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Project summary: The global aim of our studies is to optimize clinical trial methodology in the field of Traumatic Brain Injury (TBI) to maximize the chance of demonstrating benefit of new therapies. The proposal concerns a combined methodological and clinically orientated research project involving multidisciplinary expertise from the field of biostatistics, epidemiology and clinical neurotraumatology. It is organized as an international collaboration and built on the sharing of extensive TBI databases. We will create the IMPACT* database as a unique research resource, including data from a total of over 40,000 patients from five multi-center surveys and eleven clinical trials. This database will be used as culture medium for improving trial design. Specific aim 1 will address the benefits of covariate adjustment and innovative statistical approaches which better exploit the ordinal nature of TBI outcome measures. Specific aim 2 will use advanced statistical techniques for dealing with center effects and variation in patient management. We anticipate that results will also provide novel evidence in support of treatment recommendations in TBI, an area where to date evidence is woefully inadequate. Specific aim 3 will provide recommendations for better standardization and increased sensitivity of outcome measures. Specific aim 4 will address the relative merits of the large, simple "mega trial" compared to a conventional focused phase III trial and will greatly assist in choosing the best design for future trials. Our results will contribute to the broader NIH plans for reshaping clinical research infrastructure and will facilitate the efficient execution of clinical research in the field of TBI. Relevance: Traumatic Brain Injury constitutes a major health and socioeconomic problem throughout the world. Pre- clinical work has generated many promising therapies, but clinical studies have failed to show convincing evidence of efficacy in the overall TBI population. Various agents may wrongly have been discarded due to insensitive clinical trial methodology. We aim to improve trial methodology to give new therapies a better chance in the future, and thus to contribute to improved standards of care for future patients. IMPACT: International Mission on Prognosis and Analysis of Clinical Trials in TBI.

Important unanswered questions concerning B12-dependent enzymic processes are: What are the conformational changes which promote the rate of homolytic cleavage of the Co-C bond of coenzyme B12 by a factor of 10-10 and which favor homolytic over heterolytic cleavage in B12 holoenzymes? Why is the Co- N(DMBz) bond so long and what is the role of DMBz in these processes? If the contribution of the enzyme to rearrangement processes (e.g. methylmalonyl-CoA to succinylCoA) is restricted to insuring the existence of long-lived substrate radicals, how do the enzymes prevent radical combination with B12? These and other questions have not been fully answered for several reasons. The B12-dependent enzymes are large, making X-ray and some spectroscopic studies difficult. The cofactor generally lies tightly bound in a protected enzyme pocket inaccessible to probe reagents. Only four alkylcobaltcorrin X-ray structures including coenzyme B12 and methyl B12 have been reported. The molecules are complex and unique and spectroscopic methods are limited by this complexity and by a lack of background data. Therefore, the conformations of well studied coenzyme analogues are unknown. One consequence of this limited information is that there are few clear relationships between structure and spectra. Also, there remains an inadequate background with which to assess the steric factors leading to Co-C bond cleavage. To continue to address these questions, we propose a three-pronged approach. Detailed studies of (i) alkylcobaltcorrins, (ii) synthetic organocobalt species, and (iii) ribonucleotide reductase (RR) are contemplated. The range of conformations possible for alkylcobalamins will be defined via the synthesis of modified or sterically strained alkylcobaltcorrins (cobalamins, cobinamides and other side chain- modified derivatives) and additional organocobalt compounds with sterically or electronically modified equatorial ligands based on the Cosaloph and "Costa" systems. The effect of structural changes induced in these systems will be assessed by X-ray crystallography, modern spectroscopic methods (CD, 2D and HMQC NMR, Raman) and by reaction rates and mechanistic studies (DMBz dissociation, ligand exchange). Application of some of these methods has produced a wealth of information and provided new insight during this grant period. These methods will be extended to the study of RR. RR is relatively small, with a loosely bound accessible, B12 site and a mechanism of action related to the Fe enzymes. Thus, RR has special advantages and significance. Steric strain in the alkyl linkage between Co and adenine in coenzyme analogues will also be varied and the interaction of the analogues and coenzyme B12 with RR will be investigated. For NMR studies, we will employ cobalamins containing isotopically enriched sites. The spectroscopic (CD, vis, NMR (2D, HMQC)), background to be developed will be generally useful in studies on other B12 holoenzymes and on B12 transport systems.

The hypothesis is that R108512 dose dependently accelerates colonic transit in patients with functional constipation or constipation-predominant irritable bowel syndrome. The specific aim of this study is to measure gastric, small bowel, and colonic transit in 40 patients with functional constipation or constipation-predominant irritable bowel syndrome randomized to placebo, 0.5, 2.0, or 4.0 mg per day as a single dose of R108512.

Antibiotic resistant bacterial infections caused by both Gram-positive and Gram-negative pathogens pose a serious threat to human health. Resistance is increasing while research into new antibiotics and possible new targets is lagging. Both Gram-positive and Gram-negative bacteria are surrounded by a cross-linked carbohydrate polymer, peptidoglycan, which is conserved in all bacteria. This polymer is essential for bacterial survival because it stabilizes the cell membrane against high internal osmotic pressures. Peptidoglycan biosynthesis is a major target for antibiotics because interfering with this process leads to cell lysis. This research is directed towards understanding the mechanisms of action of vancomycin, penicillin, and moenomycin, important antibiotics that represent three classes of antibiotics that inhibit peptidoglycan synthesis. To understand the biological mechanisms of these drugs, an integrated program involving synthetic organic chemistry, biochemical and microbiological assays, structural studies, and bacterial genetics will be employed. A better understanding of how these drugs kill might lead to therapeutic strategies to improve their spectrum of activity and make them more effective at killing resistant microorganisms. Since these compounds target a fundamental metabolic process in bacteria, a better understanding of this process could lead to new antibiotic targets or strategies as well.

The studies of the cytochrome P450 (P450) arachidonic acid (AA) monooxygenase, now established as a major pathway for the bioactivation of endogenous AA, uncovered new and important functional roles for this enzyme system in cell and organ physiology. The most extensively characterized of the P450-derived metabolites, the epoxy- and the omega/omega-1 hydroxy-arachidonic acid, have been implicated in the regulation of tubular sodium and water transport, renal hemodynamics, and renovascular reactivity. Furthermore, associations between genetically controlled alterations in P450 function and/or expression, and the control of systemic blood pressures suggest important roles for these enzymes in kidney and body homeostasis, and in the pathophysiology of hypertension. However, the physiological significance of renal P450 and the site and mode of action of its metabolites remains to be unequivocally defined, and there is a need for advanced experimental models of P450 AA Monooxygenase, isoform-dependent, functional phenotypes. Project 2, in conjunction with the cell and organ physiology components of this Program Project, proposes to utilize molecular approaches for the development and bio-molecular characterization of models of P450 isoform-dependent function and/or dysfunction. P450 gene disruption and/or overexpression will be employed for the integrated functional and biochemical analysis of the significance and the functional role(s) of specific kidney AA epoxygenase and omega/omega-1 hydroxylases. We will apply a combination of analytical, biochemical, and recombinant DNA techniques to the analysis of P450 gene-dependent changes in eicosanoid biosynthesis, AA metabolism, and P450 isoform organ expression and regulation. The long term goals of this project are to provide a molecular understanding of renal P450 eicosanoid biological significance and mode of action. The answers to these important questions are needed for the development of meaningful approaches to the unequivocal definition of: a) their physiological significance, b) relevance to the human diseases such as hypertension, and for the development of rational strategies for future pharmacological and/or clinical intervention.

Despite the fact that student veteran populations on college campuses stand to dramatically increase following the passage of the Post-9/11 Veterans Education Assistance Act of 2008 (commonly referred to as the "new GI Bill"), little is known about the health issues, specifically alcohol-related, of veterans as they shift from military life to college life. Given that previous research suggests that veterans are at risk for developing alcohol-related problems and disorders, their entry into another hazardous environment, the heavy-alcohol consuming college campus, may exacerbate these risks and disproportionately affect their well-being and adjustment. Therefore, this study will examine the prevalence and correlates of military veterans'/student service members'alcohol use and abuse following (re)entry into college. It specifically explores whether student veterans'alcohol-related cognitions and patterns of use differ from those of their non-military peers, as well as whether they also experience a greater proportion of negative outcomes (mental health, social, and academic) as a result of their alcohol use. The sample includes 354 students (195 veterans/student service members;68 ROTC students;91 civilian students). Data collection will involve internet-based surveys addressing alcohol-related expectancies, beliefs, consumption patterns, as well as mental health (e.g., including depression, psychological distress, posttraumatic stress disorder) and academic (GPA, major, academic self-efficacy, locus of control) related correlates. Individual (e.g., combat exposure), interpersonal (e.g., social support), and institutional (e.g., campus alcohol culture) predictors of alcohol use will also be explored. Hypotheses regarding the prevalence and correlates of military veterans'/student service members'alcohol use and abuse during college will be primarily tested via multi-level structural equations modeling. PUBLIC HEALTH RELEVANCE: This study examines prevalence and correlates of student service members'alcohol use/abuse following (re)entry into college. We have particular interest in establishing whether veterans'alcohol use patterns are differentially linked to negative health, social, and academic outcomes. Identification of these factors will provide universities with necessary insight for the development of support programming to aid in successful academic and personal functioning of this special sub-group.

The long-term goal of this project is to design and evaluate practical image enhancement methods that would enable people with impaired vision, particularly those with central field loss due to age-related macular disease, to enjoy and benefit from the spread of video technology. Towards this goal, we are developing enhancement methods and investigating factors affecting patient performance with, and the appearance of, enhanced images. Our work is guided by our evolving vision model of contrast perception that we (and others) use in developing, testing, and refining image enhancement techniques. We believe that a basic understanding of contrast and image perception is essential for progress in this area, and that our clinical and basic research efforts usefully interact with each other in ways that benefit both. In this proposal we emphasize more-directly-applicable aspects of the project by developing new ways to measure preference and perceived image quality for video sequences, and by evaluating improved and new enhancement algorithms. We continue our investigation of form vision in patients with central field loss and of the possible role of brain plasticity in this process. We also continue to investigate the possible impact of short-term adaptations to blur and sharpness in reducing the perceived improvement of perception with enhancements, and possible ways to counter these effects. Specifically, we will evaluate the properties of our new method for measuring instantaneous image quality in motion videos and its value in studying image enhancement as well as the image degradation caused by digital compression. We will implement and evaluate a method of pairwise comparison to quantify preferences for enhancement, and apply these evaluation techniques in the selection of enhancement parameters and assessment of image quality. We will evaluate: our adaptive enhancement algorithm (as implemented on an inexpensive consumer product), an improved nonlinear version of that algorithm, and an enhancement for digital video designed to work directly in the compression (MPEG) domain. We will also evaluate the benefit of newly-available high brightness and high dynamic range displays. We will continue our psychophysical studies, in an effort to understand the adaptations and changes that affect patients who lose their central retina. PUBLIC HEALTH RELEVANCE: A growing number of Americans suffer from disabling age-related visual impairments. Access to the growing flow of video images presented on the screens of TVs, computers, and hand-held devices is important as a means for obtaining information and sharing in the culture of our society. The goal of this project is to design and evaluate practical image enhancement methods that would allow people with impaired vision - particularly those with age related macular disease - to enjoy and benefit from the spread of video technology.

Hypofractionated high dose radiation therapy is an increasingly important treatment approach. Image guidance is imperative to ensure accurate patient positioning and to allow the use of tight treatment margin for high dose delivery. At present, the prevailing guidance method employs x- ray imaging to provide 3D volumetric and/or 2D projection information. Unfortunately, the approach is found wanting for tumors at low radiographic contrast with the surrounding tissue. These include important abdominal sites such as prostate, pancreas and liver. Consequently, detection of implanted markers has been employed as an alternative. However, these surrogates are also less than ideal due to the lack of anatomic information and the invasive nature of the procedure. In response to PAR10-169, an academic-industrial partnership is formed between Johns Hopkins University (JHU), Elekta-Resonant and Cleveland Clinic (CC), to develop an integrated 3D x-ray/ultrasound imaging system to provide on-board guidance for hypofractionated radiation therapy. The partnership has significant expertise in x-ray and ultrasound imaging, robotic engineering and radiation therapy. We have 3 specific aims. Specific Aim 1 is to design and construct the system. A 3D ultrasound imaging system will be docked onto a medical accelerator equipped with on-board x-ray cone-beam CT (CBCT) imaging. Ultrasound imaging overcomes the hurdle of detecting low contrast soft tissue target with x-ray imaging, while CBCT provides the anatomic information for setting up the patient in the machine frame of reference and for dose re-calculation if necessary. Robotic control with force sensors will be employed to automate the positioning of the ultrasound system which will remain on the patient throughout the treatment session to provide real-time monitoring. The procedure is non-invasive. The monitoring is non-ionizing and desirable for applications with pediatric patients. Specific Aim 2 focuses on optimizing the IGRT workflow for planning, radiation delivery to avoid the interference of imaging and irradiation procedures with each other. For Specific Aim 3, we will validate the performance of the integrated imaging system at JHU and CC. We will test the hypothesis that guidance using the integrated robotic system is superior to CBCT or ultrasound imaging alone by demonstrating that the target definition is more reproducible and less user-dependent. The expertise of partners is well suited to fulfill the expectations of PAR10-169. Two commercial grade systems will be constructed. JHU and CC represent the end-users to conduct in-field validation of these systems. Elekta-Resonant is also an ideal industrial partner to pursue commercialization for dissemination in the community.

The discovery and development of RNA interference and especially the breakthrough of using small interfering RNAs (siRNA) to inhibit gene expression is revolutionizingbiology and opening new avenues for developing novel therapeutics. Despite the current usefulness of the technology, a bottleneck exists for quickly finding effective siRNA target sites in a high throughput-like manner and most researchers currently employ a rational trial-and-error design method. In this proposal, we present a novel cell-based technology to identify effective inhibitors from siRNA libraries (either random or directed). The combination of using a library to find the most effective, robust siRNAs against accessible sites on any given target within a cellular environment, coupled with the inhibitor activity intrinsic to siRNAs, holds great promises in finding effective therapeutics. We have chosen dengue virus, a NIAID Class A threat, as our initial target for developing this technology. Potential target dengue viral sequences will be linked to the HSV thymidine kinase (TK) gene and stably expressed in human cells. Addition of ganciclovir leads to cell killing of any cell expressing the HSV TK protein. Prior to addition of ganciclovir, a proprietary library of siRNAs designed to only target dengue sequences ("directed" library)will be introduced into the cells. Those cells that subsequently survive the killing challenge will be isolated and the rescued siRNAs identified. Among the purified and validated inhibitorsidentified, the most potent anti-dengue siRNAs will be further tested for their anti-dengue viral activity in cell-based and animal models. This approach could potentially be extended to any RNA target of interest, including other viruses and oncogene mRNAs.

The objective of the research project is to elucidate the structure, function and metabolism of human apolipoproteins and, in particular, those proteins present in very low density lipoproteins. The studies include an investigation at the molecular level of the interaction of pure lipoprotein lipase with its activator protein apoC-11 and glycosaminoglycans. The primary structure of the "arginine-rich" protein and its interaction with ligands and the determination of the primary amino acid sequence of apoB, the major pretein of low density lipoproteins, will be determined. The methods include techniques of protein chemistry, physical chemistry and enzymology. It is anticipated that knowledge of the mechanism of apoC-11 with lipoprotein lipase and of the structure of the arginine-rich protein and apoB will contribute to our understanding of the normal and abnormal metabolism of human plasma lipoproteins.

Much evidence implicates the involvement of the serotonin 5-HT1A receptor in anxiety and depression. 5-HT1A receptor agonists display anxiolytic/antidepressant effects, and 5-HT1A receptor function is altered after repeated treatment with anxiolytic and antidepressant drugs. While much data has accumulated about 5- HT1A receptor signal transduction in cells genetically engineered to express the receptor, much less is known about the receptor in its native environment. The potential importance of regional differences in 5-HT1A receptor function is underscored by our previous studies demonstrating regional variations in receptor/effector coupling efficiency and recent evidence that chronic treatment with anxiolytic/antidepressant drugs elicits regionally specific 5-HT1A receptor adaptation. This project therefore proposes to gain a better understanding of 5-HT1A receptor signal transduction, especially with regard to regional differences in receptor/G protein coupling, G protein specificity, and 5-HT1A receptor adaptation after repeated treatment with various classes of anxiolytic/antidepressant drugs. The following Specific Aims are proposed: 1) To determine if previously described regional differences in receptor/effector coupling efficiency are demonstrable at the level of receptor/G protein coupling. Dose-response curves for agonist-stimulated binding of [35S]GTPgammaS (in membranes and brain sections) before and after partial irreversible receptor blockade will be generated to test the hypothesis that 5-HT1A receptor activation of G protein coupling will exhibit greater efficiency at somatodendritic dorsal raphe (DR) autoreceptors than at postsynaptic receptors in hippo-campus. Coupling efficiency at postsynaptic receptors in the lateral septum (LS) and medial prefrontal cortex (mPFC) will also be determined; 2) To determine if 5-HT1A receptors couple preferentially to specific G proteins in different brain regions. Selective knockdown of pertussis toxin (PTX)-sensitive G protein alpha subunits by intracerebroventricular or intra-tissue infusion of antisense oligodeoxynucleotides (AS ODNs) will test the hypothesis that 5- HT1A receptor coupling to G proteins will be region- and agonist- specific. We predict that in the DR, 5-HT1A receptors will couple preferentially to Galphai3 to mediate agonist-induced inhibition of 5-HT synthesis; in the hippocampus, 5-HT1A receptors will couple preferentially to Galphai2 to mediate inhibition of forskolin-stimulated adenylyl cyclase. We will also test the hypothesis that some agonists may preferentially couple to different Galphai/Galphao subunits to mediate these effects; 3) To determine if repeated treatment with different classes of anxiolytic/antidepressant drugs results in regionally- specific 5-HT1A receptor adaptation and changes in receptor/G protein coupling. Agonist inhibition of 5-HT synthesis in forebrain regions will be assessed to test the hypothesis that repeated treatment (21 days) with ipsapirone, paroxetine or clorgyline (but not imipramine) will desensitize somatodendritic 5-HT1A autoreceptors and reduce agonist-stimulated binding of [35S]GTPgammaS in the DR. We will also test the hypothesis that in the hippocampus, agonist-stimulated binding of [35S]GTPgammaS will be increased by repeated treatment with imipramine, decreased by clorgyline, but remain unchanged after ipsapirone or paroxetine.

The objective of this project is to study the mechanisms involved in androgen mediated gene expression in the rat prostate and the seminal vesicle. Protein synthesis patterns have been analyzed by 2D gel electrophoresis in castrate and testosterone stimulated prostate and seminal vesicle. A major group of secretory proteins in both organs is under androgen control. Both organs have a high concentration of poly(A plus)-mRNA which code in a wheat germ translation system for polypeptides which are similar to the in vivo secreted polypepties. The two major poly(A plus)-RNA's from prostate (labeled beta and gamma) code for the subunits of the major secreted protein referred to as prostate binding protein. Likewise, rat seminal vesicle has two major poly(A plus)-mRNA's (labeled IV and V) which code for two major proteins. Double-stranded DNA copies (ds cDNA) of prostate beta and gamma poly(A plus)-mRNA and seminal vesicle poly(A plus)-mRNA for proteins IV and V were prepared using reverse transcriptase. With a variety of restriction enzymes, maps were prepared for the various ds cDNA's. Subsequently, the cDNA's for beta and gamma from prostate were cloned in the plasmid pBR322 utilizing the "tailing method".

The functional role of lymphoid cells, including the macrophages (M phi) and T and B lymphocytes and their soluble mediators in the induction of an IgA immune response will be studied in vitro. Optimal in vitro culture conditions for enumeration of IgA antibody (plaque) forming cells (PFC) following stimulation with T dependent antigens including DNP-Streptococcus mutans and TNP-erythrocytes, and lipopolysaccharide (LPS) as adjuvant from tissues including Peyer's patches (PP), mesenteric lymph nodes (MN), cervical lymph nodes (CN) and spleen (S) from CH3/HeN and C3H/HeJ mice will be evaluated. Single cell preparations or purified T and B cells from PP, MN, CN or S and M phi from spleen or peritoneum of E. coli K235 monoassociated mice will be immunized and incubated in Mishell-Dutton type cultures with LPS as adjuvant. The importance of monocyte (LAF monokine) and T cell (TRF) mediators and LPS on the induction of kinins, and their involvement in an IgA response will be established. The origin of precursor IgA B cells and T cells in mucosal tissues including the saliva glands will be determined by examination of homing patterns of lymphocytes from gut-associate lymphoid-tissue (GALT) and MN, CN and other sources. Cell tranfer experiments utilizing radioisotope methodology will be performed in groups of rats that are gnotobiotic, B cell suppressed, irradiated, or conventionalized in order to determine the origin of precursor lymphocytes. Furthermore, the efficacy of various modes of immunization in inducing antibodies in secretions will be assessed by tracing the homing patterns of antigen-sensitized cells to their sites of expression and by quantitating the immunological response utilizing fluorescent microscopy and serological techniques.

Cell and Tissue Core Introduction to revised application: We appreciate the study section recommending approval this Core with an excellent/outstanding descriptor. The following concerns or suggestions were described by the reviewers: Transport of tissue from the Vanderbilt Center to Yale: As suggested by reviewer 1, "this has likely been solved." As described in the revised description of the mouse modeling core, Dr. Osteen and fellow investigators have routinely shipped tissue and other material to collaborators throughout the world. Overnight shipping of frozen material on dry ice has been used to deliver tissue from Vanderbilt to Yale as part of ongoing collaborations between the groups. Development of non-radioactive in situ hybridization : Reviewer one suggested that "future development should work towards expertise in non-radioactive in situ hybridization." We agree that this would complement our existing techniques. We have included a plan to provide this technology and incorporate it into the center by the end of year one. The expertise is already present in the Center as demonstrated in preliminary data presented in Project I. Budget. Reviewer two correctly pointed out that we did not justify the projected 50% saving from use of the core. These savings come in the form of bulk purchases and eliminating redundant experiments and tissue collection. As all investigators in the Center will use endometrial tissue and most will perform similar experiments using cultured cells, we estimate that the cost of a single technician (rather than the duplicated efforts of two or three) will account for at least a 50% cost reduction. Long-term freezing and storage. Reviewer two suggested the use of a cryogenic system reaching temperatures below -130 C for long term storage of cells. We do indeed have access to a Yale University freezing facility operated by the Cancer Center. This facility will allow storage of cells on a fee for service basis. This facility is subsidized by the University and is more cost effective than establishing our own facility. We apologize for not including this information in the original application. We thank the study section for the constructive criticism. All of the requested information is now provided and all suggestions incorporated into the revised application.

The architectural complexity of the retina is brought about by a series of developmental events controlling" proliferation, fate determination, migration, process outgrowth, target recognition, synaptogenesis and cell death. These processes establish a precisely layered structure in which retinal neurons become positioned at different depths, connected via two intervening synaptic layers. Superimposed upon this layered organization, certain cell types are distributed as orderly arrays across a given layer so that they, and their processes, ensure a uniform sampling of the retinal surface as they establish connectivity with their afferent and target neurons. This research program is seeking to understand the cellular, molecular and genetic determinants of this patterning and connectivity in such arrays, focusing upon the population of horizontal cells. A quantitative trait analysis of horizontal cell number in recombinant inbred strains will first identify genes that control the size of this neuronal population. Chimeric mice will be produced from parental strains that differ in horizontal cell number, to determine whether dendritic field size is controlled by cell-intrinsic vs environmental instructions. A comparable quantitative trait analysis of the cone photoreceptors will also be conducted, and the role played by the afferents in specifying dendritic patterning of horizontal cells will be examined in various knockout and recombinant inbred strains in which the convergence ratio between cones and horizontal cells is modulated, or in which neurotransmissionbetween these afferents and the dendrites is altered or abolished. The roles played by both homotypic neighbors and by afferents in the establishment of this connectivity will be determined, examining the hypothesis that the onset of visual activity drives a competitive interaction between neighboring horizontal cells as they seek to colonize individual pedicles in the developing outer plexiform layer. Microarray analysis of embryonicretina from .two parental strains . showing a two-fold difference in both horizontal and cone cell number will be used to identify downstream genes critical for the establishment of these differences. These experiments will reveal the genes that regulate horizontal and cone cell number, as well as the biological mechanisms by which horizontal cells establish their morphological patterning, dendritic coverage and connectivity with cone afferents during development.

The misuse of an addiction to opioid medications has become the most rapidly increasing drug problem in the US, which has consequences for pregnant mothers and their babies. The prevalence of maternal opioid drug use at the time of delivery in California is approximately one percent. The neonatal abstinence syndrome (NAS) is a constellation of narcotic drug withdrawal symptoms that develops in 42 to 94 percent of infants born to narcotic dependent mothers. There are no preventative treatments for this severe syndrome, which can result in a prolonged hospitalization and treatment in a neonatal intensive care unit. Based upon a genetic discovery, we demonstrated that administration of a 5-HT3 antagonist (ondansetron) prevented the symptoms of narcotic drug withdrawal in experimental studies in mice and in humans. From this, we hypothesize that ondansetron administration to pregnant narcotic-using mothers just prior to delivery, followed by a 3-day period of ondansetron administration to the neonate, could reduce the incidence or severity of NAS symptoms. Ondansetron is a broadly utilized drug with a substantial safety record in pregnant women, but we do not know what effect pregnancy may have on ondansetron pharmacokinetics and it is hard to predict ondansetron pharmacokinetics at birth. Therefore, we will first perform a detailed characterization of ondansetron pharmacokinetics in pregnant mothers and in neonates. Besides providing important information about differences in drug metabolism during pregnancy and in neonates, these results will ensure that appropriate ondansetron doses are selected for treatment of the mothers and neonates in the subsequent efficacy study. In the second part of this study, we will conduct a multi-center, randomized, double blind, and placebo-controlled trial to determine whether ondansetron treatment will reduce the incidence (percentage of infants requiring narcotic drug treatment) or severity (total amount of narcotic drug required, length of hospitalization, symptom severity scores) of NAS in babies born to narcotic-using mothers. This project tests a very novel approach for preventing an important pregnancy-related complication of narcotic drug abuse. If a brief period of ondansetron administration to the mothers that use narcotic drugs, and to their babies, can prevent or ameliorate the development of narcotic drug withdrawal symptoms in neonates, this would reduce human suffering and the growing societal cost of opiate abuse. PUBLIC HEALTH RELEVANCE: This project tests a very novel approach for preventing an important pregnancy-related complication of narcotic drug abuse. After characterizing the ondansetron metabolism in pregnant women and neonates, we will determine if prenatal administration of ondansetron to pregnant mothers that use narcotic drugs followed by a 3-day period of ondansetron administration to the neonate, can prevent or reduce the development of narcotic drug withdrawal symptoms in neonates. If so, this would reduce human suffering and the growing societal cost of opiate abuse.

The goal of this research is to develop new methods for the design and synthesis of Type-III Peptidomimetic inhibitors of aspartic proteases. A Type-III mimetic, is an organic structure, distinctly different in shape and atom type from a peptide substrate, that binds so as to complement the local topography in the enzyme active site. These are considered likely to lead efficiently to orally bioavailable enzyme inhibitors. Model studies with pepsin and R. Chinensis pepsin will be continued by creating potential candidates for synthesis by applying the structure-generating program, GrowMol, to two different classes of enzyme-inhibitor complexes: (1) those formed from tight-binding transition-state analogs bound to the target enzyme; and (2) those formed from weaker binding inhibitors bound to a destabilized enzyme. Of the many potential synthetic targets generated, Beilstein's CrossFire is used to identify synthetically accessible templates or core structures from which to build the peptidomimetic. These are further refined by the chemist, synthesized, assayed against the target enzyme and when active, their binding mode to the enzyme will be determined by X-ray crystrallography. New strategies for the Design of Protease Inhibitors will result and the goal is to find ways to design Type-III peptidomimetic inhibitors directly from the structures of peptide inhibitors bound to the target enzyme. These strategies will be applied to two clinically important aspartic proteases, HIV protease and plasmepsin II.

Epstein-Barr virus (EBV), a herpesvirus, is a probable human cancer virus as it is oncogenic in sub-human primates, transforms human B lymphocytes in vitro, causes a human lymphoproliferative disorder and the tumor cells in endemic Burkitt's lymphoma and nasopharyngeal carcinoma contain the EBV genome. EBV is also strongly associated with the lymphoproliferative disorders and lymphomas which are major complications of iatrogenic and disease associated immunosuppression; thus virtually all post-transplant B cell proliferative disorders are composed of cells bearing the EBV antigens and many AIDS associated lymphomas contain the EBV genome. The above diseases are invasive, associated with cell and tissue destruction and frequently fatal; all are associated with immunosuppression. Our major long-term objective is the elucidation of the individual and interactive roles played by EBV and cellular genes and gene products, and the contributions of the immune system to the development of EBV-induced human B cell lymphoproliferative disease and lymphomas. We will address these questions in two model systems in immunodeficient mice reconstituted with a functional human immune system; one of these was described recently by Mosier et al. (1988), the other is an as yet unpublished system which we have recently developed in which the contributions of EBV, B cells, and immune surveillance can be independently assessed. In the model described by Mosier et al. (1988), which we have replicated, EBV-containing human B cell lymphomas develop spontaneously 2-4 months after reconstitution of SCID mice with EBV positive but not negative human lymphocytes. In our new model, SCID/hu chimeras develop rapidly fatal human lymphoproliferative disease and tumors 2-4 weeks after injection of EBV; furthermore, the tumor cells grow in vitro and elicit new tumors upon transfer into SCID mice. Our specific aims are: 1.) to examine the roles of EBV and the target B cell in disease induction with emphasis on the molecular processes involved, and 2.) to examine the contributions of T cell surveillance to the prevention or reversal of disease. We have extensive experience in viral immunobiology and immunology, particularly with regard to EBV, and in the contemporary techniques of molecular genetics. The two model systems described here exhibit many similarities to the lymphoproliferative disorders and lymphomas, which complicate in AIDS and organ transplantation in man. The proposed studies should yield insights into the roles of EBV and the immune system in the pathogenesis of these conditions.

Learning to communicate using natural language is a uniquely human capacity that underlies the exchange of information among people. There is as yet no consensus concerning how susceptible this process is to environmental and biological variation. The proposed Program Project addresses this issue, with a focus on how language is used for higher order thinking, a key cognitive underpinning of academic and 21st century career success. To examine the impact of environmental variation on language used for higher order thinking (Project I), 60 children, who were selected to represent the demographic range of the Chicago area and had been observed between 14 months and 10 years during the previous grant periods, will be followed until 15 years as they enter early adolescence. To explore continuity over development, higher order thinking will be coded in videotapes of spontaneous parent-child conversations early in development, and in tasks designed to elicit higher order thinking that parents conduct with their children at ages 10, 12, and 14 years. Standardized assessments of higher order thinking will also be collected. Using these data, models of cumulative parent input to higher order thinking from 14 months to 10 years will be constructed and used to predict children's subsequent skills in higher order thinking. To explore relations between language and higher order thinking in the face of biological variation (Project II), 40 children with unilateral brain injury, who had been observed from 14 months and will now be followed from 11 to 15 years, will be studied to determine how environmental input and biological aspects of their lesions combine to predict their use of language for higher order thinking, and whether input plays a similar role in this group as it does for children who have not suffered brain injury. In addition, the brain bases underlying language used for higher order thinking will be assessed in children from Projects I and II using fMRI techniques (Project III). Three cores will provide broad support to the projects: Administrative Core A, Data Collection and Transcription Core B, and Statistical Core C. The proposed work builds on and extends 10 years of longitudinal data in a diverse sample of typically developing children and children with brain injury, and thus offers a unique opportunity not only to explore the impact of early parental input on the development on higher order thinking, but also to examine how that input interacts with early brain injury.

This is a competing continuation to further localize and identify a gene for early onset primary torsion dystonia (PTD). The gene under study is DYT6, one of several genes that can cause this disease. 1)They plan to examine 30 families in which 3 or more members are affected with PTD. They will also examine 50 simplex or multiplex Mennonite PTD families. 2) They will carry out a series of genetic tests to further elucidate the genetic defect in these families. 3) Families displaying linkage or linkage disequilibrium will be used to further localize and characterize these genes by positional cloning. Families excluded from linkage with identified PTD loci will be analyzed by linkage analysis in a genome search. Once other PTD loci are identified, further localization by fine scale mapping will be performed and linkage disequilibrium will be sought. Clinical features due to different PTD loci will be determined and explanations for the variable expression and reduced penetrance of PTD genes will be investigated.

Children and young adults who were exposed to alcohol during fetal life show emotional disturbances such as depression, anxiety, attention deficit, and hyperactivity. Animal studies have linked the behavioral abnormalities to problems in the stress axis function, particularly the hyperresponsiveness of corticotrophin-releasing hormone (CRH) neuronal activity, to a variety of stressful events. Adaptation to a stressful event depends in part on an individual's ability to produce increased levels of the hormones of the stress axis and to reduce the levels of these hormones once the stressor has subsided. Recent reports indicated that hormone and toxicant exposure at crucial developmental stages results in alteration of key genes, resulting in physiological and/or behavioral changes not only in exposed individuals but also in their offspring. Hence, the question is raised whether epigenetic alterations of the CRH neuronal function are caused by alcohol exposure during the development. We hypothesize that alcohol exposure during development impairs stress axis function by altering DNA methylation and expression of proopiomelanocortin (POMC) gene in beta-endorphin neurons, which regulate CRH secretion from the hypothalamus. The objective of the proposal is to test the hypothesis that alcohol exposure during brain development produces epigenetic transgenerational effect on the stress axis function by altering DNA methylation and mRNA expression of CRH and/or its regulatory POMC genes in the hypothalamus. This will be achieved by identifying whether the stress axis hyperresponsiveness is accompanied by altered DNA methylation of gene-promoter activities in beta-endorphin and/or CRH neurons, and by evaluating whether gene-silencing activity is accompanied by the alteration in the expression of DNA methyltransferases in these neurons in the hypothalamus of offspring exposed to alcohol during the developmental period. Furthermore, whether fetal alcohol exposure induces transgenerational effects on the stress axis function will be studied. The proposed studies should provide a better understanding of the molecular mechanisms responsible for the detrimental effects of alcohol on the development of the neuroendocrine axis of stress and should reveal new putative epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets for stress axis dysfunction in fetal alcohol exposed patients. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Recent reports have indicated that hormone and toxicant exposure at crucial developmental stages cause an alteration in the function of key genes, resulting in physiological and/or behavioral changes not only in exposed individuals but also in their offspring. Hence, the question is raised whether an inheritance of epigenetic alteration of the corticotrophin releasing hormone neuronal function induced by alcohol exposure during development causing the stress axis dysfunction that leads to various affective disorders in fetal alcohol exposed patients. The goal of this proposal is to determine the epigenetic transgenerational effect of alcohol on the stress axis function in order to identify new putative epigenetic disease biomarkers as well as to develop early detection strategies and therapeutic targets for stress axis disorders in fetal alcohol exposed patients. [unreadable] [unreadable] [unreadable]

The proposal outlines the start of a methodological research program that should serve to enhance the study of chronic disability over time and the availability of such data and analyses to researchers. It focuses on disability data rising form the National Long Term Care Survey (NLTCS) and statistical method currently used for their analysis. The long-term goal of the principal investigator and her collaborators is to work with new and existing statistical methodology for the analysis for survey-based longitudinal disability data in developing new statistical tools to preserve the confidentiality of such data sources while making more broadly available for analysis by others. The goal will be achieved through extensive study of the NLTCS database, coordinated research on new statistical methodology for categorical data with the other researchers, and the extension of disclose limitation methods to this data analytic setting. The principal investigator is a New Investigator under the NIH definition. Nonetheless, the principal investigator has an established research record in data access and confidentiality, including statistical disclosure limitation, and substantial experience working with social science data.

Our objective is to study the pathogenic relationship between human hepatitis B virus (HBV) and the development of hepatocellular carcinoma at the molecular level. Our goal is to fill the gap between putative "hepatoma oncogene" and HBV chronic infection/integration. The major effort will be to examine potential amplification, expression, and transposition of oncogenes and cellular DNA near the HBV integration site. Specific aims are as follows: 1. To continue and complete our current project of HT14 and FOCUS hepatomas. Both appear to have a single site of HBV integration. This project includes DNA sequencing of both HT14 and FOCUS flanking cellular DNA, isolation of the preintegration site from FOCUS, chromosomal localization of HT14 and FOCUS HBV, expression of FOCUS HBV, etc. 2. To follow up several putative amplification loci near the site of HBV integration in hepatomas R16, HT2, HT12, HT14. 3. To trace the history of potential HBV transposition in PLC/PRF/5 hepatoma cell lines. 4. To look for specific chromosomal abnormality with the strategy of combinations of classic and molecular cytogenetics, RFLP probes will be used in this approach (Restriction Fragment Length Polymorphism). 5. To analyze HBV or oncogene specific RNA in adult or fetal hepatoma.

The recent discovery of genes responsible for the potassium leak currents, the KCNK family of potassium channels, has created the opportunity to directly target arrhythmogenic triggers of atrial fibrillation, rather than merely trying to contain them through pulmonary vein isolation. Expression of the KCNK0 gene in neurons can render these cells electrically silent by effectively shorting out any depolarizing currents. We have extended this work to demonstrate that expression of KCNK0 in cardiomyocytes renders them inexcitable. We thus propose to evaluate the KCNK0 gene for its ability to silence cardiomyocytes as a treatment for atrial fibrillation in the following specific aims: Specific Aim1: In vitro evaluation of KCNK0 for the electrical silencing of cardiomyocytes. This will involve assessment of biological effects in cardiomyocytes - establishing transfection efficiency targets, assessing cell viability, studying the electrophysiologic effects of heterogeneity from mosaic KCNK0 expression, and assessing possible cytotoxicity. Specific Aim 2: In vivo adenoviral delivery of KCNK0 to the anterior right atrium to create a line of block. This will involve linear delivery of the Ad.KCNK0 followed 5 days later by multielectrode recordings of electrical activity. Comparison of two gene delivery methods and dose response will be evaluated with assessment of gene transfer efficiency, toxicity, and protein levels. Specific Aim 3: In vivo adenoviral delivery of KCNK0 to silence pulmonary vein myocytes in a large animal model. This will involve baseline electroanatomic mapping, followed by KCNK0 gene delivery to the pulmonary veins, and then remapping of the pulmonary veins to document electrical silencing. Animals will also undergo atrial programmed stimulation and Holter monitoring to assess for proarrhythmia. Comparison will be made to Ad.GFP treated animals as well as animals treated with traditional radiofrequency pulmonary vein isolation. PUBLIC HEALTH RELEVANCE: Our primary objective is to demonstrate the feasibility of electrically silencing the pulmonary veins using gene transfer as a potential treatment for atrial fibrillation in an experimental animal model. In this project, sheep be will be used to determine whether expression of a modified potassium leak current, KCNKO, by adenoviral gene therapy approach leads to loss of electrical excitability (electrical silencing) of the cardiomyocytes investing the pulmonary vein ostia.

The objectives are: (1) to further characterize human 6-phosphofructokinase (PFK; EC. 2.7.1.11) isozymes in terms of their biochemical properties, cellular and subcellular localization, and the nature of the enzymatic lesions and their metabolic consequences in inherited PFK deficiency states; and (2) to undertake a detailed genetic dissection at the level of gene(s) localization, structure, organization, and mechanism(s) and regulation of expression with special reference to the mutant genes. These objectives will be pursued in the following specific studies: (1) Comparative structural and kinetic analyses of the three homotetramers; (2) Immunocytochemical localization of the three PFK subunits; (3) Elucidation of the biochemical and genetic bases of inherited PFK deficiency in patients with typical and atypical clinical syndromes; (4) Molecular cloning and characterization of the normal and mutant PFK genes; and (5) Elucidation of the control mechanisms involved in gene(s) expression in a number of pathophysiological states in humans; (6) Precise chromosomal localization of the PFK loci and investigation of the existence of complementation groups in glycogenosis type VII. These goals will be approached by: (1) Direct isolation and characterization of three homotetramers; (2) Immunocytochemical localization of the PFK subunits using subunit-specific antibodies; (3) Chromatographic, immunochemical and metabolic studies of the PFK-deficient humans; (4) Somatic cell and in situ hybridization methods; (5) Isolation of PFK-specific cDNAs and of natural and mutant PFK genes; and (6) Detailed structural and functional analyses of the cloned PFK genes in a number of pathophysiological states characterized by altered gene expression. These studies are expected to yield important clues as to the basic biochemical properties, genetic control mechanisms and physiological significance of PFK isozymes in human biology. These studies are expected to define the clinical syndromes, the nature of the genetic defects underlying them at the molecular level and their metabolic consequences.

Training in epidemiology and outcomes research is essential to prepare the next generation of researchers in a rapidly moving field. Discoveries in genetics, pathophysiology, diagnosis, and treatment of several Gl and liver disorders have been made at a very rapid pace over the past few years. Modern clinical and translational researchers need to be equipped with extensive training in the methods and conduct of research in areas that are becoming highly sophisticated and specialized;these include conventional epidemiology, clinical epidemiology and outcomes, genetic and molecular epidemiology, and health services research. These areas address the need for translational research both from bench to bedside as well as from bedside to clinical practice. The proposed training program has three objectives: (1) To produce physician scientists who are thoroughly trained in the content areas, methods, and ethics critical for epidemiology and outcomes research in gastroenterology and hepatology; (2) To produce physician scientists who have mastered basic research skills, including project design, planning, funding, execution, management, and reporting;and (3) To produce physician scientists who will pursue a research agenda that will new discoveries as well as make the best use of current discoveries to make the health care safer, more effective, more patient-centered, more timely, more efficient, and more equitable. The program will take advantage of outstanding expertise in research and mentoring in epidemiology and outcomes research available at Baylor College of Medicine and UT-School of Public Health including the Gl and Liver Section and the Houston Center for Quality of Care and Utilization Studies (HCQCUS). The proposed fellowship training program has four elements: 1) vigorous mentoring for each individual fellow in a self-selected area of concentration to engender the development of a feasible career research agenda that has potential for substantial impact, and 2) formal research training resulting in a master's degree to provide a foundation of research skills, 3) Applied research seminars to model use of research skills and provide a foundation in health services research knowledge and skills, and 4) participation in the research process as a co- investigator and as a principle investigator to provoke application of research skills.

The transforming proteins encoded by the type C virus, but clearly of host cell origin, provide potential transplantation antigens that will understandably be of direct importance in cancer. This project is focused on the production of monoclonal antibodies to these proteins that will result in the evaluation of these proteins as transplantable antigens; a determination of their presence throughout the phylogenetic scale; and a better understanding of the mechanisms of transformation and thus the means to biochemically or immunologically alter the events that lead to cell transformation.

This project is designed to study morphological and functional changes in the neural substrate for a learned behavior. Zebra finches (Poephila guttata) will be used as an animal model in which to investigate factors regulating structural plasticity in developing and adult brain, trophic interactions in vertebrate fore brain, neural and hormonal mechanisms underlying the development of a complex learned behavior, and sex differences in neural morphology and cellular processes. 1. Zebra finches learn the sounds used for communication during a restricted period of development, and song learning and behavior are controlled by well-defined neural circuits. The contribution of proprioceptive feedback from vocal muscles to song learning has never been evaluated, although sensory axons from the vocal organ are known to terminate throughout the trigeminal tract. We have recently shown that lesions of the telencephalic end- station of the trigeminal system disrupt vocal behavior in juvenile birds during song learning, but have no effect on maintenance of song patterns by adult birds. We will map out the precise axonal connections and topographic relationships of this telencephalic nucleus in order to determine if afferent fibers from the vocal organ project to this area, and if so whether they terminate selectively in a region that is necessary for normal vocal development. 2. We have previously demonstrated that the major thalamic relay of the song-control system initially makes an exuberant projection to its cortical target nucleus; subsequently both the target nucleus and its thalamic input regress. We will label individual thalamic axon terminals at different stages of development in order to determine whether single terminal arbors are expanding and regressing, as has recently been demonstrated in the visual system, and whether the timing of these events correlates with naturally-occurring cell death or the extent to which this circuit contributes to vocal behavior. We will also lesion this thalamic nucleus in order to examine induced cell death - i.e., we will ask whether afferent input is necessary for neuronal survival in the target nucleus in both developing and adult animals. 3. We have discovered that cell division is much higher in the germinal epithelium of the telencephalon in males than in females during late stages of post-hatch development. This is the first demonstration of a robust sex difference in neurogenesis in vertebrate brain, and suggests that sex dimorphisms in neural circuits underlying vocal behavior may emerge via regulation of neuronal proliferation. We will begin to explore this remarkable result by investigating the role of sex hormones on cell division in the telencephalon.

Loss of function through ablation of genes of importance in mice has been commonly employed in the field to generate animal model to study receptor and cpregulator function in diseases. However, many diseases are caused by gain of function through amplification or over-expression of coregulators and receptors. Thus, over expression of these genes in mice in a tissue specific manner will undoubtedly aid our understanding of the physiological function of these receptors. In this project, we propose to generate ES cells that can be used to over-express all 49 murine nuclear receptors and >10 coregulators relevant to metabolic diseases. The coregulators will include: PGC1a, SRC-3, SRC-2, SRC-1, RIP140 and other coregulators shown to be important for metabolic and cardiovascular diseases, cancer and aging. These ES cells can be: (1) used to produce mice over-expressing these factors in a tissue-specific manner in any tissue where specific Cre recombinase mouse line is available or (2) used to investigate the effect of their over-expression on stem cell function. The ES cells will be generated in a 'high throughput' fashion, since recombination frequency is very high in the ROSA locus. In addition, we will generate a limited number of adult mice from these ES cells for proof of principle. To generate knockin over-expressing ES cells, we will utilize a unique procedure. We have generated a targeting vector for the ROSA locus with a CAGGS promoter directing nuclear receptors and coregulators expression; a LoxP-Stop-LoxP cassette is inserted in between the CAGGS promoter and the coregulator/receptor coding sequence to prevent expression until recombination occurs. When mice harboring this knockin are crossed to tissue-specific Cre mice, the coregulator/receptor gene will be turned on and expressed in any tissue where the Cre is expressed. This system has many advantages: (1) no expression of gene of interest in mice in the absence of the Cre recombination; (2) a single mouse line can be used to overexpress coregulator/receptors in any given tissue of interest; (3) temporal expression can be effected if inducible expression of Cre is employed (tamoxifen or tetracycline inducible Cre); (4) ensure uniform expression level in any tissue of interest; (5) the locus/promoter limits expression of the inserted gene to no more than a few fold over the endogenous level; (6) in a similar token dominant negatives of regulatory molecules can be expressed in specific tissues. Once ES cells are generated, frozen ES cells will be made available to all investigators in the field. Similarly, embryos generated from the mice we produced will also be freely distributed to investigator upon request. In addition, easily used targeting cassettes will be made available for investigators in any field.

Monoclonal antibodies against T cell surface markers have been used experimentally in vivo in an attempt to delete T cells and allow for subsequent tolerance to foreign transplantation antigens. One such monoclonal antibody OKT3, directed against the CD3 delta chain of human T cell receptor complex has been shown to have profound immunosuppressive effects on transplantation rejection responses in vivo. We have developed a hamster monoclonal (145-2C11) directed against the CD3 epsilon chain of the murine T cell receptor complex. Like its anti human CD3 counterpart, it can significantly effect transplantation responses in vivo. Animals treated with small quantities of the anti-T3 antibody exhibit prolonged skin graft rejection by as much as three weeks. In addition, cells removed from anti-T3 treated mice are unresponsive to a variety of alloantigens including both class I and class II. Future studies will be designed to examine the role of in vivo treatment with anti T3 on bone marrow engraftment and abrogation of GVH and HVG responses.

Turnover of cells and matrix in bone occur through osteonal remodeling, wherein resorption removes and osteoblastic infilling replaces microscopic regions of compact bone that have reached the end of their functional life. Imbalances in this turnover process can lead to impaired bone quality and increased fragility. How bone remodeling targets area of bone has long been unclear. We found that inhibiting osteocyte apoptosis after fatigue prevents activation of osteoclastic resorption, demonstrating for the first time that osteocyte apoptosis is a controlling step in the activation of targeted bone resorption of microdamage. We also discovered that the spatial and temporal relationships between osteocyte apoptosis and osteoclastic bone resorption previously established for remodeling of microdamage in bone hold as well for resorption induced by ovariectomy and disuse. Finally, we found that the surviving osteocytes near areas of microdamage also appear to respond to the focal microinury and may potentially secrete regulatory factors that help coordinate the osteoclastic response. In the current studies, we will use a combination of real-time PCR, immunohistochemical and histomorphometry approaches in the rat ulnar fatigue model in vivo to determine the regulatory processes by which apoptotic and non-apoptotic osteocytes surrounding microdamage signal to osteoclasts and activate bone remodeling. We will also determine which among the osteocyte derived signals is dependent upon osteocyte apoptosis by using a pan-caspase inhibitor to suppress the apoptosis. We will focus on osteocyte expression of major regulators of bone resorption that are a) an established requirements for osteoclastic differentiation and b) have demonstrated expression by, or effects on, osteocytes. Complementary in vitro studies on osteocyte-like cells will be used to test causal relationships using specific blocking approaches to determine roles of individual signaling cytokines within the pathway linking apoptotic and pro-osteoclastogenic cells. In the second series of studies, we will use the rat ulnar fatigue model to establish the importance of osteocyte apoptosis in the activation versus progression of bone resorption around microdamage. Finally, we will test whether osteocyte apoptosis is a 'common final pathway' for initiation of bone resorption in response to diverse remodeling stimuli, by using a pan-caspase inhibitor to suppress osteocyte apoptosis in mouse models for estrogen withdrawal and disuse. PUBLIC HEALTH RELEVANCE. Maintaining bone quality and preventing bone tissue fragility depends upon bone remodeling, wherein osteoclasts remove and osteoblasts replace microscopic regions of bone that have reached the end of their functional life. Our recent discoveries indicate that this remodeling, as well as the increased turnover and bone loss in postmenopausal and disuse osteoporoses, in turn, depends of the regulated death (apoptosis) of osteocytes within the bone. These findings suggest that osteocyte apoptosis may be a 'common final pathway' controlling bone resorption in response to diverse remodeling stimuli, and also suggest that this apoptosis could represent a novel therapeutic target for modulating osteoclastic activity in aging and osteoporosis. [unreadable] [unreadable] [unreadable]

Physical and chemical techniques will be developed for the analysis of the structure of complex macromolecular assemblies or organells. The major goal is information on the conformation of individual assembled components and proximity relationship between components. Techniques will include singlet energy transfer fluorescence, breakable photocrosslinkers, phototransfer reagents and dye photosensitized chemical modification. A variety of potential uses for immunoglobulins and fragments directed against fluorescent dyes will also be explored including electron microscopy and affinity chromatography. These techniques will be applied to several different systems including the conformation of ribosome bound tRNA's, structure of several ribosomal proteins, free and in the intact ribosome, the topological arrangement of proteins in the ribosome and proteins which form the lytic lesion of the mammalian complement system, complement protein-membrane interactions, and elements of the cell surface involved in cell-cell recognition. A few shorter range projects including conformation of nucleic acids with covalently attached carcinogens and mechanism of microtubule disassembly should be completed early in the project period.

A pilot multicentered interactive data bank project restricted to traumatic coma has been implemented. The objectives of the study are: 1. To utilize a uniform clinical vocabulary and method of data input. 2. To evaluate the feasibility of a multicentered traumatic coma data bank system to examine: a. The ability of diverse institutions to participate and collaborate effectively. b. The ability to collect data of high quality. c. The ability to insure the quality and quantity of data from diverse institutions. d. The preliminary utility of the data for patient management and in indicating research leads. e. The ability to provide guidelines and protocols for expansion to additional centers and other neurologic disorders.

Up to one third of the 700,000 men and women who served in Operations Desert Shield and Desert Storm during the 1990-1991 Gulf War (GW) have developed a chronic multisystem illness known as Gulf War Veterans' Illness (GWVI). Neurobehavioral findings include memory problems, executive system deficits, slowed motor and processing speeds, sustained attention deficits, reduced visuospatial skills and psychomotor dysfunction. Given the spectrum of cognitive deficits noted above, combined with evidence of structural and functional abnormalities on neuroimaging, it is likely that neuropathological changes also occur in GWVI. Several environmental exposures have been implicated as potential contributors to GWVI including exposure to acetylcholinesterase (AChE) inhibitors such as pyridostigmine bromide (PB; anti-nerve gas pills) and organophosphate (OP) pesticides/nerve agents (e.g., sarin/cyclosarin). The pattern of deficits may be related to whether GWV were exposed to OP nerve agents as well as their premorbid vulnerability (e.g., PON1 status) to such exposures. GWV may also be at higher risk for developing a progressive neurodegenerative disorder such as Alzheimer's Disease as they age. Some studies suggest that accelerated aging may occur in GWVI related to axonal transport deficits, increased axial diffusivity, increased WM anisotropy on diffusion tensor imaging and insulin resistance and/or metabolic syndrome. Findings of reduced hippocampal volume in GWV suggest that that the pattern of deficits may be consistent the development of an Alzheimer-type dementia. This is important to investigate because the GWV cohort in general is aging; 52% of GWV are age 45 and older and 16% are age 55 to 85+. In addition, recent evidence suggests that an unexpectedly large number of GW veterans sustained traumatic brain injuries (TBI) so they may also be at risk for long term sequelae such as chronic traumatic encephalopathy (CTE). Given the issues raised above, there is a critical need for a GWVI CNS tissue biorepository that will conduct extensive ante mortem longitudinal assessments on enrolled GWV. Our first specific aim is to establish a VA GWVIB as a national resource to support research on the etiology and pathogenesis of GWRI and our second aim is to perform selected psychometric and biological assessments on enrollees to maximize the value of tissue donated to the GWVIB. Well-characterized CNS tissue when combined with antemortem health data and biological assessments (such as ApoE genotype and serum PON1 activity) will be invaluable to advance research on GWVI. The GWVIB will be a multi-site collaboration among VA Boston Healthcare System (VABHS) and the Southern Arizona VA Healthcare System (SAVAHCS). The GWVIB will utilize strengths across the Boston and Tucson sites in enrollment, tissue collection, processing, storage, neuropathological diagnosis, medical informatics and data management. VABHS will serve as the operations/data coordinating center and conduct the neuropathological diagnostic analyses, with SAVAHCS contributing expertise in CNS tissue processing and storage. SAVAHCS will also coordinate CNS tissue distribution. Notable enhancements to be initiated are the utilization of an active versus passive recruitment approach, enlarging our collection of tissue from Veteran controls through a collaboration with the National Disease Research Interchange (NDRI), and improved outreach to investigators to increase the utilization of the GWVIB in GWI research. This will allow us to leverage the substantial investment of VA resources already in place at GWVIB and to add value to existing clinical and CNS tissue resources.

The enteroendocrine cells, which comprise approximately 1% of epithelial cells in the gastrointestinal tract, represent the largest population of hormone-producing cells in the body. The enteroendocrine cells share a common lineage with other non-endocrine cell lineages and originate from primitive intestinal stem cells in the intestinal crypts. There are two complementary populations of stem cells, a self-renewing stem cell population and a quiescent stem cell population. The self-renewing stem cells highly express Lgr5 and reside between Paneth cells at the crypt base where Wnt signaling is active whereas quiescent stem cells express Bmi1 and reside at the position 4. Thus the lineage-specific precursor cells are thought to differentiate immediately from the self-renewing Lgr5+ cells. Endocrine precursor cells differentiate toward mature hormone-producing endocrine cells that are classified into at least 15 different terminally differentiated lineages (or subsets) by their expression of specific peptide hormones. Although the molecular mechanisms that regulate the differentiation from the endocrine precursor cells have not been fully characterized, key transcription factors have been implicated in enteroendocrine cell differentiation (Pax4, Pax6, BETA2/NeuorD, Pdx1, Gfi, Nkx2.2 and Sox9). There is a relationship between spatial orientation of the enteroendocrine cells and their differentiation process. Previous reports on characterization of entroendocrine cell differentiation using BrdU incorporation, morphological and immunohistochemical methods demonstrated that the majority of enteroendocrine cells complete the differentiation process within the crypt and migrate upward along the villus as mature hormone-producing cells. However, a small population of enteroendocrine cells migrates downward to the bottom of the crypt. Enteroendocrine cells are comprised of subsets, whose differentiation is determined by specific transcription factors that regulate both the specific co-expression of hormones and their location within the crypt-villus axis. The differentiation signaling pathways mediated by Wnt, Hedgehog, Notch, BMP and EphB/ephrin are restricted spatially along the crypt-villus axis in the epithelium and the mesenchyme of the intestine. Below position +4 (where the self-renewing Lgr5+ stem cells reside with Paneth cells) in the crypt, Wnt-signaling is especially active . Presently, it is unclear what determines whether a subset of enteroendocrine cells remains in the Wnt signaling rich area instead of migrating up to the villi and why. Similar to other enteroendocrine cells, EC cells, the presumed cell of origin of the SI-NETs, differentiate from the ISCs. A majority of EC cells are found at higher positions within the crypts and the villi and would eventually be sloughed as they reach the villus tips. However a small proportion of EC cells can be found below +4 position where an active stem cell niche maintains ISCs. Similar to our previous work in the duodenum of mice, we wondered if the EC cells in the distal SI that reside below +4 position were derived from reserve ISCs and were susceptible to tumorigenesis in Familial SI-NET patients. Presently, it is unknown whether there is a subset of ISC marker-expressing enteroendocrine cells in the human small intestine and whether they are related to SI-NETs. Therefore, we are presently investigating the relationship between the expression of ISC marker genes in EC cells along the crypt-villus axis of the human norman ileum and their expressions in SI-NETs. Recent results identified multifocal aberrant crypt-containing endocrine cell clusters (ACECs) that contain crypt EC cell microtumors in patients with familial SI-NETs. RNA in situ hybridization revealed expression of the EC cell and reserve stem cell genes TPH1, BMI1, HOPX, and LGR5(low), in the ACECs and more advanced extraepithelial tumor nests. This expression pattern resembled that of reserve EC cells that express reserve ISC genes; most reside at the +4 position in normal crypts. The presence of multifocal ACECs from separate tumors and in the macroscopic tumor-free mucosa indicated widespread, independent, multifocal tumorigenesis. Analyses of mitochondrial DNA confirmed the independent origin of the ACECs. We have also shown that fully differentiated EECs residing predominantly at the +4 position in the crypt have features of both label retaining cells (LRC) and intestinal stem cells (ISC) and are capable of de-differentiating to ISCs to regenerate the epithelium under basal and pathological ISC dynamics. Using lineage tracing in vivo and in ex vivo organoids, we show that the enterochromaffin (EC) cell is the predominant EEC with this potential. These studies suggest that an ISC gene-expressing subset of fully differentiated EC cells contributes to basal and pathological stem cell dynamics in the small intestine. These findings provide novel insights into the +4 reserve ISC hypothesis, stem cell dynamics of the intestinal epithelium and novel insight in the development of EC-derived small intestinal tumors. Take together, our studies suggest that Familial SI-NETs originate from a subset of EC cells (reserve EC cells that express reserve ISC genes) via multifocal and polyclonal processes. The epithelium of the small intestine renews itself every 3-5 days as a result of actively cycling intestinal stem cells ISCs residing at the crypt base. Most recently, we have shown that the maintenance of the intestinal epithelium by Lgr5-expressing ISCs occurs via an asymmetric model of ISC division under normal homeostasis in addition to the previous prevailing model supporting symmetric division. In fact, our in vivo data examining mitotic spindle orientation and lineage traced progeny pairs indicate that the majority of ISC division occurs asymmetrically. In summary, the our data support a neutral drift model dominated by asymmetric cell division of ISCs as the basis of homeostatic maintenance of the rapidly renewing intestinal epithelium.

This proposal is concerned with further studies of in vitro culture systems able to support the various developmental forms of salivarian trypanosomes (Trypanosoma brucei sspp. and T. congolense) found in the vertebrate and invertebrate hosts. First, attention will be given to the reproduction of the entire life cycle of trypanosomes in culture. Bloodstream forms from established cultures of T. b. brucei and T. b. rhodesiense, incubated at 37 C, will be used to initiate cultures of the vector forms (procyclic, epimastigote, and metacyclic) incubated at 26-28 C. In vitro-produced metacyclic forms will serve as starting material for the cultivation of the life cycle of T. congolense. In the second part of the proposal the emphasis will be on the cultivation of metacyclic forms currently (in the case of T. brucei sspp.) available only from tsetse fly tissue cultures. Studies will include: (a) the possible substitution of tissues of nonhematophagus Diptera for those of tsetse fly in the T. brucei sspp. metacyclic-form culture system; (b) attempts at cultivation of metacyclic T. brucei sspp. in association with various Glossina and mammalian cell lines cultivated at 26-28 C; and (c) the design of a culture medium based on the chemical analyses of tsetse fly saliva for the growth of the forms found in the salivary glands (T. brucei) and proboscis (T. congolense). Comparisons between the various stages of T. brucei and T. congolense life cycles produced in vitro with those developing in vivo will be studied by light and electron microscopy. Furthermore, the MVATs of the metacyclic stages of T. brucei produced in vitro and in the tsetse flies infected with the same stocks will be compared by immunological tests. There is a need for additional methods to cultivate infective forms of salivarian trypanosomes, which may ultimately be used in developing new approaches to immuno- and chemotherapy.

Validated clinical pharmacodynamic (PD) assays that reliably quantify drug effects on molecular targets in tissue specimens are critical tools for converting molecular oncology breakthroughs into targeted therapeutics, and subsequently for translating these new drugs into patients by confirming that they work as intended in early clinical trials (Phase 0, I and II). The vision and pioneering effort of DCTD to develop, clinically implement and transfer to the oncology research community highly reproducible and informative PD assays, combined with the strategic decisions of its dedicated SAIC-Frederick support program to use only assay technologies that have proven clinical applicability and to apply diagnostic assay expertise and developmental principles to PD assays, have already yielded validated assays for several molecular targets since the program's inception in mid-2005. One of these assays accelerated the clinical development of a molecular targeted agent from Phase 0 trials directly into combination Phase 1 studies with best available therapies in five CTEP-sponsored trials. The molecular data from the validated assay justified bypassing any single agent Phase I or II trials, where it was not expected to show any single agent activity in most genetic backgrounds. The DCTD expects that using additional validated PD assays to simultaneously interrogate multiple molecular targets both within and across important signaling pathways in a single biopsy specimen and/or surrogate tissue will point to rational combinations of molecular targeted agents. Because many molecular targeted therapies are used in combination regimens with best available chemotherapy, there is also anticipated value in being able to evaluate the effects of molecular targeted therapy on markers of chemotherapeutic sensitivity and resistance to guide the selection of which chemotherapeutic regimens are suitable for combination with the molecular therapy, and which should be avoided in combination because, for example, the molecular targeted therapy induces high expression of a known molecular determinant of drug resistance. The DCTD envisions using extending the proven principles of assay development, validation and clinical implementation to develop and validate clinical PD assays for known chemotherapeutic markers, and then to deploy these in the Phase 0 trial setting under the FDA's Exploratory IND Guidance in order to not only confirm the molecular therapeutics intended action on drug target but also to confirm that it is not altering determinants of chemotherapeutic sensitivity for the best available regimens with which it is planned to be combined in Phase I and II trials. In addition, the rapid development, analytical validation of assay performance, SOP-driven validation transfer from the development lab to the clinical lab pioneered a formal process that solves two of the most vexing and intransigent problems regarding laboratory studies of experimental drug action in cancer patient specimens at academic medical centers: first, because the drugs are experimental, the drug effect assays are highly specialized, and are only used during early clinical drug development, they have little intrinsic market value. So, investigators must obtain the key reagents of the assay from R&D suppliers that do not adhere to quality assurance measures required of manufacturers of diagnostic tests that are critical for achieving consistent lot-to-lot content and performance. The SAIC-Frederick clinical PD support program has solved this issue by establishing an internal quality assurance program for key reagents and centrally procuring them, qualifying them for use in the validated assay, and then making them available upon request via the DCTD Biomarker's website to the oncology research community. In addition, this website provides access to the validated assay SOP, the specimen handling SOP to achieve valid assay results, and training courses offered at the NCI-F for laboratory staff from extramural institutions that wish to conduct a validated assay on site. To qualify to conduct their own assays, institutions are required to send laboratory staff for assay operator certification classes at NCI-Frederick, which include laboratory-based training in specimen handling, conduct of the assay, and data analysis and reporting by SAIC-Frederick senior scientific staff and lectures on PD assay theory and practice by senior DCTD staff. Via the convergence of DCTD direction and portfolio management with SAIC-Frederick's application of technical expertise in diagnostic testing, a number of validated assays are already in the clinic or moving into the clinic at this time, including assays for PARP target function, PARP pathway expression, DNA damage by chemotherapeutic agents, DNA methylation, tumor cellularity, tumor stem cell identification and enumeration of and molecular changes in circulating tumor cells. These validated assays are achieved using a number of clinically useful technology platforms of the SAIC-Frederick PS program: immunoassay, quantitative RT-PCR, quantitative immunofluorescence of tumor sections and cytospins, and the Veridex Cell Search instrument a clinically validated platform for enumerating circulating tumor cells. The steps of PD assay development, clinical implementation including SOP-based specimen handling, analytical validation and transfer to a recipient clinical lab have been formalized into milestones and metrics by the SAIC-Frederick PD support program, and rigorously achieving each milestone in the process is key to the successful introduction of additional clinical PD assays into the early clinical trial setting. These steps are definable and have objective metrics that allow decisions at each stage of development and validation regarding feasibility and go/no-go decisions by management. They are amenable to formalized statements of work that could be used to outsource this process to qualified vendors in order to accelerate the rate of PD assay development and validation and expand the capacity without any compromise in the quality of the process or the resulting assays. The SAIC-Frederick PD program already has demonstrated a high level of competence managing outside vendors and specifying similarly complex, specialized technical services, such as customized antibody generation and the scaled production of purified biologics used as key assay reagents. DCTD envisions that the SAIC-F PD support program can direct such an expanded effort via directing the technical development and validation of additional assays under contracts to highly qualified vendors with the technical capabilities to follow the steps exactly. Maximum numbers of clinical PD assays could be developed by the SAIC-Frederick PD program by using a strategy of tiered technical support services, in which a small number of highly qualified vendors are selected for some particular assays, but also manage a larger number of second tier vendors to develop assays with which they have unique specializations and hold the second tier vendors to the same high level of quality assurance. This management strategy offers several advantages, not the least of which is that is minimizes the number of projects with which the SAIC-Frederick senior staff will need to interact so as not to lessen the assay development effort at NCI-Frederick, it lessens the number of transactions that SAIC-Frederick Research Contracts department will need to oversee, and the two-tier system significantly increases the number of vendors that can be contributing to the effort with a small number of contract technical managers/project officers. There are assays in development at SAIC-Frederick that could be immediately transferred to accelerate reduction to SOP-driven assays that can be validated within 6-12 months and returned to the NCI for clinical implementation in the Developmental Therapeutics Clinic. It is also envisioned that as more assays are made ready for clinical use, the emphasis of the DCTD on unmet medical need in uncommon cancers will result in a more centralized center where physicians can access these validated assays that may be very useful in personalizing each cancer patient's therapy based on molecular markers. Many of these PD assays will provide important information about molecular targeted therapy trials sponsored by DCTD/CTEP, including inhibitors of molecular targets controlling stem cell proliferation and survival, angiogenesis, cell cycle progression and novel cytoreductive mechanisms like mitotic spindle inhibitors. Other PD assays in early to mid-development in the SAIC-Frederick PD program that will benefit from accelerated development and validation include assays of direct drug action on the DNA methyltransferases that play major roles in controlling DNA methylation and abnormal gene silencing in cancers, and drug action on the enzyme topoisomerase I which is already a validated therapeutic target via the market approval of the camptothecins. Outsourcing the final steps of the defined, formal process that has yielded past assay successes along with active project management will free up the SAIC-Frederic staff to focus on the development of much needed assays in areas of multi-channel analysis of drug response in rare tumor stem cells, new assays to distinguish cells recovering from chemotherapy damage or committing to cell death via apoptosis or necrosis, and the high-value payoff of adapting as many validated PD assays as possible to the customized use of the Veridex platform instrumentation to isolate circulating tumor cells.

The third categorical seminar hosted by the Principal Investigator and Dr. Jean-Luc Urbain will be held in Toronto, Ontario on Saturday 23 June, 2001, again one day before the beginning of the Society of Nuclear Medicine Annual Meeting. A complete list of speakers has not yet been finalized. The main objective of this seminar is to educate the nuclear medicine community on those recent developments in molecular medicine which will dominate drug development in the new millennium and which are likely to influence radiopharmaceutical development as well. International experts in genomics, proteomics, pharmacogenomics and DNA therapies will provide participants with the state-of-the-art of these new technologies. A detailed description of the molecular biology of these technologies will introduce each subject. We also believe that the interaction of participants with these scientists provides a unique opportunity to bridge our community with the world of molecular biology that will impact tomorrow's nuclear medicine practice. Specifically, the aim of this seminar series was to enable the attendees to: 1) Understand the molecular biology of gene sequencing, PCR, cloning, antisense and gene therapy. 2) Recognize the implications in medicine of the human genome project and other genomics. 3) Discuss the importance of proteomics and the current methods of structural analysis. 4) Appreciate the genetic causes underlying variability in response to drugs. 5) Identify the strengths and weaknesses of antisense chemotherapy for the treatment of diseases. 6) Recognize the potential role of disease treatment by gene therapy.

The polyamines putrescine, spermidine and spermine have been implicated in the regulation of cellular proliferation and differentiation. However, a direct role has been demonstrated only for spermidine in DNA replication. We have previously shown that spermidine is required for both murine erythroleukemia (MEL) cell proliferation and induction of differentiation. Furthermore, we have demonstrated that spermidine is required for both the induction of heme synthesis and transcription of alpha- and beta-globin mRNA. The requirement of spermidine for induction of MEL differentiation may reflect the necessity of cell division for expression of the mature phenotype. The biosynthesis of spermidine and spermine is associated with the formation of 5'-methylthioadenosine (MTA). We have recently demonstrated that MTA inhibits induction of MEL differentiation. This effect was not associated with spermidine depletion or cytostasis. MTA is a known inhibitor of S-adenosylhomosysteine hydrolase and DNA methylation and may inhibit induction of differentiation by interfering with transmethylation reactions. MTA also inhibits cyclic AMP phosphodiesterase activity and potentiates the cellular cAMP response to adenylate cyclase activators. Therefore, this naturally occurring nucleoside may be an intermediate involved in the interrelationships between cellular proliferation and specific gene expression. The proposed studies will thus extend our work on the involvement of polyamines in leukemic cell proliferation and differentiation. The initial studies will monitor the effects of MTA on MEL intracellular S-adenosylmethionine metabolism, nucleic acid methylation, and intracellular cAMP metabolism. A more precise understanding of the biochemical basis for regulating MEL commitment to differentiation will be explored by using MTA analogues and other inhibitors of S-adenosylhomocysteine hydrolase. This work should provide the basis for monitoring the effects of MTA and MTA analogues on transcription and DNA methylation of specific MEL genes including alpha-globin, beta- globin and mouse c-myc. Since previous studies have also demonstrated that spermidine is required for induction of human HL-60 leukemic cell differentiation, the proposed work will thus also monitor the effects of MTA on methylation and expression of specific genes (c-myc, c-fos) in this cell line. These studies should be important in providing a basis for understanding biochemical interrelationships between proliferation and differentiation in leukemic cells.

The role of procoagulant hemostatic factors in atherogenesis has been enigmatic. Increased plasma concentrations of procoagulants in youth may reflect early plaque development, and rising levels of procoagulants may mirror the severity and extent of vessel wall involvement by the disease. Insight into these processes may be gained from a longitudinal study of clotting factors in subjects beginning at the earliest stages of atherosclerosis (prior to age 30), and continuing through ages 40 and 50, when subclinical and clinical evidence of disease may be recognized. Such individuals with disease in middle age may have had elevated concentrations of procoagulants in youth and a greater rate of increase in levels over time as compared to those without evidence of atherosclerosis. A unique opportunity to test this hypothesis is presented by the CARDIA study, which for the past twenty years has serially examined a cohort of men and women who were ages 18-30 at inception. In an ancillary study, we measured levels of fibrinogen, factor VII, factor VIII, and von Willebrand factor in blood samples collected 1990-91 (Year 5) and 1992-93 (Year 7). In these Year 5 and 7 samples, obtained when subjects were in their 20's and 30's, cross-sectional analyses revealed that fibrinogen was higher in women than in men, factor VIII and von Willebrand factor were higher in blacks than in whites, and associations were reported of factor VII with cholesterol and triglycerides, and fibrinogen with body mass index, cholesterol, and LDL-cholesterol. Certain correlations were confined to specific sex/race groups, such as fibrinogen with blood pressure, triglycerides, and cigarette smoking in white males. These associations between established risk factors for atherosclerosis and procoagulants suggest common links to atherogenesis. We now wish to extend our previous study to the year 20 examination (2005-6). We shall examine the associations of rate of change in hemostatic factors with clinical and laboratory evidence of atheromatous disease, such as coronary calcification and increased carotid intimal/medial thickness, as well as changes in CARDIA measured lifestyle factors such as diet, physical activity, body mass index, and smoking, and the development of hypertension, hyperlipidemia, and diabetes. In addition, we shall assess whether changes in factor VII and factor VIII coagulant activities over time are associated with particular factor VII or factor VIII haplotypes. Study of CARDIA participants offers a unique opportunity to examine longitudinal changes in hemostatic components linked to atherothrombosis and to correlate their evolution with that of other recognized coronary risk factors during the critical period preceding clinical manifestations of disease.

The goal of the MSK SPORE in Lymphoma is to improve the cure rate of patients with diffuse large B cell lymphoma, through a collaborative effort between three New York City institutions: 1) Memorial Sloan Kettering Cancer Center (MSK), 2) Weill Cornell Medical College (WCMC), and 3) Herbert Irving Comprehensive Cancer Center (HICCC) of Columbia University. The overall approach for this SPORE in Lymphoma seeks to shift current treatment paradigms and clinical practice by introducing, developing, and applying new concepts, methods, and technologies to address several DLBCL subgroups with a clear unmet medical need. Our overall broad aims are: Specific Aim 1. To develop novel treatments for DLBCL based on targeting specific genetic and molecular alterations that contribute to the oncogenic process. Specific Aim 2. Identify potential biomarkers of antitumor efficacy using tissue specimens from patients enrolled on four clinical trials developed in the SPORE. We plan to identify and utilize biologic, genetic, and clinical biomarkers to select patients with DLBCL for novel therapeutic approaches. In Project 1, we will develop novel treatments to target the oncogenic cooperation between Myc and Bcl2. Such therapy can subsequently be evaluated in patients enriched for high Myc+/Bcl2+ expression in DLBCL using standard immunohistochemistry methods. These patients have a clear unmet medical need, as they have a poor prognosis with standard chemotherapy In Project 2, we will investigate the safety and clinical efficacy of genetically modified T cells to express chimeric antigen receptors (CARs) targeting CD19 in elderly patients with relapsed DLBCL who are not candidates for stem cell transplant.19 These patients have a dismal prognosis, with a median overall survival rarely exceeding one year.20 In Project 3, we will investigate the safety and efficacy of the first Tumor Enriched-Hsp90 (TE-Hsp90) inhibitor PU-H71 in patients with relapsed DLBCL.21,22 A novel PET-based molecular imaging using radiolabeled I-124 PU-H71 will be used to examine in vivo targeting of HSP90 by PU-H71, and to guide dosing and patients selection.23 Because c-Myc and intrinsic apoptosis pathway proteins are client proteins of TE-Hsp90, the efficacy of this treatment will be retrospectively assessed in patients with Myc+/Bcl2+ DLBCL. Finally, in Project 4, we will elucidate the normal and pathologic role of CBP and p300 in B cells, establish pre-clinical models for their therapeutic targeting, and test the activity of the novel HDAC inhibitor mocetinostat in a phase II clinical trial. we will use targeted sequencing strategies to select patients with DLBCL that carry mutations in the CBP/p300 histone acetyltransferase (HAT) genes for therapy with novel HDAC inhibitors.7-12 Our goal is to identify safe and active new agents in biomarker-defined patients with relapsed DLBCL.

Aging is associated with progressive lose of skeletal muscle mass and strength referred to as sarcopenia, a significant risk factor for disability, fraily, and mortality. Epidemiologically, muscle weakness defined based on a single measurement of hand grip strength has repeatedly proven to correlate with subsequent adverse health outcomes, even when measured in mid-life to predict physical disability decades later. These findings have led to ongoing efforts to define clinically meaningful cut points for muscle strength in the diagnosis of sarcopenia and growing interest in using grip strength as a key endpoint in clinical trials of new interventions for frailty. However, the success of such efforts requires improved knowledge in two areas. First, whether a single measurement of strength can sufficiently capture the underlying risk? Second, given that most of the pilot trials have a short follow-up, typically 6 months; it is important to know the degree and heterogeneity of detectable change in the short term and the clinical relevance of such change to long-term health outcomes. So far, these topics have not been adequately studied in a longitudinal setting. The central hypothesis is that short term trajectory of grip strength, characterized by rate of change and intra-person variability in grip strength, predicts long term trajectory of grip strength and adverse health outcomes in older adults. To test this hypothesis, this project proposes to evaluate: (i) the heterogeneity of short-term (6-month) trajectory of grip strength, (ii) the impac of short-term trajectory of grip strength on long-term (3- year) trajectories of health outcomes, and (iii) the effect of multisystem physiological dysregulation on short-term trajectory of grip strength. The study applies state-of-the-art statistical models to analyze existing longitudinal data from the Weekly Disability Substudy of the Women's Health and Aging Study (WHAS ) I. The substudy is comprised of 102 women aged 65 years and over who were evaluated on a weekly basis over six months, followed by additional five semi-annual visits of 6 months apart for a total follow-up of three years between 1993 and 1996. PUBLIC HEALTH RELEVANCE: This study is designed to break new grounds in understanding the clinical relevance of temporal changes in hand grip strength and their likely causes and consequences in older women. The results will provide useful risk assessment information to patients, families and clinicians, and preliminary data to define target population and treatment goals for future intervention studies on muscle strength in older adults.

This research will determine the functional abilities and the architectural dimensional needs of 500 people with arthritis. Architectural dimensional needs refers to those building specifications that make the building more accessible to the handicapped. A random sample of clinics in Tucson will be used to obtain the widest distribution of functional level, age and sex of these patients as possible. The functional abilities (i.e., range of motion and other parameters) of the individual will be determined by a therapist (physical or occupational), performing a standard joint examination and then using an analysis technique developed for this research program. The individual will then proceed through a system of adjustable architectural mockups. The dimensional range of the architectural mock-ups required for satisfactory function will be determined. An optimum value, for instance, the height of a handrail, will be derived from the study of the subject population. The functional abilities and dimensional data will be recorded and processed by computer to yield statistical profiles of the arthritic population seen. This will produce a catalog of dimensional needs for each functional group in this population. The functional groupings are derived from statistical analysis of individual functional abilities. The intersection of the dimensional requirements of the largest reasonable population can then be used to evaluate the appropriateness of existing building codes and point to other specifications not stated in existing codes, but shown necessary by this research. The methods and techniques developed will be carefully recorded and freely shared so that they may be used in the future with other handicapped populations to increase the basic anthropometric data bases of the dimensional needs of the handicapped. It is hoped that this research will be a significant step toward developing "accessibility codes" based on broad-based scientific data rather than existing limited data and personal opinions.

During this past year (2011-2012) our laboratory built upon the foundation begun in 2008 to explore the interface between the malaria parasite and the host immune system. We have continued to collaborate with Dr. Rick Fairhurst (LMVR) and Dr. Mahamadou Diakite (MRTC) on a 4-year longitudinal study of 1400 children in 3 villages in Mali. In 2009 we identified a sub-cohort of these children, selecting those with the sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed from 2009 to the present, with blood samples being obtained before and after the transmission season as well as during bouts of clinical malaria infection; these samples provide a unique and valuable resource for studies on the development of humoral and cellular responses to blood-stage antigens of malaria parasites. Analysis has shown that children with HbAS are significantly protected against malaria in this population; this was not true for children with the HbAC genotype. Increasing age, a surrogate of acquired immunity, was also protective. Because long-standing data has shown that antibodies can play a significant role in protection against erythrocytic stages of infection, we have pursued a detailed characterization of the antibody populations present in the Malian children. We have already shown that children with HbAS genotype show lower responses by ELISA to a number of merozoite antigens relative to age-matched controls. We believe that the lower titers in the HbAS children may result from their lower incidence of infection and we are analyzing a study of asymptomatic parasitemia in these children. During the past year we have followed these children into a new transmission season and shown that, surprisingly, their IgG levels to specific merozoite proteins do not diminish during the dry season with little malaria transmission. We are continuing to follow the antibody profiles over several transmission seasons using both ELISA and GIA as readouts and are also conducting more in depth studies of the antibodies themselves. In addition, we have developed a flow cytometry assay to profile the antibodies in these children to antigens present on the surface of parasitized erythrocytes. We have investigated changes in these antibody populations with age, with malaria exposure, and with host genotype. Another aspect of our studies of host responses to proteins on the surface of parasitized erythrocytes, we have continued our collaboration with Dr. Kavita Singh (RTB) on a major surface antigen of infected red cells, viz., VAR2CSA. This protein is a member of the large PfEMP1 family and has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A (CSA) in the placenta. Dr. Singh has produced 6 domains of the VAR2CSA protein in recombinant E. coli and we have tested their recognition by antisera from Malian adults. Antibodies from multiparous Malian women but not men recognize some of these domains by ELISA. We have also standardized an opsonization assay using these IgGs and shown that FCR3 parasitized red cells expressing VAR2CSA can be efficiently opsonized by a human monocytic cell line. We have developed a novel procedure testing the ability of various domains to inhibit opsonization by these antibodies. We have thus identified several domains that are important for recognition by anti-VAR2CSA antibodies in this functional assay. Our second major area of investigation relates to the identification of malaria parasite-encoded antigens which could be the targets of new vaccines or drugs. We have hypothesized that there are conserved epitopes present on the infected red cell which could represent such targets. While most blood-stage vaccine candidates are from merozoites, antigens present on the surface of the infected red cell have significant advantages as targets because of their exposure to the serum for long periods. However, the antigenic complexity and diversity of the known surface molecules (e.g., PfEMP1) have proven daunting for vaccine development. To address this problem, we have pursued a new strategy using DNA aptamers. We had previously prepared several DNA aptamer libraries and performed repetitive selections on various targets, and we analyzed the selected aptamer populations using next-generation sequencing technology. We have focused on a small subset which binds to infected but not uninfected red cells; some of these aptamers inhibit parasite growth in vitro. This year we have completed broader studies on the reactivity profiles of this subset and shown that some recognize all the parasite isolates tested. More recently we have developed methods to affinity purify the parasite targets of the aptamers. This is a complex process, involving preparation of extracts from parasitized erythrocytes, binding to immobilized aptamers, and identification of target molecules by mass spectrometry. However, we now have bands on SDS-PAGE gels and are confident we will have identification of target molecules. Using our standardized blood-stage parasite growth inhibition assay (GIA), we have collaborated with others in analysis of several different human trials of various blood-stage vaccine candidates and in testing of preclinical animal sera. An important step forward has been made in collaboration with Dr. Simon Draper and colleagues (Oxford University), who are using recombinant adenoviruses encoding P. falciparum blood stage antigens. A new potential blood-stage vaccine candidate - PfRH5 - has been identified that elicits antibodies with very high levels of GIA activity. Binding of PfRH5 to the red cell protein basigin has been shown to be essential for merozoite invasion. We have tested antibodies to PfRH5 against parasites from different geographic locations and are evaluating antibodies from an immunization-challenge study in Aotus monkeys for functional activity. We are continuing to collaborate with Dr. James Burns (Drexel University) on his novel fusion protein of P. falciparum MSP1 and MSP8. We have shown that it elicits extremely high levels of antibodies with GIA acitivty in rabbits; this construct is being extended to preclinical studies in Aotus monkeys. We have continued to expand our studies on transmission blocking immunity and in collaboration with PATH/MVI, we are performing mosquito membrane feeding assays (MFA). This involves culturing P. falciparum sexual stages in vitro, feeding these parasites to laboratory-reared mosquitoes in the presence or absence of specific antibodies, and later counting oocysts in the mosquito midgut. To accelerate the development of transmission blocking vaccine, this year we have qualified the MFA to test its reproducibility. Using these results we have worked with Dr. Michael Fay of the Biostatistics group to develop a computerized model of the MFA which allows us to ask questions about assay variables and predict outcomes. In addition, it allows us to test sexual stage vaccine candidates for transmission blocking activity and obtain confidence limits. We have constructed and characterized a new set of mouse monoclonal antibodies to the full-length P. falciparum circumsporozoite protein prepared by Dr. Sanjay Singh at Gennova, India. Seven of these monoclonals have been produced in quantity, and we are working with extramural collaborators (Drs. Fidel Zavala and Chris Ockenhouse) to test these for passive protective activity using transgenic P. berghei parasites. Results have been presented at the American Society for Tropical Medicine and Hygiene (10 talks and posters-Philadelphia, PA)(2011). Results have been presented at the European BioMalPar meeting in Germany (2011), the Japanese Society for Parasitology (2012), a World Health Organization workshop in Geneva, Switzerland (2012), and at Gennova, India.

Millions of Americans now entering midlife and old age were exposed to high levels of lead, a neurotoxin, as children. Evidence from animal and observational studies suggest that children exposed to lead may be at elevated risk for dementia in old age. More evidence is needed to characterize the nature of this risk. Such information would inform future research on disease prevention and assist communities in preparing for high disease burdens following lead-exposure events. The two studies proposed in this application will increase the evidence base on the long-term neurological consequences of childhood lead exposure using neuroimaging, genetic analysis, and advanced longitudinal statistical techniques. These studies will also help prepare the applicant for a successful interdisciplinary career integrating the fields of environmental health, cognitive aging, and observational epidemiology. The applicant's long-term career goal is to become a clinical neuropsychologist and independent academic researcher who conducts public health-oriented research on the degenerative consequences of environmental exposures. The proposed project integrates training and research plans to develop the applicant's expertise in these areas, with fellowship funds allowing for mentored training in (1) the application of neuroimaging to environmental health research, (2) the assessment of cognitive health and environmental exposures, (3) the application of quantitative analysis to longitudinal and behavioral genetic studies, and (4) the ethical conduct of research in observational studies. The training plan includes coursework and workshops, one-on-one mentorship, and participation in seminars and conferences. While ambitious, the planned studies and training plan are feasible because they will utilize existing data gathered in a population-representative longitudinal birth cohort, the Dunedin Study, which lead-tested 565 Study members in childhood (56% of living cohort) who are now in late midlife. This sub-cohort offers a globally unique opportunity to evaluate the long-term consequences of childhood lead exposure because, unlike with most lead-tested cohorts around the world, lead exposure was shared equally across socioeconomic groups in the Dunedin cohort. Study 1 will involve secondary analysis of existing neuroimaging data to determine whether childhood lead exposure relates to degenerative alterations in neural structure (Aim 1) or function (Aim 2) by late midlife. Study 2 will involve secondary analysis of existing genetic data to determine whether neurocognitive sequelae of childhood lead exposure are moderated by polymorphisms in genes suspected to alter the toxicodynamics of lead (Aim 3). These projects will build the evidence base on the neurodegenerative risk facing the millions of Americans who were exposed to lead in childhood, informing future research on disease prevention and future policy on community lead-exposure response. The fellowship will give the applicant unique, dual expertise in environmental health and degenerative disease epidemiology, and prepare him for future training focused on the application of epidemiological findings to disease prevention.

Growing subjects are in a state of positive K balance and have a limited ability to excrete K. This suggests that the immature kidney has a limited capacity for K secretion and/or enhanced ability to reabsorb K. The major regulatory site of K secretion in the kidney is the cortical collecting duct (CCD). In contrast to the high rates of net K secretion observed in CCDs isolated from adult animals and microperfused in vitro, segments from neonatal animals show no significant K transport. Yet, these same neonatal segments absorb Na at a rate half that measured in the adult, suggesting there exists a fundamental difference in cation transport mechanisms between the neonate and adult. K secretion in the CCD, mediated by principal cells, is determined by a two step process: active K uptake into the cell by Na-K-ATPase and passive diffusion down a favorable electrochemical gradient through apical K channels. To determine whether a paucity of apical K secretory channels limits net K secretion early in life, we will use patch clamp analysis to compare the apical K conductances of neonatal and mature principal cells. To examine whether the absence of conducting K secretory channels is due to a low open probability (P-O) of existing channels and/or a low channel number (N), we will next determine whether exposure of the neonatal CCD to factors known to increase P-O or N induces net K secretion. The discrepancy between onset of Na and K transport in the neonatal CCD suggests that qualitative changes in the transepithelial Na absorptive pathway occur during postnatal differentiation. To test this, we will compare the apical Na conductances (patch clamp analysis), membrane transporters active in Na reabsorption (helium glow photometry), and Na-K-ATPase activity (ouabain- sensitive basolateral 86Rb uptake) in neonatal and mature principal cells; the latter findings will be correlated with measurements of basolateral membrane surface area (electron microscopy). Because clearance studies in the neonate indicate significant K retention, we will also test the hypothesis that enhanced K absorption in the neonate reduces net urinary K excretion. To assess the K absorptive capacity of the CCD and outer medullary collecting duct (OMCD), we will determine the contribution of K absorption, measured as unidirectional lumen-to-bath 86Rb fluxes, to net K transport in segments isolated from maturing animals. Should we document significant K absorption early in life, we will test whether K absorption is coupled to H secretion by H-K-ATPase, a transporter immunocytochemically identified in intercalated cells. That population of intercalated cells possessing functional H-K-ATPase will then be identified and the polarity and activity of H-K exchange measured to determine if there is a maturational change in activity of this pump. The studies proposed in this application should help us to understand the physiologic basis for the limited K secretory capacity of the neonatal CCD and provide broad insight into the regulation of the K secretory process and its interrelationship with the mechanism of Na absorption.

The goal of this research is to improve our understanding of respiratory, metabolic and cardiovascular control during exercise and to elucidate the mechanisms by which diseases limit work performance. Computerized data analysis will be used to yield accurate descriptions of dynamic physiological responses to exercise. Though exercise is the principal cardiorespiratory stress, there is no consensus on the control of exercise hyperpnea. However, evidence is accumulating which suggests that exercise hyperpnea is closely coupled to CO2 delivery to the lung. We propose to test this hypothesis during: 1) onset, 2) transition to steady-state, and 3) steady-state of exercise while perturbing certain physiologic factors. We will study effects of limiting the rate of cardiac output increase, altering the CO2 set-point, attenuating the carotid bodies with 100% oxygen, varying CO2 output by diet and continuously varying external dead space. Responses will be studied below and above the anaerobic threshold to discern the effects of metabolic acidosis. In dog, we will employ cardiopulmonary bypass techniques to separate the pulmonary from the systemic circulation, to delineate the role of pulmonary blood flow on respiration. Neurophysiologic studies in cat will be pursued in an attempt to define the reflex pathway for cardiodynamic hyperpnea. Mathematical simulations of respiratory control will aid in refining our hypotheses. A second thrust will be to study factors affecting gas exchange in health and disease. Breath-by-breath measurements of gas exchange will allow us to assess the role of arterial blood O2 content in determining the energetic pathways during exercise. The effect of altering the substrate for exercise on respiratory and cardiac demands will be determined. The role of the carotid bodies in exercise induced bronchospasm will be evaluated. The pathophysiological processes which affect both inspiratory and expiratory breathing patterns resistance to airflow, and CO2 and O2 transfer across the lungs will be studied in order to establish guidelines to assess the severity of the physiological defect in patients. This must preceed a mechanistic rationale for therapy.

Understanding peptide structure and dynamics is of great interest in chemistry and biology. EPR methods have been used to rank distances between side chains in doubly labeled peptides, [unreadable]thereby revealing local folding geometry. Recently, EPR of site-specific spin labeled peptides has been shown to be an excellent probe of-position-dependent dynamics. Although these EPR experiments have added considerably to our understanding of peptide motions, many important details remain unresolved. In particular, conventional 9 GHz EPR spectra are not particularly sensitive to the anisotropy of the spin label motion. Thorough characterization of the local label motion is necessary in order to clarify the interpretation of the position-dependent dynamics and to aid in the determination of distances in double label experiments. High frequency EPR has been demonstrated to be more sensitive than 9 GHz spectra to motional anisotropy at short correlation times. We have obtained high frequency EPR spectra of the alanine based 3K- 11 helical peptide at temperatures from 275-305 K. The 3K- I I helical peptide, shown below, is labeled at the cysteine with a methanethiosulfonate (MTSSL) spin label. Ac-AAAAKAAAAKCAAAKA-NH2 The high frequency EPR spectra exhibit greater sensitivity to motional anisotropy than low frequency spectra but remain amenable to simple line shape analysis according to motional line narrowing theory. Simulations of the experimental spectra using different models for the anisotropic motion of the spin label chain attached to the peptide backbone are in progress.

Glutamate-mediated neurotransmission is the main form of excitatory neurotransmission in the vertebrate nervous system and is largely dependent on the proper function of the AMPA receptor, one of the most commonly found receptors in the central nervous system. The research plan outlined in this proposal will directly address how the.structures of both the AMPA receptor auxiliary subunit stargazin and the scaffolding protein PSD-95 regulate AMPA receptor sensitivity and clustering. The goal .is to obtain a 3D structure.of both stargazin and stargazin in complex with PSD-95 using two related electron microscopy approaches:-2D electron crystallography and single particle reconstructions. This research is core to the better understanding of several debilitating conditions and diseases of the central nervous system as well as learning and memory, as all of these processes are dependent on the proper function of glutamatergic neurotransmission. Overactivation of AMPARs and aberrant excitatory neurotransmission are both implicated in epilepsy, stroke, Parkinson's disease and more. A better understanding of the mechanisms underlying these diseases is essential for public health. The research outlined in this proposal directly addresses the role of AMPAR auxiliary subunit structure in relation to AMPAR function towards accomplishing this goal.

Centromeres of eukaryotic chromosomes are specific regions along the chromatin fiber that play a fundamental role in chromosome movement during cell division. Centromere DNA sequences isolated from the yeast, Saccharomyces cerevisiae, enable foreign DNA introduced into yeast to function as ordinary yeast chromosomes during cell division. We will examine how the centromere DNA sequence interacts with chromatin components in the cell nucleus, including nuclear proteins and microtubules, to give rise to a functional centromeric unit. We will map the location of nuclease cleavage sites and will measure the coiling of the DNA duplex within centromeric chromatin to determine the organization of centromere DNA, histones and nonhistone proteins. The proteins that are associated with centromere DNA in vivo will be identified by direct isolation of intracellular centromere plasmid-protein complexes. The bound proteins will be characterized in terms of their DNA-binding properties by a filter binding assay and for their molecular heterogeneity by gel electrophoresis. We will then examine how these proteins interact with centromere DNA and microtubules to give rise to a functional centromeric unit. With the isolation of centromere specific DNA-binding proteins we should eventually be able to reconstitute the centromere spindle-fiber complex in vitro. Knowledge of the factors that control the organization of the centromere may provide fundamental principles governing the molecular mechanisms of cell division. Cell division is a central process of living organisms. Changes in the control of cell division may be an important mechanism that results in the production of aberrant cell populations, including cancer cells. Centromeric abnormalities, premature centromere separation and chromosome instability have been found in cases of Roberts' - SC phocomelia, ataxia telangiectasia, and a number of other syndromes. An understanding of the factors controlling cell division may be fundamental for the further understanding of these dysfunctions.

The objective of the project is to form a self-assembled monolayer containing a cyclodextrin functionalized with thiol groups on the primary side and flavin on the secondary side. The large molecular weight of this compound makes FAB-MS analysis necessary.

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. Idiopathic pulmonary fibrosis (the clinical condition associated with the pathologic diagnosis of usual interstitial pneumonitis) is a progressive fatal disease of unknown etiology. Currently, definitive diagnosis can only be made by surgical lung biopsy, although clinical diagnoses are made in certain patients with characteristic radiographic findings and a suitable clinical history. Accurate diagnosis is essential because related disorders, such as non-specific interstitial pneumonitis are responsive to immunosuppressive therapy and carry a significantly better survival than does idiopathic pulmonary fibrosis. Radiographic techniques currently available are inadequate to differentiate clinically responsive from non-responsive disease, and so many patients must undergo surgical biopsy with the concomitant risks related to these procedures. The goal of the present study is to establish a radiographic technique to quantify radiographic changes in lung parenchyma and vasculature associated with interstitial lung disease which will allow a more precise clinical diagnosis. We will compare two models of fibrosis in mice using tracheal aspiration of bleomycin or silica. Animals will be examined using micro CT at end-inspiration and end-expiration to assess shifts in histogram density which will be related to pathologic findings and degree of fibrosis. Individual animals will be assessed at multiple time points to determine the radiographic pattern of fibrosis over time, and to detect differences between the two models. In addition, pulmonary vasculature will be examined using digital subtraction techniques to assess the changes produced by the fibrotic process, while cardiac output will be estimated using stroke volume measurements. Finally, vasoactive agents will be employed to assess the degree of reversibility in anticipated pulmonary hypertension associated with fibrosis. Multiple mouse strains may be examined including: C57BL/6, C3H/HeJ, 129S1/SvImJ, A/J, BTBR T+ tf/J, FVB/NJ, NOD/LtJ, DBA/2J, BALB/cByJ, B6;129-Igf2, C57BL/6-Tg(ACTB-EGFP)1Osb/J, and C57BL/6x129 ColR/R. Vasoactive agents which may be used to assess vascular changes associated with the fibrotic process include;nitroprusside, nitric oxide, epoprostinol, diltiazem, phenylephrine, and hypoxia.

This proposal requests partial funding for a national conference, "Recommendations for independence: Prevention of Secondary Conditions in Two Primary Disabilities, Spina Bifida and Cerebral Palsy." The broad aims of the project are to support prevention of secondary conditions that have a negative impact on the ability of individuals with these disabilities to live as independently as possible and to stimulate and support secondary conditions prevention at six points of initiation by: individuals with these disabilities; their families and other significant social supports; medical and health professionals; education and vocational professionals; advocacy organizations; and research and policy institutions. The project will address three prevention goals: developing personal and interpersonal behaviors that support independence; attaining and retaining mobility; and establishing and maintaining body systems integrity and function. Each goal will be addressed with prevention best practices from four perspectives: Medical/clinical; assistive technologies; personal skills; and social/ environmental factors. The conference will produce and disseminate prevention recommendations for all points of initiation.

We greatly appreciate the opportunity to submit this Challenge Grant to NIMH under: (15) Translational Science, 15-MH-108, Screening approaches to identify pharmacologic treatments for mental disorders. The objective of this grant is to implement a high throughput screen for small molecules that potentiate the activity of FosB, a transcription factor that promotes resilience and antidepressant-like responses in several animal models and is deficient in the brains of depressed humans. FosB, which is induced in certain brain regions by chronic stress, represents a positive, coping mechanism that promotes positive adaptation to stress. 1) FosB induction in brain by chronic stress correlates with an animal's resilience to the deleterious effects of the stress. 2) Using viral-mediated gene transfer or inducible and brain region-specific bitransgenic mouse models, overexpression of FosB in these specific brain areas is sufficient to render animals resistant to subsequent stress and to reverse behavioral abnormalities induced by chronic stress. 3) Conversely, overexpression of a dominant negative antagonist of FosB in these brain regions makes animals more vulnerable to stress. 4) Chronic administration of standard antidepressant medications also induces FosB in these same brain regions, and overexpression of the dominant negative antagonists of FosB block the antidepressant-like behavioral effects of these medications in several behavioral assays. 5) Depressed humans have lower levels of FosB in these brain regions compared to extensively matched control subjects. This is the ideal time to submit this Challenge Grant, since we are poised to initiate the proposed studies immediately and they can be completed within two years. Thus, Drs. Eric Nestler and Gabby Rudenko have developed a novel high throughput screen to identify small molecule potentiators of FosB. An initial screen of ~50,000 compounds has identified ~500 hits that now require substantial validation. A series of validation assays, first in vitro and then in vivo, have also been developed. While targeting a transcription factor for psychiatric drug development is highly novel, several factors suggest that it represents a viable, albeit high risk, approach. For example, FosB is expressed at low levels throughout brain and peripheral tissues under normal conditions, suggesting considerable tissue specificity. Moreover, even if small molecule FosB activators prove unsuitable for the treatment of depression, high affinity, small molecule ligands for FosB would be invaluable tools to help us better understand FosB action in brain. Such molecules also could potentially be used to image FosB in the living human brain by use of PET or related brain imaging technologies, which would be a major boon to clinical investigations in depression and perhaps represent a novel tool to diagnose depression or other stress-related disorders or to track a patient's progress during treatment. We have recently shown that induction of the transcription factor, DeltaFosB, in brain represents a positive adaptation that helps individuals cope with chronic stress. Interestingly, the protein is also induced in brain by standard antidepressnts treatments, and depressed humans show lower levels of DeltaFosB in brain. We now propose to identify small molecule activators of DeltaFosB as novel treatment agents for depression.

DESCRIPTION: The project evaluates effectiveness of five, theoretically motivated treatment programs for young children with developmental, language-based reading disabilities. Treatments are based upon evidence that: 1) developmental reading problems are due to multiple, core linguistic and cognitive deficits, primarily in phonological awareness; naming/processing speed, and metacognitive control; and 2) data demonstrating the effectiveness of treatments that address more than one of the deficits are most effective. This project integrates three proven treatment modules (PHAB/DI-Phonological Analysis and Blending; WIST-Word Identification Strategy Training, RAVE-O-Retrieval-Rate, Accuracy, Vocabulary Elaboration, and Orthography) into a comprehensive triple-focus intervention, comparing it to two dual-focus programs (PHAB/DI + WIST, PHAB/DI + RAVEO) and a curriculum control condition. Two additional enhanced treatment programs, one that includes spelling/writing training, the other comprehensive fluency training, will each be integrated with the triple-focused program. All treatment programs employ similar format, taught by trained research teachers hired for the project, and independently monitored for treatment integrity. In addition to treatment comparisons this project also evaluates the impact of developmental timing of the intervention (starting in 1st, 2nd, or 3rd grade), differing levels of intervention intensity (hours of instruction-0, 35, 0, 105, 140, 175, 210, 245, 2800, teacher/student ratios-1:1, 1:4, 1:classroom), and the role of individual differences, particularly those related to different RD definitions, or cognitive subtypes, on reading outcomes. In all, over 432 children, selected to represent the full range of race, SES, and IQ levels, will receive interventions and be longitudinally followed until 4th grade. Three treatment sites (Atlanta, Boston, Toronto) are used to enhance generalizability and to address dialect, cultural, socioeconomic, and curriculum differences within schools. Repeated measure designs, multiple and logistic regression, and growth curve modeling will be used to evaluate treatment outcomes in relation to individual differences on standardized and experimental measures, particularly those related to word identification, fluency, and reading comprehension.

Globally, tobacco use accounts for 4.9 million deaths each year, including 400,000 in the U.S. These deaths are caused by inhaled smoke toxicants like carcinogens, carbon monoxide (CO), and nitric oxide (NO). Another smoke toxicant, nicotine, produces dependence with prolonged use. Cigarette smoke toxicant content and smoker toxicant exposure has been well characterized, as have the subjective and cardiovascular effects of cigarette smoking. However, in the U.S. and globally, millions of people use waterpipes (hookah or shisha) to smoke tobacco: the smoker fills the "head" with sweetened and flavored tobacco, covers it with perforated aluminum foil, and tops the foil with lit charcoal. Charcoal and tobacco smoke pass to the user via a half-filled water bowl, hose, and mouthpiece. Waterpipe tobacco smoking is increasing in the U.S., especially among college students for whom prevalence rates may be 15-20%. Relative to one cigarette, one waterpipe use episode can generate 100 times the smoke, with user toxicant exposure, effects, and smoke toxicant content that are largely unknown. These outcomes may depend upon an individual's use frequency and whether the individual uses the waterpipe alone or with a group. This project combines clinical laboratory methods, analytical chemistry, and cellular biology to achieve three specific aims. 1) Learn about individual waterpipe user toxicant exposure, effects, and smoke toxicant content. In two separate sessions, 130 waterpipe users (65 reporting 2-5 uses/month and 65 reporting >21 uses/month) will use a waterpipe loaded with preferred brand/flavor of waterpipe tobacco or placebo. Outcome measures include CO and NO, plasma nicotine, subjective, cardiovascular, and pulmonary response, and puff topography (puff number, volume, duration, and interpuff interval). Topography records will be replayed in an analytical laboratory to study smoke toxicant content and cytotoxicity/mutagenicity. 2) Learn how group use influences waterpipe toxicant exposure, effect, and smoke toxicant content. In an observational study, trained staff will use rigorous sampling and data collection procedures to study group waterpipe tobacco smoking in local cafs. In a laboratory study informed by observational study results, 65 waterpipe- sharing groups will use a waterpipe in the laboratory as a group in one session, and as individuals in another session, and user toxicant exposure and effects, and smoke toxicant content will be assessed as in Study 1. 3) Compare the toxicant exposure and effects of waterpipe tobacco smoking and cigarette smoking. In two sessions, 100 waterpipe users who also smoke cigarettes will either use a waterpipe or smoke a cigarette;outcome measures include exposure to CO, NO, and nicotine, as well as cardiovascular, respiratory, and subjective effects. Waterpipe use is disturbingly common among U.S. young adults. More information is needed about waterpipe effects, and it can be obtained from clinical laboratory studies and smoke toxicant analysis. This information is necessary to understand this potentially dangerous behavior, while shaping knowledge, opinions, and attitudes in a way that enhances public health. This project is relevant to public health because waterpipe tobacco smoking is a little-understood but rapidly emerging strain in the nation's tobacco use epidemic. The project will inform nascent efforts to prevent waterpipe tobacco smoking from contributing substantially to tobacco's morbidity and mortality by revealing the user toxicant exposure, subjective, cardiovascular, and pulmonary effects, and cytogenicity and mutagenicity of waterpipe tobacco smoke produced by individuals and groups;it will also address frequent but probably erroneous statements regarding waterpipe filtering and lower toxicant levels relative to cigarette smoke. The positive health impact of this type of research on tobacco cigarette smoking is well-documented;this project seeks similar positive outcomes on another potentially lethal form of tobacco use.

This program is devoted to the characterization of metalloenzyme active sites through the use of multinuclear, multifrequency (9; 35; 95 GHz) CW and pulsed electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies. Emphasis is placed on key catalytic intermediates prepared by cryoreduction and rapid freeze-quench ENDOR/ESEEM methods. The studies will focus on enzymes that carry out two, frequently correlated, processes: O2 activation; radical generation; methodology and applications development represent a third component. Particular goals include: 1) Dioxygen-activating iron enzymes: a) Mononuclear nonheme iron centers: cis- Diol dioxygenases; alpha-Keto-acid-dependent dioxygenases; Superoxide reductases; b) Diron centers: Ribonucleotide reductase; c) Heme enzymes: Cytochromes P450; Heme oxygenase; Nitric oxide synthase. 2) Enzymes generating catalytic radicals: The "radical S-adenosylmethionine" superfamily. 3) ENDOR development: Stochastic rapid-passage ENDOR; Extension of ENDOR/ESEEM to Zn enzymes/proteins through study of Co(II)-analogues.

The rise in obesity has led to increased prevalence of metabolic disease including diabetes, fatty liver disease, cardiovascular disease, and others. Veterans suffer from these diseases at a disproportional rate relative to the general population. Additionally, obesity and diabetes are strongly linked with post-traumatic stress disorder, which, based on recent research is now actually considered predictive of diabetes. Scientists now posture that obesity per se is not deleterious to health, but it is the dysfunction of adipose tissue that leads to disease. Healthy adipose tissue stores and releases energy appropriately, and secretes endocrine factors that communicate with peripheral organs and tissues to regulate metabolism. However, unhealthy adipose tissue has limited capacity for lipid storage, and is often stretched to that limit, when it becomes inflamed, leading to disruption of many important processes. What determines whether adipose tissue is ?healthy? or ?unhealthy?? It has become increasingly evident that increased adipocyte size is linked with diabetes. Adipocyte size is also inversely proportional to the hyperplastic potential of adipose tissue. That is, when new adipocytes can be made via cell hyperplasia, adipocyte size stays normal; when a limit to hyperplasia occurs, adipocyte size swells to handle the lipid load. Therefore, we might conclude that ability to proliferate adipocytes is key to metabolic health. Adipocytes arise from mesenchymal stem cells (MSCs), pluripotent cells that can also become muscle, bone cartilage, and other tissue types. How do these cells ?decide? which differentiation pathway to follow? While some pathways that regulate adipocyte differentiation (or adipogenesis) are known, the factors that regulate these pathways and thus determine cell fate are poorly understood. We have identified a potentially novel adipogenic signal, namely, sphingosine-1-phosphate (S1P). This molecule signals through g protein- coupled receptors to elicit a variety of cell outcomes. There are also receptor-independent functions for sphingosine-1-phosphate. Because our previous work led us to hypothesize that this molecule and therefore the enzyme that synthesizes it, Sphingosine Kinase 1 (SK1), may have a role in adipocytes, we made a mature adipocyte-specific SK1-deletion mouse. We found that these animals have a basal phenotype much like metabolic syndrome, but they are not obese. Specifically, they are insulin resistant, have high circulating levels of leptin and insulin, and show signs of non-alcoholic fatty liver disease. Further investigation revealed upregulation of osteo- and chondrogenic pathways and signaling in adipose tissue of these animals, which supports that they exhibit a defect in adipogenesis. We hypothesize that sphingosine-1-phosphate, intracellularly or in the extracellular milieu, participates in creating the adipose tissue microenvironment to promote adipogenesis in adipogenic precursors deriving from MSCs. In this proposal we present data supporting that SK1 and S1P downregulate anti-adipogenic signaling pathways, and that this is required for metabolic health. These studies will not only shed light on how to keep adipose tissue ?healthy? and therefore protect against metabolic disease, but the ?flip side of the coin? is that we will also discover new mechanisms regulating osteogenesis and chondrogenesis. Stimulating these pathways in adipose-derived stem cells is currently a major focus for regenerative medicine. We request funding for these studies from the VA, as both diabetes and tissue repair are of great concern for the veteran population and thus this work is highly relevant to veterans? health.

The resistance to curative therapy in multiple myeloma (MM) may be linked to its genomic instability, accentuated by survival signals provided by the marrow microenvironment (ME). Toward the P01's grand theme of MM growth control, the overall goal of this project is to increase the frequency of durable CR as a prerequisite for long-term survival and to interpret treatment failure in the context of gene expression profiles (GEP) of both MM and ME. In the previous P01 funding cycle, 600 of eventually 660 patients were randomized to Total Therapy 2 (TT2) + thalidomide (THAL) and received post-transplant consolidation therapy. CR frequency was higher with added THAL (51% vs 36%, P=.002). For the 2/3 of patients lacking cytogenetic abnormalities (CA), 74% of TT2 (vs 40% of patients receiving predecessor TT1, P<.001) remained in continuous CR at 3 yr from onset of CR. The overall hypothesis for the new Project is that the ME provides MM-dependent sanctuary mechanisms that can be inactivated by agents co-targeting the ME and MM cells (THAL, dexamethasone, Revimid(R), and VelcadeTM). Thus, this project will pursue 3 major specific aims. Aim 1 will assess the long-term outcomes (event-free [EFS] and overall survival [OS]) and obstacles to sustaining CR among TT2 patients; TT2 patients who relapse or progress will be randomized to treatment with MM and ME co-targeting salvage therapies. In Aim 2, the successor to TT2--a phase 2 trial of TT3--will attempt to improve CR by incorporating Velcade (exhibiting marked activity in end-stage disease) into induction and consolidation therapies as part of melphalan-based tandem autotransplants and intersperse THAL and dexamethasone peri-transplant to provide continuous treatment during treatment-free gaps of TT2. Functional imaging will be used as a potential early prognostic indicator of improved treatment outcome. GEP analysis, in collaboration with Dr. Shaughnessy's project 3, will be used to discern MM-associated ME signatures and their alteration by therapies, to better understand mechanisms of treatment success or failure. In Aim 3, the maturing data of TT2 and TT3 trials will form the basis of TT4, which will be developed in Year 4 with the goal of introducing GEP-based risk-adapted therapy. Future studies generated by this P01 will test, in collaboration with NCI, promising novel agents for previously treated patients, once pre-clinical studies have validated that critical signaling pathways identified have indeed been targeted. Thus, in concert with 4 projects and access to 4 cores, this project will advance comprehensive treatment for sustained disease control in MM in a highly translational fashion.

More than a decade ago, we reported that among dendritic cells, EpCAM was selectively expressed by epidermal Langerhans cells. In the past few years, we have revisited the utility of EpCAM as Langerhans cell surface marker and have extended our earlier studies and demonstrated that EpCAM expression, in conjunction with other surface markers, differentiates Langerhance cells from all other dendritic cells, including several recently described novel cutaneous dendritic cell subsets. In an effort to elucidate important aspects of EpCAM function in vivo, we identified a pre-existing targeted mouse embryonic stem cell generated by BayGenomics and, working with the CCR Mouse Knockout Core Facility headed by Dr. Lino Tessarollo, generated EpCAM +/- mice in which beta-galactosidase was inserted into one EpCAM allelle. EpCAM +/- mice were viable, fertile and indistinguishable from wild type littermates. Examination of beta-galactosidase expression as a surrogate for EpCAM revealed that EpCAM was expressed in a variety of developing epitehelial structures in skin and other organs. Mating of EpCAM +/- male and female mice gave rise to only wild type and heterozygous animals. No viable EpCAM-deficient pups were obtained. Assessment of embryos in timed pregnant females revealed that EpCAM-deficient embryos implanted and were indistinguishable from EpCAM-sufficient embryos until EGA 8.5 when they began to exhibit developmental delay. EpCAM-deficient embryos became nonviable and were resorbed soon thereafter. We subsequently determined that EpCAM was transiently expressed in conceptus-derived placentas with maximal expression at EGA 8.5-9.5. Detailed studies of placentas associated with EpCAM-deficient embryos revealed that they were small and thin with incompletely developed and poorly vascularized labyrinthine layers. EpCAM-deficient placentas also contained markedly decreased numbers of parietal trophoblast giant cells, a phenotype that has previously been associated with embryonic lethality. The findings were reported in PLoS One in 2009. Thus, although mechanistic aspects of EpCAM function remain to be elucidated, EpCAM clearly has one or more nonredundant roles in normal physiology. To gain additional insights into EpCAM function, we have generated mice with an EpCAM allele that can be conditionally deleted in a lineage-specific fashion. Working with Dr. Tessarollo and using recombineering, we developed a targeting vector that allowed loxp sites to be inserted into the EpCAM locus. Embryonic stem cells with a targeted EpCAM allelle were generated and identified, and then utilized to generate the corresponding mice. Germline transmission of the targeted allele has been confirmed. We have now crossed mice with targeted EpCAM alleles with mice that express the recombinase cre in cells of various lineages to ask and answer relevant questions. Tissues or cells of interest include Langerhans cells, keratinocytes, thymic epithelial cells and intestinal epithelia. The latter tissue is of interest because EpCAM mutations have been causally linked to congential tufting enteropathy, a rare congenital diarrheal syndrome. Experiments that have been completed to date clearly indicate that the targeted EpCAM allele efficiently recombines in several lineages and that this results in phenotypes in several tissues. We are characterizing these phenotypes and are carrying out complementary in vitro studies to gain additional insights into mechanistic aspects of EpCAM function. An initial series of investigations regarding EpCAM function in Langerhans cells has been completed and the findings were published in the Proceedings of the National Academy of Science in 2012. We determined that Langerhans cells that fail to express EpCAM exhibit decreased motility in epidermis and exit epidermis more slowly than EpCAM-expressing Langerhans cells after antigen-induced activation. This results in decreased migration of Langerhans cells from skin to regional lymph nodes and enhanced contact sensitivity reactions. These results definitively linking Langerhans cell migration to function and support the concept that Langerhans cells have a anti-inflammatory function as suggested by others. The results also suggest that EpCAM may be anti-adhesive, rather than pro-adhesive as previously suggested. In studies that are ongoing, we are additionally characterizing the role of EpCAM in Langerhans cell-keratinocyte interactions and in additional immune-related functions. Subsequent studies in mice whose Langerhans cells lacked EpCAM revealed that antibody-forming responses to proteins that were applied to skin were enhanced rather than predicted. Increased immune responses were associated with enhanced localization of antigen-bearing Langerhans cells in skin draining lymph nodes. These results have recently been published in the Journal of Investigative Dermatology. Our interpretation of the our seeming disparate results using mice whose Langerhans cells lack EpCAM is that the role that EpCAM plays in the regulation of Langerhans cells is context dependent. We have also explored mechanisms by which EpCAM regulates intercellular adhesion using human intestinal epithelial cells as a model system. We have determined that EpCAM acts in part by binding tightly to the tight junction-associated protein claudin-7. This interaction sequesters claudin-7 distinctly away from tight junctions and protects claudin-7 and associated claudin-1 from degradation in lysosomes. In the absence of EpCAM, whole cell levels of claudin-7 and claudin-1 decrease dramatically but remaining claudin-7 and claudin-1 becomes tight junction-associated. This results in increased tight junction avidity and increased trans-epithelial electrical resistance. These findings have been described in a manuscript that has been submitted for publication. These results have recently been published in the Journal of Biological Chemistry. Studies intended to delineate the mechanism(s) by which EpCAM stabilizes selected claudins are in progress. We have also begun to propagate mouse intestinal organoids and spheroids and are we assessing EpCAM-function in the these model system. We have observed a dramatic phenotype that can be reversed to large extent by treatment with a single pharmacologic inhibitor. The mechanism(s) that are responsible for this effect are under investigation, but our studies clearly argue for an important role in epithelial stem cell survival an/or proliferation. We have also identified and characterized a novel proteolytic pathway that is a dominant regulator of EpCAM function. We are in the process of submitting publications that describe our results in detail. In an effort to explore EpCAM function in other tissues, we are involved in collaborations with several developmental biologists with relevant expertise. One question that we are addressing relates to the functional equivalence of EpCAM and TROP2, an EpCAM-related molecule that is also expressed in epithelial tissues. The approach that we are taking involves use of transgenic mice that express EpCAM or TROP2 in intestinal epithelium and mice that have germline null mutations in EpCAM.

Measure CD spectra of B-hydroxy ketones to correlate and determine absolute molecular configuration. Samples were obtained by chemical synthesis and are enantiomecitally enriched.

The central hypothesis of this proposal is that there is an abnormal qualitative and quantitative distribution of gangliosides in malignant gliomas and medulloblastomas, perhaps similar to fetal brain, but different from normal adult tissues, especially normal adult neurons and glia. Biochemical analyses will first characterize and quantitate the types of gangliosides present in a large series of malignant human glioma biopsies, permanent glioma derived cell lines, and athymic mouse and rat-human glioma xenografts and detemine if unique gangliosides are present which are characteristics of gliomas. This methodology will then be utilized to similarly characterize and quantitate the types of gangliosides present in our models of human medulloblastoma. Our specific aims are: a) To produce monospecific monoclonal antibodies directed against the unique D-54 MG mono- and disialogangliosides 3'-iso-LM1, LD1, and GM3, GD3, GM3, GD2 and additional unique gangliosides defined in the course of this study; b) to define the distribution in human glioma tissue of these gangliosides qualitatively at the cellular level using immunocytochemistry with monoclonal antibodies directed against 3'-iso-LM1, LD1, GM3, GD3, GM2, GD2 and each new unique ganglioside and quantitatively at the tissue level using analytic techniques and thin layer chromatography on multiple human gliomas with varying cytomorphologies and degrees of malignancy; c) to use these monoclonal antibodies directed against 3'-iso-LM1, LD1, GM3, GD3, GM2, GD2 and each new unique ganglioside to examine the function of the defined gangliosides in cell-cell interactions, clonogenicity, and invasion utilizing in vitro and in vivo assays of cell growth, adhesion, and invasiveness; d) to use those monoclonal antibodies directed against 3'-iso-LM1, LD1, GM3, GD3, GM2, GD2 or any of the new unique gangliosides exhibiting broad reactivity within and among human gliomas and minimal normal tissue reactivity to demonstrate radioimaging and radiotherapeutic efficacies in the nude mouse - human glioma xenograft model; e) to define similarly the potentially abnormal qualitative and quantitative distribution of gangliosides in medulloblastoma, using the most complete and rigorously characterized models for this tumor extant and to execute similar studies as with gliomas. These studies will, therefore define the biodistribution of the altered gangliosides in human glioma and medulloblastoma, contribute to the understanding of the functional significance of gangliosides in adhesion, growth, and invasion, and establish pre-clinical efficacy of radiolabeled anti-ganglioside antibodies in glioma and medulloblastoma radioimaging and radiotherapy to aid in selecting monoclonal antibodies for clinical trials.

The gram-negative enterobacterium Xenorhabdus nematophila mutualistically colonizes the intestines of S. carpocapsae infective juvenile nematodes. The objective of this project is a structural and functional analysis of X. nematophila Nil A, B and C membrane proteins that are required for colonization of S. carpocapsae. We hypothesize that Nil A, B and C function as a signal transduction complex, and will test this by examining interactions between the Nil proteins and other X. nematophila proteins, by determining whether the Nil proteins regulate gene expression in X. nematophila, and by deciphering the external signals recognized by the Nil proteins. Understanding molecular events underlying this model mutualistic interaction is relevant to understanding how microbes cause disease and for developing disease therapies. This statement is based on several findings: that pathogens and mutualists share common aspects in their interactions with hosts, that vertebrates and invertebrates share common aspects of immunity, which can be targeted for disease therapy, and finally, that beneficial microbes contribute to the overall health and life-styles of animals.

Overexpression of PKC, a cytoplasmic serine/threonine kinase involved in signal transduction, may promote the development and maintenance of tumors. We have previously reported safety and activity of ISIS 3521 at 2.0 mg/kg by a 21 day CI. The present study explored an alternate schedule of 24 hr CI once weekly. Between 1/98-11/98, 11 patients with refractory solid tumors received ISIS 3521 at doses of 6, 12, 18 and 24 mg/kg in cohorts of 3 to six. 91 doses have been delivered so far. No gr 4 toxicities occurred. Gr 3 toxicities were: fever/chills (n =1) and hemorrhage (n =1) at 18 mg/kg and chills (n =1) at 24 mg/kg. Other gr 1/2 toxicities include thrombocytopenia (4), myalgias (6), chills (7), headache (3), fatigue (4), fever (7) and nausea/vomiting (4) which were transient. Steady state plasma levels were achieved within 4 hr and were proportional to increasing dose. Primate studies had identified complement activation as a potential toxicity of ISIS 3521; therefore, split products (C3a and Bb) were analyzed in all patients. Transient activation was seen at doses > 18 mg/kg when compared to < 18 mg/kg (median 3.9 versus 1.4- fold increase in C3a, p<0.001 and median 1.9 versus 1.0- fold increase in Bb, p<0.001). This correlated with a 1.5 second prolongation of prothrombin time (p<0.001) but not with grade 1-2 thrombocytopenia. Clinical evidence of complement activation was not observed. One patient had stable colon cancer for 3+ months. This regimen appears acceptable and patient accrual is currently ongoing to determine the MTD.

Diseases caused by retroviruses such as AIDS and leukemia have intensified the need to understand the mechanisms of retrovirus replication. Our primary objectives are to understand how reverse transcription of viral mRNA occurs and how the cDNA products are integrated into the genome of infected cells. Owing to their similarity to retroviruses, LTR-retrotransposons are important models for retrovirus replication. The retrotransposon under study in our laboratory is the Tf1 element of the fission yeast Schizosaccharomyces pombe. During the synthesis of cDNA, reverse transcriptase (RT) generates a series of highly specific intermediates. We identified residues of Tf1 RT that recognize specific intermediates of cDNA by screening large numbers of RT mutants. A combination of genetic assays and physical analyses identified a set of 35 mutations that inhibited integration without reducing reverse transcription. Our experiments focused on a cluster of mutations in ribonuclease H (RNase H) that included a region with five single amino acid substitutions in a five?amino acid segment. Surprisingly, these mutations in RNase H did not reduce the levels of full-length double stranded cDNA. Crystallographic studies by Dr. Edward Arnold and colleagues indicated that the corresponding residues of HIV-1 RT interact directly with the PPT. This observation led us to test whether the mutations in the RNase H of Tf1 were defective for the cleavages on either side of the PPT that generate the primer, or the cleavage that removes the PPT after it has primed plus strand synthesis. Defects in the position these cleavages would alter the sequences at the 3' end of the minus strand and as a result have a drastic impact on the ability of IN to catalyze strand transfer. The sequence from the 3? ends of the minus strand cDNA from Tf1 particles was determined using ligation mediated PCR. The mutations clearly increased the levels of cDNA that retained the PPT at the 3? end. In order to determine whether this added sequence resulted from a defect in the cleavages that generate the PPT or the cleavage that removes the PPT after synthesis of plus strand DNA, the 5? end of the plus strand DNA was analyzed. The results of primer extension provided strong evidence that the mutations in RNase H specifically inhibited the removal of the PPT RNA from the 5? end of the plus strand. As a result, our data identified a cluster of conserved amino acids in RNase H that have the specific function of removing the PPT. [unreadable] [unreadable] In addition to removing the PPT primer from the plus strand, RNase H is known to remove the primer from the 5? end of the minus strand DNA. Tf1 uses a unique mechanism of self-priming to initiate reverse transcription. Instead of using a tRNA, Tf1 primes minus strand synthesis with an 11 nucleotide RNA removed from the 5? end of its own transcript. We tested whether the self-primer of Tf1 was similar to tRNA primers in being removed from the cDNA by RNase H. Our analysis of Tf1 cDNA extracted from virus-like particles revealed the surprising observation that the dominant species of cDNA retained the self-primer. This indicates that integration of the Tf1 cDNA relies on mechanisms other than reverse transcription to remove the primer. [unreadable] [unreadable] Our analysis of the genome sequence of S. pombe revealed a strong clustering of pre-existing LTRs associated with the 5? end of ORFs. Experiments based on the production of new integration events revealed that the association of Tf1 with LTRs was the result of integration preference. To define the determinants of the target sites we developed an in vivo assay for integration using a plasmid that contained ade6 as the target and a plasmid with Tf1 that induced transposition. The version of Tf1 we expressed contained a neo gene to cause target plasmids with insertions to gain resistance to kanamycin. When Tf1-neo was expressed, the plasmid with ade6 served as an efficient target for integration. We isolated 50 separate insertions in the intact target plasmid and found ninety-five percent occurred within a 160 nt region in the ade6 promoter. To determine which sequences of Ade6 were required for efficient integration, we created a series of 10 deletions within the target plasmid. This analysis revealed that the 160 nt region of the promoter was the only sequence that was required for efficient integration. We asked whether promoter activity was required for integration by measuring transcript levels of ade6. Deletions of sequence on either side of the 160 nt region caused five to ten-fold reductions in ade6 mRNA. Nevertheless, the deletions caused no reduction in integration efficiency. These results indicated that transcription was not important for target site activity. We next considered whether transcription factors themselves were directing the integration of Tf1. To identify positions where factors bind in the promoter of Ade6, we used micrococcal nuclease mapping. We observed a strong correlation between micrococcal sensitive sites and the position of the prominent insertion sites. This suggested transcription factors played a role in directing Tf1 integration. Hoffman and colleagues showed previously that the transcription factor Atf1p binds to and activates the promoter of fbp1. We tested whether the promoter of fbp1 is a target of Tf1 integration using the target plasmid assay. We found that the fbp1 promoter was a target for Tf1 insertion and that the majority of the insertions occurred 40 nt from the position where Atf1p binds. A mutation that blocks the binding of Aft1p caused a significant reduction in Tf1 integration at the promoter of fbp1. These data indicate that Atf1p is responsible for targeting Tf1 to specific insertion sites in the fbp1 promoter.

The core will provide important resources necessary for each project's data-gathering, statistical analysis and interproject coordination. Dr. Dennis Spencer will be responsible for selecting patients appropriate for this study, providing the tissue, arranging weekly conferences to maintain interproject integrity; he will help develop hypotheses that connect clinical epilepsy issues with the basic research; he will coordinate the imaging data such that precise neuroanatomical relationships can be maintained, and is responsible for the overall program coordination. Computerized quantitative imaging is critical for patient selection criteria, and intraoperative imaging is necessary to provide important cytoarchitectonic correlation for each project. The presumptive pathological substrate must be compared to normal MRIs particularly since this is a quantitative examination. New funds are requested in this resubmission for a normative MRI volumetric study to provide this critical comparison. Dr. Gregory McCarthy will supervise the imaging research assistant in this regard. Dr. Jung Kim proves a central resource by providing qualitative neuropathological assessment and is responsible for the quantitative hippocampal neuronal, and glial cell counts which underlie all further morphometric, physiological and biochemical assessment. A patient data base has already been established, but statistical management for this program will be provided by Yale's Department of Biostatistics.

Hedgehog proteins are secreted signaling molecules that regulate the growth and differentiation of cells during animal development. Hedgehogs meet the classical definition of a morphogen by forming gradients of decreasing concentration that emanate from sites of secretion and induce differentiation of distinct cell types at different positions along the gradient. In this manner Hedgehog proteins mediate the formation of complex tissue patterns. Abnormal Hedgehog signaling can result in severe developmental defects and cancer, and clinical trials assessing the efficacy of Hedgehog signaling inhibitors are underway in cancers of the brain, breast, lung, colon, pancreas, and skin. Perhaps owing to its importance for patterning multiple tissues, the activity of the Hedgehog signaling pathway is tightly regulated. Hedgehog proteins appear to interact with multiple protein and glycan partners that modulate its activity, distribution, or both. The 12-pass integral membrane protein Patched is required for normal reception of Hedgehog signals and has been widely identified as the "Hedgehog receptor", but evidence for direct binding of Hedgehog to Patched is absent in flies and indirect in vertebrates. Several additional cell-surface proteins have been shown to bind directly to Hedgehog proteins with high affinity including Ihog in flies and Cdo, Boc, and Hedgehog-interacting protein in vertebrates. Others, including the protein cores of glypicans and a vertebrate-specific protein Gas1, associate with Hedgehog proteins with weaker affinity or as part of larger complexes and have been suggested as co- receptors for Hedgehog proteins. We have expressed and purified active fragments of Hedgehog, Ihog, Cdo, Boc, glypican, and Gas1 proteins, determined crystal structures of Hh:Ihog, Hh:Cdo, and Hh:Boc complexes, and demonstrated that these interactions depend on additional co-factors including heparin and Ca2+. We have also recently expressed and purified small amounts (~40 ug) of Patched. To investigate the molecular mechanisms governing Hedgehog signaling and understand the nature of the Hedgehog receptor at the molecular level we propose X-ray crystallographic, biophysical, and biochemical studies of these Hh pathway components and their complexes. These studies will elucidate the molecular mechanisms governing activity of the Hedgehog signaling pathway and thus the molecular strategies employed to generate complex tissue pattern. Such studies are also likely to uncover molecular interactions or processes that may be targeted for therapeutic inhibition of Hedgehog signaling. We will achieve these goals by pursuing the following specific aims: (1) determine the molecular mechanisms by which glypicans modulate vertebrate and invertebrate Hedgehog signaling, (2) determine the molecular mechanisms by which Gas1 modulates vertebrate Hedgehog signaling and the interplay between Gas1 and other modulators of Hedgehog signaling, and (3) determine the nature of vertebrate and invertebrate Hedgehog receptor complexes by reconstituting the minimal Hedgehog binding complex in vitro with purified components. PUBLIC HEALTH RELEVANCE: Hedgehog proteins are secreted signaling molecules that regulate the growth and differentiation of cells and tissues during animal development, and unregulated Hedgehog signaling has been implicated in human birth defects and cancers. By determining the molecular mechanisms governing activity of the Hedgehog pathway we will gain insight into how complex tissues and organisms develop from single cells and identify key pathway interactions that may be targeted for anticancer therapy.

Friedreich ataxia (FRDA; OMIM 229300) is an autosomal recessive neurodegenerative disease caused by reduced levels of the mitochondrial protein frataxin (FXN), which is due to large expansions of a repetitive GAA sequence located in the first intron of the FXN gene. The expanded GAA repeat tract inhibits transcription of the FXN gene by inducing heterochromatinization, and results in reduced levels of FXN mRNA and protein in all cells of the body. The length of the GAA repeat tract and FXN levels inversely correlate with disease severity, and are considered quantifiable molecular hallmarks of FRDA. Characteristic physical symptoms of FRDA include discoordination, slurred speech, muscle weakness, peripheral neuropathy, and cardiomyopathy. At the present time there is no effective treatment for this debilitating and progressive disease. Additionally, efficacy studies of therapeutic approaches currently being developed are hampered by a lack of appropriate animal models that recapitulate the complexity of FRDA molecular and clinical phenotypes. Technical limitations pertaining to the instability of long GAA repeat sequences have thus far prevented the field from attaining a FRDA GAA repeat knock-in (KI) mouse model carrying tracts greater than 230 repeats, which is considerably shorter than the average pathogenic length observed in patients (600 ? 800 repeats). As expected, the current FRDA KI models with short repeats exhibit only mild molecular and behavioral phenotypes of FRDA, and thus are of limited use in pre-clinical studies of drug candidates. Herein, we provide preliminary evidence of our innovative strategy to overcome this technical obstacle and create an improved GAA repeat model bearing 750 GAA repeats in intron 1 of the mouse Fxn gene. Based on published evidence and our preliminary data, we hypothesize that a new FRDA KI mouse model carrying long tracts of ~750 GAA repeats in intron 1 of the Fxn gene will demonstrate a robust phenotype that resembles many aspects of FRDA and will be well-suited for testing the efficacy of therapeutic approaches for this disease. To create and validate our FRDA KI expanded GAA repeat mouse model, we propose the following Specific Aims: 1.) Development of a new FRDA KI mouse model carrying 750 GAA repeats. 2.) Evaluation of new homozygous FRDA KI-Fxn750/750 and hemizygous FRDA KI-Fxn750/- FRDA mouse models to select the most robust model for internal and external validation studies. 3.) Internal and external validation of FRDA KI mouse models carrying 750 GAA repeats. We expect that by engineering a mouse model that mimics the genetic mutation of the human FRDA condition, with expanded GAA tracts of 750 repeats, the molecular and behavioral phenotypes will be apparent and quantifiable. Generation and standardized internal and external validation of this ?second generation? FRDA KI mouse will allow for comprehensive and comparative evaluation of novel drug candidates in one model system that recapitulates many critical aspects of the disease phenotype and etiology.

Increasing evidence suggests that susceptibility to cancer may be determined in part by low-penetrance genes, such as genes involved in the synthesis and metabolism of hormones or the metabolism of toxic substances. Many of these genes have been found to be polymorphic, with different actions depending on the polymorphism. In some cases, the frequencies of polymorphisms differ by race and ethnicity. Studies of gene-environment interactions which combine genetic information with standard epidemiologic data on reproductive factors, behaviors, medication use, and other exposures have the potential to greatly advance our current understanding of the etiology of human cancer. We propose to establish a DNA repository by obtaining cheek cell samples from as many as possible of the African-American women enrolled in the Black Women's Health Study (BWHS), a prospective follow-up study begun in 1995. Every two years since entry, mail questionnaires have been used to obtain data on reproductive history, personal habits and behaviors, medication use, and the occurrence of cancer and other serious illness. A recent pilot demonstrated the willingness of participants to provide a DNA sample by mail by swishing the mouth with mouthwash, expelling it into a small container, and mailing it directly to a laboratory. The DNA yields were high, and successful PCRs were carried out. We propose to seek mouthwash samples by mail from the approximately 57,000 BWHS participants who have completed at least one follow-up questionnaire; an estimated 36,000 women will provide samples. DNA will be extracted and stored at the participating laboratory. During year 05, nested case-control analyses will be carried out of breast cancer risk in relation to several genes involved in estrogen metabolism and a gene involved in the regulation of insulin-like growth factor. The DNA repository will serve as a resource for studies of gene effects and gene-environment interactions in relation to cancer in BWHS data alone and in combined or pooled analyses with other large cohort studies. The BWHS will provide the largest source of data on gene-environment interactions in black women, and will contribute substantially to the data available on young women of any race.

Phospholipase A2S (PLA2S) are essential in the proliferation and differentiation of human epidermis. Strong evidence supports their roles in regulating skin eicosanoid production, formation of the permeability barrier, protection from bacterial invasion, and wound healing. Despite their abundance and very robust activity compared with that of other cell types, PLA2s are only partially characterized in human keratinocytes. We have observed that human primary keratinocytes grown at low densities have a dramatic increase in both arachidonic and linoleic acid release compared to confluent keratinocytes. This cell morphology is correlated with a substantial number of cellular processes. The small monomeric G protein rac has an important role in process formation in other cell types. We hypothesize that a kertinocyte PLA2 is activated by the small monomeric G protein rac and that the coordinated activities of rac and PLA2 cause actin rearrangement necessary for keratinocyte process formation and migration in wound beds. Knowledge about PLA2 activity and identification of the types of PLA2, in human keratinocytes will provide a better understanding of their multifunctional roles in skin and could potentially lead to the development of agents that would improve wound healing and host defense.

Bacterial pathogens must acquire iron to replicate and survive in mammalian hosts. The iron-porphyrin heme, bound to circulating hemoglobin, contains up to 80% of bodily iron. This fact has led to the hypothesis, which is backed by experimental evidence, that heme serves as a source of iron during infection. However, in the anthrax-causing bacteria B. anthracis, deletion of iron-regulated surface determinant (Isd) genes, which code for surface proteins that bind heme, did not reduce B. anthracis virulence in animal models of infection. These results suggest other factors contribute to iron uptake in this deadly pathogen. A hallmark of Isd systems is the presence of a conserved protein module termed the near-iron transporter (NEAT) domain, which mediates the transfer of heme into Gram-positive pathogenic bacteria. In silico analysis of the genome of B. anthracis indicates a non-Isd gene, designated BAS0520, is annotated to encode for a single NEAT-domain protein. The objective of this proposal is to determine if BAS0520 represents the missing link mediating heme uptake during anthrax infection. Specifically, we hypothesize BAS0520 is a surface protein that extracts heme from host hemoglobin, thereby promoting heme transport into the bacterial cell and enhancing iron-dependent replication in mammalian hosts. This hypothesis will be tested with two aims: 1. Determine the mechanistic function of BAS0520. Biochemical approaches will define the molecular and structural factors of the NEAT domain of BAS0520. 2. Determine the role of BAS0520 in iron acquisition and anthrax disease. Growth studies and animal infection models using fully virulent strains will be used to define which mechanisms of iron uptake are important for anthrax disease.

The efforts planned for this Core, are modeled on the components that have been demonstrated in the previous period of the PPG to enable high productivity and efficient integration. This core is a hub of PPG activities and provides administrative support, and guidance without micromanaging individual project teams. The Core will be responsible for scheduling, organizing, hosting and/or facilitating on a regular basis the meetings that support the progress ofthe PPG. These meetings include (i)-plenary meetings (including Advisors), (ii)-electronic meetings, (iii)-thematic meetings of research groups enabled by participation of PPG teams in National and International conferences. The Core is responsible forthe administration of travel support for these meetings, including the allocation and accounting of funds. The Core is assuring compliance with all institutional, governmental and NIH-specific regulations and requirements, including timely communication with, and Progress Reporting to the NIH. All financial reporting and coordination aspects ofthe PPG that encompasses several major Institutions here and abroad are managed as well through the Core activities. Additional management objectives are to make certain that decisions about allocation (and reallocation) of resources, project expectations and progress, and accomplishing the goals of the PPG are addressed appropriately, fairly and in a well thought out and timely manner. The Core will thus continue to function to ensure the highest possible impact of the PPG's scientific discovery and dissemination activities by (l)-providing the necessary oversight, coordination and administrative support that will ensure the optimal performance of the PPG and its Consortium activities; (2)-providing services in the computational biology and bioinformatics aspects of the PPG, including resource sharing, and the sharing and dissemination of data generated by this Consortium; and (3)-developing an Information Management System for the area of research represented by the PPG, including the computer-facilitated expert annotation system TRansporter Annotation Center (TRAC), and the associated literature-mining tool Coupler.

The objectives of the Indianapolis Center for Sickle Cell Disease are: To screen approximately 6,000 individuals in the metropolitan area per year and to provide appropriate education and counseling to individuals found to have a hemoglobinopathy. To educate the professional and lay public for better understanding of sickle cell disease. To conduct research in the following areas: 1. a study linkage between the Hb locus and other genetic markers. 2. the clinical significance of Hb isoalleles. 3. phagocytic and opsonic function in patients with sickle cell disease.

The overall goal of this proposal is to develop the ability to detect and quantitate changes in gene expression in real-time and with a degree of sensitivity suitable to monitor minor changes at a single cell level. This will have considerable value in basic biomedical research, disease mechanism studies and diagnosis, and drug discovery. This revised proposal centers on the use of molecular beacons (MBs) for the analysis of gene expression in living cells. In particular, it focuses on the further development of MBs for studying breast cancer cells and single neurons. During our previous research in studying mRNA expression in single cells, we have encountered several limitations of MBs for cellular analysis. These included susceptibility to degradation by nucleases and single strand binding proteins, incomplete quenching of the fluorophore, and weak fluorescence intensity of conventional fluorophores. The autofluorescence of the cells also proved to limit the sensitivity of MBs for the detection of genes with a low expression. To overcome these limitations, we have proposed many novel solutions including superquenching moieties, bright amplifying fluorescent polymers, and molecular beacons comprised of locked nucleic acid bases. While these advances have not been tested in vivo they have shown promise in solution experiments. In addition to improving MBs as effective molecular probes for intracellular monitoring, we will also integrate advanced imaging methods into our analysis. In our previous results, we have demonstrated the advantages of confocal imaging when applied towards intracellular analysis. In this proposal we plan to demonstrate the advantages of multiple photon imaging and fluorescence time resolved imaging for more sensitive detection of MBs by removing the biological background. Once the MBs are improved and the new techniques are developed we will focus on intracellular applications of MBs for the detection of mRNA using these advancements. Currently, several genes, MnSOD, Cyclin D1 and survivin, have been identified as cancer markers and have been studied in cancer genomics. Using MBs, we will focus on elucidating the effects that different anti-cancer agents have on these genes in different human breast carcinoma cell lines. Given the genetic foundation of learning and memory, we will also use MBs to explore the effects of extracellular stimuli on the gene expression in neurons. The study will focus on EGR1 and Synaptotagmin IV, both of which have been linked to different stages of learning and memory.

Neuropeptide Y (NPY) is known to be co-localized, with norepinephrine (NE) and adenosine triphosphate (ATP) in vascular sympathetic neurons where it is thought to play a role as a co-transmitter/co-modulator. Recent evidence has established that NPYplays a physiological role in sympathetically mediated vasoconstriction by acting on postjunctional Y1 receptors. NPY is also known to exert prejunctional effects leading to inhibition of NE and most likely ATP release and an increase in NPY-ir release via Y2 receptors as well as inhibition of NE synthesis via Y3 and Y5 receptors. These results suggest that the process of NE synthesis and release of sympathetic co-transmitters may be differentially modulated by NPY suggesting an additional level of control in the prejunctional regulation of sympathetic neurotransmission. However the physiological role of this action has not been completely established. We have obtained evidence that an important mechanism for the prejunctional effects of NPY is through inhibition of Ca 2+channels. There is also evidence for a role of SNARE proteins in the preferential release and modulation of transmitter releaseby auto and heteroreceptors. The purpose of the present proposal is to further investigate the physiological role (Aims 1 and 2) on the NPY induced modulation of sympathetic co-transmitter release and NE release and the mechanisms involved (Aim 3) in the prejunctional modulation by NPY and other mediators. The rationale for studies proposed in Aim 1 is as follows: If there is endogenous modulation of transmitter release by NPY, then agonists and antagonists specific for the prejunctional NPY autoreceptor should alter evoked transmitter release in a manner consistent with the receptors being activated by endogenous agonist (e.g. released NPY). In other words, the effect of exogenously administered agonists and antagonists should vary with the biophase concentration of endogenous NPY. The response of the effector cell should be consistent with inhibition or stimulation ofNPY release. It would also seem necessary to demonstrate functional receptors in vivo. A similar rationale exists for Aim 2. In Aim 1 we examine the effect of a series of selective Y1 and Y2 agonists and antagonists on the nerve stimulation evoked release of NE, NPY-ir and ATP as well as perfusion pressure in the mesenteric arterial bed. This will be done before and after the endogenous NPY concentration is elevated by: 1) increasing the frequency of nerve stimulation or 2) decreasing the perfusion rate; or 3) after the NPY concentration has been reduced by depletion of tissue levels acutely or chronically. We will also examine the in vivo effect of agonists and antagonists in the pithed rat preparation. In Aim 2 similar experiments will evaluate the effect of NPY drugs on the nerve stimulation evoked increase in NE synthesis as measured by DOPA accumulations after decarboxylase inhibition. In Aim 3 we will determine if interruption of SNARE proteins by Botulinum neurotoxins (BoNTs) inhibit the evoked release of NE, NPY-ir and ATP as a mechanism for preferential release or differentiated modulation. We will also examine if signaling through SNARE proteins contributes to the prejunctional modulation by NPY. These studies will provide useful new information on the functional role and mechanism of action of NPY in the prejunctional regulation of sympathetic neurotransmission.

This project is predominantly based on a cross sectional self-administered mailed survey designed to address the type and frequency of ethical dilemmas faced by physicians, how they approach them, the types of ethical support they would find useful in addressing these ethical dilemmas, and their attitudes and practices in situations of scarce resources. The questionnaire was originally designed in English, piloted, translated into French, Norwegian, and Italian. Following IRB approval, the study sample included general practitioners and general internists from each of four European countries including England, Switzerland, Norway and Italy. In 2013 we revised the survey and administered it to a sample of physicians from Ethiopia to examine how ethical dilemmas related to bedside rationing from a developing country compare to those in developed, resource-rich countries that have already been surveyed. One publication from the Ethiopian survey has been published and two are in preparation. The principal investigator has also collaborated with non-NIH colleagues in using a similar survey among physicians across the US to determine their attitudes towards addressing costs in clinical practice. In addition, we are considering administering the survey in other resource-poor countries. The results of the European survey have been analyzed and six peer reviewed journal articles have been published and two book chapters have been published. A book based on the research findings and commentaries from philosophers, physicians, and health policy experts was published by Oxford University Press in 2015. These publications report: 1. The ethical difficulties encountered in clinical Practice by European doctors. 2. Physicians access to ethics support services in the four European countries in the study. 3. The prevalence and determinants of physician bedside rationing in four European countries. 4. Physicians views on resource availability and equity in four European health care systems. 5. An examination of whether ethics consultants should help clinicians face scarcity in their Practices. 6. The experiences of Ethiopian physicians which involve extensive resource scarcity, tough decisions and adverse consequences Results of the survey in the US have yielded valuable information about the attitudes of US physicians regarding their role and responsibilities in addressing health care costs.

The broad objectives of this work are to provide new computer simulation tools that will enable the detailed analysis of the role of molecular diffusion in biological processes at the subcellular and cellular levels, and the application of these tools to selected problems where close contact with experimental work is possible. The diffusional encounter of molecules with enzymes, antibody molecules, receptors, or other proteins; with membranes; or with nucleic acids is centrally involved in metabolism, immune and nervous system functions, signal transduction, cytoskeletal remodeling, gene expression, and a host of other processes in biology. The goals of the next project period will be to extend our simulation methods to allow the study of such processes, and to apply such simulation methods to selected problems with an emphasis on increasing scales of space and time. The methods will help in the development of antiviral agents, neurological drugs, and other materials of medical importance. The specific aims for the next project period include the following, (a) New methods will be developed for the unified treatment of polar and apolar interactions of diffusing molecules, (b) Coarse-grained models will be developed for detailed description of the internal motions of biopolymers and their intermolecular interactions in diffusional processes, (c) Continuum simulation methods for molecular diffusion will be developed as a complement to the more familiar Brownian dynamics simulation methods, (d) Applications will be made to study crowding effects relevant to antiviral drug action, and to study diffusion and reaction of neurotransmitter in model synapses. The public health relevance of this work lies in the fact that the methods to be developed will be useful in the computer-aided design of Pharmaceuticals, including monoclonal antibodies that rapidly bind to and inactivate viruses, and drugs that act at multiple sites within synapses. Training of undergraduate, graduate and postdoctoral students for careers in public health will continue to be a key aspect of the project. [unreadable] [unreadable] [unreadable]

Text4baby if a free mobile health education service that provides pregnant women and mothers with an infant less than one year of age with free health messages. An educational program led by the National Healthy Mothers, Healthy Baby Coalition (HMHB), text4baby is intended to help women,have safe and healthy pregnancies by empowering them with the information they need to be healthy and give their babies the best possible start in life. This is an evaluation of this project.

DESCRIPTION: Cholecystokinin plays a major role in the mediation of pancreatic secretion and gallbladder contraction after a meal, however little is known about the mechanisms regulating its secretion. During previous cycles of this grant, the investigator explored mechanisms responsible for feedback modulation of CCK release by intraluminal proteases and identified a trypsin-sensitive CCK-releasing peptide factor which is secreted into the proximal bowel. This has now been purified and sequenced to demonstrate its identity with the diazepam-binding inhibitor. The aims of the current proposal revolve around the hypothesis that DBI is the CCK-releasing peptide responsible for feedback regulation of pancreatic secretion and post-prandial secretion of this hormone; that secretion of DBI is under neurohormonal control with release mediated by enteric neural circuitry involving serotonin enterochromaffin cells, substance P sensory neurons, and cholinergic secretomotor neurons; and that DBI acts directly on CCK-releasing cells. Component aims are focused to demonstrate that DBI is released into the lumen during diversion of bile-pancreatic juice and nutrient stimulation, and that its secretion parallels that of CCK. It is postulated that immunoneutralization of DBI in the duodenum should abolish CCK and pancreatic secretion under these conditions. Structure-function studies are planned utilizing both the in vivo rat model as well as STC-1 CCK-releasing cells to identify key regions for biological activity. Another aim is focused toward demonstrating that nutrient-stimulated release of DBI occurs via the neural circuitry previously suggested. Finally, the localization of CCK and DBI in the intestine and the benzodiazepine receptors that may mediate this activity will be performed using immunohistochemistry and receptor autoradiography. The benzodiazepine binding sites responsible for CCK release will be characterized by both biological and binding studies. Through these studies, the investigators hope to further their understanding of the mechanisms regulating CCK secretion.

Cystic fibrosis (CF) is an autosomal recessive disease and a prime candidate for gene therapy. CF represents a major target for gene therapy research as the airway epithelium is relatively isolated and, theoretically, readily accessible by nebulized vectors. However, CF gene therapy faces the same obstacles that prevent the widespread use of gene therapy for other diseases; of 1300gene therapy clinical trials conducted worldwide to-date, barely three percent of these have been approved for phase three clinical trials. Clinical application of gene therapy is hindered by lack of safe and efficient delivery methods. Non-viral vectors can provide the requisite safety, but design of efficient materials is limited by a lack of understanding of the intracellular mechanisms of gene delivery. Many approaches to vector design are empirical, as data provided in the literature do not definitively provide robust model criteria for the design of gene delivery vehicles. Therefore, the principal objective of this proposal is to elucidate design criteria for safe and efficient gene delivery vehicles that will be used to construct a model for epithelial airway gene delivery. Specifically, this study will examine the internalization mechanisms and intracellular behavior of integrin-mediated gene transfer vectors. The specific aims of this proposal are to determine (1) the effectiveness of vector-ligand conjugation for targeted, receptor-mediated internalization at the cell surface; (2) the roles of endocytic vesicle acidification, trafficking kinetics, and processing pathways- namely clathrin and caveolin mediated vesicle transport - in vector escape and subsequent DNA unpackaging from the delivery vehicle; and (3) the interplay of molecular motors and cytoskeletal structures in endocytic vesicle trafficking of vectors. Correlating targeted delivery efficiency and intracellular trafficking studies will produce insightful structure-property relationships that will aid in the rational design of gene delivery vehicles. This study will focus on the design of synthetic, targeted, non-viral vectors that are minimally cytotoxic and easily produced. Preliminary results have established generalizable principles for the design of targeted vectors to different endocytic pathways from an epithelial (HeLa) cell model using prototypical ligands transferrin and folate. The long term goal is to use these principles to affect targeted airway gene delivery in human lung and bronchial epithelial cell lines. The clinical goal of gene therapy is to treat disease by delivering therapeutic genes to a patient in a highly efficient, specific, and safe manner. PUBLICH HEALTH RELEVANCE: The aims described herein seek to elucidate design criteria for delivery approaches in human airway tissue, and ultimately, the development of cystic fibrosis treatment.

The proteasome, located in the cytosol and nucleus of eukaryotic cells, plays a central role in cellular regulation by controlling the concentrations of hundreds of regulatory proteins, removing misfolded proteins, and producing some peptides displayed in antigen presentation. It is an approx. 2MDa cylindrical particle with its active sites of proteolysis buried deep inside the structure where they are accessible only through a narrow channel. Substrate proteins must be unfolded to fit the channel and to be degraded. The long-term goals of the research proposed here are to understand protein unfolding by the proteasome using a combination of modern biochemical, computational, genetic, and cell biological methods. These experiments will provide an in-depth understanding of proteasome function and may reveal unexpected regulatory mechanisms. The immediate goal is to understand how the unfolding step affects the specificity of the proteasome for its substrates and how it affects the nature of its degradation products. We will first define how degradation is initiated after substrate binding by the proteasome. Proteins are targeted to the proteasome by the covalent attachment of a ubiquitin tag but degradation initiates at a different site in the substrate. The initiation step contributes to the specificity of degradation and the sequence and spatial requirements for efficient initiation will be determined in vitro. The effect of these rules on the specificity of degradation of natural proteins will be tested in vivo. Second, we will investigate how the proteasome unfolds and degrades only part of a protein in a process called processing. The proteasome degrades most proteins completely to small peptides but a novel type of signal can lead to partial protein degradation. The processing signal and mechanism will be characterized and physiological examples of processing will be detected by a bioinformatics search and a genetic screen. This work is relevant to human diseases;many of the proteins degraded by the proteasome regulate cell growth and division. Since proteins must be unfolded before they are cleaved by the proteasome, understanding unfolding during degradation may open new avenues to fight cancer, for example by stabilizing specific proteins against unfolding and degradation. Some neurodegenerative diseases are associated with the accumulation of protein aggregates, which the proteasome fails to remove for unknown reasons. The investigation of partial protein degradation may lend insight into this question.

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 high prevalence of breast cancer calls for safe and efficient chemoprevention. Promising anti-breast cancer effects have been observed from an ethanol/water extract from bamboo Phyllostachys edulis, one of the most widely distributed and fastest growing plants. The bamboo extract (BEX) as a dietary supplement reduced the incidence of mammary tumors in 7,12-dimethylbenz[a]anthracene (DMBA)-treated lean Sprague-Dawley (SD) rats by 44%, enhanced tumor tissue differentiation, and up-regulated the activity of estrogen-conjugating hepatic sulfotransferases by 63% (P=0.011). BEX also reduced the tumor weight in obese SD rats by 68% (P=0.023). Moreover, BEX inhibited the proliferation of estrogen receptor (ER)-positive human breast cancer cells, possibly through relieving oxidative stress and inducing cellular senescence. Thus multiple pathways may contribute to the anti-breast cancer function of BEX, such as enhancing estrogen metabolism;accelerating the differentiation of mammary gland;ameliorating oxidative stress;inducing senescence in breast cancer cells;and inhibiting the acceleration of tumor growth in obese subjects. This investigation will focus on: (i) The influence of BEX on DMBA-induced mammary tumors, including using Magnetic Resonance Imaging (MRI) to monitor the development of micro-tumors, characterizing the molecular properties of both micro- and grown tumors, and studying tissue invasion and metastasis of the tumors. (ii) The influence of BEX on the differentiation of mammary gland and estrogen metabolism in the liver. (iii) The toxicity of BEX in SD rats as a dietary supplement. The knowledge obtained from this study will lay a firm basis for characterization of the potentially novel anti-breast cancer compound(s) in BEX.

This proposal describes a highly innovative approach for the treatment of cancer. One of the primary modalities for cancer treatment involves the use of chemotherapeutic agents. While useful, all of these drugs manifest substantial side effects. Thus, their use requires a tradeoff between eradicating the cancer on the one hand and causing long term cellular damage on the other. Here we propose to address the above problem using caged (inactive) drugs that can be uncaged (activated) by long wavelength light in a site-specific manner; such drugs could specifically target the tumor and spare normal tissue. This innovative strategy is based on recent developments in chemistry in the area of two photon removable protecting groups (including Bhc, BHQ and NDBF). These moieties can be removed by two-photon excitation using focused red (800 nm) light. Since long wavelength light can efficiently penetrate tissue, this uncaging strategy could be used to selectively release anti-cancer drugs within a tumor below the skin without affecting the surrounding tissue. Not only would this reduce the side effects from chemotherapy but it could also obviate the need for surgical tumor removal in some cases. However, before such an approach can be implemented, the key question that must be addressed is: At what depths can light activation (via two-photon excitation, 2PE) produce biologically useful levels of drugs? That question, in the context of application to Ras-driven cancers, is the focus of the research proposed here. Thus, in Aim 1, we will synthesize and study the properties of a caged fluorophore model and caged inhibitors of protein prenyltransferases including a caged farnesyltransferase inhibitor (FTI), a caged geranylgeranyltransferase inhibitor (GGTI) and a caged dual prenylation inhibitor (DPI) in solution and in cell culture models. In Aim 2, a phantom tissue model will be used to evaluate the efficiency of uncaging of a fluorphore and caged inhibitors of protein prenyltransferases at different depths. Finally, in Aim 3 we will test the efficacy of site-specific uncaging of a caged FTI, a caged GGTI and a caged DPI for inhibiting H-Ras- and K-Ras-stimulated transformation and tumor formation in both a mouse skin cancer model and an in vivo xenograft model. If successful, this approach could be a revolutionary step in improving cancer therapy.

The TRP superfamily comprises a large group of cation channels with homology to the Drosophila TRP channel, which plays a key role in phototransduction. Among the seven TRP subfamilies, the TRPP proteins are of particular interest because mutations in the founding member, PKD2, result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). TRPP channels are evolutionary conserved and in other organisms and play a key role in male fertility. A number of recent studies have demonstrated that members of the TRPP family can be found in ciliated structures where they are thought to participate in mechanosensory signal transduction pathways. Despite intensive investigation, however, there are numerous aspects of TRPP biology that remain controversial including the subcellular localization of TRPPs and the identity of proteins that participate in TRPP signaling pathways. Many attempts have been made to address these questions using heterologous expression systems. However, the results have not always been consistent and they have been difficult to verify in vivo. We have developed a Drosophila model system with a targeted mutation in the fly homologue of PKD2 (named amo for Almost There). Male flies carrying a null mutation in amo were found to have normal amounts of motile sperm but are infertile due to an inability of sperm to be stored in the female storage organs. In addition we find that Amo is concentrated at the tip of the sperm flagella suggesting a conserved role for TRPPs in axonemal structures. This Drosophila system offers us the opportunity to use a genetically tractable model organism to correlate in vitro findings with in vivo analyses. In this proposal we will use a multidisciplinary approach to address three unresolved issues in TRPP biology: 1) the developmental regulation of TRPP2 localization in vivo 2) The requirement of cation channel activity for TRPP2 function in vivo and 3) the identification of upstream and downstream components of the TRPP signaling cascade. We anticipate that these studies will provide new information about the critical functional and signaling properties of the TRPP class of ion channels.

The broad long term objective of the proposed research is to gain insight into the biological role of gamma delta T cell receptor (TCR) bearing lymphocytes. In part, lymphocyte functions are defined by the ligands their receptors can recognize. We have therefore undertaken to identify ligand for gamma delta TCRs and to define mechanisms of receptor-ligand interactions. In the proposed studies, we follow up on reports by others that an entire subset of (human) gamma delta T cells responds specifically to small non-peptidic ligands whose common denominator is their content of phosphate groups. The mechanisms and physiological relevance of this response have remained unclear, although the mycobacterial origin of some of these ligands implies a role in host resistance. We have recently found a response of murine gamma delta T cells (hybridonmas and clones) to similar anionic ligands, including certain phospholipids, polyanionic peptides and deoxyribonucleic acids. This murine response is likely analogous to the the human response, so that the murine system can be used as a model to assess physiological relevance through in vivo experimentation. Our studies on mouse gamma delta T cell responses have already revealed several intriguing features. Firstly, like the human response, the murine response involves an entire subset gamma delta T cell subset. A common denominator of the reactive cells is their expression of Vgamma1. Vgamma1contains peculiar clusters of basic amino acids which could provide binding sites for anionic ligands. Mutation of one such cluster reduced responsiveness to a phospholipid while not affecting other TCR-dependent reactivity. We now propose to investigate in more detail the role of the TCR in the response to anionic ligands. Secondly, the murine response does not appear to require ligand presentation, but is strictly dependent on the presence of serum. At least two serum factors must be involved, on that is required for the ligand response and one that is inhibitory. It is thus conceivable that serum factors control normal gamma delta T cell reactivity. We therefore propose to characterize the serum factors involved and to define their role in the gamma delta T cell response. Since most of our experiments so far have involved only hybridomas and clones, we also propose to establish conditions for reactivity of normal gamma delta T cells with anionic ligands. Thirdly, gamma delta T cells reportedly can support antibody switching, and the development of autoreactive antibodies in vivo. Having found that certain murine gamma delta T cells respond to DNA and other anionic ligands, we plan to inquire whether the same cells also promote the development of anti DNA and anti phospholipid antibodies.

ProjectSummary Themicroenvironmentplaysauniqueroleinthedevelopmentofmelanoma,whicharisesintheskin.Theskin displays externally visible signs of aging, such as decreased elasticity and a loss of collagen integrity. Much workhasfocusedrecentlyonhowchangesincollagencanaffecttumormetastasis,bothbiomechanicallyand biochemically. Data from several groups indicate that the cross-talk between stromal fibroblasts and transformed melanocytes is important for invasion, melanoma growth, and even therapy resistance. Our proteomicsanalysesindicatethatfactorsusedtocrosslinkcollagen,suchasHAPLN1aresecretedbyyoung, but not aged dermal fibroblasts, and Wnt5A is secreted by aged, but not young fibroblasts. The interplay between these molecules may contribute to age-related changes in collagen integrity. We hypothesize that changes regulated by age-related alterations in the extracellular matrix (ECM) initiate or promote a pro- metastaticprogram,andimpacttherapyresistance. We will query the contribution of aged skin biochemical and architectural contributions in governing melanoma?smetastaticprogressionaswellasresistancetotargetedtherapy.Weproposeanalternatetheory of matrix stiffness that hypothesizes that increasing stiffness will have a non-linear effect on metastatic progression,andtherapyresistance.Wewillusepathophysiologicallyrelevantinvitro3Dstromalsystems(3D skin reconstructions), animal models of melanoma, a novel simultaneous multi-channel immunofluorescent analysis (SMIA) of formalin-fixed and paraffin embedded (FFPE) human tissue cohorts in combination with a customized new software written for the bulk analysis and acquisition of SMIA-generated images, single moleculeRNAimagingcoupledwithhigh-throughputsinglecellimagingandsequencing,allofwhichwillfeed back into an increasingly intricate mathematical modeling for improved understanding and better patient personalized (i.e., metastatic and efficacy of drug treatment) prediction capabilities. We expect our data will reveal a synergistic picture of the mechanochemical interactions between the aged microenvironment and singulartumorcellsthattheindividualapproachescouldnothavedeciphered. This team project involves experts in the biology of melanoma metastasis, Wnt signaling and aging (Weeraratna), single cell RNA systems biology (Raj), tumor-stromal interactions and digital imaging quantitativeanalyses(Cukierman)andcomputationalandmathematicalpredictivemodeling(Shenoy).

The acute respiratory distress syndrome (ARDS) affects more than 125,000 patients in the US annually, and carries significant morbidity and mortality rates approaching 40%. Despite increased understanding of the initiating events leading to ARDS, therapies directed at these mechanisms have failed to improve the lives of patients with this devastating disease. Unintended bystander lung tissue injury and dysfunction from dysregulated acute inflammation are the hallmarks of ARDS. In health, acute inflammatory responses are terminated by specialized pro-resolving mediators (SPMs) that restrain the inflammatory response and signal for resolution. A new family of docosahexaenoic acid-derived SPMs from macrophages was recently uncovered in murine peritonitis and coined maresins (i.e., macrophage mediators in resolving inflammation). In work in progress, we have identified a new intra-vascular biosynthetic pathway for maresin 1 (MaR1) early after acute lung injury (ALI) that is pivotal to controlling lung inflammation and protecting lung function. Of note, at a later time point after ALI, MaR1 is produced via a different biosynthetic route that involves lung tissue macrophages. We hypothesize that MaR1 activates resolution pathways that are critical in ARDS. To investigate this hypothesis, we have proposed the following specific aims: 1) to determine restoration of lung function by MaR1 after ALI using established and innovative methods; 2) to determine the role of MaR1 on monocyte recruitment for resolution of acute lung injury; 3) to examine the role of MaR1 in macrophage programming, and the macrophage subsets that serve as the cellular source of MaR1 during resolution of acute lung inflammation. We will use a clinically relevant murine model of ARDS for discovery and mechanistic experiments, and validate these findings in human samples from a Registry of Critical Illness. Under the guidance of his mentor Dr. Bruce D. Levy, the principal investigator has developed a five-year training program that includes tailored didactic activities, and mechanisms to ensure continued mentoring and supervision, to develop the skills necessary to become a successful physician-scientist in the field of pulmonary and critical care medicine. The candidate's training will be overseen by an advisory committee with expertise related to key areas of this proposal including ALI, resolution of inflammation, monocyte/macrophage biology, and functional imaging. The proposed career development plan will establish scientific foundations and provide the additional training necessary to achieve the principal investigator's ultimate goal of becoming an independent investigator studying the biologic basis for resolution of ARDS.

ABSTRACT The delta cell of the pancreatic islet has been barely investigated despite secreting somatostatin (SST), a powerful inhibitory peptide that is essential for the homeostasis of different tissues. In the islet, SST inhibits the secretion of insulin and glucagon, but little is known about the mechanisms that activate delta cell and SST secretion. No other roles have been investigated or even proposed for the delta cell in islet biology and glucose metabolism. The nervous system, endocrine organs, and local neighboring cells, including immune cells, could potentially interact with the delta cell as a ?switch? or ?brake? to modulate the function of the whole islet. It is clear that the relevance of this powerful inhibitory component in the islet has been overlooked. The position of the delta cell as a key element in the regulation of islet hormone secretion needs to be addressed to understand how islet hormone secretion is regulated. The general hypothesis of this proposal is that the delta cell is a signaling hub where paracrine, immune and nervous signals converge and are integrated to set the level of SST secretion that ultimately modulates overall islet activity. This hypothesis will be tested through two related but not interdependent aims. In Aim 1 we will study the role of GABA as a key paracrine signal in delta cell function. Our previous results suggest that delta cell function is tightly adjusted by GABA, a paracrine signal secreted by beta cells through glucose independent mechanisms. GABA could therefore modulate delta cell responses to other local signals. We will use human islets and, when translatable, mouse islets to determine (1.1) the effects of endogenous GABA on the magnitude of basal SST secretion, (1.2) the effects of endogenous GABA on delta cell responses to glucose and other, local signals, and (1.3) how loss of endogenous GABA signaling contributes to the changes in somatostatin secretion in high BMI and type 2 diabetes. In Aim 2 we will examine the role of the delta cell and SST signaling in islet inflammation. Our preliminary findings indicate that the delta cell responds to signals from the immune and neural compartments and secretes SST to counteract inflammation and neuroinflammation. Thus, the delta cell could protect the islet from unchecked and damaging immune responses. We will test (2.1) the effects of cytokines and proinflammatory neuropeptides on delta cells, and (2.2) the effect of SST on local immune cells and sensory nerves. We will use a combination of novel in vitro (isolated islets), ex vivo (pancreatic tissue slices), and in vivo (intraocular islet grafts) approaches together with pharmacological tools, optogenetic stimulation, cell ablation, functional imaging and systemic metabolic readouts to study how delta cells are activated and how they influence the sensory nerves and surrounding endocrine and immune cells. We expect our studies to further our understanding of the circumstances under which the delta cell is recruited to influence endocrine and immune cells in the islet. If SST?s role as an immunomodulator is validated, it is likely that the delta cell will be reconsidered as a key element in the natural history of diabetes. Therefore, important advances in our understanding of the pathogenesis of diabetes could be expected.

The overall goal of this research is to develop a mechanistic understanding of TGN and vacuole membrane protein sorting and assembly in the yeast Saccharomyces crevisiae. Yeast has proved to be an excellent model system, both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. Genetic analyses have revealed that a yeast trans-Golgi network (TGN) membrane protein is retained by retrieval, and yielded a large collection of GRD genes involved in the retention and retrieval of yeast TGN membrane proteins. Using deletion and site-directed mutagenesis, the static retention signal on the yeast TGN membrane protein dipeptidyl aminopeptidase A (DPAPA) will be identified and characterized. The very large collection of Grd proteins that has been recently identified will be used to investigate the molecular basis for static retention in the TGN and retrieval of TGN membrane proteins from the prevacuolar compartment. Assays will be developed to determine functions for the individual Grd proteins in static retention or the retrieval step. Genetic analysis has also revealed a large collection of yeast genes encoding not only subunits of the yeast vacuolar proton-translocating ATPase (V-ATPase), but also three genes encoding proteins that are not subunits of the enzyme but instead are required for assembly of the V-ATPase. Using both genetic and biochemical approaches, the V- ATPase assembly factors will be characterized to determine whether they form a stable complex in the ER and whether this complex depends on the continued synthesis of substrates (the v-ATPase subunits) for the assembly reaction. The individual roles for the three V-ATPase assembly factors will be investigated by cross-linking, co- immunoprecipitation and density gradient centrifugation approaches to determine their mechanistic involvement in V-ATPase assembly. To develop a detailed molecular understanding of the assembly pathway of the V-ATPase complex, the kinetics and degree of association between the assembly factors and the V-ATPase integral membrane subunits will be monitored early in the assembly reaction. Additional functions for the assembly factors will be investigated such as escorting the V- ATPase into ER derived vesicles and chaperoning the complex to the cis-Golgi.

The purpose of this project is to develop methodology for analyzing molecular population genetics data. Work has focused on the use of nonparametric methods for localizing susceptibility loci for complex diseases in humans. A strategy for mapping disease loci is to test for association with case-control samples, and follow up with a family-based test of association to confirm positive results. One way to improve the power of the first test is to increase sample size by combining the case-control and family data. To deal with the correlation between the two tests that this strategy introduces, we have developed a Monte Carlo procedure that always gives a valid test. For late-onset diseases it is common that parental genetic data are not available. Three recent family -based tests of association and linkage utilize an unaffected sibling as a surrogate for untyped parents. We have extended one of these tests and have compared the properties of the four tests in the context of a complex disease for both biallelic and multiallelic markers, as well as for sibships of different sizes. We have also examined the consequences of having some parental data in the sample. Two family-based tests of association and linkage to quantitative traits were developed that do not use parental data. One procedure assumes a mixed effects model in which the sibship is the random factor, the genotype is the fixed factor and the continuous phenotype is the dependent variable. Covariates can be easily accommodated and the procedure can be implemented using available statistical software. The second is a permutation-based procedure. We conducted a simulation study to illustrate the relative power of each test for a variety of quantitative genetic models. Optimal marker selection for the transmission/disequilibrium test (TDT) when applied to admixed populations was investigated. We found that collapsing a microsatellite marker to two alleles can increase the power of the TDT, and a method was developed for finding the optimal collapsing the uses estimates of alleles frequencies in the admixed population.

As malaria continues to remain a major public health challenge in much of the world, the loss of effective and affordable antimalarials through resistance development in malaria parasites is a continuing grave concern. With the parasite genome sequence at hand, there is a renewed hope that a reasonable drug pipeline can be established for dealing with these challenges. A critical goal of basic molecular and cell biological research on malaria parasites is to explore appropriate targets for developing new antimalarials. Our group has previously identified and validated the parasite mitochondrion as an attractive target for antimalarial drugs. Mechanisms of action and resistance to one of the currently used drugs, atovaquone, were elucidated by work in this group. A new class of compounds being developed also appears to target mitochondrial physiology in malaria parasites with related but distinct mode of action from that for atovaquone. One of the aims for the renewed project will be to investigate this series of compounds to derive useful mechanistic details that could aid in further drug development. Biochemical and genomic insights provided by recent work suggest that the parasite mitochondrion is physiologically quite distinct from its host's organelle. For the renewed project, a series of approaches will be taken to understand roles played by some of the canonical mitochondrial processes in different stages of malaria parasite life cycle. Through improved gene disruption methods, some of the questions of long standing regarding the role of the mitochondrion in malaria parasite biology will be addressed. A surprising finding has been the ability of malaria parasites to survive for significant length of time even with inhibited mitochondrial electron transport and collapsed mitochondrial membrane electropotential. Work to be conducted in the renewed project will address mechanisms by which parasites are killed by anti-mitochondrial drugs. Overall, research to be conducted during the next funding period will aim to advance the underlying goals of this project: to derive a deeper understanding of the functional significance the mitochondrion in malaria parasites and the mechanisms by which interference with the mitochondrial functioning leads to parasite demise. [unreadable] [unreadable] [unreadable]

Despite progress in defining the pathophysiology of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and advances in supportive care, mortality remains high at 30-40%, and no specific pharmacological intervention can be recommended for blocking progression of ALI. Thus, recent attention has focused on identifying pathways that promote resolution of ALI. Our new data show that E-cadherin shedding is associated with pulmonary recruitment of a specialized population of dendritic cells (DC) expressing CD103 (aE[unreadable]7-integrin), a leukocyte cell-surface receptor for which E- cadherin is the only known ligand. In mice with ALI induced by Pseudomonas aeruginosa infection or bleomycin toxicity, mice lacking CD103 showed delayed resolution of neutrophilic inflammation and had higher mortality than wild type mice. In vitro, soluble E-cadherin altered the LPS-induced cytokine profile of bone marrow derived dendritic cells to an anti-inflammatory phenotype, and mice lacking CD103 had far fewer suppressor regulatory T cells in the bronchoalveolar lavage (BAL) than wild type mice during resolution of P. aeruginosa pneumonia in vivo. These data support our overall hypothesis that E-cadherin-CD103 interactions promote resolution of acute lung injury. Using complementary in vivo and in vitro approaches, we will define the mechanisms by which E-cadherin programs CD103+ DC to resolve pulmonary inflammation. Specific Aim 1 will assess how E-cadherin-CD103 interactions control dendritic cell function. Specific aim 2 will determine the anti-inflammatory pathways activated in pulmonary CD103+ DC in the injured lung, and Specific Aim 3 will elucidate the downstream effector mechanisms by which CD103+ DC resolve acute lung inflammation. Significance: Our long-term goal is to understand how lung epithelial injury responses promote repair, and the results of the proposed studies should form the basis for new strategies aimed at enhancing endogenous lung repair in this clinical conditions for which current therapy is quite limited. PUBLIC HEALTH RELEVANCE: Acute lung injury is a common and severe cause of lung disease with no specific pharmacologic treatment. In many patients, acute injury progresses to a chronic lung injury characterized by persistent lung dysfunction and debilitation. Although much has been learned about the biological processes that incite lung injury, little is known about the innate mechanisms that resolve acute lung injury. Consequently, the results of the planned studies should enhance fundamental understanding of how epithelial and immune cells interface to limit progression of lung injury and may identify new therapeutic strategies for this difficult clinical problem.

One of the pathological hallmarks of Alzheimer's Disease (AD) is the senile plaque, principally composed of the A? peptide. A?1-42, the 42 amino-acid peptide fragment of the amyloid precursor protein (APP), has a striking propensity to aggregate into amyloid fibrils within plaques. A? aggregation appears to be concentration-dependent;thus, mechanisms that regulate extracellular (or interstitial fluid, ISF) A? levels may be important for amyloid plaque formation. Under normal physiological conditions, A? exists in brain extracellular fluids (ISF and CSF). This steady-state level of A? is maintained by a balance between production and clearance. While genetic mutations that lead to familial AD point to A? over-production as a cause for disease, it has been suggested that the much more prevalent sporadic form of AD may be caused by impaired A? clearance. A growing list of proteases are known to degrade soluble A? (sA?) in vitro, and inhibition or deletion of some of these proteases in vivo have resulted in increased steady-state levels of brain A? in mice, including neprilysin (NEP), insulin-degrading enzyme (IDE), endothelin-converting enzyme (ECE). We have recently published that the extracellular protease, matrix metalloproteinase-9 (MMP-9), is capable of degrading sA? in addition to fibrillar A? (fA?) in vitro and compact amyloid plaques in situ. The ability to degrade fA? is an important characteristic of MMP-9 which is lacking in most other A?-degrading proteases. MMP-9 immunoreactivity is seen in reactive astrocytes surrounding amyloid plaques of aged APP/PS1 mice. Furthermore, we have recently found that gene deletion of mmp9 in APP/PS1 mice resulted in an increase in amyloid plaque load. In this grant application, we will test the hypothesis that MMP-9 directly degrades extracellular A?, thus attenuating plaque pathogenesis in APP/PS1 mice. Furthermore, we will test the hypothesis that inhibiting TIMP-1, an endogenous inhibitor of MMP-9 (i.e. disinhibiting MMP-9), will further attenuate plaque pathogenesis. If true, TIMP-1 may be identified as a potential therapeutic target for the treatment of AD. In Aim 1, we will determine if MMP-9 activity attenuates amyloid plaque accumulation by directly degrading A? in APP/PS1 mice. In Aim 2, we will determine if MMP-9 directly catabolizes brain extracellular A?, using in vivo microdialysis in APP/PS1. In Aim 3, we will determine if MMP-9 regulates the growth/regression of pre- existing compact plaques in APP/PS1 mice imaged using intravital 2-photon microscopy. In Aim 4, we will determine if TIMP-1 inhibits MMP degradation of A2, accelerating amyloid plaque formation. If true, TIMP-1 may be identified as a potential therapeutic target for the treatment of AD. PUBLIC HEALTH RELEVANCE: This R01 grant application proposes to study the role of the protease MMP-9 and its endogenous inhibitor, TIMP-1 in regulating amyloid plaque growth in a mouse model of Alzheimer's disease. The central hypothesis of the grant is that MMP-9, secreted by astrocytes surrounding amyloid plaques, regulates amyloid plaque growth/regression by degrading soluble and fibrillar A? and amyloid fibrils of which plaques are comprised. If the hypothesis is true, potential targets for intervention to degrade plaques may be identified.

DESCRIPTION: It is stated that the overall objectives of this research are to apply new methodology for the introduction of nitrogen functionality into organic molecules to the synthesis of the amaryllidaceae alkaloids pancratistatin, crinine and lycorine, aminoglycosides, aminonucleoside precursors, AZT variations, aminocyclitols, and the insecticidal alkaloid stemofoline. The principal investigator notes that all of the routes are short and make use of the alpha- and beta-azidonation chemistry and that all of the above compounds have interesting biological properties.

It was demonstrated by Geschwind and Levitsky (1968) that large left-right asymmetries exist in the cerebral cortex of adult human brains in areas which are probably involved in language functions. These results have been confirmed in hunam infants and fetuses, and in primitive man and non-human primates. This research proposes to study the brain for cytoarchitectonic asymmetries in Nissl preparations in the areas which show gross right-left differences. Furthermore the project will establish pigment architectonic methods in order to study microscopic asymmetries more effectively and quickly. Animal models will be sought, and application of asymmetry data to clinical radiological methods will be explored.

DESCRIPTION: (Applicant's Abstract) This proposal describes a population-based cohort study aimed at describing the family aggregation of age-related cataracts, visual function, intra-ocular pressure, and systemic characteristics of aging, cross-sectionally and longitudinally The population resides in Beaver Dam, Wisconsin. Subjects were 43-86 years of age at the first examination in 1988-90 Standardized protocols for interviews, examination, blood tests, and ocular photography and grading were performed at first examination and two follow-up examinations in 1993-95 and 1998-2000. Within the baseline population (n equals 4,926) we identified 587 sibling groups composed of 1,403 persons. Study participants have had standardized examinations at the three examination cycles. Information has been collected regarding cataract, other age-related eye diseases, medical conditions, medication usage; blood pressures, height and weight have been measured, as have several blood parameters baseline. At the follow-up visits, other measures of frailty (estimated by hand grip strength, slower timed walk, decrease peak expiratory flow) have been added to the protocol. Mortality has been carefully monitored and death certificate information has been obtained. We will investigate whether changes in cataract, as well as other ocular and systemic factors, aggregate in families and whether families that display greater progression of cataracts are at greater risk of systemic morbidity, frailty and mortali1y. During the course of the study, we have had follow-up interviews during which we have become aware of about 400 avuncular, parent and cousin relations in the community. We will complete our family pedigree information prior to the beginning of this grant. We propose extensive family aggregation analyses using generalized estimating equations and, when appropriate, commingling and segregation analyses. This grant will capitalize on data previously collected from grants funded by the National Eye Institute. We currently have no funds to support the proposed analyses. We believe this project offers the possibility of examining important hypotheses in a large group of families in a population-based framework.

The understanding of digestion and absorption of dietary amino acids is incomplete. This study outlines a series of studies which will investigate the availability and metabolism of amino acids given as an enteral load, either as amino acids per se or as intact protein. This protocol addresses metabolic issues with clinical consequences regarding nutrition and the use of amino acid supplements.

Programmed cell death occurs in many tissues of our bodies throughout life. The quick and efficient removal of dying cells is a critical step that protects neighboring cells, and is also an important part of tissue remodeling and wound healing. The failure to remove apoptotic bodies has been implicated as a cause for certain types of chronic inflammation and autoimmune disorders. In mammals, macrophages and dendritic cells, as well as many other cells types can carry out the engulfment of apoptotic corpses. While several surface receptors that participate in the recognition of apoptotic cells have been identified, relatively little is known about the intracellular signaling events that regulate the uptake process itself and its downstream consequences. Recent cloning of the engulfment genes in the model organism C. elegans, and the identification of their homologues in mammals, has provided an exciting opportunity to delineate this process in better detail. The major focus of this proposal will be to determine the signaling events mediated through one of the evolutionarily conserved engulfment proteins GULP and to gain a better molecular understanding of the process of engulfment of apoptotic cells in mammals. CED-6 was identified as a candidate cytoplasmic adapter protein involved in engulfment of apoptotic cells in C. elegans, ced-6 is in the same functional genetic pathway that includes two other genes, ced-1 and ced-7, both of which encode membrane proteins. Elegant worm genetic studies have shown that ced-6 expression is required in the engulfing cell and not in the apoptotic cell and that ced-6 functions downstream of ced-1. Our laboratory has cloned the murine homologue of ced-6, denoted gulp, and has performed initial characterization of GULP protein in mammalian engulfment. In Aim 1, we will use the J774 macrophages and primary macrophages to perform structure: function studies of GULP and understand the role of GULP in anti-inflammatory cytokine production following engulfment. Several lines of evidence suggested CD91/LRP 1 as a GULP interacting protein, and LRP likely represents the CED- 1 homologue in mammals. The focus of Aim2 will be to determine the biological significance of GULP: LRP interaction through the use of LRP deficient cells lines in vitro, and conditionally targeted LRP deficient mice in vivo. To understand GULP function at the whole animal level, in Aim3, we will generate GULP knockout mice, as well as knock-in mice in which the LZ region is specifically mutated. In these mouse studies, we will determine the effect on engulfment due to the absence of GULP, or disrupted GULP function, and any potential effects on autoimmunity due to defective engulfment.

Multifaceted approaches are needed to limit carbapenem-resistant Enterobacteriaceae (CRE) infections, including Klebsiella pneumoniae, which have emerged as a worldwide public health problem. These multidrug- resistant organisms are associated with high morbidity and mortality that often exceeds 50%, as highly effective and non-toxic treatment regimens are lacking. While intestinal colonization with CREs has been proposed as a potential risk factor for infections during CRE outbreaks, its actual contribution to infection remains incompletely understood. Moreover, there is a fundamental gap in knowledge on how these antibiotic resistant organisms transition from colonization to infection within affected hosts. The long-term goal of this application is to elucidate at the bacterial genome level how CRE infections emerge and spread. Understanding these processes is critical to developing intervention and real-time clinical monitoring approaches to limit the impac of CRE infections at an individual and population level. We will focus our study on patients after liver transplantation who are at very high risk for CRE infections and adverse outcomes. Our central hypothesis is that the intestine provides a microenvironment in which CRE can expand and adapt with small genetic variations and subsequently lead to infections. To address these questions we propose to establish a cohort study of adult patients undergoing liver transplant, and track CREs within affected patients and across the hospital. In this prospective cohort we will enroll 300 patients pre-transplant, collect stool samples to ascertain intestinal colonization pre- and repeatedly post-transplant, and assess patients for CRE infections over a 6-month period. Our study design will allow us to execute the following Aims: 1) Define the rate and role of colonization on CRE infection in liver transplant patients; 2) Evaluate the within-host evolutio from CRE colonization to infection; and 3) Investigate the spread of CRE between liver transplant and other patient populations in the hospital. In Aim 1 we will test the contribution of CRE colonization to infection and characterize outcomes using Kaplan-Meier survival analyses and a Cox proportional hazard model. In Aim 2 we will apply 16S and whole-genome sequencing to answer whether CRE colonization dominance develops prior to infection and how modifiable risk factors (e.g. certain antibiotics) relate to these adaptations. We will also define the clonal diversity of colonizing CRE and assess whether infectious isolates arise from dominant colonizing clones or acquire novel virulence traits. In Aim 3 we will assess the within-hospital evolution and spread of CRE infections by extending whole-genome sequence analyses to infections that occurred in non- liver transplant patients. Our multidisciplinary approach is innovative in its combination of high-resolution genomics with detailed epidemiological investigations to monitor the evolution of CRE infections in real time. This research is significant with direct translational impact in establishing a framework to track the emergence of multi-drug resistant Enterobacteriaceae and to ultimately devise novel containment strategies for CREs.

The final common pathway of electrical excitability in neurons is generation of conducted action potentials. Action potentials in nerve and muscle are initiated by activation of voltage-gated sodium channels, and the threshold and frequency of firing which encode information in the nervous system are critically dependent on sodium channel properties. The ion conductance activity of sodium channels is controlled on the millisecond time scale by two distinct but coupled gating processes: activation and inactivation. Activation controls the voltage- and time-dependence of conductance increase in response to depolarization, and inactivation controls the voltage- and time- dependence of the subsequent return of the sodium conductance to the basal level within one millisecond. Both processes are essential for normal electrical excitability of nerve and muscle cells, and elucidation of their molecular basis is a major challenge for molecular neurobiology. The essential nature of the inactivation process is illustrated by the striking effects of dominant mutations which impair this process in the periodic paralyses of skeletal muscle and long QT syndrome in the heart. One can anticipate that similar hyperexcitability syndromes may be caused by mutations in brain sodium channels and contribute to both inherited and spontaneously arising forms of epilepsy. In the current project period, we have made substantial progress on several topics related to the molecular basis for sodium channel inactivation, its modulation by second messenger- activated protein phosphorylation and by peptide neurotoxins, and its interaction with pore-blocking drugs. We have discovered the key amino acid residues in the inactivation gate which are essential for its function, elucidated the three-dimensional structure of the key region of the inactivation gate, identified candidate residues involved in formation of the inactivation gate receptor, defined the phosphorylation sites responsible for regulation of channel gating by protein phosphorylation initiated by the dopamine D1/cAMP-dependent protein kinase signaling pathway and the muscarinic acetylcholine/protein kinase C signaling pathway in neurons, and identified important components of the receptor site for scorpion toxins and local anesthetic drugs which interact with the inactivation process. In the next project period, we propose to build on this foundation of molecular information about sodium channel gating and further define the molecular basis of its physiological and pharmacological regulation. Our objectives are to elucidate the molecular interactions of the inactivation gate with the putative inactivation gate receptor, to define the three-dimensional structure of the inactivation gate, to probe the molecular mechanisms by which protein phosphorylation influences sodium channel gating and define the interactions of the relevant phosphorylation sites with the inactivation gate, to determine the molecular basis for high affinity binding of local anesthetics to inactivated channels and for molecular trapping of these drugs in their receptor site by closure of the channel activation and inactivation gates, and to analyze the coupling of movements of the S4 voltage sensors to voltage-dependent activation and inactivation using alpha- and beta-scorpion toxins as specific molecular probes. These proposed studies will give new insight into the molecular mechanisms of sodium channel gating and its modification by second messenger-activated protein phosphorylation and by drugs and neurotoxins.