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Although epidermal growth factor (EGF) stimulates the growth of various cells in vitro, the biological role of this polypeptide is not known. Evidence that EGF may function as an autocrine or paracrine factor is suggested from previous studies demonstrating submaxillary gland preproEGF mRNA in other organs. We have shown that primary cultures of mouse uterine epithelial cells possess specific binding sites (Kd approximately 1.8 nM) for 125I-EGF and approximately 50 x 10-3 receptors/cell. EGF stimulated proliferation of the uterine cells in vitro, whereas other known growth factors did not. Immunolocalization of EGF in pronase-treated sections of the mouse uterus revealed staining at the luminal aspect of epithelial cells. Localization required protease treatment and the material reactive with anti-EGF antiserum was apparently not estrogen-dependent since this pattern of staining was readily observed in uteri from animals ovariectomized for over two weeks. Protease-treatment of sections was also required for localization of EGF in epithelial cells of the mouse kidney and mammary gland (mid-pregnant). We have found that kidney EGF occurs predominantly as the apparent precursor form (140 kDa) bound to cell membranes. Thus, it is likely that the observed pattern of EGF localization in uterus represents a membrane-bound form of the precursor. Hybridization with a 32-P-labelled cDNA probe for submaxillary gland preproEGF mRNA occurred with samples of uterine mRNA, although at much lower levels than that of kidney and submaxillary gland. The level of preproEGF mRNA increased in immature mouse uteri following treatment with estrogen. Northern blot analysis of uterine A+ mRNA from estrogen-treated mice revealed a single band of 4.9 kilobases, equivalent to that of submaxillary gland preproEGF mRNA. Studies are in progress to further understand the synthesis sorting and processing of the EGF precursor in different organs and the potential for various hormones to regulate these events.

Residential histories collected during a national survey of oral health of U.S. schoolchildren conducted in 1986-1987 were used to establish two groups: those children who always lived on a public water supply in a fluoridated community (N=8,165) and those who never lived in a fluoridated community (N=8,233). Comparisons of the mean levels of Decayed, Missing and Filled Surfaces were made by age, sex and region of the country. Mean DMFS for children with lifelong exposure to water fluoridation was 2.8 compared to 3.4 mean DMFS for children who had never lived in areas with fluoridated water. Mean DMFS for mesial-distal surfaces was about one-third higher in children without water fluoridation. Both groups of children reported high use of supplemental and/or topical fluorides. Regional differences between groups varied greatly from 61% difference in Region VII to 6% difference in Region III. These findings were presented at a workshop "Mechanisms of Fluoride" and published in the Journal of Dental Research, Sp. Iss. Feb. 1990. Mean dfs for deciduous teeth was computed for those children ages 5-9 who had a lifelong history of community water fluoridation or no history of water fluoridation. The mean dfs was lower in all age groups for children on fluoridated community water supplies, with a combined difference of 23% fewer dfs.

It is well known that the aged brain undergoes a series of subtle but progressive alterations that eventually lead to altered neuronal function and, ultimately, altered behavior of the individual. Together with the well-described age related decline in cognitive performance, it has also been found that motor function undergoes a steady and progressive decline in most aged humans. The midbrain dopaminergic neurons, which are important for regulation of motor activity, appear to be especially vulnerable to age-related factors, leading to a specific increase in neurodegenerative disorders afflicting these neurons, such as Parkinson's disease (PD) with age. This program project grant is focused on understanding why midbrain dopamine neurons are selectively vulnerable to environmental and endogenous agents, leading to lost function and structure during aging. We will work along an overall "dual-hit" hypothesis, proposing that it is a combined endogenous inability to cope with oxidative stress and external factors making it worse that leads to a selective degeneration of these neurons in some individuals but not others during aging. Every project is devoted to the understanding of age-related deterioration in the function and morphology of the midbrain dopaminergic system, with a focus on motor impairment following specific intrinsic and extrinsic damaging events. To this end, all primary projects in the program (Project 0001-0005) will provide a distinct level of analysis of three specific determinants of motor impairment, namely: growth factor reduction (intrinsic, genetic alteration), endotoxin exposure (prenatal extrinsic factor, LPS), and methamphetamine exposure (extrinsic factor to young adult mice). The Animal Core will provide all subjects to guarantee consistency between each project, and thereby facilitate the capacity to unite discoveries emerging from different research approaches into a coherent understanding of the neurobiological basis of factors that might affect the impact of aging in this transmitter system. The subjects will be examined at many levels of analysis, including cellular (Projects 0001 - 0004), tissue culture (Project 0005) and behavioral (Project 0001 and 2). Project 0003 will integrate the different treatments (GDNF/BDNF partial deletion, LPS treatment, and methamphetamine) from the perspective of neuroinflammation, and Project 0004 will examine the same three factors from the perspective of in-depth analysis of neurotransmission itself. Finally, Project 0005 will examine intrinsic versus extrinsic determinants of the alterations observed by isolating the brain regions (substantia nigra and striatum) in slice cultures or transplants. This thematic integration around an animal model (GDNF/BDNF knockouts) with external factors will focus multiple dimensions of research activity on a single problem (age-related decreased motor function) with the goal of providing a relatively complete description of the neurological basis of age-related dopamine neuron cell dysfunction leading to motor impairment.

Renal osteodystrophy (ROD) is a multifactorial and pervasive disorder of bone remodeling in chronic kidney disease (CKD). The prevalence CKD in the United States is estimated at 15.1% in 2011. With the aging population, the number of patients affected by ROD, therefore, is expected to increase. Despite the widespread use of therapies, such as phosphate binders, calcium, and vitamin D, bone-fracture rates and mortality risk following fractures are markedly greater in patients on dialysis, compared with the general population. Furthermore, the effectiveness of treating dialysis patients with conventional osteoporosis drugs is not established. The high cost and numerous side effects of drug therapies have spurred the search for alternative treatment options for ROD. It has recently been shown that low-magnitude mechanical stimulation (LMMS) is osteogenic. Although many theories exist, the mechanism of action of LMMS is not well established. A more precise understanding of how vibrations work is needed to optimize the efficacy of LMMS treatment and increase its appeal as a viable non-pharmacological solution to the problem of weak bone. One major hurdle in monitoring efficacy of treatment to LMMS in particular and other interventions in general is the lack of non-invasive tools to study the anabolic response at micro-structural levels in humans. All LMMS trials conducted to date use BMD as the outcome variable, an approach which has many limitations. In high-turnover ROD, for example, increased trabecular bone volume may offset cortical bone loss, resulting in normal or increased areal-BMD despite poor bone strength. The proposed project focuses on overcoming a critical barrier towards understanding the mechanism of action of LMMS therapy in humans by developing a non-invasive high-resolution imaging based biomechanics technique to study the dependence of local-strain on bone formation at the microstructural level in response to LMMS intervention in dialysis patients. If the aims of this project are achieved, scientific knowledge of LMMS treatment in humans and technical capabilities to monitor bone's anabolic response to treatment will be improved. Most importantly, the successful completion of the aims has the potential to change the current paradigm of managing ROD by adapting LMMS as an attractive and preventive intervention.

Clostridium difficile recently has been linked to antibiotic-associated enterocolitis and pseudomembranous colitis which are severe and sometimes fatal diarrheal syndromes. This toxin-producing, strictly anaerobic bacterium was first described in 1935, but has only been recognized as a clinically significant pathogen within the last 8 or 9 years. Evidence exists that the pathogenic effect of this organism is mediated by two toxins and that adherence of C. difficile to the host may possibly play a role in pathogenicity. Conclusive studies on the genetic determinant(s) responsible for the expression of C. difficile toxins have not been reported. However, plasmids and bacteriophage mediated production of toxin and other extracellular enzymes have been previously demonstrated in other clostridial species such as Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium novyi. Because of the previously reported linkage of toxin genes to extrachromosomal DNA and the documented occurrence of adhesive or invasive genes being coded for by plasmids in other bacteria, the high molecular weight extrachromosomal DNA of C. difficile will be characterized. Preliminary work in our laboratory has demonstrated a large extrachromosomal DNA element in 16 out of 16 strains of C. difficile tested. Also bacteriophage particals were identified with electron microscopy to be associated with C. difficile. The major objectives of this two year pilot study is to determine if the large extrachromosomal DNA observed is plasmid or phage in nature and to determine if this DNA codes for assayable determinants responsible for pathogenicity.

B3. Molecular Genetics and Epigenetics The Molecular Genetics and Epigenetics component of the IHSFC, formerly called Facility Core C: Microarray and Molecular Biology, has been transformed to address the expressed current and future needs of Center members for instrumentation and expertise support in the areas of gene expression, epigenetics and functional genomics. The goal of this activity is to provide to Center members access to cutting edge technologies in the analysis of gene expression and modulation through access to instrumentation, instruction, assistance in methods and training in the analysis of data. We have added the epigenetics analysis to this iteration of the grant, replacing resources that had been previously directed towards proteomics. This decision was made because the Proteomics Core at Johns Hopkins School of Medicine is (http://www.hopkinsmedicine.org/msf/) now widely available for Center members and we choose not to duplicate these efforts. The objectives of this component of the IHSFC are to: provide state-of-the-art analysis of gene expression and analysis using the Affymetrix platform for mouse (genome 430A 2.0 array), rat (expression 230 2.0) and human (genome U133 Plus 2.0 array) gene chips that comprise the global gene profiling for these respective species; provide infrastructure so that an investigator can understand the global gene expression changes modified by environmental exposures that are manifested through epigenetic changes; integrate with the Bioinformatics/Biostatistics core for using Bioconductor and other tools for the analysis of genetic and epigenetic changes.

Project Summary/Abstract There is significant evidence that anesthetics which act at the GABAA receptor alter immune function and that multiple immune cell types express functional GABAA receptors. However, the mechanism of this immune modulation is unknown. In this proposal, we present exciting preliminary data demonstrating that mice lacking expression of the GABAA receptor ?4 subunit (gabra4), a subunit expressed in immune cells including CD4+ lymphocytes, have significantly greater lung inflammation after sensitization with house dust mite antigen, a common allergic asthma model. This heightened inflammation results in greater in vivo airway reactivity. We also demonstrate that gabra4 knockout CD4+ cells express increased inflammatory cytokines compared to wild type CD4+ cells when stimulated via the T cell receptor in vitro, suggesting the hyper-inflammatory phenotype of the gabra4 knockout mouse may be CD4+ cell-mediated. We hypothesize that GABAA receptor currents, which are depolarizing in CD4+ cells, alter calcium signaling processes key in lymphocyte activation and function, specifically calcium oscillations and store-operated calcium entry. We propose to demonstrate that ?4 subunit containing-GABAA receptors mediate currents in CD4+ cells which are augmented by GABAA receptor ligands, including XHE-III-74A, a novel ?4 subunit selective positive allosteric modulator developed by a collaborator. We will also determine if the CD4+ cell is key in producing the gabra4 KO mouse?s hyper-inflammatory phenotype by performing adoptive T cell transfer experiments. Furthermore, we propose to determine if altered GABAAR signaling affects CD4+ cell calcium dynamics (oscillations and store operated calcium entry) using a novel in situ calcium imaging technique that utilizes precision-cut lung slice technology. This offers the key advantage of maintaining the native environment of the inflamed lung. Finally, we will determine if XHE-III-74A, an ?4 subunit- selective GABAA receptor activator that does not cross the blood brain barrier, will limit house dust mite antigen induced lung inflammation and airway hyper-responsiveness when administered chronically during the sensitization process. Although the studies proposed here focus on lung inflammation, the implications of this work may be relevant to inflammatory processes of many types. Given the widespread use of GABAergic medications, this research promises to be of high clinical significance. My department and Columbia University provide an ideal environment to complete these studies, as all necessary support is already in place. In addition, my proven mentoring committee provides a perfect blend of skills to help guide the science, including expertise in ion channel physiology, lung biology, immunology, and calcium signaling. A comprehensive career development plan has been established, including coursework and additional training. Taken as a whole, this K08 proposal outlines a robust pathway to scientific independence and the foundation of a successful and sustained career as a physician-scientist.

Solid Phase Sciences Corporation (SPSC) proposes to produce a library of substituted acridine and pyridine based heterocycles. The library will consist of 5-10 milligrams each of a total of 384 compounds. SPSC will accomplish this by utilizing it's novel high-loading support and commercially available solvent delivery robot. Specifically, libraries will be made off of this high-loading support to separate the desired products from undesired starting material. A liquid handling robot will be used to add reagents and solvents. The goal of the Phase I effort will be to integrate the use of this high-loading support in solid phase synthesis with automated liquid handling into one automated system to produce a library composed of a series of acridine and pyridine based heterocyclic compounds for general screening.

PROJECT SUMMARY Pseudomonas aeruginosa is a versatile bacterial pathogen that causes life-threatening acute and chronic infections in diverse patient populations. Complicating treatment is the ability of P. aeruginosa to resist the majority of antimicrobial therapies. It is therefore critical to identify virulence properties that can be targeted for the development of novel therapeutics. Several studies demonstrate the importance of iron homeostasis for P. aeruginosa pathogenesis. Recent work from our lab shows that the P. aeruginosa prrF chromosomal locus, which encodes the iron-responsive PrrF1 and PrrF2 small regulatory RNAs (sRNAs), is required for acute lung infection in mice. The PrrF1 and PrrF2 sRNAs contribute to iron homeostasis by repressing the expression of iron-utilizing pathways when this nutrient is limiting. This ?iron sparing response? further impacts diverse virulence properties, including quorum sensing and biofilm formation. The prrF locus produces a distinct sRNA (PrrH) that is regulated by heme, an abundant source of iron in the human body, thus linking iron and heme homeostasis pathways of P. aeruginosa. While our studies have established the broad impact of this locus on P. aeruginosa physiology and virulence, the mechanisms by which the individual sRNAs transcribed from this locus mediate gene expression and pathogenesis remain unknown. Based on our preliminary and published studies, we hypothesize that the PrrF and PrrH sRNAs play critical yet distinct roles in regulating P. aeruginosa iron homeostasis and virulence. We will test our hypothesis by 1) identifying PrrF target mRNAs responsible for virulence attenuation of the ?prrF1,2 mutant; 2) determining the genetic basis of heme regulated expression via the PrrH sRNA; and 3) defining the mechanisms by which PrrF and PrrH regulate gene expression. These studies will define the specific mechanisms by which the prrF-transcribed sRNAs mediate iron homeostasis and virulence of P. aeruginosa.

This proposal aims to understand the changes in visual processing that occur when subjects shift between alert and non-alert waking states. While great advances have been made in understanding central mechanisms of visual perception of alert, attentive subjects, there is little understanding of cortical processes that come into play when alertness wanes. The awake, non-alert state is not equivalent to anesthesia, or to sleep states. When non-alert, we are capable of perception, but our perceptual capacities differ. It is commonly believed that accidents happen when we are not alert, but the extent to which early thalamic or visual cortical mechanisms may be responsible for this (as opposed to higher cognitive processes) is an open question. This proposal relies on a unique model system that is very well-suited to address this question: the awake rabbit, an animal who's inner mental life transparently and frequently shifts between alert and nonalert EEG-defined states, and who's stable eyes and diffident nature make it an ideal subject for these experiments. The proposed research will examine how changes in the brain state of awake subjects influence the multiple, sequential stages of information processing that occur within the visual thalamocortical, intracortical, and cortical output networks. The experiments will compare state dependent changes in the visual response properties of excitatory and inhibitory neurons at the input layer and within several output systems of the cortex and will investigate the underlying mechanisms leading to these changes, at the subthreshold and spiking level. This work will lead to a better understanding of cortical mechanisms of visual processing in a dynamic, awake brain. From a health perspective, these studies will have an important impact on our understanding of how alertness/vigilance deficits can impact visual perception and performance, and will provide the basis for future clinical studies of human mental health and behavioral disorders.

Our primary aim is to study the morphological, biochemical and immunological properties of the mouse mammary tumor virus (MuMTV). The viral rosettes, consisting of viral membrane fragments with attached projections, and viral projections alone, are two structural components which have been isolated and their protein composition studied. Work is in progress on the isolation of viral cores and ribonucleoprotein complexes, which will be analysed by electron microscopy and polyacrylamide gel electrophoresis in order to identify the various proteins of the virus with its architecture. Purification of the various viral proteins, including reverse transcriptase, is an integral part of our program. The MuMTV-reverse transcriptase and two of the five major viral polypeptides have been purified. Within the next six months we hope to have enough purified enzyme from RIII milk borne MuMTV for antibody production. This antisera will be used to study the degree of relatedness between the 4 known variants of MuMTV and other oncornaviruses. The techniques for the isolation of MuMTV-polypeptides are being standardised. The purified viral proteines will be used for the production of monspecific antisera in order to determine the level of expression and distribution of several MuMTV antigens in various mouse tissues by radioimmunoassay.

The long-term goal of this research is to gain a better understanding of the physiological mechanisms underlying the hormonal control of behavior. This specific project is the initial step in determining if catecholaminergic neurotransmitters are involved in mediating the effects of hormones on behavior. The first goal of this project is to establish and validate methodology for delivering small amounts of drugs to into the ventricular circulation. Once the methodology is established, two studies will be run. They will serve both to validate the methodology and determine the effects of lowering catecholamine function on behavior. The first will examine the behavioral effects of decreasing norepinephrine levels primarily in telencephalic brain areas. The second will examine the effects of a more generalized depletion of both norepinephrine and dopamine. In both studies, norepinephrine and dopamine depletion in brain areas known to be involved in controlling the behaviors of interest will be monitored by high performance liquid chromatography with electrochemical detection. This will allow examination of the relationship between catecholamine depletion in specific brain areas and alterations in normal behavior patterns. This work will serve as the foundation for a more extensive investigation of the involvement of the catecholamines in modulating hormone-sensitive behaviors. It is obvious that catecholamine dysfunction is involved in a number of serious mental health problems. This research seeks to clarify how catecholamines modulate normal behavior. The neuropharmacological studies will demonstrate how different brain areas respond to drug treatment and the behavioral consequences of neurochemical changes in specific areas.

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. SCIENCE RESEARCH CORE The Arkansas INBRE proposes to continue its Science Research Core, which will comprise services offered by three scientific research facilities at two lead institutions. These core facilities will provide investigators throughout the state with access to the instrumentation and expertise needed for three key areas of modern biotechnology[unreadable]proteomics, digital microscopy, and DNA damage and toxicology. These three areas were chosen because the technical expertise and, especially, the instrumentation of each facility are not commonly available, even in an average, well-funded research laboratory, particularly not at four-year PUIs. These three facilities were especially chosen to address the specific needs of the 13 Arkansas INBRE Project Leaders (see Table 11). With access to these technologies for specific studies, researchers can then pursue and extend research projects in their own laboratories. The use of these facilities should help researchers obtain necessary preliminary data to apply for extramural research funding. Arkansas INBRE support for the Science Research Core and its three facilities is primarily intended to guarantee access to these facilities by PUI investigators (both faculty and students). This support is not sufficient to completely sustain these facilities, which derive additional, more substantial support from other grant funding sources and from the institutions at which they are located (UAMS and UAF). The three facilities, described in detail below, have the goal of providing expertise and equipment for the following purposes: + Proteomics: To enable researchers to study protein structure and function using state-of-the-art instrumentation; + Digital Microscopy: To allow researchers to locate macromolecules within cells and tissues, thereby better assessing their function in normal and diseased states;and + DNA Damage and Toxicology: To allow researchers to incorporate DNA damage and toxicity analyses (as in cell injury and death) into their studies of normal and diseased cells and tissues. Each Science Research Core facility will also be committed to educating and training Arkansas INBRE faculty and students at both partner and non-partner PUIs, in sophisticated technologies specific to each facility. Hence, all facilities will provide workshops or demonstration sessions relevant to the instrumentation and expertise available. These offerings will be organized by leaders of all Science Research Core facilities and publicized by the Administrative Core statewide. The Science Research Core will also provide access to other Core Facilities located at the lead institutions, for example, DNA Sequencing and Flow Cytometry, for all researchers within the Arkansas INBRE. In addition, through the INBRE consortium, the Arkansas INBRE will network with other INBREs to obtain access to facilities that may not currently be available in Arkansas (see http://louisville.edu/research/genomics/content/microarray_services.shtml and letter of support from the directors of the Kentucky INBRE and the University of Louisville Microarray Facility, Louisville, KY). The Science Research Core is also committed to assisting both student and faculty participants in the Summer Mentored Research Fellowship Program (see Outreach Core), enabling summer fellows to become familiar with technologies that facilitate modern biomedical research and, ultimately, to participate more fully in the INBRE program. As evidence of this commitment, faculty investigators at non-partner PUIs have been using the current INBRE facilities (see below under each facility). Workshops offered during the summer introduce researchers from across the state to available, state-of-the-art technologies. All facilities within the Science Research Core will interact as needed with the Bioinformatics Core to provide for the needs of the Arkansas INBRE partner PUIs (see Objectives, Bioinformatics Core). At present, one of the most daunting aspects of biomedical research concerns data management and bioinformatics tools for analysis. For proteomics research, data collection for protein structure determination requires new information technologies (protein informatics) to analyze and interpret the data. Faculty associated with the Bioinformatics Core will be available to provide the requisite expertise for data analysis, including more conventional statistical analysis of all types of data and analysis of data generated by microarray or proteomics techniques.

Inflammation or peripheral nerve injury increases neuropeptide Y (NPY) expression in pain-relevant areas of the nervous system, such as large primary afferents and lamina l/ll of the dorsal horn of the spinal cord. However, the contribution of endogenous NPY to chronic pain is unclear. This NRSA proposes to test the overall hypothesis that NPY tonically inhibits nociceptive signaling in the spinal cord and brain following inflammation or nerve injury. Specific Aim #1 will test the hypothesis that genetic knockdown of NPY increases allodynia and/or hyperalgesia associated with nerve injury and inflammation. Aim #1 experiments utilize a conditional knockout mouse (Npytet) that contains a doxycycline (Dox)-regulated cassette (tTA) upstream of Npy exons to evaluate the contribution of NPY to the development (Aim 1 A) and maintenance (Aim 1B) of chronic pain, and correlate these changes with NPY expression using immunohistochemistry and western blot (Aim 1C). While Aim #1 establishes a contribution of NPY inhibition to behavioral manifestations of chronic pain, Specific Aim #2 uses direct microinjection techniques to investigate the site of action of NPY. NPY Y1 and NPY Y2 receptors decorate lamina l/ll of the dorsal horn. I propose to target these receptors with intrathecal injection of selective Y1 or Y2 receptor antagonists (Aim 2A). Previous studies indicate that inflammation up-regulates the expression of NPY and Y1 in the arcuate nucleus of the hypothalamus (ARC), and that microinjection of NPY into the ARC attenuated behavioral signs of heat hypersensitivity. In Aim 2B, I propose to microinject Y1 or Y2 antagonists into the ARC, and predict that this will exacerbate allodynia and hyperalgesia. [unreadable] [unreadable] [unreadable]

The ability to predict the response of normal tissues to a given course of radiation might allow a clinician to anticipate and avoid undue toxicity. Recent studies have demonstrated that the intrinsic radiosensitivity of skin fibroblasts from individual patients is predictive of the severity of acute and late radiation reactions experienced by these patients. While radiosensitivity assays are cumbersome, newer techniques which measure DNA double strand breaks (dsb) or the level of p53 induction (a central molecule involved in radiation-induced apoptosis) following irradiation may prove to be useful as predictive assays. The primary goal of this project will be to assess whether these assays performed on skin fibroblasts obtained form patients treated with radiation are predictive for acute or late skin reactions. Early passage fibroblast cell lines will be established from 26 breast cancer patients exhibiting severe skin reactions and from case-matched controls treated on a prospective radiation fractionation trial at the Royal Marsden Hospital. DNA dsb will be measured with pulse-field gel electrophoresis or single- cell gel electrophoresis. The levels of p53 and WAF1 (a downstream effector of p53) induction will be measured by various methods including Western blotting, enzyme linked immunosorbent assay, electrophoretic mobility shift assay and Northern blotting. We hypothesize that the level of DNA dsb induction following in vitro irradiation of skin fibroblasts should correlate with p53 induction, and that higher levels of p53 and DNA dsb induciton may predict for more severe acute or late normal tissue reactions in patients. The ability to accurately predict the severity of radiation reactions can potentially aid the clinician in planning a course of radiotherapy.

The University of Maryland requests support to develop a broad- based inter-departmental curriculum and program in Preventive Cardiology. This will be targeted to medical students and a wide range of house officers and fellow in training. A concerted effort to reach both junior and senior faculty will also be made. The program will build on a unique alliance between the Division of Cardiology and the Department of Epidemiology and Preventive Medicine. Led by the above, the Departments of Family Medicine, Pediatrics, Internal Medicine and the Office of Medical Education have prominent roles, with support from basic science departments as well. Objectives include: A) Development of a Center for Preventive Cardiology to foster education, patient care and research; B) Development of an integrated, core curriculum aimed at teaching, reinforcing and verifying knowledge, attitudes, and clinical skills with respect to prevention of cardiovascular disease; C) Training in "Health Risk Assessment" for medical students, house staff, fellows and faculty; D) Development of a Faculty Educator Development Core ensuring teaching by role model throughout the program; E) Development of a weekly conference series in preventive cardiology ; F) Preparation of a regular column in the Maryland Medical Journal, "Perspectives in Preventive Cardiology," and a preventive cardiology campus newsletter; G) Development of teaching materials and evaluation instruments; H) A program for the applicant's own educational development in preventive cardiology, epidemiology and medical education. Plans for perpetuation of the program beyond the proposed funding period are also presented.

The purpose of this study is to examine how the daily light-dark cycle in the environment affects the timing of hormone release, the timing of the daily cycle of body temperature, the sleep-wake cycle, and inner body functions which ordinarily vary with the time of day.

We will continue our investigation of the assembly of the bacterial cell wall. The main emphasis will be on the study of teichoic acid biosynthesis. We have discovered a new carrier for cell wall assembly, a lipid teichoic acid carrier, and are in the process of determining its structure. We believe this is an important key to the assembly of the bacterial cell wall by membrane bound enzymes. We will examine the structural relationship between the E. coli UDP-glucose 4'-epimerase and TDP-glucose oxidoreductase. These closely related enzymes and their structure have implications for the evolution of related enzymes to adapt to specific function. We are examining membrane recognition by embryonic cells. We will try to elucidate the membrane components involved in specific cellular recognition in embryonic cell aggregation and ultimately apply the same techniques to recognition problems involved in contact inhibition in normal and malignant cells.

The chief objective of this investigation is to clarify the mechanism of action of a simple flavoprotein oxidase, putrescine oxidase, as part of a general research interest in the mechanisms of flavoprotein catalyzed reactions. To this end, the kinetic component of the mechanism of putrescine oxidase will be studied utilizing standard steady-state and transient state (rapid-scan stopped-flow) kinetic techniques. The mode of cofactor binding to apoprotein will be explored using circular dichroism, fluorescence, and absorbance techniques together with studies of the effects of group-specific reagents on binding of the FAD. Effort will also be directed toward the design, construction and use of various types of specialized on-line instruments to be used in biochemical research.

The Project Director, Dr Rathod, will rely on the extensive infrastructure of his home institution, The University of Washington, for normal management (eg interactions with NIH and US institutions subcontractors). However, given the complexity ofthe project particularly in South Asia, the PD will emply a experienced, professional, part time Project Manager (50% effort) for the first two years ofthe Project. The Manager will go to 100% time commitment as the Project scales up in South Asia and the US in years 3-7. The South Asian part of project management will be handled through a new and innovative arrangement All South Asian contracts, and their administration, will flow through Dr Narayansamy's unique organization that has a team of business managers, scientific project managers, and technology support staff. Their scrupulous track record, and their familiarity with business and political norms in South Asia, will allow the main ICEMR scientific team to focus on the research and training aspects ofthe project.

Study of protein expression and protein-protein interaction is of central importance to understanding oncogenesis and cancer progression. Robust protein capture arrays will greatly facilitate cancer proteomics research. There is an acute need in designing novel types of affinity binders that are easy to express, smaller in size, highly stable, and extremely economical to screen and manufacture. This proposal describes a novel engineered protein scaffold for rapidly generating antibody-like molecules by using molecular selection processes. If the proposed project progresses to phase II/III, it will offer great commercialization opportunity in areas of proteomics research, molecular diagnostics and assay development for drug screening. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: This project is related to development of a type of protein capture array for cancer research and diagnostics. The focus of the proposed work is to create a novel class of affinity reagents to accelerate deciphering gene function in the post-genomic era, with broad implications in improving human health. [unreadable] [unreadable] [unreadable]

New molecular information from blood is being used to improve patient outcomes. Assays for established biomarkers have recently advanced (so-called 'high sensitivity', 'variants'and 'ultra', for examples), allowing physicians to use this additional information to provide better care. The enhanced patient outcomes result from more detailed, accurate and precise diagnostic and risk stratification, resulting in more timely and useful care (such as pharmaceuticals, treatments options, and timing of treatments) and avoiding unnecessary and costly actions. The objective of this proposal is to enable a new capability to isolate and concentrate biomarkers from blood in small volumes (200 microliters), at high sensitivity, over short periods of time, and to monitor multiple markers. We initially will focus on biomarkers for myocardial infarction and stroke. Upon completion of the entire project-which this proposal enables-enhanced information will be provided to physicians, allowing for accurate and fast diagnostics and treatments providing better patient outcomes-saving lives and money. To accomplish the proposal objective, a pair of microfluidic techniques will allow processing of small samples of blood to remove unwanted materials and isolate and concentrate the target biomarkers. These two techniques enable the objective because they keep the sample volume minimal and remove unwanted materials that could degrade detection, while quickly isolating and concentrating target species. Physically, this is made possible by exploit a unique combination of dielectrophoretic, flow and electrophoretic forces combined into the two techniques (gradient dielectrophoresis and electrophoretic capture) pioneered in the PI's laboratory. Gradient dielectrophoresis will remove cells and debris (and perhaps concentrate the targets) and electrophoretic capture will isolate and concentrate individual biomarkers away from possible interfering species. The four target biomarkers identified for proof of principle to enable the larger project are: two cardiac markers- myoglobin and cardiac troponin I, (cTnI), a stroke marker-neuron specific enolase (NSE), and an inflammatory marker-tumor necrosis factor-alpha (TNF1). These targets will provide a reasonable test for the success of the strategy and techniques.. PUBLIC HEALTH RELEVANCE: Developing an ability to isolate and concentrate biomarkers from blood using gradient dielectrophoresis and electrophoretic capture improving detection limits by at least two orders of magnitude while keeping the sample volume very modest (200 microliters).

In vitro fertilization (IVF) provides a unique opportunity to isolate and observe certain human reproductive processes, and thus potentially to differentiate among possible mechanisms of reproductive toxicity. In this clinical protocol, the woman undergoes pharmacologic hyperstimulation of her ovaries, and multiple ova are surgically extracted and fertilized in a dish, usually with her husbands sperm. Thus, the quality of each ovum is assessed, rates of fertilization and cleavage can be studied, early development of the embryos can be observed, and finally implantation into the uterus can be assessed, after the transfer of a known number of embryos. In collaboration with staff at UNC School of Medicine, we undertook a study to relate exposures to clinical outcomes in IVF. Although the patients are themselves infertile, this clinical protocol allows us potentially to demonstrate effects of certain exposures, such as caffeine, on specific reproductive processes. We have now completed the analysis of number of ova harvested, and fertilization and cleavage of the ova and the findings are being written up for publication. - infertility, in vitro fertilization, reproductive toxicants - Human Subjects & Human Subjects: Interview, Questionaires, or Surveys Only

This project focuses on the development of a coronavirus vaccine candidate for severe acute respiratory syndrome (SARS).

Description: The overall goal of the OSUEHS Centers COEP is to increase the public's ability to understand and to make informed decisions on issues relevant to the role of environmental factors in human health and disease. The COEP has eighteen current or past partnerships and collaborations with other OSU programs. The Center's COEP works actively with the Science and Math Investigative Learning Experiences (SMILE) program. These programs jointly received an NIEHS dissemination grant in 1996 to bring environmental health science education to teachers, students and community in rural Oregon. The program has reached an increasing number of students, teachers and community members since 1996. The majority of the population affected by this program consists of Native Americans (25 percent) and Hispanics (46 percent). Each year this program focuses on a different environmental health science theme, and as a portion of the program each summer teachers come to OSU to be trained in an environmental health science subject area. Of the four themes presented yearly since 1996, two were directly related to a research focus of the Center (1996-Chemicals in the Home Environment and 1998-Water Quality and Human Health). The "Lunch with a Scientist" portion of this program also involved Center investigators. Family Science Nights are also offered as a portion of this program through the COEP each fall. The final component of this program is the High School Challenge Weekend. Center members have served as environmental experts in this program by having students work in cooperative groups to develop solutions to environmental problems. Through the OSUEHS Center/SMILE Program, educational programs have been provided to over 1225 elementary, middle, and high school students, 125 teachers and 2000 community members. Rural communities (775 individuals) have received informal science education through family science nights. Center members are actively involved in programs such as Adventures in Learning, American Women in Science, and Science Education Partnerships. Teacher training components are currently being integrated into the program. Teacher workshops are being offered in the curriculum, Exploring Environmental Issues: Focus on Risk. Sixty-five teachers in 1999 have been trained in this curriculum, with future workshops planned in 2000. Teacher training in the ToxRAP program also will be a major focus of the Center through support from the Society of Toxicology. The OSUEHS Center sponsors a yearly course, Your Health and Chemical Risks. This course and environmental health science articles in the monthly newspaper reaches over 3,500 community members each month.

This application for a comprehensive Training Program in Brain Tumor Biology at UAB offers the rigorous interdisciplinary education required for the development of independent research scientists in this field. The Training Program is based in the Departments of Cell Biology, Pathology, and Surgery. The 16 faculty members of the Training Program have been selected based on their complementary research expertise in basic and translational brain tumor research, dynamic research programs, and training records. Research projects include: studies of the molecular aspects of brain tumor migration/invasion, angiogenesis, cell surface receptor-initiated signaling, the regulation of apoptosis, and novel viral approaches to therapy. The exposure of the trainees to the full spectrum of intellectual and experimental approaches to this disease will be integrated formally through a new graduate level course in Brain Tumor Biology, a weekly Brain Tumor Research Conference, a monthly Brain Tumor Research and Treatment Seminar Series, and an annual UAB Brain Tumor Biology Scientific Retreat. The Training Program will be administered by Dr. Etty Benveniste (Director), Dr. Candece Gladson (Co-Director), and Dr. Jim Markert (Co-Director), and by an Executive Committee (Drs. Benos, Gillespie, Roth and Sontheimer). This group of faculty will be responsible for development of individual training plans, integration of research and didactic components, oversight of formal requirements, recruitment, competitive review of formal applications, and oversight of the trainees'progress. An additional strength of this Training Program is the close association with the UAB Brain Tumor SPORE Grant (PI: Dr. Yancey Gillespie), and the UAB Comprehensive Cancer Center Neuro-Oncology Program. Funding is requested for both advanced pre-doctoral students and postdoctoral trainees. Thus, this Training Program in Brain Tumor Biology will prepare future investigators in this important and clinically relevant area of research.

The cytochrome P-450 dependent monooxygenase system plays a pivotal role in both the detoxification and bioactivation of drugs, environmental contaminants, and other potential chemical toxicants. The balance between detoxification and activation is largely dependent on the relative amounts and activities of different isozymes of cytochrome P-450. The long-term objective of the research proposed in this application is to design irreversible inhibitors of specific isozymes of cytochrome P-450. Such isozyme-specific inhibitors could be used in vivo either 1) diagnostically to assess the role of the various cytochromes in mediating or protecting against chemical toxicity or 2) therapeutically to redirect the metabolism of xenobiotics from potentially harmful to innocuous pathways. This proposal will focus on the mechanism, isozyme specificity, and structural requirements of the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol. As the only known suicide substrate of cytochrome P-450 which acts by virtue of the modification of the protein rather than the heme moiety, chloramphenicol or one of its analogs should prove a unique tool for studying and modulating the various functions of the enzyme. Emphasis will first be placed on elucidation of the mechanism by which a single isozyme of rat liver cytochrome P-450 is inactivated in vivo upon covalent modification of specific amino acid residues in the protein by metabolites of chloramphenicol. Subsequent studies will focus on determining the specificity of chloramphenicol as an inhibitor of different isozymes of rat liver cytochrome P-450 and on elucidating which structural features of the chloramphenicol molecule are responsible for its effectiveness and specificity as a suicide substrate of cytochrome P-450. These studies should provide the rational basis for the design of isozyme-specific inhibitors for modulating monooxygenase function in vivo.

The overall goal of this program since its inception has been to define the pathobiological response of the mammalian respiratory system to the inhalation of ambient concentrations of oxidant air pollutants. The focus of this renewal application will be on mechanisms of environmentally induced asthma in young children, using the model of environmental asthma in infant rhesus monkeys that the investigators have developed through the support of this program. Using this model over the previous five years of funding, the investigators have made a number of startling discoveries regarding the effect of chronic ozone exposure on lung development and growth during infancy, including: stunting of airway growth, postnatal loss of airway generations, impaired establishment of the fibroblast growth factor (FGF)-2 ternary signaling complex by basal cells, the failure of epithelial surfaces to innervate, impaired central nervous control, enhancement of the allergic response, airway hyperreactivity, disrupted alveolarization, and airway remodeling. The analytical framework in which all of the studies proposed for this renewal will be conducted is the epithelial/mesenchymal trophic unit (EMTU), whose cellular components establish trophic interactions via an extracellular signaling complex modulated by the basement membrane zone (BMZ). The overall hypothesis is that environmental exposure to oxidant air pollutants promotes the development of allergic asthma in the developing lungs of young children and exacerbates its severity by: 1) disrupting the homeostasis within the EMTU and 2) fundamentally compromising the establishment and differentiation of the trophic interactions that promote normal airway growth and development. These changes result from the superimposition of continual cycles of acute injury, inflammation, and repair on the immune response to allergen exposure. Each of the four projects within the program will focus on different components of the EMTU: Project 1, epithelial and mesenchymal cells (fibroblasts, smooth muscle) and the BMZ; Project 2, mucosal immune system; Project 3, innervation and neural control; and Project 4, vasculature.

Project Summary Connexin proteins form gap junction channels that mediate direct intercellular molecular communication crucial in development, physiology and response to trauma/inflammation. Defects in connexins cause human pathologies. Elucidation of the molecular mechanisms that regulate connexin channel function is essential for understanding their roles in human physiology and pathophysiology, and to identify targets for translational and basic science studies. The long-term goal of this project is to understand the cytosolic interdomain interactions that control the gating of connexin channels. This has been characterized for Cx43, in which interactions between the cytoplasmic loop (CL) and the C-terminal domain (CT) mediate channel gating by pH and other factors. The CL-CT mediated regulation of Cx43 is crucial for its physiological function, and is a therapeutic target for cardiovascular pathologies. However, the role of CL-CT interactions in modulation of other connexin channels, just as likely to be biomedically important, and their mechanism of action have not been explored. We have found that Cx26 channels are modulated by CL-CT interactions. Cx26 and Cx43 are representative members of the two largest families of connexins. Though structurally analogous, the effects of CL-CT interaction on Cx26 channel function seem to be fundamentally different from those in Cx43, suggesting that CL-CT interaction is a common modulatory mechanism in connexins, yet operate in connexin-specific ways. We propose to elucidate the molecular mechanisms of CL-CT control of channel function and properties using Cx26 and its closely related isoform Cx32. Cx26 is the only connexin channel for which there is a high- resolution structure, making it the basis for structure-based studies of all connexin channels. The proposed studies explore the basis and mechanisms of CL-CT interactions and channel properties they modulate, using strategies successfully applied to other channels, including use of competing peptides, engineered disulfide linkages, macroscopic and single channel recordings, and mutational analysis. The experiments utilize intact channels, complemented by peptide NMR. We propose to (a) determine the involvement of CL-CT interactions in channel gating, (b) identify the sites of CL-CT interactions, and (c) determine how CL-CT interaction and its effects are altered by mutations that cause human disease. Cx26 and Cx32 are widely distributed in the body. Mutations of Cx26 are responsible for over half the inherited sensorineural deafness worldwide, and also cause serious disfiguring skin disorders. Mutations of Cx32 cause a peripheral demyelination. In both connexins, many disease-causing mutations are positioned to affect CL-CT interaction. Both connexins are implicated tumor progression and a wide variety of pathological and physiological processes. The combination of a high resolution structure, a large number of disease causing mutations and the extensive experience of both PIs in studying gating, permeability and regulation of Cx26 and Cx32 channels provide a basis for productive, comprehensive investigation of CL-CT interactions.

The Administrative Core provides overall coordination, logistical support, and financial accounting for all cores and research projects. Among the tasks the Administrative Core will be responsible for are: bookkeeping and accounting; provision of grants management services; procurement of services, supplies, and equipment; coordination of and assistance in preparing progress and fiscal reports; maintenance of IRB approvals; and preservation of copies of Conte Center records and data sets. Collaborative education for the next generation of young scientists and physicians will be implemented to train students to become well versed in the art and science of interdisciplinary investigations that study genomic variations and environmental signals that promote mental health, and onset and progression of mental disorders. This is the major training goal of the center.

DESCRIPTION (adapted from the applicant's description): The overall hypothesis of this proposal is that alterations in myocyte calcium handling are centrally involved in the abnormal contractility of failing myocytes and contribute to the progressive decline of cardiac function in CHF. Two specific hypotheses will be tested. The first hypothesis is that the "signature" defects in calcium handling in CHF are due to an increase in the relative transport rate and capacity of the Na/Ca exchanger (NCX) with respect to the sarcoplasmic reticulum (SR) rather than an increase or decrease in the absolute rate or capacity of either transport mechanism. This will be tested by (1) measuring the calcium transport capacities of the SR and NCX in single myocytes from normal, hypertrophied and failing feline hearts and from non-failing, failing and left ventricular assisted device-supported (LVAD) human hearts, and (2) manipulating the calcium transport capacity of the SR and/or NCX in feline and human myocytes transfected with SERCA2a, native or mutant phospholamban (PLB), and/or NCX. The second hypothesis is that calcium influx via reverse-mode NCX makes a greater than normal contribution to excitation-contraction (EC) coupling and SR calcium loading because NCX density increases and the density of the L-type calcium channels and t-tubules decreases in CHF. This will be tested by (1) determining if calcium influx via reverse mode NCX modulates calcium-induced calcium release (CICR), (2) determining if calcium influx via reverse mode NCX replaces calcium entry via L-type calcium channels as the primary source of trigger calcium in CICR, and (3) measuring the amount of L-type calcium current, NCX current and SR calcium loading that is lost when T-tubules are disrupted by osmotic shock from the surface membranes of non-failing and failing feline and human myocytes.

The long term objectives of the research supported by this grant are to determine the novel chemical mechanisms by which vitamin B6 (PLP), Vitamin B12 (adenosylcobalamin), S-adenosylmethionine (SAM), and iron-sulfur clusters function in enzymatic reaction. The conventional role of PLP biology is to stabilize carbanionic intermediates in enzymatic reactions. A major focus of the research supported by this grant is the elucidation of the role of PLP in catalyzing reactions that involve free radical-paramagnetic intermediates. PLP-facilitated radical reactions appear to take place in reactions of aminomutases such as lysine 2,3-aminomutase and lysine 5,6-aminomutase. Radical intermediates of substrates and vitamin coenzymes will be characterized spectroscopically, and the structures of the enzymes that catalyze their reactions will be elucidated. The roles of SAM and [4Fe-4S] clusters in the initiation of radical formation will be unmasked and characterized chemically, spectroscopically, and kinetically. The relationship between the actions of adenosylcobalamin in Vitamin B 12-dependent and SAM/[4Fe-4S]-dependent aminomutases will be explored. Novel chemistry in the actions of vitamins B6 and B12, SAM, and [4Fe-4S] will be discovered. New beta-aminoacids will be produced by engineering of the genes producing aminomutases. Aminomutases are important in amino acid metabolism and the biosynthesis of antibiotics such as Streptothricin F, Myomycin, Blasticidin S, and Taxol. The contributions of beta- amino acids produced by lysine 2,3-aminomutase and arginine 2,3-aminomutase to the functions of antibiotics are not known. The beta-aminoacids may become significant in drug development.

We will study the importance of two cellular receptors for herpes simplex 1 (HSV-1), herpes viral entry mediator (HVEM) and nectin-1, in the infection of the cornea and the establishment of latency by using mice knocked out (KO) for these receptors. Studies in cell culture and in vivo murine models of HSV infection indicate that HVEM and nectin-1 are the most efficient at mediating entry and most important in HSV pathogenesis. Reactivation of HSV-1 can lead to recurrent disease in the form of oral ulcers or more serious disease including encephalitis and herpes stromal keratitis (HSK). The precise mechanism of HSK pathogenesis is not fully understood, but factors known to contribute to disease include viral replication and the resulting immune response. We plan to study the outcome of corneal inoculation as well as examine which receptors function as HSV-1 entry mediators in susceptible tissues (corneal epithelium, corneal stroma, TG neurons, non-neuronal cells of the TG, immune cells, etc.). To fully understand disease pathogenesis and develop therapeutics, it is essential to understand the target tissues of HSV-1 in the eye. Our proposed studies will lay the foundation for further studies to determine whether infection of specific cell types, such as cells of the immune system, are important in HSV pathogenesis in the eye. By determining the precise cells infected by HSV in an ocular infection and the receptors utilized by the virus, novel treatment regimens can be developed that target those receptors. Such targeted therapies might attenuate primary infection and/or reduce inflammatory immune cells thereby preventing devastating sequelae including HSK, blindness, and encephalitis. In Aim 1, we will characterize the role of HVEM and nectin-1 in primary HSV-1 infection of the cornea by determining whether HVEM and/or nectin-1 are required for primary HSV-1 infection of the cornea. We will also evaluate the importance of input viral load on the development of zosteriform disease and determine which cells in the eye and TG are infected during primary HSV-1 infection and whether or not those cells are expressing HVEM and/or nectin-1. In Aim 2, we will determine whether HVEM and/or nectin-1 are necessary for HSV latent infection of the trigeminal ganglion. PUBLIC HEALTH RELEVANCE: Our specific aims are to identify the importance of the cellular receptors, HVEM and nectin-1, for HSV-1 infection of the eye. But more importantly how the use of these receptors contribute to herpes stromal keratitis (HSK). An understanding of the role for these receptors in HSK and how they contribute to disease will provide insight for the development of novel therapeutics for the treatment HSK which is the most frequent cause of corneal blindness in the US.

Head and Neck Squamous Cell Carcinoma (SCCHN) is one of the most common cancers worldwide and a significant cause of cancer morbidity and deaths. Although SCCHN is unique among solid tumors in that the majority of patients present with local disease, metastasis remains a critical problem for SCCHN treatment: patients without metastases can be cured; patients with metastases cannot. Even for local disease, despite improvements in outcome with intensive, multimodality therapies, such treatments are highly toxic, poorly tolerated and only partially efficacious. Long-term survival is only ~ 50%, with most patients dying from their cancers. There is an urgent need to uncover the critical genetic alterations in SCCHN and to determine how these impact patient outcome and metastatic risk. To search for genes that are critical for head and neck squamous carcinoma (SCCHN) biology, prognosis and therapy, I have initially used high resolution single nucleotide polymorphism (SNP) arrays to detect copy number alterations in 684 human cancer cell line of diverse tissue origin, including SCCHN. This analysis has identified recurrent homozygous deletions involving >200 genes. Among these, PARD3, encoding a critical regulator of cell polarity, is disrupted in squamous carcinoma cell lines and primary tumors, including from the head and neck region. Since abnormal cell polarity has been linked to increased migration and metastasis, these findings may implicate loss of function of PARD3 or its downstream effectors in the risks of recurrence and metastasis of a subset of these cancers. The overall goals of this proposal are: (1) to further characterize the role of PARD3 in SCCHN tumorigenesis, migration and metastasis; and (2) to capture and genotype circulating tumor cells (CTCs) from the peripheral blood of patients with SCCHN, to determine if CTCs can identify patients at risk for metastases before metastatic disease is clinically apparent. The experiments will utilize primary tumors and peripheral blood from patients treated for SCCHN, as well as PARD3 loss-of-function and gain-of-function squamous cell lines for in vitro and in vivo studies to identify the cancer-related phenotype(s) controlled by PARD3 and the domains and activities required for tumor suppression. By characterizing PARD3 loss in the tumor type in which inactivating mutations have arisen as a primary event during tumor formation, this work will directly implicate alterations in cell polarity in the development and invasive and metastatic potential of these cancers. By capturing and molecularly characterizing CTCs in actual SCCHN patients undergoing treatment, this work will permit the real time in vivo analysis of tumor cells to quantify clinical risk and guide treatment. As a result, these studies may lead to improved biomarkers and suggest potential new targets for therapies, thus providing benefit to public health by improving the care of patients with these cancers. PUBLIC HEALTH RELEVANCE: Although cancers of the head and neck pose a significant health problem worldwide, how they develop is incompletely understood, existing treatments are only partially effective and safely measuring whether treatments actually target the tumor cells in the patients being treated is very difficult. We have discovered frequent loss of a single gene, PARD3, in human head and neck cancers that could permit cells to loosen from each other, move through the blood to different sites and thereby evade detection and resist treatment. By determining whether patients possessing cancers which have lost PARD3 do worse with treatment, how PARD3 contributes to cancer formation and spread, and whether the tumor cells present in patients' blood can be captured and used to measure the benefit of treatment, the experiments outlined in this proposal may lead to better and less toxic treatments.

The purpose of this research is to examine the antecedents, process and consequences of divorce using samples of black and white males and females from divorcing and intact families located in metropolitan Cleveland. The study uses a prospective design in which the two samples, each of approximately 200, are being followed for a 3 1/2 year period. The divorcing are interviewed 3 times: at time of filing; six months after settlement; six months to a year after the second interview. The intact family sample is interviewed twice: simultaneously with the first and third divorce interviews. Among the study objectives are: (1) to indicate which periods during the divorce process produce the highest rates of disturbance for individuals of varied characteristics; (2) to indicate what factors aid or hinder socialization to the roles associated with the divorced status; (3) to explore the influence of human support systems such as kin, friends, voluntary associations and service agencies in easing or exacerbating the process of adjustment; and (4) to compare the divorced to members of the intact family sample in order to determine the relative influence of divorce as a crisis event on health status on role functioning. The analysis involves within and between-groups comparisons and employs such techniques as analysis of variance and multiple regression.

Substantial evidence suggests that activity plays a crucial instructive role in shaping the structure and connectivity of the central nervous system, but the molecular mechanisms that translate developmental activity patterns into lasting structural changes in the brain remain open mysteries. Neurotrophins have long been implicated as candidate signaling molecules that potentially mediate activity- dependent development, but their in vivo roles have been difficult to predict based on conventional genetic approaches or culture studies. By using a novel genetic mosaic approach to generate small groups of labeled homozygous mutant cells in intact animals, this proposal will dissect the role of activity and neurotrophin signaling in brain development in vivo, with single-cell resolution. Specifically, the proposal will combine inducible genetic strategies and genetic mosaic analysis to test the function of neurotrophin receptors p75, TrkB, and TrkC in development and refinement of central somatosensory circuitry. The somatosensory system relies on activity for proper structural maturation of both axons and dendrites, and thus represents an excellent opportunity to test key hypotheses in neurotrophin signaling, activity-dependent development, and neural circuit formation. As p75 and Trk receptor dysfunction contributes to neurological disorders including Alzheimer's disease and schizophrenia, potential results will also have broad implications for our understanding of neural circuit maintenance and repair, as well as therapeutic approaches to neurodevelopmental and neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: This proposal will study the molecular mechanisms by which activity influences nerve cell structure during brain development. Proper development of cell shape and connectivity in the central nervous system is critical for perception and cognition. Accordingly, studies in nerve cell morphogenesis will considerably aid our understanding of how the brain wires itself during development, how our early experiences can generate long-lasting changes in our brains, what happens when nerve cells are injured by neurodegenerative disease or trauma, and how we can repair nervous system damage.

Eukaryotic cells dedicate significant resources to the deployment of proteins to the membrane compartments that comprise the secretory pathway. Biogenesis of all membrane proteins starts with the folding and assembly of newly synthesized proteins within the endoplasmic reticulum (ER), often with the aid of cellular chaperones. Cells must strike a precise balance between ensuring that only fully folded proteins are allowed to leave the ER and avoiding the accumulation of misfolded proteins within the ER lumen. Uptake of newly synthesized cargo molecules into ER-derived transport vesicles only occurs once proteins are fully folded. These transport vesicles, known as COPII vesicles for the cytoplasmic coat proteins that drive membrane curvature and select cargo, thus play a critical role in regulating forward transport of new proteins. We study the close relationship between protein folding and packaging into ER- derived COPII vesicles in the model organism, Saccharomyces cerevisiae. Using a combination of genetics and biochemistry, we aim to define the cellular machinery that acts at the interface between protein folding and ER export. A model for examining this process is the yeast ABC transporter, Yor1, a plasma membrane protein that acts as a drug pump to clear toxic substances from the yeast cytoplasm. Yor1 is a homolog of the human cystic fibrosis transmembrane conductance regulator (CFTR), defects in which cause cystic fibrosis. Deletion of a Phe residue in Yor1, equivalent to the major disease-related mutation in human cystic fibrosis, causes Yor1, like mutant CFTR, to be ER-retained and degraded by the cytoplasmic ubiquitin/proteasome pathway. Thus Yor1 is a useful model that allows the direct comparison of the intracellular itineraries of native and aberrant forms of a single protein. This research proposal consists of four specific aims. (1) To define the molecular mechanisms that drive uptake of Yor1 into COPII vesicles and assess how protein folding influences this event. (2) To determine how cellular chaperones contribute to Yor1 biogenesis and assess how the kinetics of chaperone/client interactions may influence COPII binding and thereby regulate ER export. (3) To identify and characterize novel factors that may facilitate Yor1 biogenesis, including specific membrane chaperones and more general folding factors. (4) To determine the mechanisms by which the unfolded protein response improves the folding and/or transport of misfolded proteins. Ultimately, a better understanding of cellular machinery that acts to regulate protein folding and forward transport may lead to novel therapeutic approaches to treat the many diseases associated with aberrant protein folding within the secretory pathway. [unreadable] [unreadable] [unreadable]

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. N-acetyl-L-cysteine (NAC), a natural antioxidant, improves the efficiency of chelating agents used against heavy metal poisoning and facilitates the excretion of Cr(III), Cd(II) and Pb(II), but not of Hg(II). Recent bio-tests on NAC complexes with transition biometals (Zn2+, Co2+, Co3+, Fe2+) showed significant anti-inflammatory activity. The goal of our proposed research is to study the structure of NAC complexes with heavy metals, and transition biometals, in solid state and in aqueous solution at physiological pH and pH = 8.5, using different [NAC]/[Mn+] ratios and [Mn+] = 10 and 100 mM. In this study, we will combine the results from different spectroscopic techniques, such as extended x-ray-absorption fine structure (EXAFS), multi-nuclear NMR, Raman and IR, to identify the local structure around the central metal ion in these complexes. We will use the method that we have developed for EXAFS data analysis of mixtures for similar Cd(II), Hg(II) and Pb(II) complexes with cysteine and penicillamine (3,3'-dimethyl cysteine), including Principal Component Analysis (PCA) of the EXAFS spectra. The aim of the current study is to provide a basis for better understanding of the detoxification treatments and the anti-inflammatory activities with the use of cysteine derivatives as complex-forming agents.

As a participating institution in the Primary Breast Cancer Therapy Group (NSABP), we are contributing data from patients entered into Protocol No. 4 of the group, which is a protocol to evaluate the relative efficacy of radical mastectomy, total mastectomy with radiation and total mastectomy alone in patients with clinically negative axillary nodes. A comparison between radical mastectomy and total mastectomy with radiation is made in those with positive axillary nodes. To date, over 900 patients have been entered into the protocol by 35 institutions of which ours is one. The present application seeks funding to permit continued patient accrual into the present study so as to bring it to completion and to permit participation in the development and implementation of new protocols which are to follow. It is premature to make statements relative to the various treatment modalities. Interesting data, however, has been obtained relative to ancillary material being collected. It has been demosntrated that patients randomized into various treatment categories are similar according to a variety of criteria. Moreover, information has been accumulated relative to the pathology of breast cancer. The submittal of data has been satisfactory. If patient accrual continues at the present rate, it should be adequate to complete the study (accrual) within the next year.

Ixodes scapularis ticks transmit Borrelia burgdorferi as well as several other pathogens of public health importance. Feeding ticks cut into host tissues and create a feeding pool from which they continuously feed for days. Host immune responses interfere with tick feeding by clogging the feeding site and producing molecules that are noxious to the tick. The host complement system is likely to pose special problems for feeding ticks because complement can directly damage non-self tissue as well as recruit arms of the immune response. The focus of this proposal is anti-complement proteins produced by I. scapularis. We plan to test the hypothesis that anti-complement proteins facilitate tick feeding and pathogen transmission. Under aim 1 of the proposal we plan to functionally characterize two novel proteins designated Salp9 and Salp20 that are highly homologous to a known tick anti-complement protein. Under aim 2 we propose to screen tick cDNA libraries prepared from salivary glands and guts for novel anti-complement proteins. Proteins produced by ticks are attractive vaccine targets because such vaccines have the potential to block tick feeding and pathogen transmission. Under aim 3 we plan to test anti-complement proteins as vaccines that block feeding and/or pathogen transmission. Our industry partner in these studies is L2 diagnostics, which will assist with further development of tick anti-complement proteins as drugs and vaccines.

Abstract The development of human enteroids as a model to study the human intestine offers tremendous opportunities to study the pathogenesis of infectious enteric disease. We propose to combine the substantial expertise from three institutions with long and impressive histories in the investigation of enteric diseases to exploit this powerful model to study the pathogenesis of four major etiologic agents of diarrheal disease: three pathotypes of diarrheagenic E.coli (enteroaggregative, enterohemorrhagic and enterotoxigenic E. coli) and Shigella. Investigators from the University of Maryland, Johns Hopkins University and the University of Virginia will collaborate in this Program Project Grant (PPG). The leadership team of this PPG comprises three internationally renowned experts in enteric diseases, with complementary training in microbiology, gastroenterology, molecular physiology, and pediatric infectious diseases. They will direct a multidisciplinary team of co-investigators with expertise in cell biology, molecular pathogenesis, and mucosal immunology. The overall goal is to increase understanding of the pathophysiology and potential treatments of these four important pathogens. The proposed studies will use normal human mini-intestines, called enteroids or colonoids, grown on monolayers to develop models that mimic human disease. Examination of pathophysiologic aspects common to the diseases studied will serve to integrate the projects. These aspects include the role of mucins, bacterial proteases called SPATES, enterotoxins and secreted cytokines and are in addition to pathophysiologic aspects specific to each infection. In addition, the contribution of cells involved in innate immunity will be examined by co-culture of the enteroids/colonoids with human macrophages, neutrophils and dendritic cells. These studies using human mini-intestines offer the possibility of revealing insights in disease pathophysiology that are specific to normal human intestine rather than the animal models and cancer cell line models used until now. Each of the four projects focuses on a specific pathogen: enteroaggregative E. coli, Shigella, enterohemorrhagic E. coli, entertoxigenic E. coli. Besides an Administrative Core, there is an Enteroid Core that provides human enteroids/colonoids and growth media, instructs all projects on how to produce enteroid/colonoid monolayers and an Immunology Core that measures cytokines and chemokines, and isolates human macrophages, neutrophils and dendritic cells. The Enteroid and Immunology Cores work together to develop co-culture systems of innate immune cells and enteroids/colonoids that will be used by the projects. The investigators will regularly interact by monthly joint laboratory meetings. The proposed project will yield many significant new insights into enteric disease caused by these important pathogens.

The Western immunoblot for the detection of 14-3-3 protein in the cerebrospinal fluid of human patients with sporadic or familial spongiform encephalopathy and chimpanzees with experimentally induced TSEs has provided for a highly sensitive and specific test in support of a clinical diagnosis of these diseases. However, it is not known how early in the course of these infections the protein becomes detectable in the CSF. We have demonstrated in spider monkeys that neuropathological lesions precede clinical signs and have hypothesized that 14-3-3 proteins are detectable due to neuronal loss at a rate sufficient to outpace the normal turnover and resorption of CSF. We have detected these proteins in the CSF of chimpanzees inoculated with CJD several weeks before clinical signs become recognizable. In humans with very early behavorial changes the test has been negative but repeat collections as the disease progresses the test becomes positive. To more definitively determine the earliest possible detection of 14-3-3 proteins in CSF we have inoculated ten chimpanzees with sporadic, iatrogenic and familial strains of CJD and are colelcting CSF and blood at intervals of every six weeks. Concurrently we have determined the stablility of 14-3-3 protein and have shown that the protein in CSF is stable at room temperature and 4C for over 35 days and indefinitely at 20C.

The objective of this project is to understand at the molecular level the mechanism by which steroid hormones affect the expression of the genome of their target cells. We have chosen to investigate the cytolytic response of murine lymphoid cell lines to glucocorticoids because this system presents unique and desirable features. Since variants resistant to the steroid can conveniently be isolated, this system offers a genetic approach to analyze the steps involved in the response. A large collection of lymphoid cell variants resistant as the result of a defect in the glucocorticoid receptor is already available. We propose to use a variety of procedures to search for new types of resistant variants. The existing collection of resistant variants, and all new variants that will be isolated, will be characterized genetically and biochemically. The genetic characterization includes complementation analysis in cell hybrids and linkage studies. The biochemical tests involve assays for analysis in cell hybrids and linkage studies. The biochemical tests involve assays for glucocorticoid receptor and for a putative nuclear acceptor site for the receptor. Enucleated cells will be prepared to examine the role of the nucleus in the lymphocytolytic response, and ouabain resistant variants will be isolated to examine a possible relationship to glucocorticoid sensitivity. We will also pursue the biochemistry of the glucocorticoid receptor. This receptor will be purified and its molecular weight, isoelectric point and subunit structure examined. We will prepare a specific anti-receptor serum. This serum will be used, in particular, to detect the production of a protein crossreacting with receptor in "receptorless" variants, to purify the receptor by immunoadsorption, and to examine its conformational change and structure. The anti-receptor serum will also serve as a tool to compare the receptor of our sensitive lymphoid cell lines with that of lymphoid cells resistant to the steroid as the result of normal differentiation, and of other types of mouse cells.

The broad objective of the proposed research is to evaluate the potential usefulness of the ferret as an experimental animal for work on the relation between changes in brain morphology and cognitive processes. It is hoped that the capability to vary the degree of chemically induced micrencephaly in an otherwise normal animal and then to observe the ensuing degree of impairment in higher mental process will provide a means of study of a form of mental deficiency. Specifically, the proposed work is aimed at determining whether the performance of ferrets made micrencephalic by prenatal treatment with methylazoxymethanol is impaired on problems that appear to require symbolic (abstractive) processes (viz., acquisition of oddity and matching-from-sample learning sets).

Biofilms found on mammalian tissue surfaces contain complex mixtures of bacterial species growing within a glycocalyx matrix. Species in biofilms have shown remarkably greater resistance to antibiotics than when grown in a planktonic state. Surprisingly little is known about the effect of systemically administered antibiotics on the microbial composition of naturally occurring, complex biofilms. Thus, the long-term objective of this proposal is to determine the effect of antibiotics on the microbial composition of biofilms as they exist in vivo and to determine if the proportion and nature of antibiotic resistant taxa is affected. Subgingival biofilms that occur on teeth will be employed as a model system because of their complexity, ready accessibility and known microbial composition. Specific Aim 1 will examine the effects of 3 systemically administered antibiotics on the microbial composition of subgingival biofilms in adult humans with periodontitis. All subjects will receive scaling and root planing and will be randomly assigned to one of 4 groups of 36 subjects each receiving one of doxycycline, amoxicillin, metronidazole or control. 28 subgingival biofilm samples will be taken in each subject at baseline, at selected time points while the agent is being taken, the same time points after cessation of the agent as well as at 3, 6 and 12 months. These samples will be evaluated individually for their content of 40 subgingival species using checkerboard DNA-DNA hybridization. Data will be evaluated longitudinally and compared with clinical parameters. Specific Aim 2 will examine the proportion and nature of subgingival species that are resistant to 4 mug/ml of the test antibiotic at the same time points. Subgingival biofilm samples will be plated on media with and without the test antibiotic and resistant isolates identified using DNA probes. Data from this investigation will indicate the kinetics of suppression of species in subgingival biofilms during antibiotic administration, the kinetics of repopulation after antibiotic withdrawal as well as the nature and proportion of subgingival species that are resistant to the antibiotics at different time points. The data should indicate the effects of antibiotics on the microbial composition of complex ecosystems such as those found in biofilms and be useful in guiding and interpreting in vitro studies of mechanisms of antibiotic resistance.

We continue our long-term study of the regulation of ribosomal RNA transcription in the yeast, Saccharomyces cerevisiae. Ribosomal RNA is transcribed by RNA polymerase I (po1I) and comprises over half of the total RNA of the cell. Both the cis-acting DNA elements, as well as the trans-acting protein factors which control po1I transcription have largely been identified in past work. The challenge now is to understand how these DNA and protein elements function to produce ribosomal RNA in strict co-ordination with the growth rate of the cell. Because ribosomal RNA production is so important to the economy of the cell, it is subject to regulation at many different levels. Thus, experiments are proposed to study regulation at the level of 1) the polymerase itself, 2) the pre-initiation complex that forms at the promoter, 3) chromatin which must be cleared for the pre-initiation complex to form. Other experiments will survey all of the po1I transcription machinery during a major metabolic shift (glycerol/ethanol to glucose) to determine which of these potential regulatory levels are actually utilized. Ribosomal RNA is a major and essential component of all living cells. Alterations in po1I transcription are not known to be associated with any disease state, but understanding how ribosomal RNA production is co- ordinated with cell growth is one of the major unsolved problems of cell biology.

LIPOPOLYSACCHARIDE INDUCED NEUROINFLAMMATION SELECTIVE INVOLVEMENT OF ARACHIDONIC ACID IN NEUROINFLAMMATION In a rat model of neuroinflammation, produced by a 6-day intracerebral ventricular infusion of bacterial lipopolysaccharide (LPS), we reported marked disturbances in brain arachidonic acid (AA, 20:4n-6) metabolism. In the present study, we demonstrated that parameters of brain docosahexaenoic acid (DHA, 22:6n-3) metabolism were unaffected in this model. Selective targeting of brain AA metabolism with non-steroidal antiinflammatory or other drugs should be considered for treating human brain diseases associated with neuroinflammation (Rosenberger et al., 2010). ANTI-INFLAMMATORY EFFECT OF ASPIRIN ON BRAIN ARACHIDONIC ACID METABOLITES Pro-inflammatory and anti-inflammatory mediators derived from arachidonic acid (AA) modulate peripheral inflammation and its resolution. Aspirin (ASA) is a non-steroidal anti-inflammatory drug (NSAID) that switches AA metabolism from prostaglandin E (PGE) and thromboxane B (TXB) to lipoxin A (LXA) and 15-epi-LXA. It is unknown whether chronic therapeutic doses of ASA are anti-inflammatory in the brain. We hypothesized that ASA would dampen increases in brain concentrations of AA metabolites in a rat model of neuroinflammation, produced by a 6-day intracerebroventricular infusion of bacterial lipopolysaccharide (LPS). In rats infused with LPS (0.5 ng/h) and given ASA-free water to drink, concentrations in high-energy microwaved brain of PGE, TXB and leukotriene B (LTB) were elevated. In rats infused with artificial cerebrospinal fluid, 6 weeks of treatment with a low (10 mg/kg/day) or high (100 mg/kg/day) ASA dose in drinking water decreased brain PGE, but increased LTB, LXA and 15-epi-LXA concentrations. Both doses attenuated the LPS effects on PGE, and TXB. The increments in LXA and 15-epi-LXA caused by high-dose ASA were significantly greater in LPS-infused rats. The ability of ASA to increase anti-inflammatory LXA and 15-epi-LXA and reduce pro-inflammatory PGE and TXB suggests considering aspirin further for treating clinical neuroinflammation (Basselin et al., 2011a). LITHIUM MODIFIES BRAIN ARACHIDONIC AND DOCOSAHEXAENOIC METABOLISM IN RAT LIPOPOLYSACCHARIDE MODEL OF NEUROINFLAMMATION. Neuroinflammation, caused by 6 days of intracerebroventricular infusion of a low dose of lipopolysaccharide (LPS;0.5 ng/h), stimulates brain arachidonic acid (AA) metabolism in rats, but 6 weeks of lithium pretreatment reduces this effect. To further understand this action of lithium, we measured concentrations metabolites generated from AA and docosahexaenoic acid (DHA), in high-energy microwaved rat brain using LC/MS/MS and two doses of LPS. In rats fed a lithium-free diet, low (0.5 ng/h)- or high (250 ng/h)-dose LPS compared with artificial cerebrospinal fluid increased brain unesterified AA and prostaglandin E2 concentrations and activities of AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2)-IV and secretory sPLA2. LiCl feeding prevented these increments. Lithium increased brain concentrations of lipoxygenase-derived AA metabolites, 5- hydroxyeicosatetraenoic acid (HETE), 5-oxo-eicosatetranoic acid, and 17-hydroxy-DHA by 1.8-, 4.3- and 1.9-fold compared with control diet. Lithium also increased 15-HETE in high-dose LPS-infused rats. This study demonstrated, for the first time, that lithium can increase brain 17-hydroxy-DHA formation, a precursor of antiinflammatory resolvins, indicating a new antiinflammatory therapeutic action of lithium (Basselin et al., 2010). PARKINSON DISEASE MODEL UPREGULATED BRAIN ARACHIDONIC ACID ENZYMES IN RAT MODEL OF UNILATERAL PARKINSON DISEASE We had reported that arachidonic acid (AA) signaling is upregulated in the caudate-putamen and frontal cortex of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, a model for asymmetrical Parkinson disease. In the present study, we demonstrated that this upregulation was associated with increased expression of two enzymes involved in AA metabolism, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2, ipsilateral to the lesion in the caudate putamen and frontal cortex. This confirms that the tonically increased ipsilateral AA signal in the lesioned rat corresponds to upregulated cPLA2 and COX-2 expression within the AA metabolic cascade;such changes, if present in Parkinson disease, may contribute to symptoms and pathology of this disorder (Lee et al., 2010). HIV-1 DEMENTIA MODEL IMAGING NEUROINFLAMMATION WITH ARACHIDONIC ACID IN HIV-1 TRANSGENIC RAT Human immunodeficiency virus (HIV)-1 associated infection involves entry of virus-bearing monocytes into brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolic enzymes. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We used our in vivo imaging method with quantitative autoradiography to demonstrate that brain AA metabolism was elevated in 6-7 month old unanesthetized HIV-1 Tg rats. Brain activities of cytosolic phospholipase A2 (cPLA2-IV), secretory sPLA2, and calcium independent iPLA2-VI, which release AA and docosahexaenoic acid from membrane phospholipids, and concentrations of proinflammatory prostaglandin E2 and leukotriene B4, also were elevated, consistent with neuroinflammation and increased AA metabolism (Basselin et al., 2011b). We now plan to use our clinical method of positron emission tomography with 1-11CAA, to test whether brain AA metabolism is upregulated in HIV-1-infected patients as a marker of neuroinflammation (AG000148). INCREASED INFLAMMATORY AND ARACHIDONIC ACID CASCADE MARKERS, AND REDUCED SYNAPTIC PROTEINS, IN BRAIN OF HIV-1 TRANSGENIC RAT Cognitive impairment has been reported in human immune deficiency virus-1 (HIV-1-) infected patients and in the HIV-1 transgenic (Tg) rat. We hypothesized that this impairment in the rat could be linked to neuroinflammation, disturbed brain arachidonic acid (AA) metabolism, and synapto-dendritic injury. To test this, we measured protein and mRNA levels of markers of neuroinflammation and the AA cascade, as well as pro-apoptotic factors and synaptic proteins, in brain from 7- to 9-month-old HIV-1 Tg and control rats. Compared with control brain, HIV-1 Tg rat brain showed immunoreactivity to glycoprotein 120 and tat HIV-1 viral proteins, and significantly higher protein and mRNA levels of (1) the inflammatory cytokines interleukin-1and tumor necrosis factor alpha, (2) the activated microglial/macrophage marker CD11b, (3) AA cascade enzymes: AA-selective Ca2+-dependent cytosolic phospholipase A2 (cPLA2)-IVA, secretory sPLA2-IIA, cyclooxygenase (COX)-2, membrane prostaglandin E2 synthase, 5-lipoxygenase (LOX) and 15-LOX, cytochrome p450 epoxygenase, and (4) transcription factor NF-Bp50 DNA binding activity. HIV-1 Tg rat brain also exhibited decreased levels of brain-derived neurotrophic factor and drebrin, a marker of post-synaptic excitatory dendritic spines. In summary, HIV-1 Tg rats show elevated brain markers of neuroinflammation and AA metabolism, with a deficit in several synaptic proteins. These changes are associated with viral proteins and may contribute to cognitive impairment. The HIV-1 Tg rat may be a useful model for understanding progression and treatment of cognitive impairment in HIV-1 patients (Rao et al., In press).

From a clinical perspective, understanding and manipulating the dynamics of T cell development may offer key insight in to the progression of various autoimmune diseases as well as present potential treatment options with regards to cancer immunotherapy and vaccination. Although existing single cell approaches are now elucidating the significance of heterogeneity, the way in which T cells evolve to achieve particular phenotypic states remains unclear. We are proposing to use novel microfluidic platforms for dynamically monitoring the response of individual CD8+ T cells over several generations to investigate differentiation, plasticity and metabolism. To accomplish this, we will utilize hydrodynamic traps to dynamically interrogate several generations of a single T cell's progeny by implementing standard immunofluorescence techniques on-chip. Since fluid surrounding the cells can be rapidly and frequently exchanged without perturbing growth, we will be able to monitor expression of cell surface markers and deliver drugs that alter cellular metabolism at precise time points. As a compliment to biochemical markers of lineage we will also measure multigenerational growth rates of single activated T cells in order to provide a physical measurement of the cell's metabolic state.

Investigating the key variables that affect exercise adoption and exercise maintenance for cancer survivors is important to begin to address the plethora of negative consequences of an inactive lifestyle on cancer survivorship. Identifying these variables and how they differ in specific disparate cancer survivor populations is an integral step in developing culturally competent behavioral interventions for adopting and maintaining a physically active lifestyle. The objective of this particular application is to examine differences in the determinants of exercise behaviors between Hispanic and non-Hispanic cancer survivors; and to conduct an efficacy culturally tailored intervention pilot specific for Mexican-American Latina breast cancer survivors. PUBLIC HEALTH RELEVANCE: There is a critical need to understand how to get people to adopt a more active lifestyle to help offset illness and increased risk for disease from a physically inactive lifestyle. This study will investigate factors that affect beginning and maintaining a more physically active lifestyle. The study will look to see if there are differences in these factors between Hispanics and non-Hispanics and based on those differences conduct a trial to increase physical activity specific for Hispanic breast cancer survivors.

The continuation of work in this area has revolved about the analysis of lymphocyte responsiveness in vitro to a putative suppressive immunogenic virus (rubeola) and the meaning of this putative suppression in relation to observations within the clinical disease in man. In addition, work has been performed utilizing laser cytometry as a method of quickly and accurately evaluating cell change in vitro as a measure of cell responsiveness to specific antigen, mitogen or allogeneic cells.

To recognize and interact with objects in an object-filled environment, an accurate perceptual representation of the surfaces that define the objects is essential. The surface representation process integrates local feature information from the early cortical processes to form surfaces. Complicating the process of representing surfaces, however, is the fact that most objects overlap one another, resulting in the occluded parts of the objects' surfaces not being encoded by the early cortical processes. Thus, to accurately represent surfaces, the surface representation process uses an arsenal of perceptual rules derived from environmental constants even as it relies on the early features of the non-occluded parts. But how these perceptual rules are implemented, at which stage of the surface representation process, and how top-down attention modulates the representation of both the occluding and occluded surfaces are not well understood. In light of these, our proposal addresses three fundamental issues in representing surfaces. The issues are: A. The boundary contour and surface property information in amodal surface interpolation B. The operational constraints in surface formation during binocular viewing C. Inhibitory mechanism and plasticity in binocular surface perception These issues will be investigated using psychophysical methods on human observers with normal vision, and those with significant sensory eye dominance. Observers in the latter group tend to suppress the image viewed by the non-dominant eye when the two eyes see different images; a situation almost resembling the extreme condition afflicting strabismic amblyopia patients. In this proposal, we will test and evaluate a new perceptual learning paradigm aimed at reducing sensory eye dominance and improving perception. Therefore, the outcomes of our investigations will not only further the knowledge of perceptual and attention mechanisms in perceiving surfaces, but also has a clinical significance.

In order to understand such important processes as enzyme-substrate binding and antibody-antigen recognition, numerous investigators have synthesized and studied artificial hosts like water soluble cyclophanes. Some of these hosts have bound guests with high affinity constants but many improvements are needed. We intend to synthesize and study a particularly rigid "molecular tweezer" which will "sandwich" aromatic guests between two DNA intercalators. The complexation is expected to be very efficient and with a predictable geometry. We also believe that the approximate binding constant can be predicted using a simple cooperative model. Molecular tweezers with functional groups converging on the binding site will be investigated. These will be the first hosts which include their guests using both hydrogen bonding and hydrophobic forces resulting in improved guest discrimination. The specificity in binding mono-nucleotides will be investigated. In addition to host-guest chemistry these molecular tweezers are expected to be novel probes of DNA structure. A tremendous array of medicinals, including the clinically useful anticancer agent adriamycin, contain flat aromatic chromophores which bind to DNA by intercalation. Many aspects of the intercalation processes are not well understood including the important neighbor exclusion principle. Our molecular tweezers are the first bis-intercalators to force chromophores into adjacent sites. We will investigate chromophores of different "widths" to determine the origin of the neighbor exclusion principle. Those molecular tweezers which obey the principle will be unable to act as mono-intercalators and therefore unable to bind the internal sites of the DNA helix. We will demonstrate that these molecules are able to selectively bind to the ends of DNA helices which are not constrained by neighbor exclusion. This type of selective binding has not been seen before and should have a number of important applications.

This project concerns mechanisms of demyelination in human disease and continues to focus on the human polyomavirus JC (JCV), the etiologic agent of progressive multifocal leukoencephalopathy (PML). PML is a fatal demyelinating disease which complicates about 5% of AIDS cases, and for which there has been no treatment known. Work this year has emphasized the detection of the virus by polymerase chain reaction (PCR) in PML tissues, in normal human brain tissues, in brain tumors, in human kidneys, in urine in infected hamster tissues, and in transgenic mice. (Parts of this work are reported under related projects in this Section.) We found that JCV DNA could be detected in 33 of 46 (72%) of sections from PML blocks. We have also found that JCV sequences could be detected in a paraffin section which had been scraped from slides following immunocytochemical staining. Detection of JCV in that biopsy tissue allowed confirmation of suspected PML which has been successfully treated with high dose AZT. This allows reexamination of large numbers of archival PML materials, even those which have already been utilized for conventional pathological studies. In a related project, two genotypes of JCV have been demonstrated following sequencing of a 610-bp fragment comprising the VP1 gene. This information has allowed design of type-specific primers which can also be applied to archival CNS tissues in paraffin. As these two types of JCV do not differ serologically, this approach is the only means to differentiate these JCV types, the clinical significance of which is currently unknown. This work has demonstrated that PCR is a powerful technique to detect JCV DNA sequences in sections from CNS paraffin blocks-allowing confirmation of diagnosis with great sensitivity, allowing distinction from SV40 DNA sequences with virus-specific primers, and allowing typing of JCV DNA as JCV-1 or JCV-2. In collaboration with a laboratory at Penn State university, JCV DNA sequences have also been demonstrated in frozen tissues from non-PML brains without evidence of demyelination or other pathology. This finding suggests that some cases of PM L may represent the reactivation of JCV which was already latent in the brain prior to the acquisition of HIV infection or another immunosuppressive condition.

1. The role of foxa2 in the survival of dopamine neurons. [unreadable] [unreadable] Animals that have only one copy of the foxa2 gene show spontaneous loss of dopamine neurons with age. This mouse will likely be very widely used because although many mutations have been identified that contribute to Parkinsons disease and related disorders, it has been difficult to reproduce the specific loss of dopamine neurons in an animal model. This animal has the potential to teach us a number of important lessons: why some dopamine neurons are more at risk than others, why the disease is progressive and why dopamine neurons are sensitive to mutations in genes that are widely expressed? [unreadable] [unreadable] Much of what is known about the relationship between the various symptoms in Parkinsons patients and the underlying defects in the nigrostriatal system comes from lesion studies in the rat. The foxa2 heterozygous mouse allows us to study for the first time, in an animal model, motor and cognitive behaviors in the context of a progressive neurodegenerative process. We are currently focused on the cellular role of foxa2 in dopamine neurons. In brain slices, we have shown that the cellular location of the foxa2 gene is controlled by inputs that signal health or stress. This result provides a way measuring the signaling logic that controls the survival of dopamine neurons. [unreadable] [unreadable] 2. Supporting dopamine neuron survival in vivo.[unreadable] [unreadable] We have shown that a single intra-ventricular injection of Notch ligands alone or in combination with other angiogenic factors promotes widespread activation of the stem cell niche in the adult brain and rescues dopaminergic neurons in a model of PD. These data suggest vascular cytokines promote regenerative responses to brain injury. A major goal of our group is to set up in vitro assays that predict the activation of the stem cell compartment in vivo. In this project, we will use our growing understanding of survival signaling in the stem cell niche to rescue injured dopamine neurons in vivo. This approach may allow a more rapid transition to clinical application than cell replacement therapy. In the past year, we have shown that the major regenerative features found in the adult rat brain are also present in adult monkeys. This result encourages our continued belief in the clinical potential of this approach.

The overall commercial aim of this project is to discover and develop novel therapeutic drug leads against diverse therapeutic targets from symbiotic marine dinoflagellate algae using multidisciplinary approaches. Using newly developed cell-based screening technologies, novel biochemical structures of antiviral and anticancer drug leads will be identified in extracts of cultured symbiotic strains of dinoflagellates isolated from diverse marine invertebrates. Culture protocols will employ various media and stress conditions to induce metabolite production. Based on knowledge of the known bioactive metabolites from free-living dinoflagellates, many or most of the bioactive leads from symbiotic strains are expected to be polyketides. Degenerate PKS primers will be used to amplify PKS gene sequences. Specific marine dinoflagellate polyketide synthase (PKS) probes will be designed and utilized to identify PKs sequences in both cultured (Phase I) and uncultured (Phase II) symbiotic dinoflagellates separated from invertebrate hosts. The PKS sequences will be cloned, sequenced, and ultimately expressed (Phase II) in a new recombinant system. This innovative research should not only yield novel drug leads, but it should also validate our methods for developing PKS libraries from the large number of uncultured (including uncultuurble) marine dinoflagellates and other microorganisms in the oceans. PROPOSED COMMERCIAL APPLICATIONS: Discovery and heterologous expression of polyketide synthase genes from symbiotic marine dinoflagellates for commercial-scale fermentation of new polyketide-based therapeutic agents for the treatment and cure of cancer and viral diseases.

The goal of this Phase 1 SBIR is to design and test a prototype of the Channel Laser Stimulator capable of stimulating single cells or part of the cell. This tool is needed to study dose dependency of heat/capsaicin-sensitive cells/neurons and "calcium" channels. Understanding how these "calcium" channels work and principles of their kinetics is important in neuroscience in general and especially in therapeutics and drug development. Further, the development of this instrument will enable the manipulation of cell activity in culture or in tissues with light, a method that could lead to important new applications for regulating cellular or physiological processes in vivo. This device may also lead to novel methods of drug screening and development as it could provide a rapid, well calibrated, and reproducible method of delivering a thermal pulse to cells or purified ion channel protein in single or multi-well format. We will design and test a Channel Laser Stimulator that (A) will deliver a consistent light intensity directly to the target-membrane and channels of the cell; (B) will enable the operator to precisely control the heat stimulus and thus activate some (predictable) amount of channel activity; and (C) will enable the operator to calibrate heat stimulus by power and pulse duration of the laser pulses. We will design this prototype of the Channel Laser Stimulator with parameters optimized for single cell level research, based on (1) the design of an infrared laser diode pain stimulator, developed by LASMED, and (2) the results of preliminary testing. We will consult with a leading scientist in the field of molecular investigations of thermo sensitive ion channels in the mammalian nervous system. All necessary tests to verify performance of the Channel Laser Stimulator will be carried out in the laboratory of this scientist by his personnel.

We are carrying out studies of the histone modifications over the insulin gene locus and its neighborhood in human islet cells, in an attempt to identify long range regulatory influences that may affect insulin gene expression. We have identified an extended domain of open chromatin structure. In addition, we are carrying out measurements of long range physical contacts within the nucleus between the insulin promoter and other genomic sites. We are particularly interested in the possible influence of downstream imprinted loci on insulin expression. We have also collaborated with the laboratory of Dr. Marvin C. Gershengorn in studies of the histone modifications over the insulin locus in human islet-derived precursor cells (hIPCs). We have shown that these cells, although they have lost expression of insulin, retain to a large extend the same modifications found in the active beta cells of islets, strongly supporting the idea that they are poised for expression of insulin. In contrast, other cell types show no such modifications.[unreadable] [unreadable] We are now exploring the long-range interactions between the human insulin locus and other sites in the genome, in order to determine the role of such interactions in insulin gene expression.

Family planning programs have become critical elements of most developing country population control policies. Despite the generally widespread acceptance of the key role of family planning programs in reducing fertility, there is little consensus regarding the programs importance relative to the roles of general economic development and improving women;s status. To address this dialogue, we propose to investigate the following questions: To what extent can be recent fertility decline in Indonesia be explained by the following factors: improved development, and improvement in women's education and economic status? Which elements of the Indonesian family planning program have the greatest impact on fertility: the distribution information, education and communication activities? Do the effects of family planning program activities vary for different groups? For example, does fertility decline more quickly when public family planning program infrastructure is expanded in communities that are in earlier stages of the fertility transition that when infrastructure is expanded in areas at later stages of the transition? How have the expansion of the public family planning program and private family planning services, economic development, and improvements in women's status influenced the major proximate causes of fertility: contraceptive use, marriage and postpartum amenorrhea? Are there time lags in the impact of family planning program components on fertility and its proximate causes? To what extent do expansion of public program components, better access to private family planning services, contraceptive methods to more effective methods? In other words, to what extent do family planning programs affect contraceptive mix versus prevalence? Our methods unify he demographic proximate determinants and economic reduced-form models of fertility. Applications of the proximate determinants model for program evaluation have often yielded different conclusions about family planning programs than have applications of reduced-form "demand for children" models. We demonstrate the compatibility of the two approaches and develop methods to directly compare their results. We will also address the statistical problem raised by the fact that policy maker allocate program inputs for optimal impact, rather than randomly. To control for this endogeneity of program placement, we will use a community fixed effects estimator. The results are interpreted as the effects of changes in the program inputs on changes in fertility within communities.

a. Search for XMRV in prostate tumors Several recent papers reported the detection of a new MLV, xenotropic murine leukemia virus-related virus (XMRV), in human prostate tumors (e.g., Urisman et al., PLoS Pathog. 2:e25, 2006; Schlaberg et al., PNAS 106:16351-16356, 2009). In collaboration with Drs. Karen Sfanos and Angelo De Marzo (Johns Hopkins School of Medicine), we surveyed nearly 800 prostate tumors for XMRV, using a combination of duplex real-time PCR and immunohistochemistry (IHC). Our studies used 22Rv1 cells, which are known to be infected with XMRV (Knouf et al., J. Virol. 83: 7353-7356, 2009), as positive controls, and uninfected human cells, such as 293T or HeLa cells, as negatives. Every PCR well was simultaneously analyzed for viral sequences and for CCR5, a single-copy human gene, as a test of the quality and quantity of sample DNA. Dilution of 22Rv1 DNA into HeLa DNA showed that we could detect the XMRV sequences in 1.5 22Rv1 cells, even in the presence of a 10,000-fold excess of HeLa cells. Our IHC assays used a rabbit antiserum raised against HPLC-purified Moloney MLV p30CA and another against HPLC-purified Moloney MLV gp70SU. Controls included not only 22Rv1, but also 293T cells transfected with a molecular clone of XMRV or mock-transfected. In all cases, the 22Rv1 cells and the XMRV-transfected cells stained with both antisera whereas the negative cells did not. Neither of these well-controlled assays gave any indication of XMRV infection in any of the prostate tumor samples. We concluded (Aloia et al., Cancer Res. 70:10028-10033, 2010) that the positive reports from other laboratories likely represented false positives. For example, positive PCR results can result from contamination with minuscule amounts of mouse DNA, and positive IHC results can arise if the antisera react with host-cell, as well as viral, antigens. Subsequent developments have revealed that XMRV was originally formed during xenotransplantation of the 22Rv1 tumor line, and that humans are almost certainly not infected by this virus (Paprotka et al., Science 333:97-101, 2011; Sfanos et al., Nat. Rev. Urol. 9:111-118, 2012). b. Development of a new MLV infectivity assay and further applications of the iGLuc infectivity assay strategy As part of our continuing studies on XMRV, we adapted a retrotransposition assay (Curcio and Garfinkel, PNAS 88:936-940, 1991; Heidmann et al., PNAS 85:2219-2223, 1988) for detection of replication-competent gammaretroviruses. In this assay (called iGLuc), an MLV-based vector is placed into 293 cells. The vector carries a Gaussia luciferase gene in reverse orientation, interrupted by an intron in forward orientation. Only when this vector has been rescued by a competent helper virus and has progressed through the entire retroviral life cycle is the Gaussia luciferase expressed. This work was performed in collaboration with Drs. Gisela Heidecker, Vineet KewalRamani, and the late David Derse (HIV Drug Resistance Program, National Cancer Institute; see Mazurov et al., PLoS Pathog. 6:e1000788, 2010). Although other reporters can be used in this assay, the Gaussia luciferase offers unique advantages: it is extremely bright, rendering the assay very sensitive, and it is secreted, so that the assay can be performed directly on culture fluids. It is easily performed in a 96-well format. Together with Drs. Sfanos and De Marzo, we screened 70 widely used cell lines for the presence of MLVs, using both PCR and IHC assays (Sfanos et al., PLoS One 6:e20874, 2011). Three lines were found to be producing replication-competent MLVs. The viruses were characterized in considerable detail: two were found to be derived from the endogenous MLV Bxv-1, whereas the third was related to another xenotropic MLV. Their replication-competence was confirmed by the iGLuc assay. In some cases, the MLVs infected the human cells during the xenotransplantation passages used to establish the cell lines; in others, the infections probably represent cross-contamination that occurred in other laboratories. We believe that the iGLuc strategy for detection of retroviral replication can have wide application. We have collaborated with Dr. Kenneth Cornetta (Indiana University) to demonstrate the utility of the iGLuc assay for testing gene-therapy vector preparations for replication-competent gammaretroviruses (Aloia et al., Gene Therapy, in press, 2012), and we intend to do the same for HIV-1-based gene-therapy vectors. In collaboration with Dr. Heidecker, we are developing the reagents for assaying mouse mammary tumor virus; a rapid, sensitive infectivity assay should greatly facilitate future in vitro studies on the biology of this virus. c. Characterization of an HIV-1 entry inhibitor Several years ago, we and colleagues at the NCI-Frederick described a small-molecule entry inhibitor of HIV-1 entry, named Stibavirin or NSC13778 (Yang et al., J. Virol. 79:6122-6133, 2005). This compound is already FDA-approved for other uses. To gain further insight into its mechanism of action, Dr. John Mellors (University of Pittsburgh) has now selected HIV-1 mutants partially resistant to the drug. We have shown that the changes he observed in and near the V3 loop of gp120 are responsible for this resistance. Several kinds of data show that Stibavirin blocks entry by binding to CD4, preventing gp120 from binding. It seems possible that this type of inhibition could be a useful addition to antiviral therapy. The mechanism of the resistance is being analyzed by molecular modeling in collaboration with Drs. Rick Gussio and Tam Nguyen (Developmental Therapeutics Program, National Cancer Institute). Patent Linked to Project: U.S. Patent #7,572,828: Identification of Anti-HIV Compounds Inhibiting Virus Assembly and Binding of Nucleocapsid Protein to Nucleic Acid; issued August 11, 2009; Robert Shoemaker, Michael Currens, Alan Rein, Ya Xiong Feng, Robert Fisher, Andrew Stephen, Shizuko Sei, Bruce Crise, Louis Henderson, and Karen Worthy. This patent describes a class of compounds with anti-HIV-1 activity, which are under investigation for use in antiretroviral therapy. [Corresponds to Rein Project 4 in the October 2011 site visit report of the HIV Drug Resistance Program]

The investigators and staff of the Immunodeficiency-Cancer Registry seek funds to host a conference on the role of immunodeficiency and cancer. The proposed evening conference will be held in conjunction with the annual spring meetings of the Society for Pediatric Research and the Society of Clinical Investigation in Washington D.C., May 1986. The purpose of the conference will be (1) to provide up-to-date information on several major topics pertaining to the association between immunodeficiency and cancer and (2) to explore the relevance of studies in patients with naturally-occurring immunodeficiencies who develop tumors, to cancer in patients with iatrogeneic or acquired immunosuppression as well as "seemingly" nonimmunodeficient persons. Case material from the Immunodeficiency-Cancer Registry will provide an introduction to discussion of the pathophysiological, cytogenetic, and virologic correlates of tumors in patients with naturally-occurring as well as therapy-induced immunodeficiencies. Experts in several areas (e.g., epidemiology, genetics, virology, and/or molecular biology), who have also had experience with immunodeficient patients or their tumors, will be invited to make formal presentations on the state-of-the art and future directions. Several clinicians who have had experience with cancer in immunodeficient hosts will be invited as discussants (rapporteurs). It is hoped that attendees, primarily clinical researchers, will gain new enthusiasm for studying prospective patient material, that new collaborations between basic scientists, pathologists, and clinicians will follow, and that the Immunodeficiency-Cancer Registry, as well as the nonprofit lay organization, Immunodeficiency Foundation, will facilitate these developments.

A new proteomics technology utilizing multidimensional (2D) ion mobility spectrometry separation steps combined with mass spectrometry will be developed to characterize the human plasma proteome. The new instrumentation provides sufficient peak capacity such that a high-throughput analysis of the plasma proteome can be performed without the need for a LC separation step. Thus the measurement can be performed on a timescale that is 30 times faster. The instrumentation will be used to generate Addressable Digital Array Maps (ADAMs) for human plasma analyses. These searchable databases will be utilized in comparative proteomics studies (Phase II) to create a cardiovascular disease ADAM (map). The rapid, high-throughput measurement provided by the 2D IMS instrumentation is groundbreaking in that it is now possible to perform plasma profiling studies on a timescale necessary for population proteomics studies. Such studies are required to identify and score multiple biomarkers to populate the cardiovascular disease ADAM. In the future the map will be used to provide an accurate assessment of health for customers in our personalized medicine business model. The research described in this proposal involves the development of instrumentation required to perform population proteomics studies in a timely fashion. These studies will allow the development of disease biomarker databases that can be used in assessments of disease predisposition for our personalized medicine business model. [unreadable] [unreadable] [unreadable]

The present proposal is to record intracellularly from cells in the posteroventral and dorsal cochlear nuclei, parts of the first integrative stage in the mammalian auditory pathway, in brain slice preparations. Brain slice preparations allow stable intracellular recordings to be made while controlled manipulations can be made of: (1) intracellularly injected current, (2) the precise temporal patterning of auditory nerve input, (3) the ionic and pharmacological environment and (4) the physical presence of tissue which might provide synaptic inputs. The voltage changes of cells in response to injected current reveal the intrinsic electrical characteristics of cells. Synaptic responses to electrical stimulation of the auditory nerve show what the properties are of synapses between auditory nerve fibers and postsynaptic cells. If there exists neuronal circuitry in the tissue of the brain slice, the synaptic responses to electrical stimulation of the auditory nerve will consist of short latency responses mediated directly through primary afferent innervation followed by longer latency responses mediated through interneurons. By cutting slices which include different parts of the cochlear nuclear complex, the position of interneurons can be used to separate synaptic inputs. Pharmacological blockers can be used to separate inputs and to obtain clues about the identity of neurotransmitters. Once separated, the characteristics of synaptic inputs can be studied unambiguously. Trains of electrical shocks will be used to give a rough indication of how the various neuronal pathways function in vivo. The physiological characteristics of cells will probably differ. After learning to differentiate cells physiologically, intracellular dye injections will be used to correlate their physiological with their morphological characteristics. To ensure that the tissue is not degenerating, both physiological and anatomical characteristics will be used to assess the tissue. In viable slices, the input resistance of cells should be high and the electrical characteristics and synaptic responses should be stable over hours. Cells injected intracellularly with horseradish peroxidase should look like Golgi-stained tissue fixed in situ.

Sjogren's syndrome in man is characterized by dry mouth and eyes often leading to stomatitis, increased incidence of caries and periodontal disease, and severe ocular problems. The salivary and lacrimal glands are infiltrated by T and B lymphocytes and antibodies to organ specific antigens can be found in the serum of patients with the disease. Experimental autoallergic sialadenitis (EAS) in the Lewis (LEW) rat shares many histopathologic features with Sjogren's syndrome and will serve as our model for the study of pathogenetic factors in the disease. It is hypothesized that EAS is due to the exposure of cell associated molecules in salivary tissues to an altered or poorly regulated immune system. This exposure, in turn, initiates a T cell mediated, autoimmune response that leads to the destruction of the salivary parenchyma. We have confirmed the reproducibility of the LFW rat model of EAS and now propose to use it to answer the following questions: 1) What is the nature of the target antigen(s) in EAS? 2) Where is the target antigen(s) in EAS located? 3) What are the abnormalities in the cellular and/or the humoral immune system that are important in the pathogenesis of EAS? 4) Can procedures that modify and/or alter the immune system affect the induction or induce reversal of EAS? We will improve the induction and immunization protocol and determine if there are clinical symptoms that correlate with the lymphocytic infiltrates seen in the glands. We will identify, isolate and localize the target antigen(s) immunocytochemically and by using established cell fractionation and protein purification techniques. Immunological analysis will include: identification and characterization of the autoantibodies and mononuclear cells present in the glands during EAS; attempts to induce the disease by adoptive transfer of immune cells and/or serum, and; attempts to inhibit the disease by modulation of the immune system and/or the target antigen(s) with monoclonal antibodies or immunomodulating agents. These results should provide a comprehensive analysis of EAS in LEW rats and may provide important insights into the pathogenesis of Sjogren's syndrome in man.

I will determine whether different RNA polymerases characterize the different morphological and functional stages of development in insects to account for the differential transcription that appears to occur. The multiple RNA polymerases will be characterized, isolated and their physicochemical properties studied so as to not only determine the nature of changes that occur during development and differentiation, but also to correlate the in vivo patterns of RNA synthesis to the variations in their levels, relative proportions and distribution. Whether both the hormones ecdysone and juvenile hormone bring about their profound effects via RNA polymerases and if so, how, will be studied in order to understand the mechanism of action of these hormones at the molecular level. The biochemical manifestations at the molecular level during the transition of cells and tissues from growth to cessation of growth and virtual non-growth to growth as occurs during induction and break of diapause in insects will be studied with particular emphasis on RNA polymerases and RNA synthesis. The existence of modifying factors which alter the transcriptive specificity, cause inhibition or activation in activity of core RNA polymerases of even cause interconversion between different molecular forms of a specific polymerase during growth and differentiation will be looked for in order to explain selective transcription by a limited number of RNA polymerases.

The primary objective of this project is to secure tissue from CaP patients (during the course of their diagnosis and treatment) for the purpose of biological studies designed to characterize cancers with respect to their biological potentials. Frozen samples as well as paraffin-embedded samples of prostate tissue will be provided for study at the University of California-Davis laboratories for DNA quantitation. It is expected that the results obtained will be helpful in the prognostication aspects of the CaP population. Ethnic differences may be defined by the studies also. Approximately 100 cancer patients are expected to enter the study.

The long term goal of the proposed project is to develop a transgenic rodent wherein activation of apoptosis can be imaged non-invasively. Strict coordination of proliferation and apoptosis is essential for normal physiology. An imbalance in these two opposing processes results in various diseases including AIDS, neurodegenerative disorders (Alzheimer's disease), myelodysplastic syndromes (Aplastic anemia, thalassemia), ischemia/reperfusion injury, cancer and autoimmune disease among others. Objective imaging of apoptosis will be a major advancement not only in the screening and validation of novel therapeutic molecules for the above diseases but also in the evaluation of therapeutic success or failure of current and future therapeutic treatment paradigms. In phase I of this proposal we will construct and test in a tissue culture a recombinant DNA molecule that codes for an inactive luciferase protein which during apoptosis becomes a functional luciferase enzyme thus enabling imaging of apoptosis. The ability to image apoptosis non-invasively and dynamically over time will be an invaluable resource to pharmaceutical companies for in vitro high throughput screening of compounds with pro- and anti-apoptotic activity, but also for target validation in vivo. PROPOSED COMMERCIAL APPLICATION: Apoptosis has been implicated in a wide variety of human diseases. The development of drugs that specifically target enzymes involved in the apoptotic cascade is a vibrant and dynamic research area. The ability to non-invasively image the effectiveness of these drugs in vivo would be a breakthrough that would have significant market applications.

In the adrenal medulla and sympathetic neuron intracellular vesicles called chromaffin granules are involved with the biosynthesis, storage, and secretion of catecholamines. The membranes surrounding these chromaffin granules uniquely contain a single b-type cytochrome of which neither the electron donor nor acceptor is known. The membranes also contain an NADH oxidoreductase activity which is inhibited by sulfhydryl-reactive reagents but which has not been further characterized. The cytochrome and NADH oxidoreductase will be isolated and purified from the chromaffin granule membrane using detergent extraction, ion exchange, hydrophobic, gel filtration, and affinity chromatography. These enzymes will be characterized in terms of molecular weight, isoelectric point, amino acid composition and amino terminus using standard standard techniques. The presence of discrete catalytic and membrane binding domains will be investigated using limited proteolytic digestion. The catalytic site orientation of the enzymes relative to the interior or exterior of the granule membranes will be determined using permeant and impearmeant labeled amino, carboxyl, and sulfhydryl reagents. The binding of these enzymes to artificial lipid membranes will be characterized in terms of protection from proteolytic cleavage. Electron transfer between the purified NADH oxidoreductase and cytochrome will be investigated with the enzymes reconstituted in phospholipid vesicular membranes containing lecithin with varying proportions of lysolecithin, cholesterol, and ethanolamine plasmalogen. A search for the function of the electron transfer system in the chromaffin granule membrane will begin with attempts to demonstrate electron transfer from the purified cytochrome to purified dopamine-beta-hydroxylase which is normally bound to the interior surface of the granule membrane. As a preliminary to this latter work the binding of dopamine-beta-hydroxylase to artificial phospholipid membranes will also be attempted.

Chronic fatigue syndrome has received considerable attention in lay and medical media. This syndrome reportedly leads to significant physical and psychological debility in a large segment of the population. Specific diagnostic and treatment regimens have been proposed and are being administered to patients. Yet, there remain few data which can validate the existence of the syndrome as distinct from a wide variety of other, largely psychiatric, disorders, and little understanding of its pathogenesis. The pilot feasibility study proposed in this application seeks to clarify and extend an understanding of the role of hypothalamic-pituitary-adrenal (HPA) axis dysregulation in the pathogenesis of this disorder. The overall objective of this protocol is to test the hyposthesis that patients with chronic fatigue syndrome manifest a characteristic neuroendocrine defect, namely, a reduction in adrenal glucocorticoid secretion mediated by a failure in the central release of corticotropin-releasing hormone (CRH). Specifically, we will confirm and extend our preliminary findings of impaired activation of the HPA axis in patients with chronic fatigue syndrome by examining the patterns of pulsatile secretion of ACTH during periods of maximal and minimal activity of the axis. The results of this proposal will be used to demonstrate the feasability of the pulsatility methodology and provide further pilot data which will enhance the likelihood for success of our federal grant application.

This renewal application for the Pediatric AIDS Clinical Trials Unit at the University of Miami School of Medicine proposes the conduct of clinical trials in pregnant women, children and adolescents with HIV infection. We propose to enroll 100 patients into various protocols placing special emphasis on the areas of perinatal transmission of HIV infection, antiretroviral therapy in pediatric populations, and the prophylaxis and treatment of opportunistic infection in children. This program, consisting of a pediatric unit, an obstetrical unit and a virology core laboratory has a distinguished record over the past five years. The program faculty have provided scientific input into the conduct of clinical trials both nationally and locally and have pioneered the treatment of children and pregnant women with HIV infection through both Phase I and Phase II/III trials. Over 90% of the women and children recruited into treatment protocols have been members of minority groups. The extensive infrastructure of social and medical services available at the medical center has facilitated the conduct of trials. In this proposal,the addition of a new subunit at the Children's Diagnostic Center in Fort Lauderdale is proposed. This subunit is located 25 miles to the north of Miami and would allow access to clinical trials for a large number of infected children. The virology laboratory component has participated in research protocols in pediatric patients since 1983 and is fully certified by the ACTG. In addition, there is an ACTG certified immunology laboratory on campus which is proposed as an immunology core. Three developmental grants in each of the stipulated areas and applications for core virology and immunology laboratories accompany the core application. Long term goals of this project include the interruption of perinatal transmission of HIV, the preservation of the immune system, delayed progression of disease and prolonged survival. The strengths of this program lie in its already existing collaborations among the program faculty, their scientific expertise, research experience and publication record, the prior experience in clinical trials, the extensive supportive infrastructure for patient care, the extensive experience in caring for women and children with HIV infection, the integration with existing community programs, and the large number of patients available for recruitment into trials.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease that causes chronic pain and joint destruction in approximately 1.3 million people in the United States. While drugs to treat RA exist, better treatments strategies are needed to prevent and treat this debilitating disease. As with a variety of autoimmune disorders, there is evidence of an elevated level of tryptophan catabolism in RA patients. This is indicative of activation of the enzyme indoleamine-2,3-dioxygenase (IDO), which initiates the breakdown of tryptophan. IDO is an immune regulatory enzyme that has been implicated in the inflammatory process associated with tumor formation. Because IDO is thought to be immunosuppressive, it has been generally assumed that inhibiting IDO would exacerbate classic autoimmune disorders such as RA. However, data from patients has suggested the opposite, that IDO activity may actually be associated with the development of disease symptoms. Consistent with this, inhibitors of IDO reduce autoantibody titers and alleviate arthritis symptoms in a B cell-mediated mouse model of RA. These data support a role for IDO in exacerbating the disease and further implicate IDO in driving autoreactive B cell responses. This proposal will explore the hypothesis that IDO does not simply promote the general suppression of immune responses, as currently thought, but rather plays a more complex role in modulating inflammation by directing the immune profile of B cell responses. Drug-like inhibitors of IDO and mice that are genetically deficient in IDO, together with the K/BxN mouse model of RA, will be used as tools to determine the mechanism by which IDO inhibition alleviates arthritis development. In this proposal, we will 1) define how the inflammatory features of arthritis induced in mice are inhibited by targeting IDO activity;2) determine whether IDO-driven B cell activation is unique to the K/BxN model or is a universal phenomenon;and 3) determine whether inhibition of IDO can be used as a co-therapeutic with current RA therapies, to increase their efficacy and reduce their side-effects in the alleviation of arthritis. The long-term goal of this project is to define the mechanism by which IDO modulates the immune response leading to autoimmunity and provide new insights into strategies that can be used to manipulate this pathway to reduce or prevent debilitating disease. If successful, the results from this proposal will suggest that targeting IDO has the exciting potential to lead to a new approach for the prevention and treatment of RA and other autoantibody mediated diseases. PUBLIC HEALTH RELEVANCE: Understanding the factors that contribute to the initiation and propagation of autoimmunity is important in the design of therapeutic strategies for the prevention and treatment of autoimmune diseases such as rheumatoid arthritis. The studies in this proposal will provide insights into the mechanism by which the immune system becomes activated to induce inflammation in the joint and identify key targets to prevent the resulting debilitating disease. In the future, this strategy could lead to a new approach for the prevention and treatment of rheumatoid arthritis.

An obstacle to the use of adenovirus as a vaccine carrier is the induction of neutralizing antibodies to hexon, the major protein component of the icosahedral capsid. The Program Project is designed to exploit the fact that human subjects have no preexisting neutralizing antibodies to chimpanzee adenovirus 68 (AdC68) that would limit its efficacy. Nevertheless, subsequent use of AdC68 for booster immunization would be limited by antibodies generated in response to the primary dose. The solution lies in using structural knowledge to design and create a series of novel AdC68 derivatives with modified neutralization epitopes. Three-dimensional crystal structures are known for human adenovirus types 2 and 5 hexons, and the overall virion architecture is known from cryo-electron microscopy. Comparison of all known hexon structures and sequences shows that there is a common molecular fold that provides an accurate and representative model for designing new adenovirus vectors. The hypervariable regions of AdC68 hexon are likely to contain the epitopes recognized by neutralizing antibodies. In this project, molecular modeling will suggest sites where modifications can be made without structural disruption. The AdC68 hexon crystal structure will be determined to confirm and refine the structural predictions. Simultaneously, the AdC68 hexon epitopes responsible for antibody neutralization will be identified experimentally by generating a panel of anti-hexon neutralizing monoclonal antibodies (mAbs). The panel will be used to find neutralization escape mutants by screening AdC68 virions with randomly-introduced mutations in the epitope regions. In addition, random pentapeptide insertions will be made in the hexon hypervariable regions to identify sites that tolerate extensive structural modification. The crystal structures for escape mutant hexons will be solved to refine structural information for each epitope. The combined information will be used to synthesize variants with disrupted epitopes. Finally, "artificial serotypes" with multiply-disrupted epitopes will be tested against the antibody panel and )olyclonal neutralizing sera to demonstrate their efficacy as booster vaccine carriers.

The flow-through centrifuge eliminates complication arising from rotating seals. Preliminary studies on plasmapheresis demonstrated negligible platelet injury and no evidence of hemolysis during 12 hours of operation. The system may provide a broad application to cell washing and elutriation, zonal centrifugation and countercurrent chromatography.

The long term objective of this project is the evaluation of the potential of liposomes as carriers of anti-tumor agents. Several model systems of antibody directed liposomes are analyzed to obtain information on the generation of functionally active targeted liposomes. The properties of the cell-liposome interactions are investigated in the presence of serum proteins. The in vitro and in vivo efficiency of drugs entrapped in targeted liposomes is evaluated.

Two major research projects will be undertaken: (1) a clinical study to elucidate the role of alterations of the various calcium and bone regulating hormones in the pathogenesis of diabetic osteopenia, and 2) a project to develop a radioimmunoassay for the isoenzyme of alkaline phosphatase derived from bone. The RIA will be used to assay serum samples from patients with the various forms of hypophosphatasia - a crippling inborn error of metabolism - to gain insight into the clinical and genetic variability of this disorder. Juvenile and adult diabetics will be studied longitudinally on the Clinical Research Center, Barnes Hospital so that changes in circulating parathyroid hormone, calcitonin, vitamin D metabolites, estrogens, etc. can be contrasted with studies of bone density and gastrointestinal calcium absorption. Differences in the degree of osteopenia, known to differ in various diabetic populations, should be reflected in dietary calcium absorption and circulating serum 1,25(OH)2D levels. Identification of hormonal differences between diabetics with and without osteopenia should provide clues to the pathogenesis of this frequent complication and suggest possibilities for corrective therapy. Normal fetal and neonatal bone will be extracted and purified for development of an RIA for normal bone AP isoenzyme. Routine methods will be used to develop the RIA once purified antigen is available. The RIA will be used to study the variability in clinical expression of this disorder, the variability in its genetic transmission, to identify "carriers", and in prenatal diagnosis.

Leishmaniasis is a major public health problem in large parts of the world, due in part to the lack of a vaccine and inadequate chemotherapy. Studies of the immune responses in humans and mice following Leishmania infection have provided an understanding of many of the cells and cytokines that contribute to the control of this disease. Nevertheless, a vaccine for human leishmaniasis does not exist. Understanding how memory T cells develop will be crucial in the development of such vaccines. Two types of memory T cells have been described: T effector memory cells, which produce effector cytokines and migrate through the tissues, and central memory T cells that do not produce effector cytokines and migrate through lymph nodes. C57BL/6 mice infected with Leishmania major are immune to rechallenge after they resolve their infections, but this immunity was thought to be dependent upon residual parasites. Now an attenuated L. major parasite that is eliminated after eight weeks has been shown to stimulate the development of long-lived protective memory T cells. These cells have the characteristics of central memory T cells, and the studies in this proposal will characterize these cells and assess whether we can generate memory T cells with an effector phenotype. The studies will include a comparison of the ability of a Listeria expressing a leishmanial antigen and L. major to stimulate effector memory T cells, assessment of the role that parasite persistence plays in blocking the development of effector memory cells, and how dendritic cells influence memory T cell generation. The experiments utilize state-of-the art tools that will allow qualitative and quantitative assessment of memory T cell development. Overall, the experiments described in this proposal will determine what type of immunologic memory can be induced in leishmaniasis, which will provide direction for the field in establishing what are reasonable goals for a leishmaniasis vaccine. PUBLIC HEALTH RELEVANCE The development of a vaccine for human leishmaniasis is a major priority, but our understanding of how to stimulate the memory T cells required for vaccine-immunity is limited. This proposal will determine how memory T cells develop, are maintained and are regulated using an experimental model of the disease. The results from these experiments will provide a foundation for the development of a successful leishmaniasis vaccine.

The physiological function and the mode of regulation of transglutaminases are being studied as to their role in the formation of temporary tissue matrix (fibrin-connective tissue matrix) during tissue or bone fracture repair and in the modulation of specific cellular processes. The physiological significance and biochemical mechanism of factor XIIIa (plasma transglutaminase) and red blood cell transglutaminase catalyzed cross-linking of plasmin inhibitor (a2PI) to fibrin and other matrix proteins are under investigation both in vivo and in vitro. The cross-linking of a2PI to fibrin plays a major role in stabilization of a temporary matrix which is vital for the initial phase of cell migration and proliferation and wound sites. In cells activated by mitogens and in transformed cells, several different molecular forms of transglutaminases have been identified. These enzymes are enriched in the plasma membranes and nuclear fractions in proliferating cells and appear to modify specific proteins in the membrane fractions.

The aim of this RO3 proposal is to examine the ability of cyclin D1 and other cell cycle regulatory proteins to promote hepatocyte proliferation in culture and in vivo. Mature hepatocytes retain the ability to function as stem cells and can repopulate the live after hepatic injury. Previous studies have suggested that up-regulation of cyclin D1 plays an important role in governing progression of hepatocytes through G1 phase of the cell cycle. To test whether cyclin D1 is capable of promoting mitogen-independent hepatocyte proliferation, we have constructed a replication-defective adenovirus encoding this protein (ADV-D1). Our preliminary studies indicate that ADV-D1 but not a control adenovirus, promotes cell cycle progression in primary hepatocytes in the absence of growth factor, and induces hepatocyte proliferation on extracellular matrices that are normally growth-inhibitory. Pilot in vivo studies indicate that ADV-D1 effectively transfects hepatocytes in the intact animal. These results suggest that transfection of hepatocytes with ADV-D1 can promote hepatocyte proliferation independent of extracellular signals, and warrants testing as an agent to promote liver regeneration. The three Specific Aims of this study will examine the mechanisms by which cyclin D1 promotes hepatocyte growth, and will examine the ability of other G1 phase cyclins and E2F transcription factors to promote hepatocyte cell cycle progression. These studies are intended to provide unique mechanistic insight in the regulation of hepatocyte growth, and to identify candidate proteins for gene targeting strategies to therapeutically enhance liver regeneration.

Short REM latency is heritable for narcolepsy and depression. Individuals with narcolepsy almost all express HLA DR2/DQw1. If short REM latency in depression is associated with HLA expression, it would indicate that REM sleep itself may be regulated by genes in or around the MHC and/or that MHC-related genes may be involved in depression.

There is a rapidly increasing awareness of the importance of titin in causing neuromuscular disorders (titinopathies), two of which we focus on in this work, centronuclear myopathy (CNM) and hereditary myopathy with early respiratory failure (HMERF). Titinopathies frequently present early-onset muscle weakness and respiratory difficulty but understanding their underlying mechanisms is held back by our still limited understanding of the functional roles of titin in skeletal muscle. Titin comprises the third myofilament of muscle and spans along the sarcomere, from Z-disk to M-band. Titin?s I-band region functions as a molecular spring that generates passive stiffness with recent studies indicating that passive stiffness affects active force at sub- maximal activation levels. However, it is not known how important titin?s stiffness is to overall skeletal muscle health and if altered stiffness can cause a myopathy. That the importance might be high is suggested by our pilot studies that reveal deranged titin stiffness and reduced active tensions in titinopathy patients with CNM. Aims 1 and 2 address how altering titin-based stiffness affects both passive and active muscle properties and if it can be disease-causing. We will perform mechanical studies on biopsies from titinopathy patients (focus on CNM) as well as study two contrasting mouse models in which the stiffness of titin?s spring region is either increased (PEVK truncation) or reduced (inactivation of Rbm20). The working hypothesis is that altering titin?s spring region alters both passive muscle stiffness and active tension and that this causes myopathy. Aim 2 also studies a novel mouse model that mimics CNM with splice site mutations in titin?s spring region and we will examine the functional consequences at the RNA, protein, structural and functional levels. We also study the A-band segment of titin, specifically the C-zone. This zone has not been studied, it is clinically important as this is where a large number of disease-causing mutations are found. We investigate in Aim 3 a mutation in the C-zone exon 343 which causes HMERF, a myopathy with respiratory muscle involvement that can be fatal. Using a novel HMERF mouse that we made the mechanistic basis of the disease will be studied. Through excision of the mutated titin exon 343, the therapeutic potential of exon skipping for treating HMERF will be tested. With its basic science and translational goals and its in-depth and integrative approach, this application seeks to continue our track record of cutting-edge titin research. Powerful techniques and novel mouse models are in place, pilot data support the guiding hypotheses, and our research team is highly experienced. The proposal will greatly enhance insights in titin biology, titin?s role in muscle disease, and titin?s potential as a therapeutic target.

"Prepostaglandin endoperoxide" is the proposed pivotal intermediate in prostaglandin biosynthesis. The ultimate goal of this research is preparation in the laboratory of this intermediate, and study of its bioconversion to prostaglandins. It is expected that this research will provide a better understanding of the biosynthesis of prostaglandins. The initial objective is the synthesis of 2,3-dioxabicyclo (2.2.1) heptane and simple derivatives. A study of the chemistry of these endoperoxides will follow. Then biogenetic type syntheses of this novel structure type will be sought. The recent isolation of minute quantities of a preprostaglandin endoperoxide lead to the discovery of physiological activity which significantly exceeds that of the related prostaglandin derivatives. Thus, it is quite possible that the endoperoxides may constitute a class of ultra- potent medically useful substances, e.g. for the treatment of heart disease. The total synthesis proposed herein would provide material in sufficient quantities for research on the medicinal utility of endoperoxide derivatives and ultimately for medicinal application.

Heart failure (HF) is characterized by an elevation in sympathetic tone. The mechanisms responsible for the sympatho-excitation of HF are not completely understood. Recent studies from this laboratory have shown that the cardiac "sympathetic afferent" reflex is enhanced in dogs with pacing-induced HF. The mechanisms by which this enhancement occurs are unclear. There is an enhancement in afferent fiber sensitivity to bradykinin and capsaicin. Preliminary evidence from this laboratory suggests that an enhanced central gain of this reflex is, in addition, responsible for the augmentation of this reflex. Furthermore, we have shown that central angiotensin II (Ang II) is at least one mediator for this enhancement. A second mechanism which may explain the increased gain of he cardiac sympathetic afferent reflex in HF is a decrease in nitric oxide (NO) production in several central sites which regulate sympathetic outflow. We hypothesize that both an increase in central Ang II and a decrease in central NO contributes to the increase in the sensitivity of the cardiac sympathetic afferent reflex and to the tonic sympatho-excitatory state in dogs with HF. Therefore, the specific aims of this project are to: 1) determine if the central gain of the cardiac sympathetic afferent reflex in dogs with HF is related to increased levels of central Ang II or to changes in Ang II type1 receptor density or both, 2) determine if acute and chronic central administration of the Ang II receptor antagonist, losartan and L-158,809 and/or NO donors prevent or reduce the enhancement of the cardiac sympathetic afferent reflex in dogs with HF, 3) determine if bradykinin prostaglandins and NO are mediators of the enhanced sensitivity of cardiac sympathetic sensory endings in dogs with HF, and 4) determine if chronic thoracic sympathetic deafferentation alters the time course and/or magnitude of the sympatho-excitatory response during the development of pacing-induced HF. These studies integrate into the overall scope of this Program Project in that the regulation of sympathetic outflow in HF is likely to be mediated by a variety of peripheral inputs with important modulation from central substances. The cardiac sympathetic understanding of neuro-humoral regulation in this disease state should include this potentially potent reflex.

Median survival of high-grade gliomas remains less than a year despite multi-modality treatment. Cure is considered anecdotal. These tumors usually have extended beyond the limits of a complete surgical resection and a dose of conventional external beam radiotherapy has been limited by surrounding brain tolerance. We believe that we can achieve a higher radiation dose to the tumor by placing radioactive seeds of Iodine 125 directly into the tumor bed, with a sharp fall-off of radiation to the surrounding normal brain. Hopefully this will achieve a much better therapeutic ratio, especially when delivered at low dose rates.

The long term goal of the Applicant's research program is to explore the molecular mechanisms underlying the etiology of age-related macular degeneration (AMD) and develop novel target-based therapeutic strategies. This K99/R00 grant will facilitate the transition for the Applicant to become an independent investigator at the Vanderbilt Eye Institute, which has a rich environment of scientific collaboration and nurturing career development of junior scientists. AMD is the leading cause of blindness in elderly Americans. The majority of AMD patients has atrophic (dry) form of the disease and has only limited treatment options at the present time. Atrophic AMD is likely resulted from gene/environmental interaction causing progressive degeneration of the retinal pigment epithelium (RPE). Aging is the most well defined environmental risk factor of AMD. We hypothesize that the mammalian target of rapamycin (mTOR)-mediated signaling pathway plays key roles in controlling the aging process of the RPE. The hypothesis is supported by recent literature data suggesting that mTOR plays key roles in integrating various environmental signals and linking them to altered tissue function and organism's life span. In our preliminary studies, we found that rapamycin reversed the senescent phenotype of primary human RPE cells in vitro. To further test our hypothesis, we have proposed three specific aims in this application. Aim 1 is to determine how the aging process regulates mTOR pathways in the RPE. Aim 2 is to determine how modulating the mTOR signaling affects RPE aging in vitro by a potential mechanism of regulating autophagy. Aim 3 is to determine whether mTOR regulates aging of the RPE in vivo using SOD1 knockout mice which develop AMD-like phenotype in the retina. During the mentored phase, the Applicant will conduct the proposed experiments under the supervision of a mentoring committee, which is consisted of mentors with expertise in AMD etiology and pathogenesis (Dr. Paul Sternberg), animal models of neurodegeneration in the retina (Dr. David Calkins) and mTOR/autophagy (Dr. Lu Bo). Members of the committee will meet regularly, monitor the research progress and assist the Applicant to advance her career towards independence. Research at the R00 phase will test the potential therapeutic effects of mTOR inhibitors in an animal model relevant to AMD. Results from these studies will provide novel mechanistic information on aging and age-related degeneration of the RPE and retina. PUBLIC HEALTH RELEVANCE: This project focuses on characterizing a novel signal transduction pathway which may control the aging process of the retina and contribute to age-related macular degeneration (AMD). Results from the studies can potentially be translated into new therapeutic approaches for treating the atrophic form of AMD.

: The overarching goals of the Lincoln University- Fox Chase Cancer Center Partnership in Cancer Research and Training are to create a cadre of well trained and experienced cancer researchers at Lincoln University oriented toward reducing cancer health disparities, to foster such research at the Cancer Center, and to create a pipeline of students with laboratory experience who choose to further their education in cancer biology, population science, and medicine. This cooperative effort will accomplish the following objectives: 1) to expose Lincoln University faculty and students to research conducted at Fox Chase in the areas of cancer biology and population science; 2) to enhance Fox Chase and Lincoln's collaborative research efforts and networking opportunities through increased participation in scientific conferences; 3) to introduce basic cancer principles through workshops, seminars, lectures, community/university outreach, and academic instruction; 4) to publish joint research publications; 5) to incorporate team-based projects that will generate a steady pipeline of students to continue advanced studies in the nation's top graduate institutions; and 6) to create competitive minority scientists that are successful in receiving funding for addressing cancer health disparities. [unreadable] [unreadable] The Partnership will conduct its activities over four years in three stages. The first year will be a planning stage, where an Administrative Leadership Group, Research Training and Research Project committees will be established, and Internal and External Advisory Boards constituted. The second year will be the priority-setting stage during which time a competition for joint pilot research projects will be held, and the training program established. The third and fourth years will constitute the implementation stage, wherein two pilot research projects and a research training program will be conducted. Summative activities of the Partnership will be the development of competitive research and training grants directed by a team of Lincoln faculty who have achieved leading roles in this endeavor. [unreadable] [unreadable] The ultimate ambition for this partnership is to establish a strategic academic collaboration with Lincoln University to implement research and education that highlights populations with health disparities. The instruction of minority and majority scientists at Lincoln and Fox Chase about cancer health imbalances will increase the sensitivity toward this issue and augment the number of capable researchers available to address this present dilemma. [unreadable] [unreadable] [unreadable]

Enterococci are commensal organisms that inhabit the gastrointestinal tract and rarely cause infections in the healthy host. However, in immunocompromised individuals or in individuals on antibiotic therapy, these organisms can cause serious disease. Hospital-acquired infections by Enterococcus faecalis, especially those associated with intravascular catheters, biliary stents and other implants have increased dramatically in recent years. This trend has likely resulted from high level antibiotic-resistance within this genus coupled with the acquisition of virulence determinants and the ability to form biofilms. It is recognized that biofilm associated bacteria are phenotypically different from planktonic cells and tend to be more resistant to antibiotics and to host immune clearance mechanisms. Innate immunity plays a crucial role in the host response to microbial infections but little is known about the immune response to enterococci and enterococcal biofilms. Our laboratory has identified and characterized two important biofilm enhancing loci in E. faecalis;one specifying the enterococcal surface protein Esp and the other the Bee pilus. Both these factors significantly enhance biofilm formation and adherence of E. faecalis to abiotic surfaces in vitro, but much remains to be learned about their functions in vivo. Our underlying hypothesis in this proposal is that the host innate immune response to biofilm and planktonic E. faecalis is quite different and we will examine this through two specific aims. In aim 1, we will compare in vitro the interaction of professional phagocytes and enterococcal biofilms versus planktonic cells from two different genetic backgrounds, to document bacterial survival, host cell response and influence on biofilm architecture. In aim 2, we propose to examine host-pathogen interactions in vivo using two murine models of biofilm-associated infections. The novelty of these studies will be the utilization of a positron emission tomography /computed tomography (PET/CT) approach to image and follow the time course of infections in real time. These exploratory studies are designed to lead to a better understanding of the host response to biofilm-related enterococcal infections and could ultimately lead to new therapeutic strategies to prevent serious enterococcal infections. PUBLIC HEALTH RELEVANCE: Hospital-acquired infections by antibiotic-resistant Enterococcus faecalis have risen dramatically in recent years. Greater understanding of the host defense mechanisms that protect against invasive enterococcal disease could provide new ways of controlling enterococcal infections.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of this study is to determine the role of caveolae in airway remodeling in asthma. This chronic inflammatory disease of the airways affects an estimated 15 million people in the United States and 100-150 million people worldwide. Among this population at risk, a subset of subjects will develop irreversible remodeling and airway obstruction. Airway remodeling is characterized by an abnormally large number of myofibroblasts in the subepithelial basement membrane associated with significantly increased deposition of connective tissue proteins leading to a thickening of the lamina reticularis. Interleukin 4 (IL-4), a Th2 cytokine important in the control of allergic response, has also been proposed as an effector influencing airway remodeling. We have recently demonstrated that IL-4 regulates the expression of caveolin-1 (cav-1). Cav-1, main structural and functional protein of caveolae, notably regulates transforming growth factor (TGF)-beta activity by dampening its transduction signal process initiated by the TGF-beta receptor complex. TGF-beta1 is a pivotal pro-fibrotic cytokine in asthma and fibrosis. Our goal, therefore, is to demonstrate that Th2 cytokines, and more specifically IL-4, mediate caveolae deficiency by regulating cav-1 gene transcription in fibroblasts resulting in enhanced TGF-beta responses in airway remodeling. We will: 1) Evaluate the hypothesis that the reduced expression of cav1 is associated with the expression of inflammatory markers, more specifically IL-4 and TGF-beta, and the development of airway remodeling in allergen challenged mice. 2) Investigate the hypothesis that among the Th2 cytokines expressed in asthma, IL-4 regulates caveolin-1 expression and caveolae formation;3) To test the hypothesis that the reduced expression of caveolin-1 protein enhances TGF-beta effects.

Our goal is to identify the biological parameters that control myelin metabolism. We are using an in vitro culture system of suspended, spherical cellular aggregates prepared from fetal mammalian central nervous systems. With these cultures we are following the development of myelin synthesis by measuring several myelin membrane specific molecules such as sulfatides, myelin basic protein and myelin associated enzymes. Preliminary studies indicate myelin synthesis does occur in these cultures and follow the same temporal pattern normally occurring in vito. Cultures from animals exhibiting genetically determined demyelinating disease (e.g., quaking and jumpy mice) will be analyzed for the same parameters and compared to normal cultures. Experiments will be performed to test the effects of hormones on myelination in these cultures. Measles virus, because of its purported relationship to multiple sclerosis, will be carefully investigated for its affects on this myelinating system. In addition, we will determine if any protein kinase activity is associated with the measles virus as well as characterize the protein substrates of the phosphotransferase enzyme(s). These brain aggregate culture studies should increase our understanding of the biological signals and mechanisms controlling myelination and may lead to the development of novel preventative, diagnostic and therapeutic methods treating demyelinating disease, such as multiple sclerosis.

Current methodologies for diagnosing infection with Neisseria gonorrhoea require specimens that are obtained by invasive means: urethral swabs from men and cervical swabs from women. These specimens are usually cultured onto Thayer-Martin media; alternatively a variety of other methods can be employed to diagnose gonorrhea, but none is as sensitive as culture. The objective of this proposal is to create an inexpensive, rapid, simple and non-invasive immunologic test for gonorrhea. It must be as sensitive as culture and applicable for use in developing countries. The project will be accomplished through 3 specific aims: (l) to optimize an already existing prototype assay called the HygEIA GC Test to use with non- invasive specimens, and to convert this ELISA format to an immunochromatographic (ICT) format with a membrane read-out. Initially, the assay will be developed using two existing antibodies, one directed against the H8 antigen (a current component of the HygEIA GC Test) and a gonococcal anti-lipooligosaccliaride antibody that reacts broadly with N. gonorrhoea; (2) to create polyclonal antibody libraries using a molecular cloning system that produces antibodies that recognize multiple epitopes on gonococcal surfaces exclusively; simultaneously these libraries will be immortalized and can be utilized time and again to produce, antibodies with reproducible specificities and; (3) to use patients at risk for gonorrhea to test prototype formats. Through overlapping strategies, beginning with a prototype that already functions, it is anticipated that a final product will emerge.

Cdk5 regulates pain signaling: Our present studies are primarily focused on delineating the roles of Cdk5 in the central and peripheral nervous system with a special emphasis on nociception and pain. We have earlier identified expression of Cdk5 and p35 in nociceptive neurons, and discovered that this expression is modulated during peripheral inflammation. We also discovered that induced inflammation results in increased calpain activity in sensory neurons, which activates cleavage of p35 to p25 and subsequently increases Cdk5 kinase activity. The p35-/- mice, which exhibit significantly decreased Cdk5 activity, showed delayed responses towards painful thermal stimulation compared to their wild-type controls. In contrast, mice overexpressing p35 with elevated levels of Cdk5 activity were more sensitive to painful thermal stimuli than controls. We analyzed TRPV1 for potential phosphorylation by Cdk5 and found that Cdk5 can directly phosphorylate TRPV1 at threonine 407, and this in turn modulates agonist-induced calcium influx. We also found that inhibiting Cdk5 activity resulted in attenuation of capsaicin-induced calcium influx in cultured DRG neurons, and this attenuation was reversible. These observations suggest that Cdk5-mediated phosphorylation of TRPV1 is important for capsaicin-mediated calcium influx through this receptor. Since Cdk5-/- mice die perinatally, we generated primary nociceptor-specific Cdk5 conditional knockout (COKO) mice to identify the precise role of Cdk5 in primary afferent pain signaling. In the basal state, the conditional knockout mice had significant hypoalgesia, confirming the direct role of normal Cdk5 activity in primary afferents. Collectively, our findings show a novel molecular mechanism for the functional regulation of TRPV1 by Cdk5 and suggest that Cdk5/p35 may be a target for development of analgesic drugs.[unreadable] [unreadable] The aim of our current study in FY08 was to identify the proinflammatory molecules that regulate Cdk5/p35 activity in response to inflammation. We constructed a vector that contains the mouse p35 promoter driving luciferase expression. We transiently transfected this vector in PC12 cells to test the effect of several cytokines on p35 transcriptional activity and Cdk5 activity. Our results indicate that tumor necrosis factor-alpha (TNF-alpha) activates p35 promoter activity in a dose- and time-dependent manner and concomitantly upregulates Cdk5 activity. Because TNF-alpha is known to activate ERK1/2, p38 MAPK, JNK, and NF-kB signaling pathways, we examined their involvement in the activation of p35 promoter activity. MEK inhibitor, which inhibits ERK activation, decreased p35 promoter activity, while the inhibitors of p38 MAPK, JNK, and NF-kB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The mRNA and protein levels of early gene response 1 (Egr-1), a transcription factor, were increased by TNF-alpha treatment, and this increase was dependent on ERK signaling. In a mouse model of inflammation-induced pain in which carrageenan injection into the hind paw causes hypersensitivity to heat stimuli, TNF-alpha mRNA was increased at the site of injection. These findings suggest that TNF-alpha-mediated regulation of Cdk5 activity plays an important role in inflammation-induced pain signaling.[unreadable] [unreadable] Phosphoproteomic analysis of Cdk5 targets: The human genome encodes over 500 different protein kinases, the key regulatory enzymes that catalyze the phosphorylation of proteins at about 100,000 different sites to reversibly control their functional activities. Defects in specific protein kinases have been linked to over 400 diseases, and about 25% of all pharmaceutical industry research and development is now focused on the discovery and evaluation of protein kinase inhibitors for therapeutic applications. Cdk5 has become a target of high interest to the drug industry because of its key role in neuronal homeostasis. So far more than 40 different Cdk5 substrates have been identified, and abnormal Cdk5 activity has been implicated in several disease processes, including neurodegenerative disorders, cancer, and diabetes. However, a global profiling of protein phosphorylation mediated by Cdk5 is still not available. Our current knowledge about such profiling comes from experiments performed in different laboratories mainly based on 2-dimensional gel electrophoresis or yeast 2-hybrid screening. Because of the limitations of these techniques at the point of validation of targeted proteins, we took a different approach to resolve this issue. We compared phosphorylation status and total protein levels of 258 different proteins by simple Western blotting analyses of Cdk5-/- and WT wild-type brains. The antibodies used in this analysis are already proven to be highly specific for their targeted sites in different biochemical pathways. We based our selection of these proteins on some known and predicted functions of Cdk5 and further categorized them into 6 different groups. The first group contained 25 different proteins involved in apoptosis, the second consisted of 77 different other kinases, the third consisted of 39 different substrates for these kinases, the fourth consisted of 43 different proteins involved in the cell cycle, the fifth contained 37 different proteins involved in numerous neurobiological functions, and the sixth consisted of 37 different proteins controlling different kinase pathways. This phosphoproteomic screening gave us a broad overview of Cdk5 targets involved in these biochemical pathways and cellular processes.[unreadable] [unreadable] We will develop strategies to help us identify the role of Cdk5 in tooth pain and develop potential analgesics. Work on other potentially important questions involving Cdk5 have been shifted to ongoing major collaborations with a number of leading laboratories in neurobiology. This shift will benefit us in our increasingly predominant program on Cdk5 and pain signaling.

The objectives of this trial is to determine the toxicities of SPI-77 when given concurrent with radiotheraphy in patients with advanced head and neck cancer; to determine the maximum tolerated does (MTD) of SPI-77 when given once every 2 weeks for 2 and 3 doses concurrent with radiotheraphy; to determine the pharmacokinetics of SPI-77; and to gather preliminary response information that may result from the treatment of SPI-77 when given concurrent with radiotheraphy.

The overall objectives of this research are to develop monoclonal antibodies to distinct determinants on human prostate carcinoma-associated antigens (PCAA) in order to: (1) develop double determinant immunoassay (DDIA) to study the shedding of PCAA by prostate cancer cells in vitro and in vivo; (2) study the cellular heterogeneity of human prostate tumor cell lines and tissues; and (3) analyze the molecular heterogeneity of PCAA. In the next year, efforts will be focused on the following activities: (1) production of new antibodies to distinct determinants of PCAA will be continued using new immunization procedure, e.g., by injecting mice with purified PCAA covered with one antibody to the antigen; (2) the DDIA will be developed with the antibodies that have already been obtained in the past year that show preferential activity for prostate carcinoma cells to study the shedding of PCAA by cultured prostate tumor cells; (3) the same antibodies will be applied in immunohistochemical study of human prostate tissue using peroxidase staining methods; and (4) the antibodies will be used in immunochemical analysis of the PCAA by two-dimensional gel analysis, i.e., isoelectrofocusing and SDS polyacrylamide gel electrophoresis. (AG)

The long-term objective of this research program is to understand the roles of key coagulation and fibrinolytic factors in inflammatory host defense and bacterial virulence. Since a rigorous understanding of these complex processes can only be achieved in an in vivo experimental setting, our proposed studies focus on innate immune surveillance and bacterial pathogenesis in gene-targeted mice with specific alterations in core hemostatic factors. The aims of this project center on the following specific hypotheses: i) host factors mediating fibrin deposition and dissolution are important determinants of the inflammatory response in vivo, ii) bacterial factors known to bind/activate host prothrombin, fibrinogen and plasminogen serve to subvert host defense, iii) leukocyte engagement of fibrin within challenged tissues is an important cue in "target recognition" leading to the implementation of effective antimicrobial functions, and iv) hemostatic factors control leukocyte activation events as well as macrophage egress from inflammatory sites into lymphatics. These hypotheses will be tested through detailed studies of S. aureus virulence/host defense in mice lacking fibrin(ogen) or expressing mutant forms of fibrinogen that either lack the leukocyte integrin receptor alphaMbeta2 binding motif, lack platelet integrin receptor alpha(IIb)beta3 binding motif, or cannot be converted to a fibrin matrix (Aim I). Further, the role of procoagulant and fibrinolytic factors in establishing S. aureus virulence/host defense will be determined through investigator-imposed genetic changes in prothrombin, plasminogen and their respective microbial-derived activators: coagulase and staphylokinase (Aim 2 and 3). Finally, the mechanistic role of neutrophils and macrophages in the fibrinogen/prothrombin-dependent bacterial clearance will be established in the context of S. aureus peritonitis (Aim 3). The proposed studies will provide a detailed understanding of the cross-talk between the hemostatic and inflammatory systems in the implementation of effective antimicrobial response in vivo. Further, these studies may illuminate therapeutic strategies centering on specific hemostatic system components that are effective in the treatment of life-threatening microbial infection/sepsis. Lay Description: The bacterial species that are the most common causes of human suffering and death have uniformly evolved the means to engage coagulation factors, apparently as a means of subverting host defense. The goal of this research is to develop a detailed mechanistic understanding of the role of host hemostatic factors in bacterial virulence and the inflammatory response as a step toward novel therapeutic interventions for life-threatening infectious disease.

This project will examine how specific neuronal connections are established between vestibular sensory organs and their central nervous system targets. In adult vertebrates, primary vestibular afferent axons serving the different vestibular end organs form distinct though overlapping regions of axonal arborization in the vestibular nuclei. While the presumed role of this specific connectivity in sensory processing is obvious, little is known about the developmental mechanisms that are responsible for its establishment. The proposed experiments will be a first step towards analyzing the development of organotopic specificity in the primary vestibular afferent projection to the vestibular nuclei. Vital-dye fiber-tracing techniques will be used to label populations or single axons in the zebrafish, Brachydanio rerio. In the initial experiments, the pattern and extent of axonal growth from each vestibular sensory organ in young zebra fish will be examined in fixed specimens both in whole-mounts using confocal microscopy and in cryostat sections. In addition, the dynamic rearrangements of axons in each projection will be studied in living animals using confocal and low light level video microscopy. Such direct observations will establish the normal time-course of axonal patterning, and permit simple perturbation experiments that will test the relative roles of axon-axon and axon-target interactions in forming specific connections. These experiments will be the first to follow the development of the neuronal connections that subserve vestibular senses. At present, little is known about the relative roles of inherent positional labels, environmental influences and patterns of neuronal activity in the formation of the specific neural connections. It is not clear to what extent the lessons learned from studies of the visual system can be applied to the vestibular system. Thus, without such knowledge of vestibular development, it is difficult if not impossible to completely understand its improper development observed in birth defects or its abnormal function after trauma.

We will study a relatively genetically homogeneous population to test the hypotheses that genetic factors are linked to schizophrenia. The study will focus on two populations. The first is from the Azores, a nine island archipelago in the Atlantic ocean that is a Portuguese state. The Azores have a centralized health system. All ten psychiatrists on the islands are collaborating with us on this project. The second population is from continental Portugal. The majority of the Azorean population is derived from this population base. Families with multiple affected members with schizophrenia, will be studied employing both parametric and non-parametric analytic strategies. We projected approximately 100 families segregating for schizophrenia, including over 300 affected family members. A complementary strategy will be used to study candidate loci. We will study a sample of 225 subjects suffering from schizophrenia and their parents (Total n=675) employing the haplotype relative risk and the transmission/disequilibrium test strategies. This strategy insures that we control for all ancestry for each subject, using the uninherited haplotype derived from the two parents. The third sample will include all other Azorean patients with schizophrenia, who agreed to participate in hopefully, achieving close to complete ascertainment of patients with schizophrenia in the Azores. This will be a valuable sample for linkage disequilibrium approaches given the nature of the Azorean population and provide us with a unique epidemiologic frame. These complementary strategies will allow us to cross validate any positive results. The careful diagnostic definition of phenotype will be based on detailed structured clinical data employing the Diagnostic Interview for Genetic Studies (DIGS), which we have translated into Portuguese. Our project is designed to capture a very complete history of the patients illness, as well as to be able to follow most subjects prospectively for a long period of time. This will be extremely valuable for achieving diagnostic certainty and minimizing false positives. An important new addition to this proposal is the Whitehead/MIT Center for Genome Research, that will perform a genome-wide scan and collaborate on all data analysis for the project.

It is porposed to study cell mediated immunity in transplantation. The interaction between sensitive lymphoid cells and antigen will be investigated under defined in vitro conditions in man and in animals. Factors which may influence the expression of cellular immunity, such as uremia, will be investigated. In these studies, the migration inhibition assay will be used as an in vitro correlate for cell mediated immunity. It is also planned to study the effect of soluble mediators of cellular immunity on the behavior of skin homografts in rodents.

Epilepsy is a neurological disorder in which normal brain function is disrupted as a consequence of intensive burst activity from groups of neurons. Synchronized population spikes are key concomitants to seizure, but the phenomenon has remained a paradox because it cannot be explained by any known neuronal synaptic mechanism. Several lines of evidence suggest a key role of glutamate in the pathogenesis of depolarization events, which in turn trigger synchronized firing. The observation that astrocytes release glutamate via a regulated Ca2+ dependent mechanism prompted us to hypothesize that glutamate released by astrocytes plays a causal role in epileptogenesis. Our recent study snowed that chemoconvulsive agents including 4-AP and bicuculline triggered TTX-insensitive paroxysmal depolarization shifts in hippocampal slices which were closely correlated with astrocytic Ca2+ oscillations. Photolysis of caged Ca 2+ in astrocytes, but not in neurons, was sufficient to trigger local depolarization events. Furthermore, agents that blocked astrocytic glutamate release reduced epilepiform activity, with no effect on baseline EEC in adult rats. The next critical step is to expand the analysis to reactive astrocytes in epileptic animals. We propose here to analyze astrocytic Ca2+ signaling in epileptic mice with cranial window using 2-photon imaging concomitant with EEC recordings. We will correlate astrocytic signaling in reactive astrocytes with neuronal firing in epileptic mice. Reactive astrocytes are easily identified in live exposed cortex based upon the intensity of GFP emission in transgenic mice expressing GFP under the GFAP promoter. The hypothesis that the efficacy of antiepileptic drugs is better correlated with the potency by which they reduce astrocytic Ca 2+ signaling and glutamate release, than with their direct effects on synaptic transmission, will be tested. These experiments offer a new conceptual and operational approach to understanding the cellular basis of seizure disorders. If a dysregulation in Ca2+ signaling in reactive astrocytes indeed proves causal in epileptogenesis - as our preliminary data strongly suggest - then the implications of this new perspective to pharmacotherapy could be profound. By more specifically targeting the glial cause of neuronal excitability, we might be able to achieve more specific, less variable and less toxic treatment options for patients with epilepsy.

This project will capitalize on, extend, and enhance the Chicago Community Adult Health Study(CCAHS), (P50 HD 38986) which is designed to become a major prospective multi-level study of the impact of individual and social environmental factors on health, their role in understanding socioeconomic and racial- ethnic disparities in health, and the biological and behavioral pathways that are involved. This project has completed a major survey of a probability sample of 3105 adults age 18+ in the city of Chicago, with a response rate of 72% and including physical measurements of height, weight, waist, hip length, and blood pressure. In addition, saliva and/or a blood sample have been collected for 661 people (60% of respondents in a subsample of 80 of the 343 neighborhood clusters (NCs) covering the entire city of Chicago, developed and characterized by the Project on Human Development in Chicago Neighborhoods (PHDCN) (http://phdcn.harvard.edu/), from which the CCAHS sample is drawn. In addition to utilizing existing and collecting new archival data on these areas, the CCAHS carried out Systematic Social Observations of 1664 blocks containing sampled households for the study. Thus, the current proposal seeks to utilize all these data to extend and enhance the CCAHS as follows: 1) to estimate via state of the art multi-level analysis the role in health and health disparities of the immediate social contexts or "neighborhoods" in which people live; 2) to estimate how a wide range of aspects of immediate residential contexts and broader social contexts (assessed by multiple methods and measures and considered both singly and in interaction with each other and individual and household factors) affect health and health disparities and help to understand or flesh out the role of social context in health; 3) to estimate via innovative new methods the role in health and health disparities of the broader social contexts which surround more immediate residential contexts; and 4) to begin to convert the existing cross-sectional study into a longitudinal and prospective study via respondent tracking and mortality follow-up, which will allow initial prospective analyses of the predictive association with mortality of individual, household, and contextual psychosocial risk factors for health assessed in the CCAHS, as well as laying the foundation for a second wave of data collection in 2008-9, which will be funded by a subsequent competing renewal proposal, and allow for prospective analyses with respect to the full range of health outcomes in CCAHS. This study will importantly advance the understanding of socioeconomic and racial/ethnic disparities in health - a major priority of the Public Health Service and National Institutes of Health for the new millenium.

Each year in the United States, 5-20% of the population display symptoms of infection with the influenza virus. More than 110,000 of these persons are hospitalized and about 36,000 die from complications. While the spread of infection is generally avoidable through immunization, vaccine may be in short supply, logistics may limit distribution, compliance and lack of concern limit the immunized population and the vaccines are not as efficacious in the very old and very young. This year, a highly virulent avian influenza strain, designated H5N1, has spread rapidly throughout Asia and into Europe. Reports of human infections with H5N1 have increased the concern that this strain, or a recombinant with a virus that commonly infects humans, may expand its host range to infect humans and result in a global pandemic. A moderate pandemic could kill more than 30 million people around the world. A vaccine could be available within six months of recognition of a pandemic. However, influenza vaccine production is limited - only 300 million doses can be manufactured worldwide due to the difficulty in cultivating virus and capacity will take years to expand. Further, in the event of any widespread disease, rapid distribution to the population, insufficient healthcare personnel to administer the vaccine, lack of compliance due to needlestick phobia, and other issues may result in a poorly immunized population. The research proposed by Rocky Mountain Biosystems, Inc. in this application will demonstrate an innovative solution to achieve an immunized, protected population, addressing issues of limited supply, lack of access and compliance. The Self Administered Vaccination Electromechanical (SAVE) device gently and quickly reduces the stratum corneum, administers efficacious and consistent amounts of vaccine in minutes, and avoids creation of "sharps" that constitute biohazardous waste. The device is designed to increase the efficacy of drugs and vaccines, while reducing dosage and extending vaccine supplies. The device is small, inexpensive, self-administrable and can be stockpiled for rapid deployment in the case of a pandemic. The device is expected to 1) extend the vaccine supply as much as 20-fold by adaptation to the more efficient transcutaneous route of vaccine administration, 2) greatly improve compliance through self-administration without needles, and 3) be capable of rapid deployment to the populace. Furthermore, SAVE's transcutaneous delivery is expected to enhance cellular immunity versus intramuscular (IM) inoculation, potentially improving immunity in the very young and old. The SAVE technology may also extend the benefits of transdermal drug delivery to new drugs, such as the growing number of macromolecule drugs and fragile protein or peptide bond biopharmaceuticals. In this proposal, the investigators propose to build pre-clinical prototypes of the patch and test it in vitro and in vivo in a swine model, as well as testing impedance in humans. [unreadable] [unreadable] [unreadable]

Positron emission tomography is a sensitive and specific noninvasive approach for delineating abnormalities of myocardial perfusion and for characterizing the efficacy of interventions designed to enhance nutritive flow. Nonetheless, the use of PET has been limited by the availability of cyclotron-produced flow tracers. Accordingly, development of generator- produced, positron-emitting flow tracers is highly desirable. In the initial two years of this award, we developed and implemented approaches to quantify regional myocardial perfusion with 82/RbCl and 62/Cu- pyruvaldehyde bis(N/4-methylthiosemicarbazone) (PTSM). We demonstrated however that 62/Cu-PTSM binds to human albumin limiting the ability to quantify flow after pharmacologically-induced hyperemia. In the proposed continuation we plan to evaluate the myocardial kinetics of two alternative 62/Cu flow tracers: n-propylglyoxal bis(thiosemicarbazone) (Cu-n-PrTS) and its ethylglyoxal congener (Cu-ETS) (tracers which we have demonstrated in preliminary studies permit quantification of regional myocardial perfusion over a wide range of flows and which do not exhibit avid binding to human albumin) is isolated perfused rabbit hearts in which flow can be altered precisely. We will then evaluate these tracers in intact dogs with PET during selected physiological and pathophysiological conditions including permanent and transient ischemia and coronary hyperemia and compare estimates of regional flow to those obtained with microspheres. Estimates of absolute regional flow with 82/Rbcl and PET, validated during the current grant interval in experimental studies, will be extended to evaluations in humans compared with estimates made with H/2/15/O. Since recent studies have suggested that the backward efflux of 82//RbCl initially trapped by the myocardium (k/2) may delineate viable myocardium (heart muscle that will improve function after revascularization procedures) from nonviable myocardium, an additional goal of the proposed continuation is to evaluate the ability of k/2 (estimated with a compartmental model) to predict viable myocardium in dogs with transient myocardial ischemia referenced to sequential echocardiographic measurements of regional function. The most promising approaches will be evaluated in normal human volunteers and in patients with coronary artery disease. Mathematical models will be evaluated with computer simulations and error analysis and modified as required. The results of this research will enable PET centers to use generator- produced, positron-emitting flow tracers to quantitatively estimate regional myocardial perfusion and, potentially, to delineate myocardial viability.

The initiation mechanism of DNA replication in Escherichia coli is unstable and inactivated after having used only once. Thus, the initiation of a new round of replication does not occur in the absence of protein or RNA synthesis. This inactivaton of DNA initiation activity appears to be a built-in property of the hypothetical DNA replication complex since an altered replication apparatus can be formed after a period of thymine starvation, leading to continued DNA replication in the absence of protein or RNA synthesis (Stable DNA Replication). The long-range goal of the proposed research is to determine the molecular mechanism of this inactivation reaction. The objective is to isolate mutants which are capable of stable DNA replication without the inductive treatments such as thymine starvation and to use these mutants as tools for genetic and biochemical analysis of the regulatory mechanism. In fact, we have already isolated such mutants (Sdr-c mutants) and partially characterized some of them. In this proposed research, we will determine whether the regulatory mechanism is indispensable for normal growth of the cell. To this end, we will attempt to isolate non-leaky Sdr-c mutants after M5 phage mutagenesis. Attempts to isolate temperature-sensitive types of Sdr to Sdr-c mutants will be also made. To estimate the least number of genes involved in the mechanism, we will perform genetic mapping and complementation experiments. The biological and biochemical properties of the DNA synthesized by the Sdr-c mutants in the absence of protein and/or RNA synthesis will be examined using transformation and DNA-DNA hybridization techniques. Finally, to determine the replicon specificity of the sdr mutations, we will examine the replication of one of the stringent type of plasmids, F plasmid, in Sdr-c mutants in the absence of protein and/or RNA synthesis.