects will be used to determine the time course of ear acupuncture treatment. This preliminary study will be a single subject multiple baseline design which will be blind to the participants and the observer of treatment. They will attempt to see how long it takes for the treatment effect to begin and how long it lasts. If no effect is observed, the study will be discontinued. The main study, the second part, will be a within- subject crossover placebo-controlled blind design. It will investigate the effects of acupuncture point therapy, methylphenidate, placebo and the two active treatments combined. The order of entry will be determined by a 4x4 Latin square design so that after drug-free baseline, the children will be randomly rotated through placebo, acupuncture point therapy, methylphenidate and acupuncture plus methylphenidate, according to one of four experimental sequences. The study is under blind conditions. Neither the child, the parents, the data collectors, nor the research staff will know whether the target child is on medication or placebo, or whether the seed pellet is on the correct point or sham point. Following the baseline assessment, each experimental phase will last two weeks or longer as is determined by the first study. The dependent measure will be scores on the Conners Parent rating scale and the baseline measures will be used to describe the subject population, compare responsiveness of the three active conditions and to check randomization to treatment groups.

Understanding the mechanism of acid secretion by the parietal cell depends on the convergence of knowledge about the structure of the H+ transporting, K+ - activated ATPase with the detailed characterization of its function at the molecular level. The proposed investigation is planned to obtain new information relative to four major molecular aspects of the Gastric ATPase: Active - site structure; Molecular weight and subunit structure; Principal conformations and their relation to ATP hydrolysis and ion (H+, K+) translocation; Organization in the membrane. 1. The nature of substrate binding and phosphorylation sites and their structure will be characterized by using fluorescence and affinity labelling techniques. 2. Studies on the subunit structure will center on the determination of the molecular dimension of the native and crystal lattice - induced enzyme preparation by electron microscopy and computer - assisted image analysis. 3. The conformational states of the enzyme induced by specific ligands and the various rate constants associated with these transitions, will be investigated by using fluorescence and radioisotopic techniques. 4. We will determine the hydrophylic and hydrophobic peptide topology of the enzyme in the membrane by proteolytic digestion techniques coupled with site - specific reagents and lipophilic photo affinity labels. The topics selected for investigation are central issues in the areas of membrane transport process. Health - related significance of these studies will derive from the expansion of our information about basic molecular mechanisms for HCI secretion.

In animals, the hormone, melatonin, plays an important role as a chemical mediator of the effects of season on behavior. In many instances, changes in the length of the night serve as the environmental signal that triggers seasonal changes in behavior. Melatonin is secreted exclusively at night, and the duration of its secretion varies with seasonal variations in the length of the night. Because of these properties, many organisms use changes in the duration of nocturnal melatonin secretion as a chemical cue to trigger changes in functions, such as breeding, that vary on a seasonal basis. Our recent research (Z01 MH 02424-01 CP) showed that humans, in the course of their evolution, have conserved brain mechanisms similar to those that exist in animals that enable them to detect seasonal changes in night-length and modify the duration of nocturnal melatonin secretion. Human responses to change in photoperiod were detected in experimental conditions in which individuals were exposed to long and short artificial "days". In a subsequent experiment (Z01 MH 02424-01 CP) we investigated whether healthy individuals living in a modern, urban environment (metro Washington, DC area), in which they are routinely exposed to artificial light, are still able to detect seasonal changes in the duration of the night and to respond to these changes by modifying the duration of the nocturnal period of melatonin secretion. We found that patterns of nocturnal melatonin secretion in women responded to seasonal changes in the duration of the night, while those in men did not. This finding suggests that there may be gender differences in responsiveness to artificial light of mechanisms that track seasonal changes in the length of the night. Because of the importance of the duration of nocturnal melatonin secretion as a transducer of the effects of seasonal changes in the length of the night on animal behavior, it would be important to investigate whether melatonin plays a role in the pathogenesis of seasonal affective disorder (SAD) with recurrent winter depression. Accordingly, in this project, we are assessing whether or not the duration of nocturnal melatonin secretion varies on a seasonal basis in women with SAD, as it does in healthy women. In 28 patients and 30 matched controls, melatonin was abnormally unresponsive to change of season in women with SAD, raising the possibility that SAD results when the pineal gland fails to exhibit a seasonal response that normally occurs in healthy women. In contrast, 9 patients and 15 matched controls, we found that melatonin is responsive to change of season in men with SAD but not in healthy men, a result that is consistent with the classic hypothesis that changes in duration of melatonin secretion trigger winter depression in SAD. The number of male subjects will be increased during the next two years to rule out Type II error. An important post hoc finding in these studies was that seasonal changes in the intrinsic duration of nocturnal melatonin secretion are almost entirely a function of seasonal changes in the timing of morning offset of secretion. This finding indicates that the degree to which individuals' melatonin secretion responds to seasonal changes in night length as a function of their exposure to, or processing of, morning light. This finding provides an important clue in our quest to understand why some individuals respond to change of season while others do not.

Chromosome-specific DNAs will be prepared for all mouse and rat chromosomes by combining the technologies of chromosome microdissection and degenerate oligonucleotide primed PCR (DOP-PCR) amplification. Difficulties with flow sorting of chromosomes have prevented complete sets of chromosome-specific DNAs or libraries other than human from being made available. Microdissection will circumvent these difficulties and allow pure chromosome-specific DNAs to be prepared from all chromosomes. A single degenerate primer will allow amplification of DNA from both mouse and rat. Cot1 DNA for use as competitor in hybridizations will be prepared for both species. Chromosome-specific DNAs will be tested for specificity by painting. Selected DNAs will be tested for representation by hybridization to a battery of unique chromosome-specific probes. Chromosome-specific DNAs will be cloned using conventional techniques. Chromosome-specific DNAs, Cot1 DNA, and protocols for use will be made available to the research community.

Although large numbers of individuals seek treatment specifically for a principal problem of marijuana dependence, little is known about effective strategies to treat this population. Young adult marijuana users who have been referred by the court system for treatment are characterized by poor retention and outcome in standard outpatient approaches. Targeting this population and addressing their low level of motivation and facilitating treatment engagement may be an effective strategy for early intervention with a population at risk for progression to more severe drug use and associated legal problems. Motivational Enhancement Therapy, directed to fostering motivation for change in this highly ambivalent group, and Contingency Management, directed to foster treatment retention and abstinence, are promising strategies, given their high level of empirical support in other substance-abusing populations and their suitability of the population targeted here. In this study 150 young adults referred by their probation officers for evaluation and treatment of marijuana use will be randomly assigned, using randomization, one of four conditions: (1) standard psychoeducational drug counseling, (2) contingency management plus drug counseling, (3) Motivational Enhancement Therapy (MET), and (4) Contingency Management plus MET. Primary outcomes will be retention in treatment, reduction in marijuana use, and induction to long-term treatment. Study treatments will last 8 we4eks and will be delivered to subjects on an individual basis. Follow-ups at 1,3,6,9 and 12 months after cessation of study treatment will assess the durability and/or delayed emergence of treatment effects.

Study the nature of the humoral immune response to syngenic mouse mammary carcinomas. Primary tumors or carcinomas in early transplant in a strain and C3H mice will be studied. Major emphasis of the studies will be the evaluation of the humoral assay for the early detection of antibodies in response to mammary tumor associated antigens. In addition, the specificity of the detected antibodies will be defined. In the first year work will be restricted to the mouse mammary tumor system. In years 2 or 3, if suitable rat carcinoma models are available, the work will be extended to this area.

As described in the previous annual reports, we expressed and purified two recombinant preS1 peptides (rpreS1) of hepatitis B surface antigen, i.e., a 91 amino acid (AA) wild-type peptide and a tyrosine-substituted 90 AA mutant peptide. Binding of either 125I-labeled rpreS1 peptide to a crude plasma membrane (pm) preparation from an autopsied human liver specimen was not significant. However, in the presence of anti-rpreS1 (which was produced by immunizing rabbits with the wild-type rpreS1 but recognizes both wild-type and mutant peptides), a high affinity binding was observed. The binding to pm appeared to be due to immune complexes formed between rpreS1 and anti-rpreS1. This Ag-Ab binding reached equilibrium within 20 minutes at room temperature. The binding was saturable with labeled immune complexes at 10-9 M when the pm preparation was kept constant. When the labeled rpreS1 was kept constant, increasing levels of either pm or anti- rpreS1 enhanced the binding. Neither hepG2 cells nor monocytes isolated from human blood showed any binding. The binding was blocked when pm was preincubated with unlabeled rpreS1 but not with anti-rpreS1. However, no binding was observed with (Fab')2 produced by digesting the intact IgG of anti-rpreS1 with pepsin. Thus, the binding appeared to be Fc receptor mediated. Preliminary results indicated that preincubation of pm with monoclonal antibodies to FcRI, FcRII and FcRIII had no effect on the binding of the labeled rpreS1-anti-rpreS1 complexes to pm. We have yet to determine whether such binding is specific to human hepatocytes. The significance of such binding of preS1 in the presence of its antibody remains to be investigated.

PROJECT 1 - Improving Clinical Assessment of Diagnosis for Latinos The goal of this proposal is to develop and test a mixed methods approach to helping clinicians in public health psychiatric settings decrease clinical uncertainty by more accurately diagnosing patients with diverse cultural backgrounds and improving, the matching of clinical services to the needs of Latinos. Our approach combines methods from cultural anthropology with statistical methods and psychiatric epidemiology. The specific aims of this study focus on generating improved cultural formulations and diagnostic accuracy using information from qualitative and quantitative perspectives, iterating between the methods in a complementary fashion. The specific aims are to: Aim 1: Identify the information about symptoms and cultural and social context gathered in the initial diagnostic interview, that clinicians use to make determinations about three disorders: major depression, drug abuse and alcohol abuse;Aim 2: Use data from an epidemiological survey to assess the potential sensitivity and specificity of the clinical determinations identified in Aim1 in detecting the underlying diagnosis. We will test the hypothesis that clinical diagnosis is less accurate for Latino than non-Latino white patients;Aim 3: Assess clinician and patient reactions to the recommended improvements to diagnostic assessments developed in Aim 2. Using focus groups and consensus panels, prioritize the kinds of information to be collected to improve diagnostic determinations in safety net settings. Aim 4: Apply the findings of Aim 3 to make concrete recommendations to improve the efficiency and fairness of diagnostic decisions for Latino and non-Latino white population groups.

Abstract / Summary This proposal aims to extend the work performed under a recent R21 exploratory grant to detect and validate measures of functional connectivity in the human cervical spinal cord (SC) using resting state functional MRI (rsfMRI). The delineation and characterization of neural circuits within the cord may provide a valuable imaging biomarker of functional integrity of the spine applicable to a wide range of disorders. The identification of patterns of highly correlated low frequency blood oxygenation level dependent (BOLD) signals in a resting state has provided a powerful approach to delineate and describe neural circuits in the brain. We recently reported the first reliable detection of similarly correlated low frequency signal fluctuations in SC in a resting state in normal subjects, and showed how functional connectivity may be quantified in the SC both within and between segments. Moreover, in parallel studies in non-human primates we have shown that these spine circuits are selectively and specifically altered by injury and revert back over time in a manner that correlates with functional recovery. We have also shown how multi- parametric MRI can be used to derive quantitative indices of tissue composition and structure which can be related to the functional changes. We hypothesize that the intrinsic neural circuits revealed by rsfMRI in the SC are an important representation of neural synchrony within spinal segments that in turn are an essential feature of normal functions; and that alterations in the patterns of functional connectivity may be used as non-invasive imaging biomarkers of the effects of injury and of therapeutic interventions. We aim (1) to further develop robust, reliable methods to detect and quantify functional connectivity in human SC by optimizing the acquisition and analysis of images at 3T; (2) to implement a novel, multi-parametric spine MRI protocol incorporating diffusion tensor imaging and quantitative magnetization transfer imaging which provide maps of quantitative indices of tissue microstructure and composition; (3) to validate the interpretation of functional connectivity measurements and accompanying changes in white matter composition and microstructure as objective biomarkers of spinal integrity and for guiding clinical management decisions. Imaging data will be correlated with a battery of physical assessments of function in subjects with a wide range of functional impairments to demonstrate their clinical relevance. We will also evaluate their capacity for monitoring and predicting outcome of treatments in patients with cervical spondylotic myelopathy (CSM) and with traumatic spine cord injuries (SCI). The significance of the work is that it will use novel MRI methods that have proven successful in studies of the brain to objectively evaluate functional circuits within the SC, and show that connectivity measures can assess and predict clinically-relevant functions and symptoms. The ability to assess functional integrity has widespread potential for characterizing injuries to the cord, their changes over time, and for assessing novel therapies.

PROJECT SUMMARY/ABSTRACT Shared decision making (SDM) has the potential to improve quality of care and reduce health disparities. To engage in SDM, patients must have both (1) knowledge of the treatment options, and (2) power ? the self- perceived need and capacity ? to influence decision making. SDM interventions, e.g. decision aids, increase knowledge. However, barriers to patient empowerment hinder engagement, including patients? perceptions that their personal input is not valued and doctor-patient power imbalances. Importantly, socioeconomically disadvantaged patients disproportionately experience these barriers. Understanding how decision aids address barriers to disadvantaged patients? engagement in decision making and identifying persistent barriers which can be targeted by adjunct interventions are critical steps towards reducing health disparities. In this study, we use breast cancer surgery as a model to identify and characterize barriers to socioeconomically disadvantaged patients? engagement in SDM. In order for a decision aid to be effective, patients must be able to access and review it; it is therefore critical to consider barriers to access. Yet even after successful review of a decision aid, persistent barriers may limit patients? engagement. The objective of this study is to test the effectiveness of a decision aid in increasing patient engagement in SDM and identify barriers to engagement not mitigated by the decision aid that could be targets for adjunct SDM interventions. We propose a multi-site cluster randomized trial using a stepped wedge design to enroll clinics serving a high proportion of socioeconomically disadvantaged patients within an established national community cancer research network. We will deliver a web-based decision aid via email directly to patients prior to their surgical consult. We will use mixed methods to accomplish the following specific aims: Aim 1, test the effectiveness of a breast cancer surgery decision aid in increasing patient engagement in decision making (measured by power and knowledge) in clinics serving a high proportion of socioeconomically disadvantaged patients; Aim 2, test the extent to which the effect of a decision aid on patient engagement is mediated through the mitigation of barriers, and determine if persistent barriers are disproportionately experienced by socioeconomically disadvantaged patients; and Aim 3, characterize how persistent barriers influence patient engagement in decision making in order to identify targets for adjunct interventions that could implemented in clinics serving a high proportion of socioeconomically disadvantaged patients. By understanding barriers to engagement in SDM that persist despite receipt of a decision aid, we will identify targets for adjunct interventions. Combining the routine pre-consultation delivery of a decision aid with the tailored delivery of adjunct interventions addressing persistent barriers to engagement is a sustainable model of SDM that maximizes clinics? finite resources. If proven effective, this approach will have far-reaching implications across a variety of healthcare decisions.

The medial temporal lobes (MTL) are the critical substrate for episodic memory. It is undisputed that a division of labor exists within the MTL. However, there is very little consensus as to how to best characterize these distinctions. Although some convergent evidence exists for a division of labor, particularly concerning the distinction between perirhinal and hippocampal encoding processes, the nature of the distinction between perirhinal and parahippocampal cortex has been underspecified. Although there is strong convergence in the literature supporting a role for the hippocampus in relational processing, the role of the MTL cortex in item processing/familiarity is less well defined, and only partially supported by the literature. Importantly, investigation into the extent and form of domain specificity within MTL cortex will contribute substantially to our understanding of episodic memory function. A fundamental hallmark of normal and abnormal aging, as well as some psychopathologies, is the alteration in episodic memory. Through an understanding of the functional architecture of the episodic memory system, great insight can be gleaned into these memory related deficits that, for example, occur during disease progression as a consequence of Alzheimer's disease or have been seen in the pathophysiology of neuropsychiatric disorders such as depression and schizophrenia. Understanding the neural mechanisms underlying the formation of human memory is an important step in efforts to describe and treat conditions that impact memory and will, thus, have a great impact on public health issues regarding treatment of such conditions. The research proposed will use functional magnetic resonance imaging to elucidate the functions of the MTL considering informational domain as being critical to a complete model of MTL organization (Aims 1 and 2). In addition, whether relational encoding and retrieval, thought to be a hallmark of hippocampal function, extends to the binding of episodic events in time will be explored (AIM 3) [unreadable] [unreadable] [unreadable]

Stroke is the leading cause of serious disability among adults in the United States.1 Aphasia, a language disorder caused by damage to the speech and language regions in the left hemisphere, is one of the most devastating results of stroke and affects individuals' ability to communicate effectively. Broca's aphasia is one of the most common types of aphasia and is characterized by restricted speech output, often not exceeding 1- 3 words per utterance, and relatively preserved auditory comprehension. Once in the chronic phase, most patients with Broca's aphasia experience very limited improvement in speech production. In a recent study2, we showed that patients with Broca's aphasia could produce fluent speech while mimicking an audio/visual speech model. In short, patients were able to mimic a one-minute script that was prerecorded and presented so that the speaker's mouth was seen on a computer screen and the speech was heard via headphones. We refer to this effect as speech entrainment, where the audio/visual speech model yokes the speech of the non-fluent patient, allowing him/her to produce fluent speech. Whereas speech entrainment might be important for understanding normal speech production, we propose that it could have important implications for rehabilitating patients with Broca's aphasia by allowing them to practice fluent speech production, something that is inherently very difficult for this population. The purpose of this pilot project is to estimate effect sizes associated with improvements in speech production as a result of speech entrainment treatment (SET). If SET results in medium or large effect sizes for improvements in speech production, we will move on to a larger trial where the effects of SET can be established and compared to other kinds of treatments for speech production in aphasia. The second goal is to understand cognitive-linguistic factors in relation to patients' ability to speak with the aid of speech entrainment. This will allow us to identify factors that are contraindicative for SET (to define inclusion/exclusion criteria in a larer trial) as well as relate our findings to contemporary models of speech processing. The long-term goal is to develop a treatment approach that can be used to improve speech production in Broca's aphasia, something that has been shown to be particularly resistant to treatment. We emphasize that the treatment approach presented here represents a starting point in our development; more data will allow us to modify SET and better tailor it towards specific patients.

The purpose of this two-year R-21 project is to develop and test an occupation-based intervention for fostering successful adaptation of elders who relocate to long-term care facilities (LTCF). The aims of this mixed methods study are: (1) to describe selected person factors of elders that contribute to adaptation to LTC settings, (2) to develop an occupation-based cultural heritage intervention based on the results of Aim 1, and (3) to test the effect of an occupation- based cultural heritage intervention on the adaptation of elders in LTCF. The hypothesis for Aim 3 is: LTC residents, 60 years of age and older, who participate in an occupation-based cultural heritage intervention will report significantly higher measures of adaptation as noted by activity engagement, social participation and quality of life compared to LTC residents who participate in a usual activity group. Twenty-four elders will be recruited and qualitative methods employed to achieve Aim 1. To accomplish Aim 3, 192 elders will be recruited and randomly assigned to receive intervention or control conditions. Quantitative analysis will compare measures of adaptation for those assigned to the cultural heritage intervention and those who receive a usual activity group. All elders, African-American and Caucasian, will have relocated within the past six months to a licensed nursing care unit in one of six LTC settings. The intervention will incorporate findings about valued aspects of residents' cultural backgrounds, as well as existing models of intervention based on cultural heritage. The intervention will be provided on a group basis to participating elders, who will be compared to control groups of elders matched on gender, age, and ethnicity. The intent of this project is to test an intervention strategy that incorporates diverse cultural values and beliefs that can be replicated in future large-scale multi- geographical studies. [unreadable] [unreadable] [unreadable]

Regulation of the microvascular response to inflammatory stimuli is highly dependent upon the interactions between neutrophils and the endothelium. We have shown a significant role for dietary copper in various endothelial interactions in the inflammatory process, including nitric oxide (NO)-mediated vasodilation, macromolecular leakage, and thrombosis. Recently, we have studied the role of dietary copper in microvascular control mechanisms with an emphasis on leukocyte/endothelial adhesion. We have demonstrated that dietary copper restriction causes tissue-specific changes in neutrophil/endothelial adhesion and transmigration. Neutrophil accumulation, the expression of the adhesion molecule ICAM-1 and the transcription factor NF-kappaB are all greater in the lungs of copper-deficient rats compared to copper-adequate controls. Based on these results, we hypothesize that dietary copper deficiency has a priming effect on leukocytes and pulmonary vascular endothelial ceils such that the lung becomes hypersensitive to inflammatory stimuli and more susceptible to the development of acute lung injury. Therefore the specific aims of this study are to: 1) determine the role of dietary copper in leukocyte/endothelial cell interactions in the lung microcirculation; 2) determine the mechanisms by which copper deficiency primes cells for acute inflammation and 3) determine at what concentration of dietary copper the inflammatory mechanisms become enhanced in the in vivo animal microcirculation. Aim #1 will use both in vivo and in vitro models of the lung microcirculation to study leukocyte/endothelial cell adhesion and chemotaxis. Aim #2 will study the role of copper in the NF-kB signaling pathway and in neutrophil priming. Aim #3 will examine these mechanisms under conditions of copper-marginal diets. These experiments have particular relevance since analysis of typical Western diets suggest that 1/3 to 1/2 of those diets may provide less than the RDA of 900mg Cu/day.

We have been interested in the role of K+ channels in neurons in general and in their role during hypoxia in particular. Some of the work that we have recently done pertains to the BK(Ca) channel in neocortical cells of mice. We have shown that the BK(Ca) present in these cells (sensitive to voltage, Ca, insensitive to charybdotoxin and Iberiotoxin, with a conductance of about 210 pS) is markedly inhibited by low 02 states in the cell-attached configuration, but not in the excised patch. Since 1) it is well recognized that the depolarization-induced by hypoxia can play an important role in neuronal cell fate, and 2) it is likely that the BK(Ca) channels we are studying are at least partially responsible for this depolarization, we propose 3 specific hypotheses/specific aims to study these channels and understand how they sense 02 lack and the role they play in hypoxic responses in both brainstem and neocortical neurons in mice and humans. These are: a) BK(Ca) channels play an important role in the hypoxia-induced depolarization of neocortical (NEO) and brainstem (hypoglossal, XII) neurons in mice and in NEO neurons of humans; the role of these channels increases with age in early mouse ontogeny. b) The hypoxia-induced inhibition of these BK(Ca) channels is secondary to changes in cytosolic factors such as phosphorylation, redox potential and intracellular pH and not related to a direct effect of 02 (or lack thereof) on the channel itself. c) BK(Ca) channels have binding domains that are essential for the cellular response to hypoxia. In order to address these hypotheses, we use single channel and whole-cell recordings, cell lines transfected with BK channel subunits, RT-PCR and in-situ hybridization to determine the BK(Ca) channel structure-function relationships. Since we have shown that K+ channel activity represents some of the early events in hypoxia, and since these events are important in setting the stage for subsequent events that lead to survival or injury of central neurons, we believe that this work can have far reaching implications on our understanding of, and possibly treatment for, hypoxic injury, stroke and epilepsy.

The specific objective of this project is to characterize an unusual transposable element recently discovered in S. cerevisiae. The 341-bp transposable element, sigma, is present in about 30 copies in the haploid genome. In five cases of sigma insertions which have been sequenced, the element is at position -17 or -19 relative to the 5' end of the sequence coding for a mature tRNA and therefore quite close to the point of initiation of transcription. Since these tRNA genes and their flanking sequences are different, it seems likely that the insertion specificity of sigma depends on the conserved internal sequences of the tRNA-coding region. Preliminary studies also indicate that transcription from the tRNA gene can be modified by sigma insertion. Thus a characterization of this system could improve our understanding of transposable elements and provide an opportunity to study the role of flanking DNA in polymerase III gene regulation. The experimental approach to these problems will combine classical yeast suppressor genetics and recombinant DNA technology, including yeast transformation. The experiments are organized around five specific issues: (1) identification of common structural features and tRNA genes associated with sigma in one strain of yeast; (2) search for RNAs homologous to sigma; (3) quantitation of sigma effects on tRNA gene expression; (4) development of an experimental system for detection of sigma transposition and definition of the essential features of the tRNA gene target; and (5) selection of mutations in potential sigma controlling elements. The existence of a transposable element which is clearly associated with a set of genes transcribed by polymerase III and which also affects the activity of those genes gives us an unexpected opportunity to ask very specific questions about the interdependence of two important cellular phenomena--transposition and transcription. The long-range goal of this project is to understand the contribution of transposable elements to transcriptional regulation in eukaryotic cells.

Protein or peptide biomarkers offer great promise in early detection, monitoring and targeted treatment of cancer. Two main strategies have been employed in proteomic biomarker discovery. Identity-based methods use high quality tandem mass spectrometry and identify potential biomarkers among sequenced peptides. Pattern-based, or label-free, approaches, on the other hand, look for discriminating peak patterns in mass spectra, without regard to their identity-enabling higher throughput analysis. In spite of the potential for biomarker discovery afforded by these methods, efficient discovery of robust biomarkers has remained a significant and unfulfilled challenge. Here we propose to develop a robust, high throughput analytical platform for biomarker discovery that combines identity and pattern obtained at high resolution and high mass accuracy. A key innovation of our approach is the use of sequence identified peptides to guide the alignment of unidentified m/z peaks (both obtained in the same LC-MS experiment) and to correct for chromatographic variation. The software will employ mathematically and statistically sound algorithms to match unidentified peaks across multiple samples, integrate peptide intensities into associated protein abundance, and use advanced pattern recognition tools for differential marker selection and quantitation. Importantly, we intend to adapt and extend the algorithm to derive quantitative data from samples that have undergone additional levels of fractionation such as strong-cation exchange at the peptide level. We anticipate that the methods we develop will provide at least an order of magnitude increase in the number of peaks detected as differentially regulated and subsequently sequence identified relative to existing identity- centric biomarker discovery approaches. Successful development and validation of the proposed platform has the potential to significantly accelerate biomarker discovery efforts for cancer as well as for other diseases, both at the Broad Institute and elsewhere. Furthermore, deployment of the biomarker discovery platform as a caBIG service will provide wide access to the platform, thereby maximizing impact in the research community.

The NCCTG Data Monitoring Committee reviews all phase III cancer treatment and cancer control trials coordinated by NCCTG. The primary functions of the Committee are: a) to assure safety of the treatment regimens, b) to examine efficacy results and determine whether or not the trial should be continued, modified, or discontinued, c) to review changes in study design in order to promote scientific integrity of the trial, d) to review accrual information in order to assure that the trial will yield meaningful results, and e) to review requests for release of data to investigators prior to general release as specified in the trial design.

The degree to which non-adrenergic vascular resistance increases operate in conditions of external circulatory support is unknown. Although increased venous content of norepinephrine is well recognized from organs with adrenergically innervated vasculature, e.g. spleen and kidney, no actual secretion rates of norepinephrine have been described from these or other peripheral locations. In addition, no direct correlations have been made between peripheral norepinephrine release and increased resistance to blood flow. Norepinephrine content of splenic venous, left renal venous and arterial blood will be determined simultaneously with splenic arterial and left renal arterial flow measured electromagnetically. Gradients between arterial and venous pressure across the left kidney and spleen will be monitored. The data will permit calculation of true norepinephrine secretion rates from spleen and left kidney and calculation of simulataneous vascular resistance in the same two organs. Norepinephrine will be analyzed by a trihydroxyindole technique. Resting conditions and acutely denervated preparations will be evaluated first. Reflex responsiveness of norepinephrine release and vascular resistance to acute hypovolemia will then be studied. Next, reflex norepinephrine release and vascular resistance patterns will be studied in response to graded changes in pulse amplitude and mean carotid sinus pressure reaching isolated, bilateral, innervated carotid sinus loops from an external pump circuit under independent control. Finally, the same pulse parameter changes will be directly introduced to the splenic and left renal circulation to determine any local effects pulse parameters exert on vascular resistance and norepinephrine release. Data from comparable experiments will be grouped and statistically evaluated by analysis of variance using Students T-test. Where it is appropriate to the evaluation of graded pulse parameter changes, regression analysis will be carried out and significance levels derived from the associated correlation co-efficients.

Human embryonic stem cells (hESCs) may provide an unlimited source of insulin-secreting beta-like cells for cell replacement therapy of type 1 diabetes, a disease in which insulin-secreting beta cells in the pancreatic islets of Langerhans are destroyed by autoimmunity. In spite of the recent progress in deriving early definitive and pancreatic endoderm-like progenitors from hESCs, a robust generation of later functional pancreatic beta-like cells has not yet been achieved in vitro. It is increasingly recognized that profound alterations in chromatin structure, including changes in epigenetic DNA methylation and histone modifications, contribute to the control of cell fate decisions. To harness the capability to efficiently differentiate hESCs in vitro, it is necessary to further define epigenetic signatures during pancreatic development. In this Beta Cell Biology Consortium application, we propose to identify and develop novel research resources and tools to advance our long-term goal of enhancing the yield of clinically-applicable human pancreatic beta-like cells from hESCs in culture for the purpose of cell replacement therapy for type 1 diabetes. Specific Aim 1 is to perform epigenome profiling for DNA methylation and key histone marks on two cell populations: mature adult human beta cells, obtained from cadaverous organs, and human ESC-derived pancreatic endocrine like progenitor cells that express neurogenin 3, a transcription factor necessary for the formation of the endocrine pancreas. We will then compare the epigenetic signature of these two populations, which will lead to identification of epigenetic targets and allow development of strategies to manipulate cell differentiation. Specific Aim 2 is to improve the yield of functional beta like cells from hESCs in vitro. We will use three emerging technologies, i.e., macromolecular hydrogels, protein transduction domains, and polyamides, to deliver or inhibit specific molecules that are known, in the literature, to affect beta cell commitment, maturation and function. If successfully completed, the proposed work will be the first data set describing the epigenetic profiles of different human pancreatic cell populations. The novel technologies to be employed will also be valuable for the community to further explore in the field of beta cell biology. Finally, the proposed work will allow us to assess hESC-derived cells in a novel manner, increasing our understanding about whether these cells may be suitable for future clinical applications. PUBLIC HEALTH RELEVANCE: In spite of the recent progress in deriving early definitive and pancreatic endoderm-like progenitors from human embryonic stem cells (hESCs), a robust generation of later functional pancreatic beta-like cells has not yet been achieved in culture. We propose to identify and develop novel research resources (such as epigenetic signatures of beta cells and progenitors) and tools (such as macromolecular hydrogels, protein transduction domains, and polyamides) to advance our long-term goal of enhancing the yield of clinically-applicable human pancreatic beta-like cells from hESCs in culture for the purpose of cell replacement therapy for type 1 diabetes.

PROJECT SUMMARY Over 80% of People Living with HIV/AIDS (PLWHA) globally, have access to combination Anti-retroviral Therapy (ART). The effective scale up of ART in sub-Saharan Africa (SSA) has dramatically reduced morbidity and mortality due to HIV/AIDS in LMICs over the past decade. Today, PLWHA live longer lives and have improved health related quality of life that is equivalent to that of people without HIV/AIDS. However, the growing burden of non-communicable diseases (NCD) secondary to HIV/AIDS and increased longevity (ageing) among this population threatens to undermine health gains related to the epidemic in LMICs. Uganda has been the site of numerous successful research collaborations for over 2 decades which have contributed to the knowledge of the pathogenesis, epidemiology and management of a range of infectious diseases, including HIV, TB and malaria. As a result of these studies, there is a cadre of successful independent investigators who have participated in the control of these conditions. However, there remains little locally- generated data, evidence and research at the intersection of HIV, NCD and ageing. A comprehensive research-training program is urgently needed that integrates priority aspects of HIV, NCD & ageing. The goal of this planning grant is to develop the required components for an application for a comprehensive multi- specialty research-training program concerning HIV, NCD and ageing at Makerere University in Uganda. The specific objectives of this grant are to: (1) Formalize a planning committee to develop and apply for a D43 research-training program at the confluence of HIV, NCD and ageing, (2) To conduct a systematic survey to assess the existing research and research training capacity for research at the confluence of HIV, NCD and ageing at Makerere University College of Health Sciences and collaborating institutions, (3) To define a pool of potential trainees at Makerere University that could be recruited and the selection process for future research training and (4) To develop a comprehensive D43 research training grant application based on needs identified at Makerere University College of Health Sciences.

The primary aim of this proposal is to identify factors that contribute to healthy aging and exceptional longevity in man. With optimal environments and behaviors, an average person has the ability to live to around age 85. Centenarians on the other hand live 15-25 years beyond what might be considered average. Many escape lethal diseases associated with aging (Alzheimer's disease, stroke, cancer, cardiovascular disease, and diabetes) or their age of onset is delayed. In order to live to such old age, centenarians are less likely to have genetic and environmental exposures that would cause at least lethal diseases at younger ages. We propose to study a cohort of over 2,500 centenarians, offspring of centenarians, and control subjects who were recruited by the New England Centenarian Study. We have selected a series of genes to study that have been shown to determine longevity in multiple model systems including C. elegans, D. melanogaster and mice. The genes include members of the insulin-like growth factor signaling pathway and the sirtuin cell signaling system. This pathway is critical for induction of enzymes and proteins involved in protecting organisms from reactive oxygen species which, when present in high amounts, can have a deleterious effect on cell and tissue health. Overall, the discovery of these pathways and their interrelationships have been key findings in longevity research that connects a large number of factors that are known to be important in longevity including disease pre-disposition, developmental factors, metabolism, environment, oxidant stress and tissue damage. Our specific aims are to (1) genotype key SNPs in each of these candidate genes in Centenarians, their offspring and controls and analyze the data using both association and linkage to identify genes that determine longevity in man. In aim 2, we will fine map those genes that show a positive association to determine the exact genetic variant that is responsible for determining longevity. Thus, the results of our work is not only contribute to understanding the factors that permit humans to live longer, but even more importantly, live longer without the disabling diseases associated with advanced age. [unreadable] [unreadable] [unreadable]

Following a glucose challenge, both insulin-dependent and -independent mechanisms contribute to the return to baseline blood glucose concentrations. Referred to as glucose effectiveness (GE), the insulin- independent component contributes as much to overall glucose homeostasis as insulin, but it has been viewed as a fixed and largely unregulated process and hence has not been a research focus for investigators. Several recent observations, however, offer evidence of the brain's capacity to potently induce glucose lowering via insulin-independent mechanisms. Adding to this work is our preliminary data showing that in leptin-deficient ob/ob mice, intracerebroventricular (icv) injection of the ani-diabetic hormone fibroblast growth factor-19 (FGF19) rapidly normalizes glucose tolerance despite having no effect on either insulin secretion or insulin sensitivity. Instead, this effect i mediated entirely by a selective, 3-fold increase of GE. Although the peripheral mechanism underlying this effect is unknown, our data strongly implicate a process whereby glucose is taken up into peripheral tissues via an insulin-independent mechanism, followed by its metabolism to lactate that is subsequently released back into circulation. With this background, we propose Specific Aim 1: To determine how FGF19 increases insulin-independent glucose disposal. Studies in this aim will 1) quantify this brain- mediated increase of glucose uptake and metabolism to lactate in response to FGF19, 2) determine the extent to which it explains the associated increase of GE, and 3) identify the tissues in which it occurs. These goals will be accomplished in mice using a combination of methods ranging from hyperglycemic clamp to metabolomics and biochemical analyses. Could a similar process contribute to the anti-diabetic effects of bariatric surgery? Rodent data implicate the brain in the glucose-lowering effects of bariatric procedures, and in some cases, glucose lowering involves insulin-independent as well as insulin-dependent mechanisms. Moreover, bariatric procedures increase FGF19 secretion from the GI tract. Based on these considerations, we propose Specific Aim 2: To determine if increased GE contributes to the anti-diabetic effect of bariatric surgery. Studies in this aim will determine if bariatric surgery activates CNS mechanisms analogous to those engaged by FGF19, including stimulation of insulin-independent glucose uptake, followed by conversion to lactate, which is then released into circulation. Our finding that FGF19 action in the brain rapidly, potently and selectively increases insulin-independent glucose disposal identifies a novel, CNS-driven mechanism with translational implications for both the pathogenesis and treatment of human diabetes. Studies in this proposal seek to clarify how this occurs and the extent to which it explains the anti-diabetic effect of bariatric surgical procedures.

The long term objective of this proposal is to work toward the systematic identification and characterization of protein-serine kinases expressed in a growing HeLa cell population. A thorough survey of this imporant class of regulatory enzymes in a proliferating human cell population should provide new insight into our understanding of cellular control mechanisms in general and specifically should help to define the chain of events involved in the ordered duplication and segregation of the genetic material which underlies cell division. This informalion, in turn, will suggest pathways for transduction of signals induced by binding of growth factors. A more complete understanding of the enzymatic and metabolic events involved in the regulation of mammalian cell reproduction will lead to more effective therapies for the treatment of human cancers. cDNA clones encoding members of the protein-serine kinase family will be identified by screening a HeLa library with oligonucleotide probes directed at highly conserved regions within the catalytic domain. Familial relationships will be confirmed by DNA sequence analysis. Structural and functional information about the putative protein-serine kinases encoded by the clones will be acquired through an analysis of deduced amino acid sequences as well as through heterologous eukaryotic expression systems. Antisera will be produced against the encoded proteins for use in 1) immunoprecipitation studies to allow enzymatic and further structural characterization, 2) indirect immunofluorescence studies to determine intracellular localization, and 3) immunoblotting studies to measure cell and tissue specificity of expression. Preliminary studies have clearly demonstrated the utility and value of the oligonucleotide homology screening approach. A cDNA clone has been identified which encodes a protein with considerable homology to protein-serine kinases encoded by cell cycle regulating genes in yeast. A major thrust of the proposed studies will be to further explore the structural and functional relationships between these proteins.

In the early 1970's the clinical effects of human fetal alcohol exposure were described as Fetal Alcohol Syndrome (FAS). Mental retardation, visual deficits, and changes in muscle coordination and reflex actions are characteristics of FAS. Research has been focused on the effect FAS has on the neurons in the central nervous system (CNS). FAS has been shown to decrease neuron numbers and alter their migration pattern. Recently, research has also been studying the effect that FAS has on the glial cells which support neuron activity and myelinate axons. Limited research in animal models has shown that fetal alcohol exposure can delay maturation of these cells and alter myelination of axons. The goal of this proposal is to investigate the effect of developmental alcohol exposure on the superior colliculus (SC) which is important in relaying visual sensory input to motor areas for coordination of head and eye movements in reflex activity. To simulate an equivalent human exposure in an animal model, pregnant rat dams will be fed a diet containing alcohol. After birth, rat pups will be reared using an artificial rearing technique to expose them to alcohol during postnatal days 1-10. Animals will be sacrificed at different ages and tissues taken from the SC for study. Maturation of glial populations, myelin development, and axon terminals will be analyzed using a combination of techniques to include light and electron microscopy, morphometric analysis, immunohistochemistry, and axon terminal labeling. Potential findings could help explain some of the neurological dysfunctions involving visual motor coordination.

The main study entitled "A Phase I Study to Evaluate the Safety and Immunogenicity of Recombinant HIV -1 Envelope Antigen in Children Born to HIV-Infected Mothers (ACTG 230 version 7.0, 5/5/97) closed to accrual. ACTG 707 is a substudy of ACTG 230 in which the safety and immunogenicity of HIV vaccines were being evaluated. Participants in ACTG 707 were selected from a subset of approximately 90 children who were in ACTG 230 and are eligible to participate in ACTG 707 because they had a positive response to the vaccine antigens while participating in ACTG 230. The presence of immune responses to vaccine antigens at the final study point (week 104 in ACTG 230) suggested that the study of the durability of these responses beyond 104 weeks would be useful. ACTG 707 will continue to study the lymphoproliferative and humoral responses to vaccine antigens once each year for two additional years past the study period (i.e., weeks 156, and 208) in those patients identified as having a stimulation index (SI) on the LPA assay of 3.0 or greater on at least 2 occasions post-baseline during the ACTG 230 two-year study period.

The Airflow Perturbation Deviie (APD) to measure resiratory airways resistance will be tested to give assurance of validmeasurements. The APD will then be used to measure airways resistance on humans who are exercising until they reach their minimum exhalation times. Concurrently, modeling will continue to predict exhalation time during exercise. Correlation of minimum exhalation time with conventional pulmonary function data will be attempted.

PROJECT SUMMARY: Complement component C3 is important for removing pathogens and plays a role in synaptic refinement during brain development. Complement accrues in Alzheimer's disease (AD) plaques (Stoltzner et al., Am J Pathol 2000). We have found that C3 and its downstream signaling fragments play important roles in synapse loss during normal aging in hippocampal CA3 of WT mice (Shi et al., J Neurosci 2015) and in the response of glia to A? and subsequent synaptic and neuronal changes in AD mice (Shi et al., Sci Transl Med 2017). We have shown that microglial uptake and clearance of A? is at partially mediated by the interaction of C3b with its receptor, CR3, on the surface of microglia (Fu et al., GLIA 2012). Together with our collaborators, Dr. Hong and Stevens, we recently reported that complement C1q and C3 mediate very early synapse loss induced by A? oligomers in the absence of A? plaques in mice (Hong et al., Science 2016). We further reported that lifelong, germline C3 knockout (C3KO) was neuroprotective in aged APP/PS1dE9 mice, despite increasing the plaque load (Shi et al., Sci Transl Med 2017). C3-deficiency in APP/PS1dE9 mice dampened the response to A? plaques, which correlated with normalization of cognition to that of WT mice. However, it remains unknown if C3 suppression after the onset of AD pathogenesis will be protective and the cell type by which this effect is mediated. Therefore, we generated C3 floxed mice and used them to generate a novel inducible C3 conditional KO model (UBC-Cre-ERT2;C3fl/fl), named ?C3iKO?, in which tamoxifen treatment at any age induces global C3 deletion. We hypothesize that C3-deletion in early stages of AD pathogenesis will protect the hippocampus against downstream neurodegeneration and, that this effect is cell- specific for microglia. Here, we propose the following Aims: 1. Determine whether induction of global C3- deletion shortly after the onset of plaque deposition in APP/PS1dE9 mice (6 mo) protects against late hippocampal changes (16 mo) in female and male mice. 2. Determine whether induction of global C3-deletion shortly after the onset of tau pathology in P301S;hApoE4TR (TE4) mice (2 mo) protects against late hippocampal changes (9 mo) in female and male mice, and if C3 effects are related to ApoE4 signaling. 3. Cross our unique C3 floxed mice to generate 2 novel, cell-specific inducible C3 cKO mouse models in which tamoxifen treatment induces C3 deletion in either microglia or astrocytes (2 mo) and determine the functional response (hippocampal LTP) to aging, fibrillar A?, tau aggregates and human ApoE4 (4 and 12 mo). Microglial and astrocyte gene expression will be assessed in all Aims to better understand signaling mechanisms in C3- mediated neurodegeneration. The overall goal of our study is to determine whether C3-deficiency shortly after the onset of AD pathogenesis is protective against neurodegeneration and cognitive decline, and whether such protection is mediated by microglia or astrocytes. These studies may guide complement-lowering therapies for AD and other neurodegenerative diseases and will provide novel research models to many fields.

Both exosomes and oncosomes are extracellular vesicles produced by normal and cancerous cells respectively, and are found in most bodily fluids. Oncosomes are of increasing clinical and scientific importance because they are involved in cancer metastasis. A device is presented to meet the challenge of high throughput and high-volume processing of clinical fluids, by continuously separating oncosomes/exosomes from the clinical fluid, and then separating the oncosomes from the exosomes. The device separates based upon physicochemical differences (such as size, density, and charge) of the exosomes/oncosomes rather than upon affinity techniques. Field flow fractionation will show feasibility and discover the physical property differences that exist between oncosome and exosome from cultured cells of melanoma and melanocytes respectively. Serum will be used with several split-flow lateral-transport thin separation (SPLITT) instruments. SPLITT will be used to separate exosome from oncosome and compared against standard techniques for exosome refinement. Upon successful completion of these technical objectives, a detailed cost/time analysis will be used to justify the evaluation of development of beta prototype, additional clinical samples, and cancer types in Phase II funding.

DESCRIPTION (Verbatim from the Applicant's Abstract): Experimental work done to date indicates that Opto-Acoustic Tomography will provide breast images with greater contrast and sensitivity to cancer than the currently preferred methods. In a Laser Optoacoustic Imaging System (LOIS) short near-infrared laser pulses are absorbed preferentially in tumors to generate pressure profiles resembling distribution of absorbed optical energy in the breast. Variation of the laser wavelength permits targeting either blood-rich malignant carcinoma or fibrous-tissue-rich fibroadenoma. Therefore, pressure waves emanating from tumors deliver information to the breast surface not only about the location and dimensions of tumors, but also diagnostic information. At the breast surface signals are detected by wide-band, piezoelectric transducers and analyzed to generate an image. Extensive laboratory research has proven the practicality and significant advantages of LOIS. LOIS is suitable for examination of all breasts independently on skin color, age and radiological density. The goal of the work proposed is to develop a commercial prototype instrument. The work during the Phase I project will focus on the general design, design of system components and fabrication of an advanced opto-acoustic array detector. Fabricated system components will be employed in the clinical research system at UTMB and tested with five breast cancer patients. PROPOSED COMMERCIAL APPLICATION: The proposed work will ultimately lead to a commercial device that will be useful as a primary tool in screening for breast cancer and as a secondary imaging tool for reduction of the large number of false positives generated by other screening procedures, such as X-ray mammography. The estimated cost per examination will be comparable to that for an ultrasonic image. The device will be suitable for guiding needle biopsies of suspicious lesions and new thermal therapeutic methods (such as focused ultrasound or laser, fiber-optic ablation) for eradication of those lesions. A potential for eventual application of the equipment in various diagnostic, monitoring and therapeutic procedures will guide further design considerations.

The objective of this proposal is to demonstrate biologically significant images with a new type of microscope of a novel and very simple design. In general, this microscope uses soft x-rays as the imaging radiation. The geometry of the microscope is very similar to the field emission microscope. Instead of using a solid emitter, the instrument uses a special cone-like, hollow emitter. The specimen to be examined is placed inside the hemispherical emitter tip. Soft x-rays are beamed through the specimen and onto the tip membrane. The tip membrane is transparent to x-rays and is coated with a high efficiency photoemitter. Photoelectrons are liberated Into the vacuum, outside the emitter tip, in a pattern corresponding to the x-ray absorption of the specimen (contact image). The photoelectrons are accelerated radially to a detector by a high voltage on the emitter. A magnified, high resolution (100 _10001), real time image results. This instrument has the potential for valuable use in biomedicine. Specimens can remain wet, unstained, and unsectioned. This technology has already been demonstrated with harder x-rays. Phase I research will address the more demanding technology needed for utilizing the desired ultrasoft x-rays and biological specimens.

It is generally accepted that an overdenture may be indicated when the remaining teeth and periodontium cannot support a conventional partial denture. The patients' best interests are often fulfilled by not rendering them edentulous. The purpose of this clinical and histological study of 20 patients is to evaluate the feasibility of utilizing vital pulpotomy procedures as a substitute for complete endodontic treatment in single-canal teeth to be retained for overdenture support. A method is proposed that eliminates the need for endodontic treatment on overdenture abutment teeth. This approach reduces treatment time and cost, yet provides increased efficiency in mastication and sensory perception as well as a state of well being and increased confidence to the patient. Three teeth that satisfy criteria for overdenture abutments are identified in one arch of each patient. These teeth will be treated, as necessary, to achieve a healthy periodontium. Immediate dentures are fabricated and at delivery, the abutment teeth each have a pulpotomy performed utilizing a selective sterile technique. Following removal of pulpal tissue to a selected depth, hemorrhage is controlled until stopped. Dentinal chips are obtained from the pulpal wall and placed over the pulp. Dycal is placed over the dentinal chips to approximately a 1 mm. depth and when set, silver amalgan is gently layered in using lateral condensation to avoid pressure on the pulp. The remaining pulp chamber is filled and root surface contoured to meet the dimensions of conventional overdenture abutments. Complete root canal therapy will be performed if any of the pulpotomized teeth exacerbate. Post-insertion evaluations will be performed at 1 day, 2 weeks, 3 months, 1 year and 2 years. Histologic evaluation will be made when the third tooth (control) is extracted at the third month after insertion of the overdenture. The remaining two roots will serve as long-term overdenture abutments.

We have attempted to elucidate the mechanisms of the neuropathogenesis of HIV-1 infection. We have investigated the interaction between HIV-1 and cerebellar granule cells in culture using two HIV isolates from AIDS patients with and without overt neurological symptoms. Upon exposure to HIV-1, progressive disruption of neuronal network and destruction of cells occurred in a time- and virus-dose-dependent manner. Immunostaining revealed that only neurons, not astrocytes or other proliferating cells, are killed. Viral proteins and HIV-1-specific DNA and RNA were detected in the culture of cerebellar neurons. HIV-1 isolates from the brain of an AIDS dementia patient is consistently more potent than other isolates from patients with immunological disease. This is the first evidence that cultured neuron is a direct target for HIV-1. In addition, we have preliminary results showing that three protein species isolated from various plants have anti-HIV-1 activity when tested in human peripheral blood lymphocyte systems.

The long-range objective of this project is to elucidate the basic mechanisms of drug allergy in man. The prototype chosen for study is penicillin hypersensitivity, the most prevalent drug allergy. An ongoing clinical study evaluates the usefulness of skin testing with penicillin derivatives in assessing the risk of immediate allergic reactions. For the major penicilloyl antigenic determinant, we have developed a radioallergosorbent test (RAST) for measurement of IgE in the sera of allergic patients, and two radioimmunoassay procedures for penicilloyl IgG. These serological assays are being applied to immune response studies in patients receiving penicillins therapeutically. Protocols are proposed to investigate the possible technical, metabolic, and genetic explanations for an apparent restriction of penicilloyl immune responsiveness. This project will pursue similar studies of the immune response to heterologous insulins administered to patients with diabetes mellitus. The frequency of IgG and IgE immune responsiveness will be determined, and the persistance of such antibodies will be evaluated in an effort to understand the pathogenesis of insulin allergy and insulin resistance. Additional studies to be pursued with these techniques include evaluation of cross-reactivity among penicillin and cephalosporin antibiotics and the development of serological tests for minor determinant penicillin antibodies. Patients with insulin or penicillin allergy who are subjected to desensitization will be studied in detail including serial IgG and IgE antibody determinations and evaluation of changes in basophil and skin sensitivity. The role of IgG antibody in protecting against IgE mediated allergic reactions will be explored. The goal of this work is better understanding of the mechanisms of clinical desensitization to insulin and penicillin, and of protective mechanisms which prevent clinical reactions in patients who have mounted a drug-specific IgE response.

We hypothesize that a donor dendritic cell (DC) population in the rhesus macaque can be identified and purified to allow its effective employment in tolerance-enhancing strategies in renal transplantation. Using technologic approaches that we have applied successfully in humans to identify circulating (P)DCl and pDC2, we have generated preliminary data that demonstrate presumptive counterparts of these DC precursors/immature DC in the rhesus macaque. In AIM I, we shall further characterize these cells to validate their relationship to human (and mouse) DC subsets, and to establish their influence on allogeneic T cell responses, particularly in relation to their postulated tolerogenic effects and underlying mechanisms. Use of hematopoietic growth factors (Flt3L and G-CSF) will greatly assist in addressing this Aim, Since. in vivo generated immature myeloid (M)DC presently represent the "standard" tolerogenic DC, we shall compare and contrast the influence of in vivo-mobilized pDCl and pDC2 with that of their immature, in vitro-generated MDC counterparts. Based on the outcome of AIM I, we shall in AIM II, test the most promising putative tolerogenic rhesus DC subset in allogeneic recipients using established and cutting edge assays of anti- donor reactivity .We shall also investigate underlying mechanisms, in particular whether infusion of putative tolerogenic DC results in immune deviation. Although other non-exclusive mechanisms may be operative, we shall focus on the ability of rhesus DC to skew towardsTh2 predominance as immature murine DC and human pDC2 have been shown to promote this mechanism. In AIM III, we shall work closely with our collaborators at Emory University Transplant Center and the Yerkes Primate Research Center in Atlanta. We shall test the donor DC population shown to exhibit the maximal tolerogenic potential in AIM II, in an established; MHC-mismatched rhesus renal transplantation model. Transplantation will be performed in conjunction with anti-CD40L mAb treatment of the recipient that, we hypothesize will markedly potentiate the tolerogenic effect of immature donor DC. Since deoxyspergualin has been shown recently to prevent DC maturation in rhesus renal allograft recipients in a tolerance- promoting regimen, we shall retain the option of incorporating this additional agent to further potentiate the tolerogenic action of immature donor DC combined with costimulation blockade.

The Animal Core serves as a centralized resource that provides mice with genetic deletions or mutations to the members of the Center, other research groups at the university, and the general scientific community. The animal lines that we have focused on thus far have deletions of genes in three systems: opioid receptors (mu-, delta- and kappa-receptor subtypes), cannabinoid receptors (CB1, CB2 and CB1/2 double knockouts), and chemokine receptors and ligands (CCL2, CCR2 and CCR5). Significant progress to date has been made by multiple laboratories associated with the Center using these lines. In addition to continuing to breed the existing animal lines, in the next grant period we will breed combinatorial mutant mice with genetic deletions of multiple genes such as two opioid receptors (Le. mu/delta) or an opioid receptor together with a chemokine (Le. mu/CXCR2). In addition, we will include two new models: mice with conditional gene deletions using the Cre-LoxP system and mice that express specific proteins tagged with enhanced Green Fluorescent Protein (EGFP). Conditional gene deletions allow investigators to evaluate the function of specific proteins of interest within a specific cell type or brain region or within a specific time window without the embryonic lethality and/or developmental compensatory responses that limit the utility of constitutive knockout strategies. The first of these mice that the Animal Core will breed will be a CXCR4-floxed mouse line. EGFP-tagged mice allow researchers to monitor the expression and localization of the tagged protein of interest in vivo or in vitro without impacting protein function and without reliance on antibodies. The first two strains of EGFP transgenic mice that we propose to breed are the CXCR4- and CX3CR1-EGFP lines. Multiple proposals for the use of the existing and new animal lines are detailed in the application. In-house breeding and use of the same animal strains by multiple investigators enhances collaborative studies and increases scientific synergy. Furthermore, because some of our existing and proposed mouse strains are unavailable commercially, the Animal Core can serve as a national resource, providing unique mouse models to the scientific community.

The research project to be completed during the proposed pre-doctoral fellowship is focused on the role of long term diet and dietary inflammation in cancer etiology. Given the evidence that dietary factors have either anti- or pro- inflammatory properties, and the idea that no nutrient is consumed alone but in conjunction with other nutrients and non-nutrient components of food, we developed and validated a dietary inflammatory index (DII) to assess the overall quality of diet with regard to its inflammation potential. We recently applied the DII to the Women's Health Initiative (WHI) Food Frequency Questionnaire (FFQ) data and evaluated the association of the DII with three markers of systemic inflammation: high-sensitivity C- reactive protein (hs-CRP), interleukin-6 (IL-6) and tumor necrosis factor alpha receptor 2 (TNF- 2) at baseline. We inferred from the findings that the FFQ-derived DII is likely to detect significant associations between the inflammatory potential of overall diet and common chronic diseases, including cancer, in the WHI and similar populations of postmenopausal women. Our current objective is to use data from the WHI observational study and clinical trial to: (I) investigate the stability of the DII over time in postmenopausal women, (II) evaluate the association between repeated measures of the DII and risk of breast cancer in postmenopausal women, and (III) evaluate the association between repeated measures of the DII and risk of colorectal cancer. The short terms goals that I will achieve at the end of the training period include the following: (1) an improved mastery of diet assessment methods, including the ability to critically evaluate the epidemiologic literature relative to diet and cancer; (2) an increased understanding of translational issues relevant to cancer prevention and control, including a better understanding of the mechanisms by which diets/dietary patterns predispose to, or prevent cancer. (3) an acquisition of a rich experience in cancer- and nutrition-related racial disparities including the ability to better communicate research findings to both the scientific and lay communities, and (4) an increased participation in many career development activities such as ethics seminars, online courses, preparation and submission of a career development grant to help establish myself as a responsible and productive investigator.

The experiments proposed in the projects of this application involve the study ofthe immune responses directed to the viral vector capsid and the transgene product in AAV-mediated gene transfer. Thus, the Vector Core is an essential constituent of this proposal, as it will provide support to the experimental activities outlined. All the projects require several different transgenes, reporter genes, and expression cassettes to be packaged in alternate AAV serotypes 1 through 9, together with other capsid variants, including variants with mutations at surface-exposed tyrosine residues, or with insertions of immunomodulatory viral peptides into the N-terminus of VP2, a vector capsid protein. Furthermore, the projects involve in vitro (cell culture) and in vivo (small animal models) experiments. Accomplishing the goals of each individual project will need a high degree of flexibility in terms of quality, number, and scale of each individual AAV preparation. It should be noted that differences in capsid structure of newly isolated serotypes and/or capsid mutants may require fine-tuning ofthe purification and formulation protocols, therefore it is preferable to have expertise in AAV vector production within the program rather than relying on contract organizations. During the previous funding period we established a Vector Core with proven production capability (~8E15 vector genomes produced), able to achieve economies of scale, and, most importantly, providing reliable vectors with a high level of purity and potency. The Vector Core will continue to produce AAV vectors with consistent and comparable quality in the new proposal using a helper virus-free transfection system and gradient centrifugation purification techniques, to support proposed studies in vitro and in animal models. The Core will produce, purify, quantify, aliquot, and store the vectors and provide the requested quality controls, depending on users'request. Quality ofthe vector will be carefully monitored to maintain titer, purity, and potency. Additionally, as in the previous funding period, the Core will manage other services for the projects, including characterization of AAV vector preparations, DNA plasmid production, peptide library resuspension and storage, and other activities that serve the scope ofthe proposed application.

The long-term objective of the proposal is to investigate the role of neurotransmitter dopamine (DA) in colon tumor neovascularization. Although substantial amount of endogenous DA is present in normal colon tissues, in contrast there is loss of DA in malignant colon tumor tissues. Furthermore, as we have recently demonstrated that DA inhibits vascular permeability factor/ vascular endothelial growth factor (VPF/VEGF) induced angiogenesis and because VPF/VEGF mediated angiogenesis promotes growth and metastasis of colon cancer, it is therefore possible that DA may also inhibit tumor neovascularization and thereby growth of colon cancer. The experiments proposed here are designed to investigate the role of DA on angiogenesis and vasculogenesis in preclinical colon cancer model. Finally, the role of endogenous colon DA on colon tumor angiogenesis and growth will be ascertained. Aim I. To investigate the effect of DA on neovascularization and growth of metastatic colon cancer. Experimental Design: The anti-angiogenic and anti-tumor efficacy of DA either alone or in combination with conventional anticancer drugs in mice bearing highly metastatic orthotopic human colon cancer. Aim II. To elucidate the molecular mechanisms of DA mediated regulation of tumor neovascularization. Experimental Design: The signaling pathways (PKA and MAPK) through which DA inhibits angiogenesis and vasculogenesis will be elucidated. Aim III. To investigate the role of endogenous DA on the angiogenesis and growth of colon cancer. Experimental Design: the rate of tumor angiogenesis and growth will be determined in DA depleted mice and mice treated with specific DA D2 receptor antagonists. Finally, the mechanism (synuclein mediated) of depletion of colon tissue DA will be determined. The knowledge generated from this proposal may not only identify a novel regulator of malignant colon tumor neovascularization and growth, but can also suggest a newer and an effective therapy for the treatment of advanced colon cancer patients. Most importantly, DA (a small molecule and a relatively cheap drug) is already in clinical use with an established safety record;therefore any positive findings from this proposal can be rapidly translated to the clinics for undertaking a clinical trial to treat advanced colon cancer patients.

Aging correlates with a marked susceptibility to infectious diseases. The progressive decline with age of the immune system - immunosenescence - is the primary underlying cause of this increased susceptibility. Important gaps remain in our understanding of the fundamental nature of immunosenescence, as well as in our practical ability to protect older adults against infectious diseases. Some of these result from the insufficient integration of the available models in which to research immunosenescence, and incomplete validation of the relevance of obtained data to the human aging. This proposal seeks to reduce this gap by a concerted effort to integrate human and mouse models and study memory T cell response in the skin of healthy young and old donors. In a novel human model, we have identified a significant defect in older adults in delayed-type hypersensitivity (DTH) responses to tuberculin antigens (Ag) by cutaneous CD4 memory T-cells. This has led to our central hypothesis: that one of the main age-related defects in immunity is the inability to efficiently direct Ag-specific memory responses to the sites of initial microbial challenge (skin, mucosa). We will test this hypothesis using a combination of complementary mechanistic mouse and human studies, designed to cross-inform each other, and always ultimately verifying the mechanisms in humans. PUBLIC HEALTH RELEVANCE: Infectious diseases inflict heavy toll upon the rapidly aging society with regard to lost productivity, mounting health costs and loss of life. At the conclusion of these studies, we expect that they not only will strongly contribute to our mechanistic understanding of age associated defect in immunity, but that their translational nature will offer insight into how to boost the attenuated immune responsiveness in old humans.

Glucocorticoids are the most common cause of nontraumatic osteonecrosis of the hip, a crippling disorder that often leads to total hip replacement. Osteonecrosis, the in situ death of a segment of bone, develops in up to 40% of patients receiving systemic glucocorticoids, especially after the administration of intensive parenteral courses. Although the pathology of the end-stage disease has been partially described, the cellular and molecular mechanisms responsible for the development of glucocorticoid-induced osteonecrosis remain unidentified and there is little consensus on optimal intervention strategies. This is because the sequential pathological changes in clinically asymptomatic patients are unknown and an animal model that replicates the progression of the human disorder is absent. Glucocorticoid therapy causes a decline in bone strength that surpasses the decline in bone density, but the mechanism behind this phenomenon remains unknown. Although it is widely appreciated that bone is composed of cells, mineral and collagen, it is seldom realized that water is another major component accounting for more than one fourth of the wet weight of bone. Fracture resistance of hard tissues is defective without water and water confers to bone much of its unique strength and resilience. The objective of this proposal is to use a murine model of osteonecrosis of the femoral head to determine whether glucocorticoid- induced deterioration of bone water and vascularity may account for the disproportionately greater decline in bone strength than in bone mass typical of glucocorticoid-induced osteonecrosis and whether the bone strength and vascularity may be compromised through the direct actions of glucocorticoids on osteocytes. Studies aimed at the cellular and molecular mechanisms of the pathogenesis of glucocorticoid-induced osteonecrosis and investigation of potential preventative therapy would increase motivation to protect the hip as early as possible. We hypothesize that glucocorticoid-induced osteonecrosis of the femoral head is primarily due to adverse effects on femoral head osteocytes because of reduced bone vascularity and canalicular fluid. To achieve this goal, osteocytes in transgenic mice will be shielded from administered glucocorticoids to determine if these animals are protected from osteonecrosis. In addition, mice expressing a hypersensitive glucocorticoid receptor in osteocytes will be examined to determine if osteonecrosis is exaggerated. Next, the impact of the administration of alendronate or intermittent PTH to prevent glucocorticoid-induced osteonecrosis will be examined. The studies proposed in this application are timely and by capitalizing on modern concepts and innovative methodology offer the opportunity for new insights that are sorely needed for the prevention and treatment of glucocorticoid-induced bone disease and are, therefore, immediately relevant and vital to the VA health care mission.

This proposed research is a revised competing continuation of a previous award. Our previous research showed that surface neuromuscular electrical stimulation (SNMES) can markedly increase muscle mass in individuals with complete spinal cord injury (SCI). We found pilot data that suggested that SNMES can also increase insulin action after complete SCI who are glucose tolerant. The general aim of this proposal is to extend our research to test whether exercise therapy can improve insulin action in complete SCI individuals who are either diabetic (DIA) or have impaired glucose tolerance (IGT). The specific aims are: 1) Can 3 months of resistance or endurance exercise therapy be affective at treating IGT or DIA in complete, SCI individuals; and 2) What are the physiological adaptations (increased muscle mass, decreased fat mass, increased muscle endurance) are responsible for the improved insulin action following SNMES-induced exercise therapy. 100 subjects (84 to start the program, 78 expected to finish) with complete SCI (either paraplegic or quadriplegic) who are either IGT or DIA, 18 to 55 yrs of age, will be studied over the next 5 yrs. The resistance training will consist of knee extensions with ankle weights, two sessions of 40 lifts per week for 3 months. The weight used will be increased as needed. Endurance exercise therapy will use twitch stimulations for 30 minutes three times per week. A control group (no exercise) will be used for comparison. The following techniques will be used to assess the efficacy of the therapies; an oral glucose tolerance test for insulin action, magnetic resonance imaging for muscle size and lipid deposits, and SNMES for the fatigue tests. Rejection of the null hypothesis will show that those with a complete SCI can use either residence-based, self-administered resistance or endurance exercise therapy to treat IGT/DIA and reduce the risk of cardiovascular disease. In addition, physiological adaptations that best predict changes in insulin action will be determined. A blocked design will be used to separately evaluate IGT and DIA. This research will address methods to prevent diabetes which is an important health problem in SCI. This study will also use practical self-administered exercise protocols, and will test links between insulin action and modifiable physiological functions. Thus, this research has the potential to significantly benefit people with spinal cord injuries.

As our society continues to age, it struggles with a host of ethical issues with which previous generations were never confronted. Included are decisions about the extent to which succeeding generations should be responsible for one another, how care is provided and how decisions about end of life care are made, and how advances in genetic technology influence the kinds of lives we lead. This application requests support for three conferences designed to advance interdisciplinary research on aging and ethics. By providing a forum for scientists from diverse backgrounds and ethicists who are committed to understanding the intersection of aging and ethics to gather, a unique opportunity would exist for stimulating critical thinking, defining the salient questions that remain unanswered, and crafting the foundation for the next generation of empirical research studies to be conducted. Each conference would have an applied focus as it struggles with a major issue at the intersection of aging and ethics. Conference topics would include: (1) Responsibility and community across the generations, (2) End of life, and (3) Aging, genetic technology, and the future. Invited presenters would come from disciplines including, but not limited to, philosophy, ethics, theology, gerontology, sociology, anthropology, psychology, medicine, and law. Commissioned papers would have as their goal generating discussion and new empirical research based on theoretical and empirical knowledge bases. The proceedings from each of the conferences would form the basis of an edited volume.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) and one of the most common causes of nontraumatic disability among young and middle-aged people. One of the hallmarks, however, is the progressive neurodegeneration that plays a key role in the progression of neurological disabilities. Little is known of the link between neuroinflammation and neurodegeneration. Recent biochemical studies suggested that there is defective oxygen metabolism in mitochondria due to increased nitric oxide (NO) as a result of vascular inflammation, which may play a crucial role in neuronal/axonal injury. In addition, NO is a strong mediator of neurovascular coupling that is responsible for increased blood supply during transient neural activation. In MS, the presence of a tonically high NO level (even during resting) may desensitize the vascular smooth muscle over time with a consequence of decreased vasodilatory capacity or cerebral vascular reactivity (CVR) and limited blood supply when neurons perform a demanding task. The Overarching Goals of this proposal are to detect and characterize abnormalities in oxygen consumption and vascular reactivity in early MS and identify tissues at risk using several advanced metabolic/vascular MRI techniques. These include a recently developed T2-Relaxation-Under-Spin-Tagging (TRUST) for the evaluation of global cerebral metabolic rate of oxygen (CMRO2) and a patient-comfortable blood-oxygen-level-dependent (BOLD) paradigm using CO2 inhalation to measure CVR. We will quantify CMRO2 and CVR abnormalities in patients with early relapsing-remitting (RR) MS and subsequent advanced stage of secondary progressive (SP) MS that are associated with clinical disability and disease progression. We hypothesize that oxygen metabolism abnormality in conjunction with impaired blood flow regulation is a key factor causing early degeneration. We also hypothesize that the combined functional index of CMRO2 and CVR has the potential to be an objective marker to predict neurodegenerative progression and its clinical outcome in MS. The Specific Aims are as follows: 1. To assess global CMRO2 abnormalities using TRUST MRI in patients with early RR and SP patients as compared with age/sex matched normal controls; 2. To measure CVR using inhalation of 5% CO2 and BOLD imaging in order to elucidate the nature of cerebrovascular dysfunction in MS patients; 3. To measure CVR and CMRO2 changes in healthy controls between groups with low and high intake of dietary nitrate (corresponding to higher NO level); 4. To determine longitudinal changes of CMRO2 and CVR in early RRMS patients and their relationship to imaging and clinical outcomes over a 5-year period. Health Relevance: By providing in vivo MRI confirmation of our working hypotheses, this application could have profound consequences for our understanding of disease pathogenesis and progression (neurological disability and cognitive decline) in MS, and for the future design of novel therapeutic strategies.

The proposed study will evaluate effects of an enhanced and expanded version of the Alcohol Risk Management (ARM) program, a four-session responsible beverage service training program for managers of on-premise alcohol establishments (i.e., bars, restaurants). ARM is a one-on-one training program that provides managers with the knowledge and skills necessary to develop responsible, establishment-specific, alcohol service policies. By influencing establishment policies, the ARM program aims to reduce illegal alcohol sales to obviously intoxicated patrons-and ultimately, to lower rates of related problems such as violence and traffic crashes. In our previous randomized trial that evaluated ARM, we observed reductions in likelihood of illegal alcohol sales to obviously intoxicated patrons one month following the training, but the effects decayed within three months. We propose to expand and enhance the training program based using Social Cognitive Theory to magnify immediate effects and sustain the effects by creating a hybrid version of ARM that includes both in-person and online training (e-ARM). e-ARM will address limitations of the earlier version of the program in several ways, including by involving more managers per establishment in the training, including ongoing online interactions (e.g., tips/reminders, discussion boards with other participants, news updates, etc.), and an online server training tool. We will conduct a randomized, controlled trial to assess the short and long-term effects of e-ARM on likelihood of reducing illegal alcohol sales to obviously intoxicated patrons and risky practices that lead to heavy consumption of alcohol. PUBLIC HEALTH RELEVANCE: Alcohol use is related to many public health problems. To prevent these alcohol-related problems, we have developed a training program for managers of bars and restaurants to prevent illegal sales to patrons who are obviously intoxicated. We are proposing to evaluate this training program to see whether the program decreases the likelihood of alcohol sales to obviously intoxicated patrons and promotes establishment environments that may lead to lower consumption of alcohol.

Objectives To evaluate currently used radiopaque agents in a wedge catheter system as a means of achieving parenchymal damage in a variety of endocrine tumors. Methods Employed Prolonged straining of parathyroid adenomas following injection of moderate doses of contrast material through a wedge catheter system has led to permanent control of hyperparathyroidism in a small number of clinical subjects. We are investigating in laboratory animals the potential for using this wedge catheter system to permanently ablate organ function without the use of particulate embolizing agents or plastics. Balloon catheters are used to obstruct arteries into which undiluted contrast material is perfused into the vascular bed of the target organ.

Chem Trak has demonstrated feasibility and is currently in Phase II development of a noninstrumented, quantitative device which accurately and precisely measures total cholesterol from a whole blood sample. ChemTrak now seeks to expand that technology and develop a noninstrumented quantitative test for HDL cholesterol. Such a test would be of immense value to both medical professionals as well as their patients for assessing risk for coronary heart disease. Total cholesterol and HDL cholesterol tests that can be performed in the doctor's office or by patients at home would provide for an inexpensive means to more closely and more conveniently monitor these independent risk factors for coronary heart disease. These products could serve as tools to educate the public as to the significance of elevated blood cholesterol and the importance of treatment, diet and exercise in the early detection and treatment of coronary heart disease. The research will evaluate methodology for the separation of HDL using the AccuMeter format and evaluate the ability to quantitatively measure the cholesterol over the range of 25 to 100mg/dL of HDL cholesterol.

The long-term goal of our research program is to elucidate the in vivo pathological significance of a novel molecular mechanism, which may be important for the regulation of genes in response to over activity of the angiotensin type 1 receptor (AT1R). Angiotensin II (AngII) is the classical mediator of the effects of the renin- angiotensin system on the cardiovascular homeostasis. This receptor regulates gene expression targeted by the AT1R blockers (ARB), a widely used class of anti-hypertensive drugs that are currently in trial for heart failure (HF) prevention. Inhibition of AT1R in vascular, renal, neuronal and cardiac cells by ARBs protects, but unregulated AT1R activation causes disease states such as hypertension, renal failure, cardiac hypertrophy and progression to HF. We have discovered a novel AT1R signaling paradigm, wherein, Gaa2?12 mobilizes into the nucleus. In the nucleus, G[unreadable]2 functions as an epigenetic modulator of gene expression programs. Thus, G[unreadable]2?12 appears to function as a novel AT1R-to-nucleus messenger that mediates AngII-induced regulation of genes. The goal of this project is to understand the in vivo significance of hither-to-unknown consequences of G[unreadable]2 functions in the nucleus which may be useful for targeted therapy. Currently, it is unknown whether G[unreadable]2 translocation is prevalent in human disease states. No experimental models for studying enhanced G[unreadable]2 functions in the nucleus exist and there are no pharmacological tools to modulate G[unreadable]2 interactions with nuclear targets. To overcome these barriers would require high-risk innovation. The overall objectives of this application are to validate the relevance of the phenomenon in a human disease state;develop new experimental models to study the role of G[unreadable]2 in the nucleus and to develop small molecules to modulate nuclear functions of G[unreadable]2. Our central hypothesis is that exaggerated nuclear translocation of G[unreadable]2 contributes to sustained or "chronic" transcriptional activation leading to pathophysiological responses. We will pursue the following specific aims;(i) Determine interactions of G[unreadable]2 in the nuclear proteome of in vivo disease models including human heart failure samples;(ii) Evaluate pathological consequences of enhanced G[unreadable]2 function in the nucleus in a novel transgenic mouse model;(iii) Develop small molecule probes for disrupting G[unreadable]2 interaction with transcription factors. If the AT1R activity is not regulated properly, AngII stimulus becomes chronic and can damage the tissue, as well as contribute to chronic disorders of myocardium. A clear understanding of novel transcription regulatory mechanisms is important to improve the therapeutic application of ARBs. These proposed studies will advance our knowledge of AT1R biology. PUBLIC HEALTH RELEVANCE: The angiotensin type 1 receptor (AT1R) for the vasoactive hormone AngII is a regulator of gene expression targeted by the angiotensin receptor blockers (ARB), a class of anti-hypertensive drugs. Inhibition of AT1R by ARBs protects against, but unregulated AT1R activation causes disease states such as hypertension, renal failure, cardiac hypertrophy and progression to heart failure. We have discovered a novel AT1R signaling paradigm, wherein, G[unreadable]2?12 mobilizes into the nucleus. In the nucleus, G[unreadable]2 functions as an epigenetic modulator of gene expression programs. In this proposal we will be investigating the novel epigenetic control of transcription linked to disease states. These studies are necessary to understand mechanisms of progression of cardiovascular diseases, and to identify new drug targets for intervention. Fatalities from cardiovascular diseases remain a public health concern and adequate treatment for their reversal is currently lacking.

The initial biochemical step in the mechanism of action of Epidermal Growth Factor (EGF) and polypeptide hormones in general, is reasoned to be its binding to specific receptors on the cell surface. Our main objective, therefore, will be to investigate EGF-receptor interactions in defined experimental systems. This will require the use of cell free systems in which EGF forms a hormone receptor complex which initiates a defined biochemical reaction so that binding can be coupled to biologic responses. We have chosen as our model the human epidermoid carcinoma cell line, A-431 because it has an extraordinarily high concentration of specific membrane receptors for EGF (2-3 million/cell). We have found that binding of EGF to these membranes in vitro produced a marked immediate stimulation of the phosphorylation of both endogenous and exogenous protein substrates in the presence of (Y-32P) ATP due to a cyclic nucleotide independent, tyrosine residue specific, protein kinase. In A-431 cell membranes two forms of EGF receptor-kinase have been observed (170K, 150K daltons). EGF stimulated kinase co-purifies with EGF receptor as the 150K from isolated membranes but as the 170K from vesicles. An endogenous calcium activated protease generates the 150K from the 170K. Prolonged receptor occupancy and internalization appear to be required for the mitogenic effects of EGF. It is likely that receptor processing and its subsequent proteolysis generate several forms of the EGF receptor-kinase which have altered kinase activity, substrate specificity and linkage to more than one regulatory pathway affecting cellular functions. Therefore, we propose to study: 1) role of specific membrane and cytoplasmic components in initial events occuring after EGF binds to cells or cellular subfractions; 2) role of autophosphorylation in regulating EGF receptor-kinase; 3) structure of EGF receptor-kinase using labeling, selective digestion, peptide mapping and sequence analysis methods; 4) by biochemical and immunological techniques the relationships between normal and abnormal differentiation and EGF receptor-kinase.

Herpes simplex encephalitis (HSE) of childhood is a life-threatening central nervous system (CNS)-specific complication of primary infection by herpes simplex virus 1 (HSV-1). We showed that HSE may result from a novel group of primary immunodeficiencies (PIDs), with autosomal dominant (AD) TLR3 and autosomal recessive (AR) UNC-93B deficiency. We recently discovered an AR form of TLR3 deficiency, as well as AD TRIF, TRAF3 and TBK1 defects (unpublished). Moreover, other children with mycobacterial disease and lethal HSE bear mutations in STAT1 (AR) or NEMO (X-linked recessive, XR). These results indicate that lesions in the TLR3-dependent, interferon (IFN)-inducing pathway predispose to HSE. The pathogenesis of HSE involves a specific pathway in the CNS, as the lesions observed are restricted to this system. Our observations in skin-derived fibroblasts from patients further suggest that the molecular pathogenesis of HSE involves impaired viral dsRNA-triggered, TLR3-dependent IFN production in the CNS, resulting in increased viral replication and enhanced cell death. However, the cellular basis of the pathogenesis of HSE remains unclear, as TLR3-IFN and anti-HSV-1 immunity have not been investigated in CNS cells. TLR3 was reported to be expressed and functional in neurons, oligodendrocytes, astrocytes, and microglial cells, leading to IFN production. Moreover, these four cell types can be infected by HSV-1 in vitro. We hypothesize that the pathogenesis of HSE involves impaired TLR3-IFN immunity in at least one of the four CNS resident cell types, particularly non-hematopoietic neurons, astrocytes and oligodendrocytes. To test this hypothesis, we intend to investigate TLR3-IFN and anti-HSV-1 immunity in CNS cells. As we have no access to primary cells from the patients' CNS, we will take advantage of the fibroblasts available from healthy controls, from HSE patients bearing all known HSE genetic etiologies (mutations in UNC93B1, TLR3, TRIF, TRAF3, TBK1, STAT1, and NEMO) and from patients with other inborn errors of IFN immunity (mutation in TYK2) to derive induced pluripotent stem cells (iPSCs). These iPSCs will then be differentiated into neurons, oligodendrocytes, and astrocytes. TLR3-IFN and anti-HSV-1 immunity will be assessed in the three iPSC-derived CNS cell types from healthy controls and from HSE patients. Also, cells from selected HSE patients with an intact TLR3-IFN pathway in fibroblasts will be investigated, to search for a CNS-specific cellular phenotype. We have already successfully generated iPSCs from one UNC-93B-deficient patient with HSE and from one healthy control. These iPSCs possess stemness and pluripotency gene expression signature. We have also shown that stem cell-derived CNS cells from healthy controls can be tested for TLR3-IFN and anti-HSV-1 immunity. In sum, this approach will allow us (i) to specify which CNS cell types are responsible for HSE in patients bearing genetic deficiencies in the TLR3-IFN pathway, (ii) to search for CNS-specific HSE-causing cellular phenotypes in other patients and (iii) to provide proof-of-principle for the iPSCs-mediated investigation of the cellular pathogenesis of non-hematopoietic PIDs.

The objective of this project is to study the inhibition of colon carcinogenesis by green tea polyphenols and their combination with atorvastatin (ATST, trade name Lipitor). Tea and ATST are commonly consumed or used by humans. Both agents have been shown to inhibit colon tumorigenesis in animal models and have been suggested to reduce colon cancer risk in humans. Based on our preliminary results, we hypothesize that when the two agents are combined, the cancer preventive activity would be higher. In this project, the inhibitory activities and the mechanisms of action of (-)-epigallocatechin-3-gallate (EGCG), alone and in combination with ATST, will be studied in a rat model and related cell lines. The specific aims are as follows: 1. Determine the inhibitory actions of EGCG and its combination with ATST in an AOM-induced rat colon carcinogenesis model. We will use different concentrations of EGCG (0.08, 0.16, 0.32, &0.48% in the diet) to determine the dose-response relationship in the inhibition of colon carcinogenesis. Then we will study the combination of EGCG and ATST at different doses to determine whether the inhibitory effect is synergistic or additive. The inhibitory action will be correlated with the levels of EGCG and ATST in colonic tissues and blood. 2. Elucidate the mechanisms of inhibition of colon carcinogenesis by EGCG and its combination with ATST in AOM-treated rats. Using samples from Aim 1, we will examine the effects of the different treatments on cell proliferation and apoptosis, on key oncogenic signaling molecules (such as (3-catenin, Akt, Erk1/2, and COX-2), key tumor suppressive signaling molecules (such as E-cadherin and RXRcc), and on membrane association of small G-proteins in the tumorous and non-tumorous colon samples. Short-term animal experiments with adenoma-bearing rats will be used as a direct approach to obtain mechanistic information in vivo. Combined immunohistochemical (IHC) and biochemical analyses will be used for these studies. 3. Delineate detailed mechanisms of colon cancer prevention by EGCG and its combination with ATST in integrated studies with human colon cancer cell lines and the animal model. More detailed mechanistic studies on the actions of EGCG and its combination with ATST will be pursued in human colon cell lines and then in colon tumor samples from animal experiments. We will study possible direct targets of EGCG action and related novel mechanisms for the inhibition of carcinogenesis. From these studies, we hope to develop a better understanding of the chemopreventive activities of EGCG and their combination with ATST against colon carcinogenesis. as well as promising biomarkers for future studies.

Here we propose to continue our studies on elucidating cis-acting regulatory elements in the mouse kappa immunoglobulin (Ig) gene locus, namely, the elements that target locus accessibility to the recombinational and transcriptional machinery in B lymphocytes. We plan on focusing on the three following regions that are both conceptually attractive as well as experimentally proven to contain accessible chromatin structures and in some cases novel regulatory elements: (1) The upstream sequence (US) 5' of the most distal variable (V) region, which is always preserved after any type of V gene rearrangement; (2) The intervening sequence (IS) between the joining (J) region and the most proximal V region, which upon V-J joining is either deleted or inverted and far removed from the rearranged V gene destined to be expressed; and (3) Germline V region promoter and recombination signal sequences (RSSs). During the previous period of support, our previous results established that these regions in chromatin contain pre-B specific nuclease hypersensitive sites. In addition, we have evidence that both the US and IS contain recombination enhancers and that the IS possesses a developmentally regulated transcriptional silencer. This proposal presents the following three major aims: 1. To functionally elucidate the regulatory DNA sequences within the US. 2. To functionally elucidate the regulatory DNA sequences within the IS. And 3. To investigate possible mechanisms responsible for generating chromatin accessibility within germline V region promoters and recombination signal sequences (RSSs).

Because it is an energetic pure beta emitter with a half-life on the scale of days (64.1 hr) 90Y is seeing increasing interest as an isotope for use as a radiation source for radioimmunotherapy (RIT). Current processes for obtaining 90Y involve extracting and purifying the 90Y produced by the decay of 90Sr in solution using a liquid-liquid extraction process. The isotope is produced at a centralized location and shipped to users worldwide. The generator proposed here will permit the production of the desired isotope locally in a hospital or regional radiopharmacy where it can be used immediately. Phase I identified two inorganic ion exchange materials that can be loaded with a "cow" of 90Sr and "milked" to produce a carrier-free solution of 90Y ready for immediate use. In Phase II this technology will be further refined and used in the design of a generator capable of delivering a useful quantity of 90Y on a weekly basis in a chemical form suitable for radiopharmaceutical applications. The generator will be fabricated and its efficacy will be demonstrated. The superior long-term stability of the inorganic ion exchanger compared to organic ion exchange materials will be demonstrated. PROPOSED COMMERCIAL APPLICATION: Radioimmunotherapy, where an antibody selectively delivers a radiation source directly to a tumor, is seeing increasing interest as a means of attacking tumors by delivering a therapeutic dose of radiation to the tumor with minimal exposure for other organs. Currently 90Y is produced by a complex multistep process process and supplied from a central source, with clinicians dependent on reliable delivery to have the isotope they need when they need it. The generator proposed here would make 90Y as readily available as 99m Tc is now and greatly increase the use of this valuable therapy. As an alternative, the developed technology could be utilized on a larger scale to simplify the current 90Y production method.

The Medical Intensive Care Unit (MICU), administered by the Critical Care Medicine Department in the NIH Clinical Center, receives critically ill patients from clinical programs of NIH. The research goals of this project include the development of techniques for automated patient monitoring and noninvasive measurements of the cardiovascular and respiratory systems. Catheterization studies are performed as necessary to obtain data that are available only through invasive methodology. The automation of the MICU has aided the medical staff by managing the large amount of data needed for the care of the critically ill patient, performing desired calculations, and allowing measurements that would not otherwise be possible. The multiple-computer system is utilized in support of research protocols, in addition to direct patient care.

This research will provide a basis for quantifying the relationship between contaminant exposure history and regulation of microbial biodegradative capabilities in natural porous media under flowing conditions representative of field scale plume contamination scenarios (low substrate concentrations and low levels of mixed electron acceptors [oxygen and nitrate]. Processes influences natural attenuation of four model compounds, representing three classes of priority contaminants (fuel hydrocarbons, chlorinated solvents, and polynuclear aromatic hydrocarbons) will be investigated. Well-characterized microbial/contaminant/porous media systems will be used to develop validated mathematical tools for the description of pollutant elution, transport, and microbial transformation. The experimental design is based on a series of integrated studies that range in scale from molecular (regulation of biodegradative pathways), to batch (kinetics of contaminant utilization), to bench-scale (flowing column studies), to field scale (stimulations of field conditions). Batch and column studies are proposed to: 1) evaluate the influence of substrate composition, porous media characteristics, and concentration exposure history of biodegradative gene expression under natural attenuation conditions; 2) investigate links between contaminant exposure history and regulation of biodegradative functions; 3) evaluate the influence of surface attachment on expression of biodegradative capabilities under natural attenuation conditions; and 4) develop state-of-the-art mathematical models to validate conceptual models, test hypotheses, and explore the implications of laboratory findings at the field scale. at the field scale. Knowledge gained from this Project pertaining to processes influencing microbial transformations will provide the foundation for improved Superfund site remediation at chlorinated solvent and hydrocarbon contaminated sites. In addition the work proposed here, in conjunction with Project 4e, will allow for development of approaches for assessment of 'alternative endpoint' clean- up standards under intrinsic and engineered remediation conditions.

Oligodeoxyribonucleoside phosphorothioates (PS-ODNs) have been, and are still, extensively studied as potential therapeutic agents against various types of cancer and infectious diseases in humans. Given that these oligonucleotide analogues are P-chiral, each of the internucleotidic phosphorothioate linkages adopts either a Rp or a Sp configuration. Stereopure Rp-oligodeoxyribonucleoside phosphorothioates have been prepared enzymatically and exhibited a lower stability to nucleases endogenous to human serum than the parent PS-ODNs with undefined P-chirality. Conversely, chemically synthesized stereopure Sp-oligodeoxyribonucleoside phosphorothioates have demonstrated superior stability to these nucleases than P-diastereomeric PS-ODNs. Thus, to further investigate the biological, pharmacokinetic, and toxicologic properties of P-stereopure PS-ODNs improved chemical methods are required to synthesize these biomolecules and increase their availability for clinical studies. We have discovered that deoxyribonucleoside cyclic N-acylphosphoramidites are efficient monomers for the stereospecific synthesis of PS-ODNs. Indeed, base- assisted condensation of the 5'-OH function of a nucleoside or a nucleotide covalently linked to a solid support with deoxyribonucleoside cyclic N-acylphosphoramidites led to rapid and efficient formation of an internucleoside phosphite triester linkage. The phosphite triester function can be either oxidized to a phosphate triester(PO)by treatment with tert-butyl hydroperoxide or sulfurized by a sulfur transfer reagent to a P-stereodefined thiophosphate phosphotriester(PS)function to permit, for example, stereocontrolled synthesis of chimeric PO/PS-ODNs. In this context, the stereospecific synthesis of the PS-ODNs [Rp,Rp]- and [Sp,Sp]-d(CpsCpsC), [Rp,Sp,Rp]-d(CpsCpsCpsC), and [Rp]11- d(Tps)11T have been successfully accomplished. The method is currently being optimized to enable the incorporation of the four different nucleobases into DNA chains of at least 20 bases long. During the course of our investigation, we have discovered that phosphate/thiophosphate protection when using cylic N-acyl phosphoramidites was heat-sensitive. This discovery led to the development of a number of novel thermolabile phosphate protecting groups. We have also recently found that these unique phosphate protecting groups could also be adapted to the 5'-OH protection of nucleoside and oligonucleotides. These findings, along with the fact that deoxyribonucleoside cyclic N-acylphosphoramidites can, unlike conventional deoxyribonucleoside phosphoramidites, effectively produce oligonucleotides under relatively wet conditions, are attractive features for parallel synthesis of oligonucleotides on arrayable surfaces. Oligonucleotide microarrays can be invaluable for analyzing gene expression from, for example, tumorigenic versus non-tumorigenic human cell lines, and high-throughput screening of point mutations and single nucleotide polymorphisms in genomic DNA that predispose human to diseases.

This R25 application requests 5 years of funding to support a 6-site, interdisciplinary training consortium in Child Intervention Prevention, and Services mental health research (CHIPS). The CHIPS program will provide early career scientists (ECSs) who are postdoctoral fellows or junior faculty, with career enhancement and direction through an annual 5-day intensive summer research institute (SRI), and the development of mentoring relationships with institute faculty. The overarching goals of this program are to: (1) recruit and retain promising ECSs to child and adolescent mental health intervention, prevention, and services research; (2) prepare researchers who can conduct research that spans the boundaries of intervention, prevention, and services research; and (3) increase the success rate and decrease the time to successful acquisition of external NIH funding for ECSs. To achieve these goals the CHIPS training consortium will: (1) recruit a new cohort of 15 ECSs each year, who have demonstrated a strong interest in and potential for developing an academic career in child intervention, prevention, and/or services research; (2) provide an annual 5-day intensive educational workshop that will integrate mentoring activities with didactic teaching on research methods and processes that span the boundaries of intervention, prevention, and services research; (3) develop a one-year career enhancement plan that involves regular contact with a mentor from the CHIPS faculty consortium by phone at the summer institute and at least one visit to the mentor's center; and (4) evaluate the impact of CHIPS by monitoring participants' satisfaction, career development, contact with the mentor (and satisfaction with that contact), and academic productivity as measured by publications, presentations, and external funding. This application supports a training consortium of 6 sites, namely, University of Pittsburgh (the coordinating site, David Brent, MD), Arizona State University (Irwin Sandler, PhD), Children's Hospital, San Diego (John Landsverk, PhD), Johns Hopkins University (Phil Leaf, PhD), Oregon Social Learning Center (John Reid, PhD), and University of California, Los Angeles: Bonnie Zima, MD, MPH). These sites have successful track records in helping early career scientists develop independent research careers. Collectively, they span the breadth of intervention, prevention, and services research, and thus can promote the development of a broader training perspective to program participants than any single training site.

The principal aim of this research is to extend our understanding of the acute effects of graded doses of three central nervous system depressants on human cognitive functioning. The drugs studied in the original proposal were secobarbital sodium, methaqualone HC1, and meprobamate. Since the highest dose of meprobamate (20.5 mg/kg) produced little effect in sensitive information processing tasks, we propose to discontinue the study of that drug and substitute ethyl alcohol. Each of the drugs will be tested in six multiple factor experiments. The several experiments are designed to converge on the question of the locus (or loci) of drug effects on specific theoretical operations (stages) in current models of information processing and memory. Our present data suggest that secobarbital and methaqualone slow very early processes on the stimulus input side, whereas alcohol disrupts operations on the output side (e.g., response selection and organization). All three drugs seem to impair memorial processes related both to short-term store and long-term store. The proposed experiments should provide a finer-grained analysis of the drug's effects while providing some integration of the previous findings from information processing and memorial tasks. The range of proposed doses is sufficiently wide to separate drug effects from dose effects.

The goal of this project is to define the mechanism that determines Xist mRNA stability. Based on previous work by the investigator, the structure of the murine Xist gene has been revised to include new information on the 3' end of the gene. The investigator has shown that this area of the gene is significantly larger than previously reported. Sequence comparison between mouse and human revealed sequence similarity in the new 3' ends. Using both Northern analysis and RNAse protection experiments, the investigator has confirmed that both Xist mRNA isoforms are produced by a mechanism involving differential polyadenylation. These polyadenylation sites are located in the new 3' sequences identified. Additional expression studies have shown that Xist mRNA isoforms are developmentally regulated, and therefore show different stabilities. This pattern of expression suggests that regulatory elements may interact differentially with each mRNA isoform, which in turn may influences Xist expression early in development. The data suggest that Xist mRNA isoforms change independently of Tsix and that the developmental regulation of the Xist isoforms is influenced by mRNA stabilization during the period in which upregulation of the chosen X chromosome is observed. The specific mechanism responsible for Xist stability remains poorly characterized. Although early in development Xist is unstable, after development Xist is exceptionally stable (T1/2 = >5 hr). Recent data have placed in doubt the importance of the 5' end of Xist in this regulation. The investigator plans to explore the role of the new 3' end of Xist in gene stability. He also proposes to pursue the mechanism of developmental regulation of Xist stability from two directions. First, a complete a mutational analysis of Xist and the regions immediately adjacent to Xist will be done. Second, the investigator plans to evaluate genes in the methylation pathway and define their role in determining Xist mRNA stability.

As microwave energy becomes increasingly present in our environment, an unmonitored population is placed at risk while we must admit to insufficient data regarding the biologic effect of such exposure. The basic aim of this project will be to determine the effects of a long term (4 months) exposure to microwave radiation (MWR) at low incident power densities. Our intent will be to examine the behavioral effects of administratively safe MWR (i.e., less than 10 mW/cm2 incident power density). Data will be obtained from several behavioral variables, and we will define a dose/response curve over the range of 0-20 mW/cm2. The chronic MWR environment will comprise individual RF chambers into which control and experimental animals will be individually introduced for 3-4 hours of whole body MWR exposure per day. Pulse modulated MWR at 1.3 GHz will be used. In the first part of this study, the effects of 0, 2, 5, 10, and 20 mW/cm2 MWR on open field behavior and on instrumental behavior for food reward will be determined in rats. Besides the 0 mW/cm2 group, additional control groups will be exposed to a mild thermal and acoustic stress. In the second part of this study and to initiate the extrapolation of the results to human exposure, the changes in instrumental behavior relevant to learning will be determined. Again, dose/response curve will be defined, via radiation at 5, 10, and 20 mW/cm2 with dual control groups, as above. These studies will provide new and needed data regarding the origin of chronic low level MWR effects on cognitive behavior and learning.

Thin filament-associated actin-binding proteins have dual function, controlling actomyosin-based contractility and cytoskeletal assembly in a variety of muscle and non-muscle systems. In striated muscles, the regulatory protein complex, troponin-tropomyosin, linked to thin filaments, controls contractility by sterically blocking and unblocking myosin-crossbridge binding on actin in response to changing Ca/2+ levels. In smooth muscle, thin filament-associated caldesmon and calponin may function similarly to modulate actomyosin-based motility and/or the state of cytoskeletal assembly. To accomplish our goal to determine the regulatory mechanisms by which these proteins function in striated and smooth muscles, it is crucial to assess their structural interactions on isolated and reconstituted thin filaments and their respective responses to Ca/2+, myosin-crossbridge binding and phosphorylation. For a fuller picture of filament function, it is also essential to understand the structural interactions of muscle and related non-muscle actin- cytoskeletal proteins State of the art electron microscopy, computer- assisted image analysis and three-dimensional reconstruction will be used to determine the arrangement of thin filament components on F-actin and to evaluate their position and influence on actin domains. Reconstruction will be fitted to the atomic map of F-actin to detail contacts with specific amino acid clusters on actin monomers. Our own published reconstructions and those of our colleagues demonstrate the feasibility of these goals. Our continued structural studies on troponin-tropomyosin regulated filaments will lead to an elucidation of the molecular mechanism of relaxation and activation in skeletal and cardiac muscle. Our studies to smooth muscle filament swill contribute to understanding the fine tuning of the smooth muscle contractile response and the construction of its cytoskeleton. A general understanding of the molecular mechanisms involved in the regulation of contractility in healthy muscle tissue will aid in our evaluation of defects occurring in disease. The control of smooth muscle contractility, for example, is of great importance in the regulation of vascular tone and pulmonary airway resistance, determinants in a number of disease processes such as hypertension and asthma. The wider significance of our goals is underscore by possible participation of caldesmon, calponin and proteins with consensus calponin homology-domains in such diverse cellular processes as cytokinesis, exocytosis, cortical cytoskeleton modeling and signal transduction modulation, i.e. processes that are essential to all normal cells and that can become aberrant in malignancy.

Fiscal Year 2019 has seen significant progress toward accomplishing both Specific Aims; some of this progress is detailed here. For Aim 1, the first project focuses on the early development of MS lesions. Previously, we studied two critical aspects of lesion development: the small veins around which white matter lesions form, and the short-to-medium-term outcomes of acute lesions. To understand whether the presence of a central vein may help distinguish MS lesions from their mimickers -- an idea that remains controversial but to which we mostly subscribe -- we previously developed a rapid imaging approach for clinical 3-tesla MRI called FLAIR*. Two studies to assess the utility of FLAIR* for diagnosis and characterization of MS lesions have been published in the last year (9, 27), and a patent incorporating this technology has been submitted (22). We found that FLAIR* is able to significantly improve diagnostic confidence in a variety of settings, including the so-called radiologically isolated syndrome. Our technique is now in use at more than 30 around the world, and with the North American Imaging in MS (NAIMS) cooperative, we are conducting a multicenter pilot clinical trial to assess whether FLAIR* allows earlier and more confident diagnosis of the disease. A U01 grant to extend this to a full-scale trial has been submitted to NINDS extramural, and we will collaborate should it be funded. In related work, we have established the long-term deleterious consequences of perivenular inflammation in the white matter, finding that perivenular collagen type I deposition is widespread in the MS brain and may impair the proliferation and differentiation of myelin-repairing cells (1) With respect to lesion outcomes, we previously established that there are two spatiotemporal patterns in MS lesions: a centrifugal pattern, in which serum contents leak from the center of the lesion and then proceed outward, over the course of minutes to hours, to fill the entire lesion; and a centripetal pattern, in which serum contents first appear on the periphery of the lesion and then proceed inward. These findings have important implications for understanding lesion development and its association with blood-brain-barrier permeability. In further work, we described how these permeability patterns help to determine the fashion in which acute MS lesions evolve into their chronic counterparts. Among other things, we found that very early events, perhaps occurring within the first month after lesion formation, appear to determine the efficacy of tissue repair, possibly including remyelination. We also showed that we can reliably identify chronically inflamed lesions on clinical MRI systems. In the past year, we have published a large cross-sectional and longitudinal study showing that such lesions can expand slowly over time and are associated with clinical disability progression (2). With the NINDS Neuroimmunology Clinic, we are conducting a clinical trial to test whether corticosteroids prevent the evolution of acute to chronically inflamed lesions, and we have recently initiated a new trial to assess whether the inflammation in these lesions can be abrogated. We have also increased our focus on the characterization of MS lesions affecting the cerebral cortex, which have proven difficult to detect by MRI (unlike their white matter counterparts). Our approach here has been to evaluate new MRI approaches with potentially higher sensitivity than previously described methods, taking advantage of the 7-tesla research system at NIH and of our collaborations with MRI pulse sequence developers at NIH, in the extramural community, and in industry. In prior years, we described and are routinely using a method that more than doubles the sensitivity for cortical lesion detection, and we recently submitted a patent application for a new technique to visualize these lesions on clinical MRI scanners (20). We have also followed up our prior discovery of the imaging correlate of inflammation in the leptomeninges (the membranes that surround the brain) by collaborating with colleagues at Johns Hopkins on a clinical trial of intrathecal rituximab, which was unfortunately unsuccessful (4). Additionally under Aim 1, we have continued our work on improving methods for image acquisition and analysis (8, 10, 11, 13, 14, 18, 19, 23, 24, 26). For Aim 2, we described the imaging and cellular/molecular events in early inflammatory demyelinating lesions that develop in the brains of marmoset monkeys with experimental autoimmune encephalomyelitis (EAE). We previously suggested, using coarse MRI techniques, that the blood-brain barrier becomes locally permeable up to four weeks prior to the onset of demyelination, and we showed that this permeability is associated with a perivascular lymphocytic and mononuclear infiltrate with parenchymal activation of microglia and astrocytes. We have since shown directly that the plasma protein fibrinogen leaks into the brain parenchyma prior to demyelination, and that fibrinogen can also be seen in chronically inflamed lesions in people with MS. In the past year, we have published the first radiological-pathological correlative study of spinal cord lesions in marmoset EAE 15). We also collaborated with NINDS colleagues to describe the effects of herpesvirus inoculation on the course of experimental disease in this model (16). The results will pave the way toward using the marmoset model in preclinical studies to predict the response of people to novel treatments. Finally, we continue to contribute to review and position papers with various national and international consortia (12, 17, 18).

The glycine receptor (GIyR), is a member of the nicotinicoid receptor superfamily, which include the homologous nicotinic acetylcholine receptor, the 5-HT3 serotonin receptor, and the GABAA receptor, all of which act in rapid mediation of signal transduction at the synapse. This receptor is a glycine-gated anionic channel, and is the major inhibitory neurotransmitter channel in spinal cord and lower brain. The overall goal of this CEBRA proposal is the determination of human alpha1 GlyR structure at high resolution. These investigations of GlyR structure aim to provide structure-function information at a molecular level. Since this receptor family is targeted by anesthetics, alcohols, inhaled solvents and other narcotics, these structural determinations will impact on our understanding of the basic mechanisms underlying ion channel inhibition, desensitization, and activation. The CEBRA mechanism of funding is especially relevant since in the absence of diffractable microcrystals, funding is typically unavailable. Yet, dedicated higher-risk efforts such as those described in this proposal require support for successful high-resolution determination of membrane protein structure. The proposed studies exploit the previously developed expression system (Cascio et al., 1993). Successful overexpression and reconstitution of homomeric GlyR (Cascio et al., 2001) presents a unique opportunity to undertake structural studies of the GlyR. Preliminary studies noted a cholesterol-dependent conformational change in GlyR. Additionally, our coupled proteolysis and mass spectrometry studies (Leite et al., 2000) and spectroscopic studies of reconstituted GlyR (Cascio et al., 2001) have determined that the four-transmembrane helix model for the nicotinicoid receptors may be inappropriate. We propose to use recent advances in membrane protein crystallography exploiting lipidic cubic mesophases, co-crystallizations with monoclonal antibodies and/or crystallographic studies of a soluble form of the ligand-binding domain of the receptor. Given the difficulties in determining high-resolution structures of membrane proteins by crystallographic methods, we also alternatively propose to further refine receptor topology via determination of experimental constraints. These distance constraints will be determined using an EDTA-strychnine reagent or site-directed Cys mutagenesis coupled with chemical modification studies and mass spectrometry. Overall these investigations aim to provide insight into the general conserved structure of nicotinicoid channels.

Vaccines are a proven and effective approach for assuring widespread protection of large populations prior to or immediately following a bioterrorism attack. Our goal is to address two significant problems currently raised by bioterrorism threats: 1) the need for an easy method of mass vaccination and 2) the shortcomings of the current anthrax vaccine (NIAID "category A" agent) which requires needles and health professionals for administration, an extended vaccination schedule for protection (6 doses over 18 months) and the concern over adverse effects. To address this challenge, we have exploited the extensive safety record of the existing live, oral Salmonella Typhi Ty21a vaccine by utilizing it as a vector to develop a live oral vaccine carrier stably expressing rPA. We hypothesize that Ty21a is a suitable vaccine carrier for the stable expression of the rPA gene harbored on a low copy number plasmid. Further, we hypothesize that this vaccine can be formulated so that it will be safe, stable, and highly immunogenic and can be easily administered orally. The current proposal is aimed at completing the necessary final development steps (e.g. removal of antibiotic resistance gene from vector plasmid) and full characterization (genetic and microbiological) of the candidate prior to constructing a genetic seed bank and small-scale (5-10 liter) manufacture and lyophilization. Finally, the candidate will be studied for genetic stability, safety, immunogenicity and efficacy in a mouse model of anthrax disease. Successful completion of this Phase I SBIR will provide the foundation for development of an oral anthrax vaccine in a Phase II proposal, and for a platform technology for oral vaccines against multiple other infectious agents. PUBLIC HEALTH RELEVANCE: Anthrax bacteria produce spores that can be processed to become easily airborne. Vaccines are a proven and effective approach for assuring widespread protection of large populations prior to or immediately following a bioterrorism attack. Our goal is to address two significant problems currently raised by bioterrorism threats: 1) the need for an easy method of mass vaccination and 2) the shortcomings of the current anthrax vaccine (NIAID "category A" agent) which requires needles and health professionals for administration, an extended vaccination schedule for protection (6 doses over 18 months) and the concern over adverse effects. Further, we hypothesize that this vaccine can be formulated so that it will be safe, stable, and highly immunogenic and ultimately can be easily administered orally.

Since the inception of this project we have determined the complete structures of the two groups of phosphoproteins in human saliva which inhibit precipitation of calcium phosphate. These proteins are represented by statherin, a tyrosine rich acidic phosphoprotein, and the anionic proline rich protein, PRP-4, the most potent of the PRP's. During the second year of the project we have also obtained a tentative complete sequence of PRP-2, a 150 residue PRP, and have localized the active site of all the human PRP's to the amino terminal 30 residue tryptic peptide and the 38 amino terminal tryptic peptide in Macaca arctoides. In addition the statherin-like peptide in M. arctoides has been purified to homogeneity. With respect to statherin of human origin, we have been able to demonstrate that the NH2-terminal hexapeptide, containing the two phosphoserine residues, contains the part of the sequence required for phase-transformation inhibition but not for spontaneous precipitation. In fact the hexapeptide is 4 fold as active as intact statherin in this assay system. Similarly other N-terminal peptides have been purified following enzymatic digestion; these include the N-terminal heptapeptide, nonapeptide, decapeptide and N-terminal 18-amino acid peptide. With increasing C-terminal extension, phase transformation inhibition decreases. Research performed this last year included solid phase peptide synthesis of the phosphoserine dipeptide, NH2-Pser-Pser-COOH, the N-terminal pentapeptide and the N-terminal decapeptide of statherin. These peptides are also being synthesized via the classical solution approach. Initial results indicate, suprisingly, that the phosphoserine dipeptide is inactive but that the penta- and decapeptide have considerable activity suggesting that the peptide backbone contain part of the statherin structure is required for biological activity. These peptide analogs will be studied by 31P NMR to determine the calcium binding site and to learn the conformation of these interesting macromolecules. Finally in collaboration with Frederic Coe, at Michael Reese Hospital in Chicago, we are beginning to study the structure of an inhibitor of calcium oxalate precipitation in human urine.

The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, of which the Toll-like Receptors (TLRs) play a prominent role. The molecular basis for the recognition of PAMPs by TLRs is a main interest of my laboratory. In collaboration with Dr. David Davies (LMB, NIDDK) and with the help of the Protein Expression Laboratory (SAIC/Frederick), we expressed mg amounts of the extracellular domain (ECD) of TLR3 and we continue to develop ways of expressing ECDs of other TLRs. The ECD of TLR3 crystallized and we determined its structure by X-ray crystallography, with and without its ligand, dsRNA. The structure of the TLR3-ECD consists of a 23 turn coil, bent into the shape of a horseshoe. The molecule is heavily glycosylated, except that one lateral face of the horseshoe is totally devoid of glycan. In solution, purified TLR3-ECD binds dsRNA at mildly acidic pH with an affinity that increases with dsRNA length. TLR3-ECD is monomeric in solution, but it forms dimers when bound to dsRNA. These dimers bind to 45 bp segments of dsRNA and multiple TLR3-ECD dimers bind to long dsRNAs. To determine the molecular basis for ligand binding and signaling, we isolated, crystallized, and solved the structure of the TLR3 signaling complex, consisting of two TLR3-ECD molecules bound to one 46 bp dsRNA oligonucleotide. The glycan-free surfaces of two TLR3-ECDs in the complex face one another on opposite sides of the dsRNA molecule which lies between them. The overall structure of mTLR3-ECD:dsRNA complex contains a two-fold symmetry axis, dictated by the inherent two fold symmetry of the ligand. No conformational change was observed in either the receptor or the ligand upon ligand binding, suggesting that dimerization per se is the activation signal for TLR3. Three intermolecular contacts on the glycan free surfaces of the two TLR3-ECDs stabilize the complex, two protein-dsRNA interactions, and one homotypic interaction between the two TLR3-ECD molecules, that is responsible for dimer formation. Using a novel ELISA to analyze dsRNA binding by mutant TLR3 constructs we identified the essential interacting residues and showed that the simultaneous interaction of all three sites is required for ligand binding. In addition, we found that TLR3 is unable to bind dsRNA when dimerization is prevented by mutating residues in the dimerization site or by immobilizing TLR3 at low density. Our results indicate that dimerization of TLR3 is essential for ligand binding and that the three TLR3 contact sites individually interact weakly with their binding partners but together form a high affinity ligand-receptor complex. The three other endosomal TLRs, TLRs 7, 8, and 9 form a closely related family of TLR paralogs that, like TLR3, recognize nucleic acid PAMPs, but differ from TLR3 in primary structure. It can be surmised from their amino acid sequences that these TLRs have several extended loops protruding from the glycan-free and convex surfaces of the LRR horseshoe, and an extended undefined, poorly conserved region between LRRs 14 and 15. Unfortunately the ECDs of these TLRs are secreted from insect cells in very low amounts, and the secreted material forms large, undefined aggregates. Currently we are investigating TLR9, which recognizes DNA that contains unmethylated CpG sequences. Our current hypothesis is that TLR9-ECD forms a complex with other proteins in vivo, and this complex recognizes CpG DNA. We have previously shown that most TLR9 resides in the ER, then migrates in small amounts to endosomes where it encounters its ligand. We found that TLR9 interacts with the gp96 chaperone and with Prat4A, a co-chaperone, as expected for ER proteins. Our immediate goal is it to obtain cells in which most of the TLR9 has migrated to endosomes, and then to examine the endosomal TLR9, both full length and ECD for possible proteins that form a complex with it. We have been partially successful in enhancing the amount of endosomal TLR9 through the use of inhibitors that block protein synthesis and TLR9 degradation, and we are employing several strategies to improve upon these results. We are also trying to piece together the TLR9 structure by making chimeras that contain parts of TLR9 grafted onto the TLR3 framework.

The goal of this Mentored Patient-Oriented Research Career Development Award is to allow the applicant to develop the clinical research skills needed to pursue an independent, productive career in rehabilitation research. The applicant is a licensed physical therapist with a clinical doctorate completing her postdoctoral training in clinical investigation. This award will enhance the applicant's ability to investigate mechanisms underlying pre-arthritic hip disease (PAHD) by combining the applicant's existing expertise in musculoskeletal disorders with additional training in the kinematic assessment of lower extremity movement and advanced methods in patient-oriented research. The long-term objective is to develop effective rehabilitation strategies for people with PAHD that will prevent or delay the need for surgical or pharmacological intervention. The specific aims of the proposed studies are designed to test the overall hypothesis that abnormal movement patterns contribute to the onset and persistence of PAHD. The proposed studies will 1) determine musculoskeletal differences between people with PAHD and people without PAHD, 2) determine the association among lower extremity movement patterns, muscle strength, bony abnormalities and self-report measures of functional ability, and 3) obtain preliminary data on the effectiveness of movement pattern training on measured impairments and functional ability in people with PAHD (exploratory treatment trial). People with PAHD and people without PAHD will participate in completing self-report measures of functional ability, tests of hip muscle strength, 3d movement assessment (kinematic) of specific functional movements and magnetic resonance imaging (MRI) to assess bony structure. Baseline data will be compared to determine if differences exist between people with PAHD and people without PAHD. To determine the best predictive variables of self- report functional ability, stepwise multivariable linear regression modeling will be used with the self-report measures as dependent variables and movement pattern abnormality, muscle strength and bony abnormalities as independent variables. Subjects with PAHD who wish to participate in a rehabilitation program will be provided motor pattern training including education to correct abnormal movement patterns during daily activities and strengthening of weak muscles thought to contribute to the abnormal movement patterns. Pretreatment and posttreatment measures will be compared to determine if improvements in impairments (abnormal movement pattern, muscle strength) correlate with an improvement in subjects' self-report of function. Results of this study will provide data to inform future mechanism-based studies of PAHD rehabilitation and injury prevention. In addition, data from the exploratory treatment trial will lead to a larger clinical trial to assess the effectiveness of movement pattern training.