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Genetic or mutational theories of cancer are supported by the mutagenic nature of most nonviral carcinogens, the age distribution of many cancers, the existence of genetic cancers such as bilateral retinoblastoma and the increased cancer incidence in some diseases as xeroderma pigmentosum. The development of techniques of gene isolation by DNA-mediated gene transfer in cultured animal cells provide a powerful tool for the analysis of the genetics of cancer. It has been shown that DNA from some human tumor-derived cell lines can transform normal cells in culture. We have also found that tumor cell lines derived from different human lung and colon carcinomas contain the same or very similar transforming gene. This gene has been found to be the human homologue to the Kirsten murine sarcoma virus oncogene (vi-Ki-ras). Our experiments describe the isolation of the transforming gene present in human colon and lung carcinomas. At present, we have molecularly cloned in 47.1 vectors over 40Kb of the c-Kirsten ras-2 oncogene from NIH3T3 tertiary transformants induced by DNA of two human lung tumors propagated in nude mice. The cloned sequences include the 5' end of the oncogenes (containing adjacent mouse host sequences) and four coding exons. Work is in progress to isolate the 3' end of the oncogenes including the fifth exon. We are currently sequencing the cloned exons of the genes in order to determine the mutation(s) which could be responsible for the activation of this oncogene in the human tumor cells. The availability of these genes will allow the comparative study of the types of human carcinomas containing the same transforming gene; the study of their structure and function, in an effort to understand the mutational events involved in these particular types of tumors; and the study of their role in the tumorigenesis in humans.

(Adapted from investigator's abstract) The reliability of surgical and examination gloves has become a major concern among health care professionals as HIV and other viral diseases have spread. Current testing regulations and methods are not adequate to insure the level of protection which is now being demanded by the industry. The proposed research will test the feasibility of a production test using volatile halogenated compounds to detect and quantify leaks in surgical and examination gloves. Theoretical calculations show that these gases can detect holes that are one order of magnitude smaller that viruses. A halogen tracer gas test could provide a cost-effective and reliable way to test surgical and examination gloves. Research will focus on the development of the apparatus and methodology to test gloves, quantification of the technique parameters, and proposals for new standards.

The Tat gene of human immunodeficiency virus (HIV) plays a central role in the activation and life cycle of HIV. Tat exerts its effects at the level of transcriptional initiation and elongation. We have previously reported the direct physical interaction between the HIV-1 Tat protein and the basal transcription factor, TBP/TFIID. Affinity chromatography demonstrated that wild type Tat, but not a transactivation mutant of Tat, was capable of depleting TBP/TFIID from cell extracts. These experiments represented the first demonstration of a basal transcription factor that binds, in an activation-dependent manner, to Tat. We now report that the Tat-TBP interaction can be detected in HIV-1-infected cells. The domain of TBP interacting with Tat has been mapped from amino acids 163 to 196 using deletion and site-specific mutants of TBP. This domain of TBP, which includes the H1 and S2 domain, is distinct from the H2 binding site for other activator proteins such as E1A. The interaction of Tat with TFIID regulates the binding of accessory proteins to TFIID. Tat stabilizes the interaction of TFIID with TFIIA in a gel shift assay. In addition, Tat competes for Dr1 interaction with TBP. Our results suggest that the basal transcription factor, TBP/TFIID, represents an important regulatory molecule in HIV transcription. The human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein is essential for efficient viral gene expression and virus replication. Tat interacts with the basal transcription factor TFIID. This interaction is mediated through the amino acid 36-50 core domain of Tat. We now demonstrate that soluble peptide analogs of the Tat core domain are able to effectively block LTR transactivation. In cotransfection experiments, Tat peptide analogs inhibited Tat transactivation of an HIV-1 LTR-CAT reporter construct up to 80-fold. Inhibition of control promoters was approximately two-fold. Tat peptide analogs inhibited HIV virus replication by 85 percent in latently infected U1 cells induced with Tat. While both short and long peptide analogs (amino acids 36-50 vs 36-72) inhibited Tat transactivation in transient assays, the short peptides were more effective inhibitors of virus replication in U1 cells. Furthermore, U1 cells treated with the Tat peptide analog showed markedly delayed virus transmission when co-cultivated with parental U937 cells. The Tat peptide analog did not decrease expression of cellular genes including beta-actin, GAPDH and histone H2Bb.

Influenza, a vaccine-preventable disease, remains as an important cause of morbidity and mortality worldwide. All age groups are affected, but children experience the highest disease incidence while adults suffer the most serious disease complications and related mortality. The burden of seasonal influenza epidemics and the continuous threat of a potential pandemic influenza highlight the need for effective preventive strategies. During influenza epidemics, children are typically affected early and they serve as disease vectors, introducing influenza into the community. Influenza vaccines have proven to be efficacious in the prevention of disease in vaccinated individuals and it has been hypothesized that immunization of a significant proportion of children could have major beneficial effects in the community, through the interruption of influenza transmission. Therefore, school-based influenza immunization programs have been proposed as prevention models. Nevertheless, the effectiveness of these approaches has not been convincingly demonstrated in large scale interventions. If proven effective, school-based influenza immunizations could become one major strategy for prevention of both seasonal and pandemic influenza. In October 2005, a large school-based influenza immunization campaign was initiated in Knox County, TN. The campaign was successfully implemented, immunizing 24,281 (46%) school-aged children attending public schools in the county. The campaign achieved at least similar coverage in the subsequent 2006-2007 influenza season. Although this campaign was set up as a feasibility demonstration project, its impact on disease outcomes has not been examined. We hypothesize that this large 2-year intervention decreased the incidence of influenza related diseases in both vaccinated and unvaccinated Knox County residents. Vanderbilt investigators are currently performing active viral surveillance in Knox County (intervention) and Davidson County (control) to compare viral activity during the second year of the campaign (2006-2007 season). We propose to perform additional and complementary analyses using alternate data sources to measure the overall intervention effectiveness (direct and indirect effects) over both intervention years. We will utilize large electronic databases that systematically record healthcare encounters in the State of Tennessee, and the infrastructure and expertise of the New Vaccine Surveillance Network (NVSN), a prospective surveillance system for viral infections in children that is currently operating in Tennessee. The proposed research and career development plan complement Dr. Grijalva's training in Medicine, Public Health and Epidemiology. Furthermore, this proposal supports his efforts to develop the expertise needed to become an independent investigator focused on prevention of influenza-related morbidity and mortality and on vaccine program and policy evaluations. [unreadable] [unreadable] [unreadable]

We propose a high risk/high impact study to develop an innovative circulatory support strategy using an implantable pump for the treatment of single functional ventricle. The most common form, hypoplastic left heart syndrome, in which the left ventricle fails to form in a way that is ever functional, is the leading cause of death from all birth defects in the first year of life. Currently, surgical repair is performed with three separate operations. In the first operation, blood flow to the lungs must be derived from a systemic-to-pulmonary arterial shunt to overcome the potentially elevated newborn pulmonary vascular resistance. The shunt, however, makes this procedure notorious for instability and mortality. In the second and third operations, the shunt is disconnected, and blood flow to the lungs is accomplished by connecting the vena cava to the pulmonary arteries. Stability and survival are greatly improved. Unfortunately, the second and third procedures cannot be safely performed in the newborn due to the unsolved obstacle of achieving passive blood flow through the reactive pulmonary circulation. We propose a unique application of a blood pump to temporarily augment flow from the great veins through the lungs until the potential for reactivity resolves. This process normally occurs most rapidly by 2 weeks and is nearly complete at 8 weeks of life. The problematic shunt is avoided, and the newborn pulmonary vessels are perfused by a low-pressure, high-volume flow from a systemic venous source similar to normal right ventricular hemodynamics. We hypothesize that under these conditions, the neonatal pulmonary arteries will mature normally. Once the transitional pulmonary vasculature has remodeled sufficiently to permit a systemic venous source to perfuse the lungs passively, the pump can be safely removed. We further hypothesize that the neonate will tolerate the systemic venous pressure elevation required to permit passive venous flow through the lungs. Using a newborn animal model of univentricular circulation, we will test these hypotheses through the following specific aim: To transition an assisted univentricular circulation (cavopulmonary pump + systemic ventricle) to an unassisted univentricular circulation in which the lungs are perfused by passive vena caval flow alone. We have generated pilot data which support feasibility of this innovative technique using currently available technology. We expect that successful completion of this approach will convert the current high risk three-stage surgical strategy to a much safer single-stage paradigm of newborn Fontan palliation of single ventricle cardiac disease.

Humans harbor a substantial Natural Killer (NK) cell compartment in their secondary lymphoid organs. These NK cells are enriched for immunoregulatory CD56brightCD16- cells, which are preferentially activated by dendritic cells (DCs). Our preliminary data demonstrate that NK cell activation by DCs restricts B cell transformation by the Epstein Barr virus (EBV) in vitro, especially when the NK cells are derived from tonsils, the secondary lymphoid organ of primary EBV infection. EBV is a human tumorvirus, which establishes latent infection in more than 90% of the human adult population. It causes tumors of epithelial and B cell origin in immune competent, and at increased frequencies in immune compromised individuals. The characterization of innate immunity to EBV is of particular interest, because failure of this initial immune control might result in increased viral titers and massive T cell expansion during primary immune responses, resulting in infectious mononucleosis. In order to characterize the role of DC/NK cell interactions during EBV infections, we plan to: 1. Characterize innate NK cell activation during EBV infection. We will dissect DC/NK cell synapse formation, DC activation by EBV derived dsRNA, and tonsillar DC subsets for their activation by EBV. 2. Analyze the protective effector functions of tonsillar NK cells. We will focus on cytokine production, cytotoxicity and assistance in protective T cell polarization by NK cells after DC activation via EBV infection. 3. Investigate EBV infection in vivo. In order to translate our in vitro findings into an in vivo model of primary EBV infection, we have reconstituted human immune systems in immune compromised mice, which could mount primary protective immune responses against EBV. This model will be explored to investigate NK cell expansion and activation during primary EBV infection, the contribution of NK cells to innate immune control of EBV, and their assistance in T cell polarization of adaptive immune control of EBV. These studies will characterize the mechanisms of NK cell activation by DCs, and protective NK cell functions against B cell transformation by oncogenic EBV in vitro and in vivo. Knowledge of the establishment of protective EBV specific immune control might suggest vaccination strategies against common EBV associated tumors like nasopharyngeal carcinoma and Hodgkin's lymphoma. PUBLIC HEALTH RELEVANCE: Epstein Barr virus (EBV) causes tumors of epithelial and B cell origin in the human population, like Hodgkin's lymphoma and nasopharyngeal carcinoma. This proposal plans to investigate how two cell types of the innate immune system, dendritic cells and Natural Killer cells, interact to initially control EBV and influence other immune cells to establish life-long protection from the development of EBV associated malignancies. An understanding of these initial events during the establishment of EBV specific immune control will help us explain why some individuals develop symptomatic acute infection with EBV, called infectious mononucleosis, and identify immune compartments that should be harnessed to efficiently vaccinate against EBV associated tumors.

Approximately 28,000 individuals in North America have a subarachnoid hemorrhage from ruptured aneurysm each year. Most are young, healthy individuals with good neurological function after the hemorrhage who have the potential for returning to a vigorous and productive life. Many will develop devastating neurological deficits or perish, mostly from vasospasm and bleeding. Less than 60% of those who reach hospital will have a favorable outcome. There is considerable room for improvement. The long term objectives of this study are to decrease the mortality and morbidity of aneurysmal subarachnoid hemorrhage, principally by obtaining information which will lead to more effective management strategies for vasospasm and rebleeding. One approach entertained for decreasing these complications is early surgery which has many theoretically attractive features but remains one of the most controversial topics in neurosurgery. Accordingly, the principal aim of this study is to determine at which interval after subarachnoid hemorrhage surgery results in the most favorable outcome. Other specific aims include: determination of risk factors for rebleeding and the timing of these disastrous events; determination of the safety and effectiveness of antifibrinolytic agents and development of an administration schedule which optimizes the actions in preventing rebleeding and minimizes ischemic neurological complications; documentation of the diagnostic and prognostic value of CT scanning in subarachnoid hemorrhage; and development of a more reliable and universally acceptable neurological grading scale for patients with ruptured aneurysm.

Although the fine structure of malarial parasites has been studied by many investigators, there remain many unanswered questions. Re-examination of the morphology of the parasites by the high voltage and scanning electron microscopes has been rewarding for understanding of the various subcellular organelles of malarial parasites. During the past year, we studied by electron microscopy the effect of immune serum on sporozoites of P. cynomolgi and P. berghei. When the sporozoites were incubated in immune serum, the surface coat became very thickened, while normal serum showed little effect on the sporozoite. Scanning electron microscopy indicated that the anterior end of the parasites was free of coat deposition and that the circum-sporozoite precipitate (CSP) reaction was resulted from coat deposition at the posterior end of the sporozoites. For the coming year, we will study microgametogenesis of P. gallinaceum and P. berghei and the effect of immune serum on the microgametes by electron microscopy.

The development of safer and more effective antipsychotic drugs requires the availability of sensitive probes of the dopamine (DA) receptor, which is a site of action of these agents. Research Biochemicals proposes a program to prepare novel substances with selective agonist or antagonist action at specific DA receptors for use by researchers in neuropharmacology. The objectives of Phase I research are: 1) the synthesis of novel aporphines and related compounds with irreversible binding activity at DA receptor sites. Structures will be formulated with particular emphasis on selective activity at presynaptic DA autoreceptors. Irreversible binding will be accomplished with alkylating and/or photoactivated functional groups. 2) the incorporation of highly selective DA receptor ligands into a practical affinity column. The prototype of this product will be a clebopride-functionalized support for use in isolating DA D2 receptors. 3) the preparation of hitherto unavailable enantiomerically pure pairs of known DA agonists and antagonists to permit stereochemical exploration of receptor surfaces. The new products generated in Phase I studies will be evaluated for commercial introduction to the expanding neuroscientific community.

Recent studies in our laboratory indicate the presence of reactivity in normal and abnormal human sera by radioimmuno-precipitation proteins from type-C viruses of mice, cats and primates, analogous to reactivities that have been observed in normal sera of mice. We propose to further characterize these reactions to learn if there is any specificity for their presence or absence in malignancy and autoimmune disease. Reactivities of additional mammalian type-C viruses with human sera will be tested and family studies involving patients with diseases which might be related to type-C viruses based on animal models will be undertaken. Absorption with various leukemia and other tumor cells as well as normal cells will be performed, as well as further characterization of the reactant components in sera and viruses. A search for viral glycoproteins by surface radioiodination of leukemia cells, and viral studies of cultured leukemia cells are also planned. These studies will be conducted in parallel with a basic research program of regulation of type-C virus expression in the mouse. We feel there is considerable complementarity and synergy between the human and mouse programs.

ViraCore is a biotechnology company that is creating a drug discovery tool that is capable of presenting complex receptors in a nanometer-sized particle format. Specific ally, ViraCore is using "lipoparticles", nanometer -sized particles surrounded by a lipid bilayer and embedded with membrane-bound receptors, to purify and solubilize complex receptors from the cell surface while maintaining their structuraI integrity. The purpose of this proposal is to use lipoparticles to develop monoclonal antibodies directed to conformationally complex epitopes of membrane- bound receptors. Many of the receptors and epitopes that we will target are difficult or, in some cases, impossible to target using traditional methods, including activated receptor conformations and intracellular structures. Lipoparticles present a unique technology for the development of important new classes of antibodies and vaccine candidates. This proposal focuses principally on antibodies to chemokine receptors, a class of membrane-bound receptors involved in HIV infection, AIDS pathogenesis, and immunity. However, the results of this proposal can have a broad impact on the development of antibodies, vaccines, and therapeutics against a wide variety of complex receptors.

The principal objective of these studies is to investigate the potential role of astrocytes in the pathogenesis of hepatic encephalopathy (HE). Since the primary morphological change in HE is characterized by astrocytic degeneration, a derangement in astroglia may be a contributing factor to the development of encephalopathy. Given that astrocytes possess membrane receptors which influence glial function, as well as provide the chief mechanism for neuronal-glial communication, we propose that encephalopathy results from a toxin-induced breakdown in glial-neuronal interaction. Accordingly, we plan to investigate the potential involvement of astrocytic receptors and their second messenger' systems (cAMP, inositol phosphate) in primary astrocyte cultures. The effect of toxins implicated in HE (ammonia, short-chain fatty acids, mercaptans, phenol), their possible synergism and the effects of serum from patients with HE, on the affinity (Kd) and number (Bmax) of receptors will be determined. We have chosen to study the adrenergic, benzodiazepine, serotonin and histamine receptors, as there is evidence of their specific involvement in HE. These experiments will consist of saturation studies of specific ligands for each of the listed receptors. Where appropriate, the ability of the receptor to form high-agonist affinity complexes will also be examined. In addition, the effect of toxin exposure on basal and receptor agonist-stimulated levels of cAMP and inositol phosphate will be evaluated. Finally, the ability of agonists, antagonists and agents that act directly on the second messenger systems to modify toxin-induced changes, will be determined using light and electron microscopy. These experiments will, for the first time, delineate the effects of HE-related toxins on astrocyte receptor systems. It is anticipated that the characterization of specific receptor systems changes will elucidate possible mechanisms involved in HE and may lead to the formulation of new therapeutic strategies.

Significance Borna disease virus (BDV) infects a broad range of warmblooded species (mammals and birds) to cause neurologic dysfunction. Evidence from several laboratories indicates that BDV may be associated with human neuro-psychiatric disorders. Preferential infection of limbic circuitry, disturbances in D2 and D3 neurotransmitter systems, and the behavioral and motor disturbances that accompany acute BDV infection in adult, immunocompetent rodents and ungulates suggest parallels with serious psychiatric disorders such as schizophrenia, bipolar affective disorder, and autism. Attempts to demonstrate BDV infections in humans through serologic and molecular analysis of peripheral blood samples have produced inconsistent results. Objectives We hypothesize that perinatal infections of rhesus macaques will manifest as subtle neurobehavioral and neuropathologic disturbances in a primate counter part to the neonatal rat model of persistent BDV infection. The objective of this pilot study is to develop a nonhuman primate model of persistent BDV infection, and determine if the clinical, behavioral and neuropathologic sequelae are consistent with human psychiatric disease. Results Intracranial inoculation of BDV in 2 juvenile rhesus macaques resulted in infection and fatal clinical disease at 5 and 8 weeks pi, respectively. Both animals developed the acute, encephalitic form of BDV infection, indicating that animals of this age are already immunologically mature enough to preclude immunologic tolerance and persistent infection. Both animals developed strong humeral immune responses to BDV-specific antigens, thus providing valuable primate reagents for refining and validating available serologic tests. In one animal, preliminary PCR assays have detected viral nucleic acids in samples of peripheral blood mononuclear cells. Two neonatal rhesus were inoculated intranasally. One developed encephalitis and was euthanatized 9 week p.i. The second neonate did not become infected following intranasal inoculation. To better approximate the immune competence of the neonatal rat, intraventricular inoculation of 2 fetuses (~90-100 day gestation) have been pe rformed in utero, using ultra-sound guided techniques. One pregnancy resulted in live-born infant with severe congenital abnormalities and neurologic deficits that was euthanatized at 5 days of age. BDV was detected in tissues and the infant had high titer of anti-BDV antibodies. The second pregnancy resulted in a near term fetal loss. Ultrasound guided inoculation (IP or IC) was performed on 2 additional fetuses of 50 days gestation. The IP inoculation resulted in fetal death ~60 days p.i. The second fetus (IC inoc) is viable and appears to be developing normally at ~43 days p.i. Future Directions The last pregnancy will be allowed to go to term, and the neonate will be monitored for persistent BDV infection. Other strategies for inducing immune tolerance to BDV in macaques will be investigated. KEY WORDS borna disease virus, neurologic dysfunction FUNDING NIH RR00169 Pilot Study - In Progress

This is a proposal for the continuation of a Research Scientist Development Award, now termed an Independent Scientist Award (KO2). During the first term of this award 60 experimental and control monkeys were created that had either normal or atypical prenatal exposure to androgen. Androgen exposures were of 40 days or less duration and critically timed to the start of the second or third trimester of pregnancy. Prenatal androgen exposure was reduced by twice daily administration of flutamide, an androgen receptor blocker. Weekly injections of testosterone enanthate were used to increase androgen. The proposed K02 Award will allow the PI to devote the majority of his time to investigating the behavioral, neuroendocrine, and cognitive consequences of these alterations in prenatal androgen exposure in socially-living rhesus monkeys. These studies will provide fundamental information about how early hormone exposure affects the development of sexual and sex-typed behavior in a primate with a complex social organization and a long period of sexual development. One of the most striking human cognitive sex differences is in aspects of spatial cognition. The proposed support will allow the PI to develop computerized approaches to studying spatial cognition in group-living monkeys to take advantage of a large sample of rhesus monkeys with atypical androgen exposure created during the previous funding period. In addition, the increased time available for research will allow investigation of possible sex differences in brain structure using magnetic resonance imaging. If neuroanatomical sex differences are demonstrated they can then be pursued in the experimental subjects as they near adult development. During the term of this award the experimental and control subjects will go through puberty and enter adulthood. A goal of the proposed plan is to explore how alterations in the prenatal androgen environment affect the process of pubertal change and adult reproductive behavior. Because none of our hormonal treatments substantially altered the genitalia of experimental females there are no apparent physical barriers to pregnancy. It will be particularly interesting to determine whether substantially less drastic alterations in prenatal androgen exposure than those previously used produce marked changes in reproductive and maternal behavior. In males that have experienced reduced prenatal androgen exposure the PI will determine whether the timing of such hormonal alterations differentially affects their sexual behavior. These studies of nonhuman primates have particular relevance to issues of human gender development, particularly the occurrence of gender dysphoria. The last goal of the proposal is to allow the PI to gain a deeper understanding of human gender dysphoria by spending time at the Clarke Institute of Psychiatry in Toronto Canada, which has one of the world's most active research and treatment programs for childhood and adult gender dysphoria. The combination of these efforts will allow the PI to expand his research into new areas, while increasing his understanding of its relevance to important human mental health conditions.

To optimize isolation, transduction and expansion of lymphocytes from HIV-1 infected individuals under the conditions which will be used for a phase 1 study, to examine kinetics and stochiometry of ribozyme protection of primary cells, to determine if HIV antigen driven proliferation can be used to expand transduced cells, to obtain populations enriched in effector lymphocytes for reinfusion and to explore alternative methods of controlling HIV replication in infected cells during transduction of vectors.

The objective of this proposal is to localize the various types of DNA (A:T-rich, G:C-rich, and repetitive sequences of DNA) and the various types of chromosomal proteins on the chromosomes of Drosophila and mammals. Several methods for staining chromosomal DNA according to its base composition at the electron microscope level of resolution are suggested. An immunological approach to protein localization of chromosomes is outlined. The location of specific proteins will be correlated with the location of specific types of DNA. These findings will be related to the biochemical structure of chromosomes. The information derived from this study should give some insight into the mechanism of gene control in eukaryotic organisms, the mechanism of chromosomal condensation, and the function of chromosomal proteins and unusual sequences of DNA.

There are numerous factors that determine the biological response of tissues that contain radioactivity such as radiosensitivity, distribution of radioactivity, type and number of radiations emitted by the radionuclide, biokinetics of the radionuclide, repair time, etc. Traditionally, the mean absorbed dose to the tissue is calculated to correlate the biological response with mean absorbed dose. However, nonuniform activity distributions in tissue at the multicellular and subcellular levels result in nonuniform doses and therefore have made it difficult to adequately correlate biological response with mean absorbed dose. This is an important problem in diagnostic and therapeutic nuclear medicine. In the case of diagnosis, the risk of the radiation insult can in principle be drastically underestimated and potentially lead to increased risk of inducing cancer. In contrast, patients can be over- or under-treated in radionuclide therapy of cancer. Over-treatment or under-treatment in radionuclide therapy of cancer can have very adverse consequences in the final outcome for the patient. While calculation of absorbed dose at the cellular level has been advocated as a means to address this problem, this has largely remained a theoretical exercise. The applicants hypothesize that the biological response of tissues containing incorporated radionuclides can be correlated with absorbed dose when calculated at the cellular level. To test the hypothesis, a novel in vitro multicellular cluster model will be used which allows tight control over variables. Multicellular clusters will be assembled with mammalian cells containing radioactivity and the cell survival fraction as a function of cluster activity will be determined for several different radiopharmaceuticals which emit alpha particles, beta particles, or Auger electrons. Different percentages of the cells will be labeled with the different radiochemicals to ascertain the impact of nonuniform distributions of radioactivity at the cellular and subcellular levels. By controlling the percentage of cells labeled, this model will also be used to ascertain the role of bystander effects in the biological effects of incorporated radioactivity. These data and cellular dosimetry calculations will be used to develop a theoretical model to predict response based on cellular absorbed dose and bystander effects. The outcome of this research is expected to have a major impact on understanding and predicting the biological response of tumor and normal tissue to nonuniform distributions of radioactivity.

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 TrialNet Natural History Study of the Development of Type 1 Diabetes is a part of the TrialNet research program, in which a group of National Institutes of Health-sponsored researchers from the United States and Canada are trying to learn more about the development and prevention of Type 1 Diabetes.

The Mouse Phenotyping Shared Resource (MPSR) was established in 2001 to provide expert, readily available, and affordable experimental pathology support to OSUCCC investigators utilizing animal models of human cancer. The MPSR specializes in the morphologic characterization of newly produced lines of genetically engineered mice, but also provides a variety of other customized pathology services, including necropsy, slide preparation, semi-quantitative histopathology for experimental studies, morphometry, hematology, clinical chemistry, consultation and referral. The MPSR is headed by Donna F. Kusewitt, D.V.M., Ph.D., a board-certified veterinary pathologist and NCI-funded Investigator and staffed by Deborah Devor Henneman, B.S., each with more than 20 years of experience in carcinogenesis and experimental pathology. The MPSR is strongly supported through the excellent veterinary resources (clinical pathology, histology, and immunohistochemistry service) and veterinary pathology expertise (10 board-certified veterinary pathologists) available in the Department of Veterinary Biosciences (VBS) at The Ohio State University (OSU). Since its inception in 2001, the MPSR has provided high quality experimental pathology services tailored to meet the needs of the growing number of OSUCCC investigators who employ animal models of human cancer. During this time, 23 funded CCC members consulted on 38 projects requiring 1,042 hours of MPSR service, representing 75% of its entire workload. The MPSR has also provided requisite information for grant applications and manuscripts for many of OSUCCC members. The volume of work performed in the MPSR has steadily increased since the service was initiated and, given the projected expansion of OSUCCC research programs, a continued increase in MPSR usage is anticipated. Indeed we provide documentation of 37 CCSG-funded investigators with over 40 projects requiring MPSR support in the near future. Finally, the MPSR also serves as an essential venue for training future mouse pathobiologists as part of an NCRRfunded T-32 training grant, "Mouse Pathobiology: Models of Human Disease".

Project Summary/Abstract Reversible protein lysine acetylation is a fundamental posttranslational modification observed in histone and non-histone proteins. Lysine acetylation can alter protein-protein and protein-DNA interactions, protein stability, and enzyme activation/deactivation. Among the major regulators of lysine acetylation is the histone deacetylase (HDAC) family. Of the 18 known human HDACs, 11 are metal-dependent hydrolases related to the acetylpolyamine amidohydrolases (APAHs). The HDACs contribute to the regulation of gene expression and many other critical cellular processes. Notably, abnormal lysine acetylation is observed in multiple human disorders, including cancer; thus HDACs are a validated drug target. Despite their critical biological functions and clinical roles as drug targets, little is known about the molecular basis for HDAC substrate specificity and inhibition. This is particularly the case for HDACs 10 and 11, which are the least well characterized of the metal-dependent HDACs. Our preliminary studies coupled with phylogenetic comparisons suggest that HDACs 10 and 11 may function as dual acetyllysine and acetylpolyamine deacetylases with unique substrate binding site architectures. However, how HDAC10 and 11 could accommodate small polyamine substrates as well as large protein substrates containing sissile acetyllysine moieties is unclear. In addition, while classic HDAC inhibitors such as SAHA are known to inhibit HDACs 10 and 11, the molecular basis for this inhibition is unknown as no HDAC10-inhibitor or HDAC11-inibitor complex structures are available. In fact, no structure of HDAC11 is available, despite the fact that HDAC11 represents a unique class of HDAC due to its limited sequence identity with other HDACs. We propose to study structure-function relationships for HDACs 10 and 11 to establish a molecular foundation for understanding substrate recognition, catalysis, and inhibition. Due to a lack of structural and mechanistic studies focusing on HDACs 10 and 11, we are currently unequipped to design HDAC isozyme-specific inhibitors. I propose to study the molecular mechanisms of HDAC substrate recognition and inhibition by (1) exploring the structural basis of HDAC10 substrate specificity; (2) defining the structural basis of HDAC10 inhibition; and (3) determining structure-function relationships for HDAC11. As mentioned above, aberrant lysine acetylation is a hallmark of certain cancers and other human disorders; therefore HDACs are critical drug targets. Currently, four broad-specificity HDAC inhibitors are FDA-approved for cancer chemotherapy, but isozyme-specific HDAC inhibitors are mostly unavailable. Our studies aim to better understand the structure and function of poorly characterized HDACs with the goal of facilitating the design of specific HDAC inhibitors for use in human disorders, particularly cancer.

There are many diseases for which there are no vaccines and others for which the vaccines are not optimal or have significant side effects. The objectives of this project are to characterize viral antigens, determine targets of humoral and cell mediated immunity, and use this information to develop candidate vaccines. Live recombinant viral vaccines, DNA vaccines and recombinant protein vaccines are being developed. The vaccines are first tested in small animals and then in non-human primates. Presently we are working on vaccinia virus-vectored vaccines for HIV.

This project is designed to test the hypothesis that specific areas of the brain such as substantia nigra and basal ganglia are especially vulnerable to oxidative stress and that their response to biochemical oxidative reactions (or oxidative stress) is defective in Parkinsons disease (PD) as well as in normal aging. Mitochondrial and synaptic membranes may lose their function due to inadequate response to oxidation an-or from direct damage induced by cytotoxic products of oxidation (e.g., cholesterol oxidation products). Levels of oxidation products and loss of function could be small under steady state conditions. Therefore, we will perturb the system by inducing oxidation of membranes in vitro (using primarily endogenous oxidants) in order to amplify existing differences. Our preliminary studies show that mitochondrial cholesterol is more oxidizable in PD than in controls. Mitochondrial and synaptosomal fractions will be oxidized by incubation with oxidizing agents (e.g., arachidonic and linoleic acid hydroperoxide plus metal ions like iron, mixtures of iron and ascorbic acid, nitric oxide with and without superoxide etc.). Different levels of oxidation of membranes can be defined by the extent of oxidation of two membrane lipids - tocopherol and cholesterol. Since the profile of cholesterol and tocopherol oxidation products would be characteristic for various types of oxidations, information on the mechanism of oxidation will be obtained. Synaptosomes and mitochondria from young and old rats, and PD and control human autopsy brain samples will be compared in all studies. Similar studies on oxidation of platelets (as an accessible model of neuronal membranes in humans) from PD and control subjects will be conducted. The extent of oxidation of membranes will be monitored and quantitated by determining the concentrations of tocopherol and cholesterol and their oxidation products as well as by estimating thiobarbituric acid reactive substances and the decline in total suIfhydryl concentrations. Oxidizing conditions which yield maximal differences between the control and experimental groups will be established first and then the cholesterol and tocopherol oxidation products will be isolated, identified, and quantitated by GC and GC-MS. A study of the effect of monoamine oxidase(MAO) on mitochondrial oxidation will be done by incubating mitochondria with dopamine, norepinephrine or serotonin (substrates for MAO) which releases in situ hydrogen peroxide and/or other oxidants derived from it. The oxidation will be followed and oxidation products characterized as with brain fraction& The effect of MAO inhibition by deprenyl upon the oxidation will also be studied. Number of similarities exist between the uptake of serotonin by platelets and brain. Serotonin uptake by platelets and synaptosomes at various levels of oxidation will be studied to examine the effect of oxidation upon membrane function. The data will be used to determine whether membrane damage induced by oxidative stress exists in PD and/or in normal aging. The data from the platelet studies may also provide a laboratory tool for the preclinical diagnosis of PD.

Transformation in retroviral systems is characterized by abnormal cell growth and differentiation. Expression of the v-abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation in pre-B lymphocytes. These transformed cells are arrested at the pre-B cell stage based upon their immunoglobulin (Ig) gene rearrangements and cell surface antigens. Cells transformed by a temperature sensitive (ts) mutant of Ab-MLV undergo a high frequency of Ig light chain gene rearrangement soon after inactivation of the v-abl kinase at nonpermissive temperature. In addition, up-regulated expression of at least three genes important to Ig gene rearrangement (RAG-1, RAG-2 and germline kappa gene) occurs at the nonpermissive temperature. This system provides an opportunity to study the pathways mediating differentiation arrest as a component of retroviral transformation. The goals of this study are: (1) To identify functional domains of v-abl protein which mediate differentiation arrest and the pathways used to transmit these signals. A series of abl mutants and protein tyrosine kinases will be used in genetic complementation studies to determine which functional domains mediate this process. To identify pathways by which differentiation arrest is mediated, over-expression of c-ras, c-raf and c-myc will be tested, as will dominant negative forms of these genes. Proteins to be tested will be co-expressed with ts v-abl in a single pre-B cell; the differentiation phenotype of these cells will then be assessed after inactivation of the ts v-abl kinase. (2) To determine what genes are up-regulated upon release from differentiation arrest. The v-abl protein appears to block light chain rearrangement by suppressing expression of genes important for this process. The increased expression of RAG-1, RAG-2, and germline kappa gene in ts transformants at nonpermissive temperature supports this idea. These events are necessary, but probably not sufficient, to initiate rearrangement. The differential display technique will be used to identify other genes with altered expression prior to Ig rearrangement. Combining efforts to investigate both mechanisms of regulation and their targets may contribute to a better understanding of normal pre-B cell differentiation and how disruption of this process contributes to the transformed phenotype. This may provide clues to therapeutic strategies for human diseases of abnormal B lymphocyte maturation, including immunodeficiencies and both abl and non-abl related lymphoid malignancies.

This three-year project tests certain strategies for inducing program changes and innovations in community mental health centers (CMHCs). The first is the improvement of evaluation technology through development of specialized program evaluation techniques specifically aimed to be more relevant and compelling in program decision-making and planning. Termed "option-evaluation techniques," these methods involve the seeking out or clarification of program alteration opportunities, which are then evaluated with respect to expected output ("yield") and associated costs or resource factors. Shaping of specific evaluation techniques for needs-assessment, process studies and outcome studies to fit this approach are proposed. The second strategy is the development of program-change potential in other CMHCs through the use of technical assistance aimed at increasing their evaluation capability and use of evaluation findings. The technical assistance program involves intensive workshops, on-site visits, and computerized data analysis via remote terminal for a number of the nation's CMHCs. This effort also involves developing measures of "evaluation achievement" to use in testing for effects of the assistance program over time. A second feature is the use of measures of organizational "readiness for evaluation" as variables mediating the effectiveness of assistance in helping generate evaluation capability and utilization. A variety of non-experimental and quasi-experimental methods are used to determine relationships between evaluation achievement and readiness measures.

This proposal requests funds to continue studies of the reinforcing or euphoric property of opiates. The studies are designed to test the hypothesis that this property is mediated by the pathways activated during self-stimulation behavior and that these pathways constitute the "normal" channels for mediating motivational effects in the mammalian central nervous system. The studies fall into two categories, neurophysiological-behavioral and strictly behavioral. In the first group, single and multiple unit recordings are made in the awake, behaving rat during and after intraventricular or intracerebral application of opiates, opiate-like natural substances, and opiate antagonists. In these neurophysiological studies the neuron populations studied include those at rewarding sites, those whose spontaneous rate of discharge is depressed or enhanced during self-stimulation behavior, those that respond to naturally rewarding stimuli, and those containing monoamines. The goal is to sample for opiate action that correlates with behavioral stimulant effects and thus might be indicative of hedonic effect, in nerve cell populations defined on anatomical and functional grounds as possibly implicated in organizing the drive-reward underprinnings of motivated behavior. In the second group, rats are tested for self-stimulation after single or repeated opiate treatment at reward sites selected on the basis of a variety of behavioral parameters, for escape behavior from brain stimulation after opiate administration, for self-administration of opiates at reward sites, and for gross activity. Doses of opiates, schedules of drug administration are designed to produce a stimulant effect; so far as the activity test is concerned, the aim is to determine whether a stimulant action or dependence can be established in rats depleted neonatally of brain monoamines. The ultimate aim is to contribute to an understanding of opiate action on the one hand and to a central motivational mechanism on the other.

DESCRIPTION: This application is for the renewal of an NEI Core Grant in support of the research programs of 16 principal investigators holding 23 active NEI individual research grants and an NEI Institutional Training grant. The previous funding cycle generated 100 publications in the major areas of aging and diabetes, extraocular muscle biology and diseases, ocular immunology and inflammation, and retinal biology and diseases. The current investigators form the nucleus of the Case Visual Sciences Research Center (VSRC), a broader group of 40 vision researchers in 19 different basic and clinical science departments at the Case Medical Center (Case Western Reserve University School of Medicine and University Hospitals of Cleveland). Since our initial Core Grant award in 1997 and its renewal in 2002 with five Modules, these modules have continued to evolve over the current funding cycle due to a changing investigator group and scientific direction, expanded services and new technologies. The five modules that are proposed are: 1) Tissue Culture and Hybridoma, 2) Histology, Microscopy and Imaging, 3) Molecular Biology, 4) Specialized Animal Resource, and 5) Proteomics. Major new functions since our previous submission include hybridoma production, live cell imaging, laser capture microdissection, and a full service proteomics facility to acquire and analyze protein expression in ocular tissues. With the recent renewal of the T32 Training grant, these modules will also continue to serve as an integral resource for pre- and postdoctoral trainees in the Case Visual Sciences Training Program. The Visual Sciences at Case has receives very strong institutional support, including, construction of a centralized facility for Core Grant modules and functions, recruitment funds and supplemental support for technical salaries and supplies in the core facilities. The change in P.I. from Dr. Lass to Dr. Pearlman will ensure continuity in leadership and direction of the Core Grant, which will enable collaborative goals and objectives while enhancing individual programs, and facilitate Case VSRC investigators'continuing efforts toward analysis of fundamental mechanisms in ocular and visual system development, function, and pathogenesis of diseases of the eye and visual system.

DESCRIPTION: DNA damage in the form of apurinic/apyrimidinic (AP) sites is induced by cytotoxic agents resulting in base substitution mutations and blocks to DNA replication. The DNA base excision repair (BER) enzyme, AP endonuclease (APE), is a multifunctional protein involved in DNA base excision repair, oxidative signaling, transcription factor regulation, cell cycle control, and apoptosis. APE is essential for the repair of AP sites thus maintaining cellular and genetic integrity. Deficient expression of APE results in a heightened sensitivity to radiation and alkylating agents with resultant tissue damage. Logically it follows that APE may also play a role in the sensitivity to malignant cells to DNA damaging therapeutic agents. Many cancer therapeutic agents will induce apoptosis or programmed cell death, however little is known about the relationship of DNA repair enzymes (particularly BER) and apoptosis. Preliminary observations indicating that APE expression can be suppressed in myeloid leukemia cells by retinoic acid or DMSO while inducing apoptosis, has led to the hypothesis that: Decreased expression of APE is functionally related to apoptosis in myeloid leukemia cells. The specific aims are: Specific Aim #1 What is the relationship of APE expression to myeloid leukemia cell differentiation and apoptosis? Specific Aim 2: What is the role of APE protein phosphorylation in APE expression, function and apoptosis. Specific Aim #3: Determination of the molecular mechanisms that are responsible for down-regulation of APE expression upon induction of apoptosis. We propose to initiate studies on the relationship of APE expression and phosphorylation to cell differentiation, apoptosis, and sensitivity to cytotoxic agents, with the long-term goal of developing ways to enhance the chemo/radio sensitivity of leukemia cells by manipulating expression of APE.

Previous studies supported by NIH grant HL46213 ("Molecular Interactions of Factor XI"), for which continued support is sought, have focused attention on FXI and its interactions with other plasma proteins, i.e., thrombin, FXIa, FXIIa, prothrombin, high molecular weight kininogen (HK), and FIX, with the platelet plasma membrane and with various inhibitory molecules, e.g., protease nexin II (PN2), in the initiation and regulation of blood coagulation. These studies, utilizing NMR and x-ray crystallography, provide three separate structures, one of the catalytic domain of FXIa in complex with the KPI domain of PN2, one of the Apple 4 (A4) domain dimer and one of the zymogen FXI dimer that provide the basis for the future proposed investigations. The long-term goals of the present proposal are to elucidate the molecular mechanisms involved in the interaction of FXI/FXIa with protein and cell surface ligands involved in its activation and with plasma protein and cell surface ligands involved in the expression and regulation of FXIa enzymatic activity. Specifically these studies propose to determine the mechanism and physiological importance of FXI homodimer formation by a mutational analysis of the A4 dimer interface to prepare a monomeric FXI molecule;to study the role of the A4 domain in FXI dimerization in equilibrium unfolding studies;and to determine whether the dimeric structure of FXI is required for FXI activation to FXIa by thrombin or by FXIIa in the presence or absence of activated platelets or glycocalicin. They will examine the hypothesis that the activation of FXI to FXIa is accompanied by a major conformational change that promotes the efficient activation of FIX by exposing a substrate-binding site in the heavy chain (A2 and/or A3 domains) of FXIa for efficient binding and activation of FIX. Finally they will determine whether the dimeric structure of FXIa is required for efficient FIX-activation and to differentiate between alternative mechanisms to account for the failure of monomeric FXIa to activate FIX on the activated platelet surface. Recent observations resulting in the recognition of the essential role of FXI in hemostasis and thrombosis concern the relationship of its domain structure to its biological function, its molecular and cellular interactions, the elucidation of pathways for activation of FXI, and the expression and regulation of its enzymatic activity. It is hoped that information gleaned from the proposed studies will result in novel approaches to targeting FXI/XIa in the development of new anticoagulants that prevent thrombosis (heart attacks, strokes and pulmonary emboli) without bleeding complications.

There continues to be substantial controversy concerning neural control of the cerebral circulation. Over the past several years, our laboratory has demonstrated that there are several factors which modulate the response of cerebral vessels to stimulation of peripheral sympathetic nerves. Of these factors, species variation and temporal course of the response are of the utmost importance. The cerebral vessels of cynomolgus monkeys are far more sensitive to sympathetic nerve stimulation than cerebral vessels of cats or dogs. Furthermore, in the cynomolgus monkey, cerebral vasoconstriction induced by continuous maximal electrical stimulation of the sympathetic nerves persists in most areas of the brain for less than 90 seconds. Thus, the escape phenomenon is very important in cerebral vessels. In addition to these new observations concerning factors which modulate the response of cerebral vessels to sympathetic nerve stimulation, we have recently applied a new method--the pulsed Doppler flow meter--to measure cerebral blood velocity in small cerebral vessels on the surface of the brain. The major objective of this proposal is to further elucidate the role of peripheral sympathetic nerves in the control of cerebral vascular resistance. The proposed studies will be performed in both awake and anesthetized cynomolgus monkeys and the temporal course of the response of cerebral vessels to sympathetic stimulation will be explored in detail under a variety of experimental conditions. Responses of cerebral vessels to sympathetic nerve stimulation will be assessed with the pulsed Doppler method and with carefully timed injections of labeled microspheres. In the proposed experiments I will study possible mechanisms that permit cerebral vessels to escape from the vasoconstrictor effects of sympathetic nerve stimulation. I will also study effects of sympathetic nerve stimulation on cerebral vascular resistance in awake and anesthetized monkeys during hemorrhagic hypotension, severe hypertension, and chemoreceptor stimulation.

Acquired immunodeficiency syndrome (AIDS) is an immunoregulatory disorder characterized by the presence of Kaposi's sarcoma or opportunistic infections. CD4+ T cells and mononuclear phagocytes are the target cells for the human immuno- deficiency virus (HIV), the etiologic agent of AIDS. Monocyte/macrophages (M-phi) are the major reservoir of HIV and, therefore, a critical concern for the host is the functional capacity of M# from HIV+ individuals to destroy opportunistic pathogens. Disseminated histoplasmosis is seen with increasing frequency as a complication of AIDS, particularly in areas where Histoplasma capsulatum (Hc) is endemic. AIDS patients with disseminated histoplasmosis have a high rate of relapse even after apparently successful therapy with amphotericin B. Therefore, it is imperative that we gain a better understanding of the interaction of Hc with M-phi from HIV+ patients. The goal of the proposed study is to characterize the intrinsic defects in the function of monocyte/M-phi from patients with HIV infection with respect to their ability to defend against Hc. The specific aims of this proposal are: l) To quantify the capacity of M-phi from HIV-infected patients to bind and to phagocytose Hc yeasts and microconidia compared to M-phi from HIV- individuals; 2) To determine if M-phi from HIV-infected patients provide a more than normal permissive environment for the conversion of Hc microconidia into the pathogenic yeast phase, or permit more rapid multiplication of yeasts than normal M-phi; and 3) To quantify the capacity of M-phi from HIV-infected patients to be activated by colony stimulating factors (CSFs) to inhibit the intracellular multiplication of Hc yeasts compared to M-phi from HIV- individuals. As changes in M-phi function may correlate with disease progression and not merely HIV infection, monocyte/M-phi will be studied from different patient groups, from those who are at risk for HIV infection but who are not infected to patients with AIDS. In addition, studies will be performed on normal M-phi infected with HIV in vitro to determine if HIV infection alone can cause M-phi dysfunction with respect to its interaction with Hc.

We have developed a high pressure liquid chromatographic technique for determination in human urine concentrations of antipyrine and its metabolites, norantipyrine, 4-hydroxyantipyrine, and 3-hydroxyantipyrine. This method involved studies to determine optimal buffer pH and molarity, ratio of solvents in the mobile phase of the system, and effects of centrifugation temperature during assay. Experiments performed disclosed interesting variations from one subject to another both in the amount of each metabolite excreted with unit time, as well as in the time of appearance of each metabolite. We are now in a position to perform family studies to determine which of these factors that exhibited interindividual variations are heritable and what the mechanism of heritable transmission is.

The proposed research has two objectives: 1) to investigate the genetic and physiological control mechanisms of tetrahydrofolate metabolism; 2) to determine the role of proteases in the metabolism and differentiation of yeast. Mutants altered in THFA metabolism will be analyzed genetically and biochemically. The THFA interconversion enzymes will be purified to see whether they occur in an enzyme aggregate. The intracellular localization of these enzymes will be determined. Mutants with alterations in the function or regulation of proteases will be isolated and characterized both with respect to genetics and biochemistry. The consequences to the cell of such alterations in protease function will be examined. This research should contribute to our understanding of regulatory mechanisms underlying transport of proteins through membranes, and may contribute to our understanding of the mechanisms governing transitions between cell states (meiosis and mitosis).

As a resubmission, the proposed five-year plan will train me and foster my launch into an independent interdisciplinary research career. My multidisciplinary background in Biomedical Engineering, Electrical Engineering and Mechanical Engineering fields and my outstanding experience in advanced biomechanical modeling and electrophysiological modeling research have fostered my interest and given me unique advantages in conducting interdisciplinary biomedical research in Urinary Incontinence (UI). The immediate goal is to prepare for a career as an independent scientist and to develop a novel subject-specific electromechanical pelvic model based urethrovaginal support and urethral function assessment (UUFA) technique to minimally invasively and quantitatively assess these two etiologic factors and to investigate the specific changes associated with aging effects that cause the increase in prevalence of SUI in women. The central hypothesis behind this research is that urethrovaginal support and urethral function in women and aging effects on female SUI can be minimally invasively and quantitatively assessed and characterized using a subject-specific pelvic modeling approach. Primary Hypothesis #1 is that urethral function in women can be minimally invasively and quantitatively assessed using a subject-specific electrophysiological pelvic modeling approach. Primary Hypothesis #2 is that urethrovaginal support function in women can be non-invasively and quantitatively assessed using a subject-specific biomechanical pelvic modeling approach. Primary Hypothesis #3 is that aging will cause poor urethrovaginal support and/or urethral dysfunction which will lead to SUI. Primary Hypothesis #4 is that a female continence profile can be created to predict the status of continence in women in women clinically and to suggest optimal therapeutic modalities for specific patients. We propose four Specific Aims (SA) to test these Hypotheses: SA #1: To develop a subject- specific electrophysiological pelvic model based periurethral muscle fatigue assessment (UMFA) technique to minimally invasively and quantitatively assess urethral function in women. Primary Hypothesis #1 will be tested. SA #2: To develop a subject-specific biomechanical pelvic model based urethrovaginal support function assessment (USFA) technique to non-invasively and quantitatively assess urethrovaginal support function in women. Primary Hypothesis #2 will be tested. SA #3: To investigate aging effects on urethral function and urethrovaginal support in young/elderly female subjects with/without SUI using the UUFA technique which consists of the UMFA and USFA techniques. Primary Hypothesis #3 will be tested. SA #4: To develop a female SUI profile as a clinical standard of female SUI by utilizing the UUFA technique to quantitatively characterize SUI etiologic factors in urethrovaginal support and urethral function in women. Primary Hypothesis #4 will be tested. At the completion of this project, it is our expectation that we will have established and validated the UUFA technique and the feasibility of its application in clinical studies, and have achieved a better understanding of the contributions of specific changes associated with aging and menopause to these etiologic factors in women.

Utilizing a 4 pound fully automatic portable blood pressure recorder with a conventional sphygmomanometer cuff, a group of hypertensives (essential, labile, renal types) will have their blood pressures and heart rates recorded at 15 minute intervals during an 8 hour period while engaged in their usual work activities. A voice channel allows the subject to indicate with each pressure recording, the time, whether standing, sitting or lying, his physical activity, his emotional state and any physical symptoms. Such recordings will be made prior to and during treatment with single or multiple antihypertensive agents. Similar recordings at work will be made for apparently healthy controls, not on medications, of comparable age and sex. Where possible and feasible, some patients and controls will have 24 hour recordings including sleep at home. Such data would be unique and of great potential value in understanding blood pressure behavior in normals and hypertensives and the benefits and side effects of chemical treatment in the latter group. Patients referred for unexplained dizziness or for atypical headaches or angina will wear the device to determine if unusual blood pressures correlate in time with such symptoms.

Although there is significant evidence that 2,3,7,8-tetrachlorodibenzodioxin (TCDD) alters lymphocyte function in mice, there have been very few studies which examine the effects of TCDD on human lymphocytes. While there have been a number of inadvertent exposures to TCDD, via industrial accidents or environmental contamination, there have been no consistent immunological defects observed in exposed individuals. Studies in German chemical workers have demonstrated alterations in lymphocytes subpopulations and functional parameters 20 years after exposure to TCDD. As part of a continued effort to assess the health effects of environmental exposures to TCDD it is imperative that we examine endpoints which have the potential to be sensitive predictors of human exposure and to correlate these biomarkers with blood TCDD levels, determine influence of potential confounding variables on biomarker expression, and evaluate inter- and intra-individual variation. Identification of additional biomarkers may aid in identifying individuals or populations at greater risk of developing disease or neoplasia after exposure to dioxins and related compounds. Samples of peripheral blood lymphocytes were obtained from several cohorts of control or TCDD-exposed individuals. These samples were mitogen-stimulated in culture, alone or in the presence of TCDD, and the cells and culture supernatants harvested after 72 hr. To date we have examined supernatants from a group of workers who were employed at a chemical plant in Hamburg-Moorfleet, Germany operated by Boehringer-Ingelheim from 1951-1984 that produced organochlorine herbicides and pesticides (including 2,4,5-trichlorophenoxyacetic acid) and opioids. TCDD contamination at this plant was confirmed in 1984 and significant increases in mortality have been observed in a cohort of workers employed at this plant for at least 3 months from 1952-1984. Peripheral blood for lymphocyte isolation and serum dioxin analysis was obtained from a cohort of 110 individuals in 1992, in collaboration with clinicians at the University of Mainz (Detlev Jung and Johannes Konietzko) and a biostatistician at the German cancer Research center. In vitro treatment with TCDD resulted in a significant decreases in the mitogen-stimulated production of the T Helper 1 type (TH1) cytokines Interleukin 2 (IL2) and Interferon gamma (IFNgamma) in ymphocyte cultures from this cohort. We believe the alterations TH1 cytokines are a result of the in vitro exposure as there is limited correlation with standard biomarkers of TCDD exposure such as total TCDD plasma levels or total plasma TEQ. In contrast, there appears to be a dose-related correlation between individual TCDD body burden and decreased secretion of the T Helper 2 type cytokine Interleukin 4. Significant correlations were also seen between in vitro TCDD-inducible ethoxyresorufin-O-deethylase activity and secretion of tumor necrosis factor alpha. While it appears that there may be some significant effects on immunoglobulin (Ig) secretion, particularly for IgA and IgG2, interindividual variability is quite high, and sophisticated statistical analyses will be required to tease out exposure-specific effects. In collaboration with Dr. Joseph Haseman, the dataset is undergoing multivariate analysis to examine correlations between immunologic effects and in vivo and in vitro biomarkers of exposure, and to determine the effects of confounding variables such as gender, smoking status, etc.

Parathyroid hormone (PTH) is a major regulator of mineral homeostasis and bone metabolism. Intermittent PTH therapy is approved for treatment of osteoporosis, yet the cellular mechanisms underlying the biologic effects of PTH are incompletely understood, including the molecular basis for the observation that low-dose, intermittent administration of PTH elicits net bone formation, whereas continuous administration of high-dose PTH causes predominantly bone resorption. The actions of PTH on bone and kidney are mediated by the Type 1 PTH receptor (PTH1R), a G protein-coupled receptor (GPCR).beta-arrestins (1 and 2) are cytoplasmic molecules that regulate GPCR activity. We previously reported that arrestins inhibit PTH-stimulated cAMP signaling and trigger cellular internalization of PTH and its receptor in vitro. Thus, our primary hypothesis is that beta-arrestin2 is a key modulator of the activity of PTH in bone. In support of this hypothesis, our preliminary data indicate that compared to wild-type (WT) mice, beta-arrestin2 null (beta-arr2-/-) mice have reduced bone mass and architecture, and importantly, an altered skeletal response to intermittent PTH that is characterized by increased bone resorption leading to decreased net anabolic effects on trabecular bone. Moreover, compared to WT, osteoblasts from beta-arr2-/- mice exhibit increased and sustained cAMP signaling in response to PTH. Thus, to further explore the role of beta-arr2 in mediating the skeletal response to PTH we will pursue three specific aims: 1) Determine the skeletal response of ovariectomized beta-arr2-/- and WT mice to intermittent PTH, thereby allowing examination of the role of beta-arr2 in the context of high bone remodeling induced by estrogen deficiency; 2) Investigate the arrestin-mediated regluation of PTH-stimulated intracellular signaling and gene expression by comparing primary osteoblastic cells from beta-arr2-/- and WT mice; and 3) Determine the skeletal response to intermittent and continuous PTH administration in transgenic mice with targeted overexpression of beta-arrestin2 in well-differentiated osteoblasts (OC-beta-arr2-GFP-Tg+). In summary, the overall goal of this project is to improve our understanding of the mechanisms regulating the activity of PTH in bone. By conducting complementary in vivo and in vitro experiments in mice deficient for beta-arrestin2 and in mice overexpressing beta-arrestin-2 in mature osteoblasts, we will provide novel insights into the mechanisms that underlie the distinct skeletal response to intermittent versus continuous PTH administration. Information gained from the proposed studies will be instrumental for developing new PTH1R ligands with improved signaling and biologic activity profiles for treatment of osteoporosis and other metabolic bone disorders.

Cocaine abuse is a major public health problem in the United States with no accepted pharmacologic treatment. We propose a placebo-controlled, randomized, double-blind outpatient study evaluating imipramine as a treatment for cocaine abuse in 80 chronic users. The study is designed to: 1) determine the efficacy of imipramine as an outpatient medication treatment for cocaine abuse 2) determine the efficacy of imipramine in blocking the cocaine euphoria and/or diminishing the craving for cocaine in abusers 3) examine the physiologic response to cocaine via a controlled laboratory challenge with cocaine before and after imipramine 4) determine the prevalence and types of psychiatric disorders in chronic cocaine abusers and contribute to the determination of whether "cocaine dependence" should be a part of the official nosology of mental disorders.

Growing evidence indicates a reciprocal relationship between the HPA axis and the immune system which may be relevant to disease expression. Since patients with AIDS exhibit abnormalities in the HPA axis, a thorough understanding of the basic physiology of neuroendocrine-immune interactions is an important prerequisite for determining the potential role of neuroendocrine function in the development of this disease. Nevertheless, adrenal steroid hormones, the final product of HPA axis activation, have complicated and inconsistent effects on in vivo immune function. Recent studies in our laboratories suggest that further understanding of these complex yet important immunologic effects of adrenal steroids will be greatly enhanced by focusing on adrenal steroid receptors in immune cells and tissues. Our results indicate that Type I and II adrenal steroid receptor binding is differentially expressed in various immune tissues and there is considerable heterogeneity between immune tissues in receptor subtype activation following in vivo hormone exposure. Moreover, we have found a highly significant relationship between adrenal steroid receptor activation in vivo and immune function. These findings in conjunction with the proposed studies will advance our knowledge of the mechanisms involved in the interaction between adrenal steroids and the immune response. The specific aims of the proposed project are as follows: Except where indicated, all experiments will be conducted on Sprague Dawley rats. For Specific Aim 1, the distribution of Type I and II receptor positive cell types in sections of spleen, thymus and mesenteric lymph nodes will be antibodies. In addition, these antibodies will be used in conjunction with antibodies to specific cell surface markers to determine receptor subtype expression in defined immune cell subpopulations using flow cytometry. For Specific Aim 2, selective Type I and II receptor agonists and antagonists will be administered in vitro and in vivo and the immunologic effects of these agents on the following immune functions will be determined: T cell proliferation, II-2 secretion, T cell independent B cell proliferation, NK cell activity, monocyte II-1 secretion. Specific Aim 3 will examine the relationship between receptor activation and the noted immune variables in a range of in vivo physiological conditions known to differ in steroid bioavailability. Effects of diurnal variation, sex, strain, and duration of endogenous corticosterone exposure will be evaluated. Finally, Specific Aim 4 will examine immune function in animals with downregulated adrenal steroid receptors secondary to chronic steroid exposure or aging. Taken together, these studies, which are a major component of the PI's RSDA, are designed to provide a foundation for understanding the role of neuroendocrine function in immune-related diseases such as AIDS.

Umbilical cord blood (CB) is used increasingly to restore hematopoiesis in transplant patients lacking sibling, or unrelated donors. A major disadvantage of CB transplantation (CBT) is the low cell dose with resultant delays in neutrophil and platelet engraftment, as well as a high rate of engraftment failure. A major hypothesis of this grant is that ex vivo-expanded CB progenitors will provide more rapid hematopoietic reconstitution and less engraftment failure than unmanipulated CB. In the two clinical CB expansion trials conducted during the last RO1 funding period, CD133+ progenitors were isolated and cultured ex vivo in a liquid culture system targeting committed, or more primitive hematopoietic progenitors. In those sequential trials, trends in faster time to engraftment were seen, but significant losses of CD34+ progenitors occurred. To avoid the need for positive selection, which results in a substantial loss of CD34+ cells, we have developed an alternative approach that involves the ex vivo co-culture of CB mononuclear cells (MNC) with bone marrow-derived mesenchymal stem cells (MSC). We hypothesize that MSC, by functioning as a surrogate hematopoietic 'niche', will provide a more suitable microenvironment for the expansion of lineage-committed CB hematopoietic progenitors than afforded by current liquid suspension culture systems. As accrual to the current CB expansion trials nears completion, this competitive renewal application will focus on the clinical evaluation of CB expansion in MSC co-cultures, with the long-term goal of improving neutrophil and platelet engraftment in CBT patients. Although a low cell dose is clearly the chief limitation of CBT, a number of investigators have also reported a deficit in the homing of CB cells to the marrow. Thus, it is conceivable that even with optimal ex vivo expansion, inadequate homing may limit the rapidity of engraftment which is the goal of this proposal. The homing defect has been attributed to low levels of fucosylation of cell surface molecules responsible for binding to P- and/or E-selectins expressed by the marrow microvasculature. This interaction is a key component of the recruitment of hematopoietic progenitors to the marrow. We hypothesize that increasing the level of CB cell surface fucosylation will improve interactions with selectins, thereby improving homing and engraftment. Aim 3 will evaluate the modification of unmanipulated and expanded CB progenitor cells with a fucosyltransferase, as a means to facilitate their recruitment to the marrow. PUBLIC HEALTH RELEVANCE: Umbilical cord blood is being used increasingly for many patients with high-risk hematological malignancies and genetic disorders who do not have a bone marrow donor, but is associated with a longer time to engraftment compared to marrow. The expansion of cord blood in the laboratory prior to infusion appears to shorten time to engraftment; additionally the treatment of the cord blood with a sugar molecule prior to infusion appears to improve its ability to home to the bone marrow quickly. Both these strategies will be explored in the grant thereby making the transplant safer for patients in terms of infectious and bleeding complications and will thus improve survival.

This proposal deals with the development of a radiation detector system that will make possible the direct measurement of the structure of charged particle tracks in a gas. Such a detector will permit the measurement of energy e.g., the diameter of a DNA molecule. This information, which is critical to development and evaluation of mechanistic biophysical models for understanding radiation effects, has hereto fore not been measurable. When a charged particle penetrates a gas, electrons produced during ionization events quickly become thermalized. The proposed detector system will determine the positions and number of these electrons by imposing a suitable RF field, causing the electrons to agitate and excite molecules of the gas which will then fluoresce producing photons that will be detected by the external optical detector system. Thus, each point of light will represent the position of a single electron. In Phase I experimental proof of the concept was made that a single charged particle track can be detected optically. In this proposal the operation of the system would be greatly enhanced so that CCD detectors can be used to obtain quantitative information from images of all of the electrons produced in the track of a single particle.

The purpose of this investigation is to identify the effect of single-dose disulfiram on human cytochrome P4502A6 activity in vivo, using coumarin 7-hydroxylation as the in vivo probe for human P4502A6. Our hypothesis is that disulfiram will have no effect on in vivo P4502A6 activity and we expect to find that coumarin clearance and 7-OH-coumarin formation clearance will not be significantly diminished by disulfiram.

Work from our laboratory has focused on the pathophysiology of Epstein-Barr virus (EBV), a gamma herpesvirus which is associated with several human malignancies including B-cell lymphoma and nasopharyngeal carcinoma. We have also explored the role that psychological stress plays in the modulation of the steady state expression of EBV early proteins and how stress could be a factor in the risk for EBV associated disease. This proposal will focus on how EBV can induce inflammation in vitro and in mice. We have explored the hypothesis that EBV-encoded early proteins can induce immune changes observed in patients infected with EBV independent of their role in viral replication. We discovered that EBV-encoded deoxyuridine triphosphate nucleotidohydrolase (dUTPase) up-regulates the production of several proinflammatory cytokines including TNF-a, IL-1, IL-6, IL-8, as well as IL-10 in macrophages. When purified EBV-encoded dUTPase was inoculated into mice the protein was capable of producing symptoms compatible with cytokine-induced sickness behavior. The data demonstrate that EBV-encoded dUTPase can induce sickness behavior in mice resulting in increased body temperature and decreased body mass and physical activity. Furthermore, there is new and extensive literature linking chronic inflammation with an increased risk for cancer. Our data provide a new perspective on how a latent herpes virus, such as EBV, when reactivated by stress or other factors, could cause immune dysregulation, the activation in NF?B in macrophages and the upregulation of proinflammatory cytokines with possible implications for EBV associated clinical symptoms and disease, including EBV associated tumors. PUBLIC HEALTH RELEVANCE: This multidisciplinary research grant will provide new information on how stress can have an impact on the pathophysiology on herpesvirus associated diseases.

We propose to study the synthesis, physical properties and utility in synthetic organic chemistry of several classes of compounds which share the distinction of having multi-center electron-rich bonds of the type usually called hypervalent. Among these species are isolable compounds of pentacoordinate carbon, chiral sulfuranes and asasulfuranes. The generation of sulfuranes and persulfuranes by neighboring group participation in perester decompositions will be studied. The generation of radical pairs, and larger arrays of radicals, within the solvent cage by such reactions will receive attention. The study of planar triarylmethyl radicals and cations is to be continued with particular emphasis on the complexes formed in the solid state between such radicals and cations and their possible electrical conductivity. Analogues of compounds from earlier studies in these areas which have shown activity in the NCI Cancer Chemotherapy Screening Program will be prepared and submitted for testing.

This application requests funding to identify risk factors for chronic back pain disability. The proposed study will draw on our existing data base of physical, psychological, and work-related premorbid data. This data base is unequalled in population size and scope of independent variables. Furthermore, this study will provide the longest follow-up of any such study to date. A better understanding of risk factors would provide a solid foundation for establishing appropriate programs to prevent chronic back pain disability, to enhance return to work, and to reduce the impact back pain has on the industrialized nations of the world. Our goal is to continue monitoring our subject population of 3,020 individuals so that risk factors for the development of chronic back pain disability can be identified. Our efforts to establish this data base have been supported to date by NIOSH; however, NIOSH has stated that the evaluation of chronic disability is beyond their scope of interest and has limited our funding to analysis for the prediction of acute industrial back injuries. To stop without evaluating chronic back pain disability would ignore the 10% of back injuries that cause the most suffering and account for approximately 80% of the total cost for back problems. We have already found that the first 26 subjects who developed disabling back problems of at least a three-month duration have a significantly different fitness level than their age-matched controls. With two more years of follow-up we estimate another 15 - 21 subjects will develop chronic back pain disability. An increased number of subjects in the chronically disabled category would add to the statistical power for the evaluation of other variables. Our results to date indicate that it is highly probable that analysis of premorbid data can predict chronic back pain disability and greatly increase our understanding of this expensive healthcare problem.

The liver is an organ with strong innate immunity, which plays an important role in host defense against microbial infection and tumor transformation. Emerging evidence suggests that innate immunity as well as a variety of cytokines produced by innate immune cells also contribute to the pathogenesis of acute and chronic liver diseases. Our laboratory has been actively studying the role of innate immunity and its associated cytokines in liver injury and repair. During the fiscal year, we have generated floxed STOP-CD59 knockin mice (ihCD59), in which expression of human CD59 only occurs after Cre-mediated recombination. By using this model, we studied the effect of cell death on activation of innate immunity and liver regeneration. Cre-inducible human CD59 mediates rapid cell ablation after intermedilysin administration. Cell ablation is a powerful tool for studying cell lineage and/or function; however, current cell-ablation models have limitations. Intermedilysin (ILY), a cytolytic pore-forming toxin that is secreted by Streptococcus intermedius, lyses human cells exclusively by binding to the human complement regulator CD59 (hCD59), but does not react with CD59 from nonprimates. Here, we took advantage of this feature of ILY and developed a model of conditional and targeted cell ablation by generating floxed STOP-CD59 knockin mice (ihCD59), in which expression of human CD59 only occurs after Cre-mediated recombination. The administration of ILY to ihCD59+ mice crossed with various Cre-driver lines resulted in the rapid and specific ablation of immune, epithelial, or neural cells without off-target effects. ILY had a large pharmacological window, which allowed us to perform dose-dependent studies. Finally, the ILY/ihCD59-mediated cell-ablation method was tested in several disease models to study immune cell functionalities, hepatocyte and/or biliary epithelial damage and regeneration, and neural cell damage. Together, the results of this study demonstrate the utility of the ihCD59 mouse model for studying the effects of cell ablation in specific organ systems in a variety of developmental and disease states.

Numerous neurological diseases are characterized by a sex difference. The neuropathology often includes infiltration of immune cells, with this immune infiltration potentially contributing to disease pathogenesis. Since it is known that sex differences exist in the immune system, this confounds investigations into sex differences in the CNS. Thus, we will use bone marrow chimeras to investigate sex differences in the CNS. By varying sex chromosomes or sex hormones in hosts reconstituted with a common immune system, one can ascertain the role of sex chromosomes and sex hormones on the brain response to injury. We will use one of the most inflammatory of all CNS disease models, the multiple sclerosis model, experimental autoimmune encephalomyelitis (EAE), to show applicability of this approach to a variety of neurological diseases. We will employ mice which differ in the complement of sex chromosomes (XX vs. XY), while having the same gonadal type, to determine the effect of sex chromosomes in the absence of confounding effects of exposure to different types of sex hormones. Specifically, in aim #1 we will determine whether the greater severity of EAE in XX, as compared to XY-, mice is due to sex chromosome effects in the CNS. In aim #2, we will determine if the sex chromosome effect in the CNS during EAE is due to the dose of X or Y genes. Finally in aim #3, we will use mice which differ in gonadal type, female vs. male, while having the same sex chromosome complement (XX vs. XX Sry) to determine whether the greater severity of EAE in female, as compared to male, mice is due to sex hormone effects in the CNS. PUBLIC HEALTH RELEVANCE: This is an exploratory (R21) grant to determine the effect of sex chromosomes and sex hormones on the central nervous system's response to an immune attack using the multiple sclerosis model, experimental autoimmune encephalomyelitis. This proposal will establish a model system to determine the effect of sex chromosomes and sex hormones on a variety of neurological diseases characterized by a sex difference.

DESCRIPTION(Adapted from applicant's abstract): The conversion of adenosine to inosine by RNA editing represents an increasingly important mechanism for generating diversity in neurotransmitter receptor expression. Such RNA modifications have been shown to alter both the ion permeation and electrophysiological properties of ligand-gated ion channels and to modulate the efficacy of receptor G-protein interactions. Recent studies in our laboratory have demonstrated that ADAR2, a double-stranded RNA-specific adenosine deaminase involved in the editing of glutamate and serotonin receptor transcripts, can selectively modify its own pre-mRNA, suggesting a negative autoregulatory mechanism by which ADAR2 can modulate its own expression level to prevent editing at aberrant sites. The long-term objectives of these studies are to define the cellular processes by which such RNA editing events can modulate neurotransmitter receptor expression and function in the central nervous system. To identify the cis-active regulatory sequences necessary for ADAR2 editing, a tissue culture model system will be utilized that exhibits RNA processing patterns analogous to those observed in vivo. Analyses of RNA from cells transfected with a variety of mutant ADAR2 minigene constructs will serve as the primary methodology for these mapping studies. An in vitro RNA editing system, utilizing purified, recombinant ADAR proteins, will also be used as a mapping technique and to further define the ADAR proteins responsible for site-specific modifications of ADAR2 pre-mRNA. The effect of ADAR2 protein levels on the specificity of adenosine modification will be examined using an inducible tissue culture system and an in vitro editing system to test whether over expression of ADAR2 can lead to the modification of known ADAR2 substrates(GluR-Band 5-HT2CR) at aberrant sites competition studiesbetweenADAR2 pre-mRNA and otherADAR2 substrates will be performed to determine if such competition represents a cellular mechanism for maintaining appropriate ADAR2 protein levels. To examine the molecular and phenotypic consequences of ADAR2 misregulation, genetically modified strains of mice will be generated in which ADAR2 levels have been altered by over expression or by selective ablation of ADAR2 auto editing. Multiple, independent mouse lines will be assessed for gross alterations in animal phenotype, changes in the level of ADAR2 expression/activity, alterations in editing patterns for previously identified ADAR2 substrates and increases in mRNA-derived inosine content. It is anticipated that these studies will provide new insights concerning the cellular processes modulatingADAR2 expression as well as the effects that such modulation confer upon the expression of neurotransmitter receptor isoforms.

The objectives of this research effort are to determine the natural history of diabetic retinopathy, and to evaluate the effect of photocoagulation therapy on specific forms of diabetic retinopathy. The Contractor will perform the function of a participating clinical center under the Diabetic Retinopathy Study.

The project documented the frequency of primary intracranial neoplasms in the pediatric populations of Rochester, Minnesota, and the state of Connecticut. In addition, using the records-linkage system available for residents of Rochester, Minnesota, we investigated the magnitude and risk factors for cerebrovascular disease in infants and children. The same Rochester, Minnesota records-linkage system was used to determine temporal trends in the incidence rates of cerebral palsy as well as the distribution of clinical subtypes and survival by clinical subtype, for the year 1950-1976. For the state of Minnesota, sex-specific neonatal mortality rates (NMR) in gestational age/birthweight risk subgroups were delineated for the years 1970-1976, and sex- and birthweight-specific NMR trends were determined for the years 1967-1976. The same record linkage system has been used to identify all possible cases of complex partial seizures occurring in the years 1960-1980. A case-control study is being designed to identify risk factors associated with the occurrence of such seizures.

PROJECT SUMMARY/ABSTRACT Electronic nicotine delivery systems (ENDS) have emerged in the US market, with use and awareness rapidly increasing in recent years, particularly in young adults. While ENDS may facilitate harm reduction in smokers, ENDS represent significant health risks, including addiction in the nicotine-nave (e.g., young adults). From a socioecologic perspective, the literature regarding tobacco retail indicates that place characteristics such as neighborhood demography and policy context influence retailer location and marketing, and these factors impact individual tobacco use. However, this literature is in its infancy in regard to ENDS and particularly to vape shops, which are stores exclusively devoted to ENDS sales. Vape shops have proliferated in the US and are unique in their product offerings, marketing, and overall retail environment (e.g., tasting bars). Vape shops, as well as the 2nd and 3rd generation ENDS they sell, have particular appeal to young adults. A particularly important and timely macro-level factor that may impact ENDS use and distribution channels is the impending FDA Deeming Regulation on ENDS and other tobacco products. The Deeming Regulation involves a range of policies implemented in the next 3 years (e.g., mandatory age verification and prohibiting free samples beginning in Aug 2016, mandatory health warning labels effective Aug 2018, manufacturers required to submit a new tobacco product application by Aug 2018, etc.). These regulations are likely to impact vape shop survival as well as their marketing and the overall vape shop experience, given young adults are the biggest segment of vape shop clientele (who will require ID), the social experience of tasting bars (which will no longer be allowed), and the history of ENDS being promoted as safe and for cessation or harm reduction (with products and ads requiring health warnings). A well-integrated program of research is needed to examine the multilevel impact of regulation on ENDS retailers, as well as on ENDS marketing, specifically among vape shops, given that they are a unique retail settings that have a particular impact on young adult ENDS use. Leveraging a Socioecological Framework, we will draw data from 6 metropolitan statistical areas (MSAs) representing the CDC-defined regions and the gradient of tobacco control in order to address 3 inter-related aims: 1) examine density and survival of vape shops over time and across contexts in relation to FDA regulation, local policies, and other sociocontextual factors (e.g., neighborhood context, density/proximity of convenience stores); 2) examine vape shop marketing and POS practices (e.g., age verification, free sampling, health warnings) over time and across contexts in relation to FDA regulation, local policies, and other sociocontextual factors (e.g., neighborhood context, density/proximity of convenience stores); and 3) examine young adult ENDS use over time and across contexts in relation to spatial access to vape shops and convenience stores, ENDS advertising exposure, local policies, and sociocontextual factors. This research will document the impact of regulation on this industry and provide an evidence base for legislation regarding zoning and vape shop marketing/POS practices to protect high-risk populations. We will disseminate findings with an explicit focus on informing public health policy and practice regarding ENDS, as well as future research.

The JAK-STAT (Janus Kinase - Signal Transducer and Activator of Transcription) signaling pathway activated by more than 35 different cytokines or growth factors has been implicated in a variety of cellular functions in the hematopoietic, immune, neuronal and hepatic systems. Ethanol inhibition of JAK-STAT activation may be a mechanism by which ethanol causes a wide variety of disorders in many organs. Acute exposure of rat hepatocytes to 25-100 mM ethanol significantly inhibits interleukin 6-, interferon gamma- or growth hormone-induced STAT activation. Chronic ethanol consumption significantly attenuates hepatic STAT3 activation induced by partial hepatectomy or interleukin 6. Acute ethanol (25-100 mM) exposure also significantly attenuates thrombopoietin- or interleukin 6-induced STAT activation in hematopoietic cells (BAF3/mp1 cells, platelets, U937, and AF10 cells), and ciliary neurotrophic factor-induced STAT activation in neuronal cells (NBTF neuroblastoma). The target sites of ethanol in the JAK-STAT signaling pathway will be explored by analyzing the effects of ethanol on cytokine-induced STAT phosphorylation, dimerization, translocation, JAK phosphorylation, receptor phosphorylation and ligand-receptor binding. The effects of chronic ethanol on the JAK-STAT pathway will be explored by examining JAK-STAT activation in vivo in rats fed with an ethanol containing diet. Identification of ethanol inhibition of the JAK-STAT signaling pathway in various tissues will enhance our understanding of the pathogenesis and progression of a wide variety of disorders caused by ethanol, such as antiregenerative effects, thrombocytopenia and fetal alcohol syndrome, and may also prove helpful in the design of novel drugs with therapeutic potential in alcohol-related disorders.

Project Summary: Aim #1. The function of Cx43 in the ciliary epithelium will be studied in three mouse models. In the nestin-cre /Cx43flox/flox line, Cx43 is selectively deleted from the pigmented (PE) layer of the ciliary epithelium resulting in a decrease in aqueous humor secretion and backflow of plasma proteins into the aqueous compartment. In the GjaUrt line, Cx43 immunostaining in the ciliary epithelium is globally reduced and also shows backflow of plasma proteins. The pax6alpha-cre/Cx43flox/flox line shows a loss of Cx43 from the non-pigmented epithelial (NPE) cells and a decrease in intraocular pressure. The three models will be studied in parallel using light and electron microscopy, immunohistochemistry, dye transfer methods and measurement of mouse intraocular pressure. Aim #2. Epithelial cells in CxSOnull lenses exhibit a striking reduction in mitotic index during the first postnatal week. Since replacement of Cx50 with Cx46 does not restore the normal growth rate, Cx50 must provide a unique functionality relating to epithelial cell proliferation. We will test two hypotheses regarding that function. The first is that Cx50 channels exhibit unique properties of selectivity, permitting the exchange of regulatory signals between cells. The second hypothesis is that Cx50, but not Cx46, regulates mitotic progression by C-terminal domain interactions with cytoplasmic proteins. To distinguish between channel properties and signaling involving connexin cytoplasmic domains, we will employ "channel-dead" Cx50 mutants that traffic normally to the plasma membrane and assemble into gap junctional plaques. One mutant with these properties is already characterized and others will be identified. The mutants will be introduced into CxSOnull mice as transgenes using a novel lentivirus approach. This strategy insures an ability to test multiple mutant genes very quickly. If these mutants rescue lens growth, this will strongly implicate the cytoplasmic domains of Cx50 as critical for the regulation of mitotic rate. If not, the properties of the intercellular channel are key and a rescue of mitotic rate will be tested by the introduction of tailless Cx50 mutants. Relevance: These studies will investigate the functional roles of gap junctions in the formation of aqueous humor and lens growth in the eye. They will permit a better understanding of the pathologies involved in glaucoma and cataract.

Differentiating cultures of embryonic skeletal muscle are being studied from two points of view. (1) A protein factor in embryo extract that is required for differentiation is being purified and characterized by standard biochemical methodology. (2) It has been found that the permeability of myoblasts to some fluorescent dyes changes abruptly as the cell differentiates. The molecular basis of this change is under investigation.

The adenylate cyclase and guanylate cyclase of cultured cells have been further characterized. Prostaglandin El activates a.c. in a highly cooperative manner. The calcium transport activity of the internal membranes of fibroblasts has been found to increase as cells crowd together and fall when they are dispersed. Two phosphoproteins, filamin and microtubule-associated proteins have been purified and characterized. The former binds to actin, the latter to tubulin. BIBLIOGRAPHIC REFERENCES: Davies, P., Shizuta, Y., Olden, K., Gallo, M., and Pastan, I.: Phosphorylation of filamin and other proteins in cultured fibroblasts. Biochem. Biophys. Res. Commun., 74: 300-307, 1977. Thomopoulos, P., Kosmakos, F.C., Pastan, I., and Lovelace, E.: Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes, (in press) 1977.

The transcription factor NF-kappaB plays key roles in regulating gene expression in both normal physiological processes and in diseases. It is required for normal embryogenesis as well as immune responses to infections. Disregulation of NF-kappaB has also been implicated in a large number of human diseases from inflammation to cancer. Although much progress has been made in the past decade on understanding the early signaling mechanisms leading to NF-kappaB activation, how NF-kappaB transmits its signal after its binding to DNA in the nucleus has only begun to attraction attention in recent years. A marine natural product named pateamine A was found to be a potent immunosuppressive agent, inhibiting the T cell receptor-stimulated transcription of interleukin-2 gene. Preliminary studies revealed that pateamine A selectively inhibits NF-kappaB signaling and to a lesser extent, that of AP-1. A systematic examination of the known steps in the NF-kappaB signaling pathway revealed that pateamine A does not affect early signaling steps up to the DNA binding by NF-kappaB. Instead, pateamine A was found to block the transactivation activity of NF-kappaB, making pateamine A a valuable probe to study the nuclear signaling mechanism of NF-kappaB. To identify the molecular targets of pateamine A, a biotin-pateamine A conjugate was synthesized, which enabled the detection, isolation and identification of two pateamine A-binding proteins. These pateamine A-binding proteins have not been previously shown to be involved in NF-kappaB regulation. The main objective of this application is to confirm the molecular interaction between these proteins and pateamine A both in vitro and in vivo and to verify the physiological relevance of these proteins as mediators of the inhibition of NF-kappaB by pateamine A. The physiological functions of these proteins in regulating nuclear signaling by NF-kappaB will be investigated. As these proteins have been implicated in several cellular processes, whether pateamine A affects the other known cellular activities of the putative pateamine A-binding proteins will be investigated. Lastly, gene chip analyses will be performed to determine the specificity of pateamine A for the IkappaB-NF-kappaB signaling pathway by comparing the gene expression profiles of mouse embryo fibroblasts treated with pateamine A and those derived from NF-kappaB and IkappaB kinase knockout animals. In addition, crystal and solution structures of the complexes between pateamine A and its binding proteins will be obtained to facilitate the future design and synthesis of novel and simplified pateamine A analogs as candidates for development of anticancer and immunosuppressive drugs.

Many medicinal reagents for the treatment of cancer originate from natural resources. To obtain sufficient quantities of these molecules and to improve their biological profile it is often necessary to produce these compounds synthetically. Monanchocidin, a member of the crambescidin family, is an attractive target for synthesis due to its complex structure and promising inhibition of cancer cell growth. An innovative strategy is proposed to address the synthesis of this molecule employing palladium catalyzed asymmetric allylic alkylation (AAA) and a novel hydroguanidination/cyclization sequence. The objective of this proposal is to develop a concise enantioselective synthesis of monanchocidin using newly developed reactions. We hypothesize that the pentacyclic guanidine can be rapidly assembled stereoselectively by developing a transition metal catalyzed hydroguanidination reaction. The specific aims of this project are; (1) to utilize palladium-catalyzed asymmetric allylic alkylation to construct the pyrolidine core of monanchocidin and other crambescidin family members, (2) develop and implement a novel transition-metal catalyzed hydroguanidination-spirocyclization method to assemble the pentacyclic guanidine, (3) develop an expedient synthetic route to access the fatty acid side chain in monanchocidin, and (4) develop a fragment coupling strategy that provides an efficient and modular route toward the first total synthesis of monanchocidin. The proposed research will be executed in an efficient manner by synthesizing key fragments enantioselectively and coupling them to the pyrolidine core of monanchocidin; assembled via palladium catalyzed asymmetric allylic alkylation (AAA). A novel method for construction of the pentacyclic guanidine of monanchocidin will be developed using a transition metal catalyzed hydroguanidination. An examination of transition metals typically used in hydroamination reactions will be conducted and the most efficient of these will be further optimized and employed in the proposed synthesis.

Recently, a family of five muscarinic acetylcholine receptor genes was identified. The encoded receptors are novel targets for more specific and effective cholinergic therapies for individuals with Alzheimer's disease (AD), but, because of their pharmacological similarities, functions of the subtypes are not well defined. The abundance and localization of individual receptor proteins in the brain, their regulation by cholinergic input, and their involvement in AD have never been studied. In the proposed investigations, subtype-specific polyclonal antisera will be used to evaluate the five native muscarinic receptor proteins in control rat and human brain as well as alterations in receptors in a model of experimental cholinergic deafferentation and in cases of AD. By immunoprecipitation, the relative abundance of proteins will be determined in basal forebrain, neocortex, and hippocampus in rats. By immunocytochemistry, the proteins will be precisely localized and colocalized with cholinergic forebrain neurons. The assessment of receptor number and localization after lesioning of the fimbria-fornix and basal forebrain in rats will provide infomation about the pre- and postsynaptic localization of the subtypes and the cellular and molecular bases for changes induced by deafferentation. Lastly, muscarinic receptor proteins in human brain will be identified, and changes that occur in cases of AD compared to age- and postmortem-matched controls will be measured. These studies will identify the best targets for subtype-specific cholinergic drugs and will provide new neurochemical markers for selective populations of neurons that are potentially at risk in AD.

The aim of this study was to determine the immunogenicity of recombinant hepatitis B vaccine when administered intramuscularly to premature infants. Ninety percent of preterm infants responded to the hepatitis B vaccine when the first dose of vaccine was given just before hospital discharge. This study is now closed, and the data are being analyzed.

Particulate matter (PM) and its adsorbed contaminants are associated with adverse health effects, including an increased risk of cancer. The level of PM10, particulates 10 um or less, in the Paso del Norte (PdN) air basin regularly exceeds federal guidelines, potentially exposing people to harmful toxicants. To date, research has not been undertaken to study biological effects of PM locally. In the proposed study, PM filters will be collected from air monitoring stations in El Paso, TX and Juarez, Chihuahua; organic material will be extracted and evaluated for biological effects. Cultured hepatoma cells will be exposed to extracts to determine Ah receptor mediated biological activity. Ah receptor ligands are known to cause numerous adverse health effects. Further, extracts will be used in the Ames test for mutagenicity, with and without metabolic activation. Finally, cells will be incubated with extracts and DNA damage will be measured with the Comet assay. The results of the study will also compare the use of a number of cell types with the goal of identifying the most sensitive and cost effective model system. This study will provide valuable baseline data on the potential for adverse health effects of PM in an airshed that has received almost no scientific attention.

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. This project focuses on the kinetics, metabolism and human toxicology of dichloroacetate-DCA and chloral hydrate-CH, potentially harmful metabolites of trichloroethylene-TCE. Humans exhibit polymorphisms of MAAI that may possess different kinetic properties toward DCA. Human haplotype variability may influence DCA's kinetics and toxicology. These hypotheses will be tested by accomplishing the following Specific Aims: Specific Aim 1: Quantify the influence of DCA, at exposure levels ranging from environmental, [unreadable]g/kg/d to clinical, mg/kg/d, on human liver MAAI and tyrosine catabolism. This aim tests the hypotheses that: there is a dose-dependent effect of DCA on human hepatocellular tyrosine metabolism in general and on the accumulation of potentially hepatotoxic tyrosine intermediates in particular; DCA inhibits hepatic MAAI expression in vivo in humans in a dose-dependent manner;and DCA, tyrosine and/or their metabolites form adducts with MAAI. Specific Aim 2: Establish the relationship between human MAAI haplotype and DCA and tyrosine metabolism. This aim tests the postulates that MAAI haplotype determines, and thus can predict: dose-dependent DCA kinetics and biotransformation and DCA's effects on tyrosine metabolism. Specific Aim 3: Determine the in vivo kinetics and biotransformation of CH in healthy adults and the influence of CH and DCA on each other's metabolism. This specific aim will examine three postulates: CH is metabolized in adults to DCA; CH, via DCA formation, inhibits its own metabolism and that of tyrosine;and these effects are dependent upon exposure level but not upon gender.

Uterine leiomyomas (fibroids) are the leading indication for hysterectomy in the United States. Despite the morbidity and high medical costs associated with fibroids, there has been little epidemiologic study of this condition in the United States. Uterine leiomyomas are histologically identifiable as benign smooth muscle tumors with varying amounts of associated fibrous tissue. Many women have more than one uterine leiomyoma, but each appears to be clonally distinct. Several specific cytogenetic changes have been identified in tumor tissue, but most show no chromosomal abnormalities. These benign tumors are hormone-dependent. They develop after puberty and regress after menopause. Both estrogen and progesterone are considered important stimulants, or at least permissive factors for tumor growth. To address the research needs in this field we have designed four studies. The first is a large epidemiologic study, the NIEHS Uterine Fibroid Study, designed to 1) estimate the age-specific cumulative incidence of leiomyomas in black and white women, aged 35-49, 2) identify risk factors for the condition, 3) compare growth mediating factors in tumor and matching myometrial tissues collected at time of hysterectomy, and 4) to identify factors associated with development of fibroid symptoms including pelvic pain and uterine bleeding. The second study (Fibroid Growth Study, Shyamal Peddada, PI) is a clinical study of fibroids designed to describe fibroid growth and compare the growth-mediating factors in growing vs nongrowing tumors. The third study, Postpartum Uterine Regression, monitors fibroid change with pregnancy and postpartum uterine regression. The fourth study, a prospective study of fibroid incidence, is currently under development. In this study we will enroll women before they have fibroids and follow them over 5 years for fibroid incidence. After estimating the age-specific incidence of uterine fibroids for black and white women, we began to examine risk factors for uterine fibroids. Pregnancy is protective, though not those that occur before the mid twenties. Alcohol appears to increase risk. In two cases we have replicated findings from animal models of fibroids. We find that the location of fibroids is somewhat different for parous and nonpauous women, and that prenatal exposure to DES is associated with increased development of fibroids. Increasing LH is associated with increased prevalence of the tumors, though LH may not be having direct proliferative effects, as we had hypothesized. We find no evidence for increased risk of fibroids with oral contraceptive use or with variability in menstrual-cycle length. We also explored our data on body fat and exercise. We find a small increase in risk with increased BMI (similar to other studies), and we also find that exercise is protective. As in the recent cohort analyses, smoking was not associated with risk in our data. We also collected questionnaire data for exploratory analyses on early-life exposures and several environmental/occupational exposures. While few factors showed associations with fibroid development, we did find an association of childhood use of insect repellent with fibroids. This may merit further investigation given the possible link between insect repellents and breast cancer. We measured fasting insulin and IGF-I in blood specimens collected from participants, hypothesizing both would be risk factors for fibroids. Surprisingly both tended to be protective, and diabetics were actually significantly less likely to have fibroids. We examined vitamin D status in relation to prevalence of fibroids both with the biomarker of hydroxylated vitamin D and with questionnaire data on time outside. With both methods, women with low vitamin D status had higher fibroid prevalence, and the findings were consistent for blacks and whites. We are beginning to examine dietary factors that may be related to fibroids. Regardiing fibroid symptoms, we found that urinary incontinence was significantly associated with fibroid size. We are currently examining the relationship between fibroid size and/or location and menstrual bleeding. The Fibroid Growth Study data have been analyzed and we conclude that: 1) spontaneous regression of fibroids occurs, 2) fibroids from the same woman grow at different rates, despite a uniform hormonal milieu, 3) fibroid size does not predict growth rate, and 4) age-related differences in fibroid growth between blacks and whites may contribute to the higher symptom burden for black women. We are currently examining short-term changes in growth of fibroids. In our study that monitored fibroid change during pregnancy and/or postpartum uterine regression we found that 36% of solitary fibroids were lost during the pregnancy/postpartum. Tumors that remained tended to have lost volume. We are now analyzing data to identify factors affecting the extent of fibroids reduction. We starting enrollment for the prospective study of fibroid incidence in October of 2010.

This project will address methodology development needs that are important in chronic disease population research. These include non- parametric estimator of multivariate survivor functions as relevant to disease prevention trials with multiple clinical outcomes and to family studies in genetic epidemiology. The closely related auxiliary data problem under which one uses data on multiple short-term response variables to strengthen the analysis of a primary clinical outcome will also be considered. Both estimators based on Peano series representations of the survivor function and likelihood-based estimators will be considered. Measurement error in key exposure and confounding variables can be a critical factor in determining observational study reliability. A more flexible than usual measurement model will be studied for application to such difficult to measurement exposures as nutritional and physical activity factors. Regression calibration estimators and empirical score function estimators of relative risk parameters will be studied based on data from this new measurement model in conjunction with an objective measures (biomarker) subsample. A third aim combines aspects of multi-variate failure time analysis and co-variate measurement modeling to develop new estimators of pairwise dependency in ages at disease onset of family members in the context of genetic epidemiology studies with measurement error in environmental factors. This project will also continue our studies of the role of various population study designs in the chronic disease research agenda as a function of the biases, measurement issues, and other limitations of ecologic studies, analytic epidemiologic studies, small intervention trials and full-scale intervention trials using the Women's Health Initiative database as a principal motivating setting.

Our ability to translate new scientific knowledge rapidly into improvements in patient and population health continues to be limited by numerous challenges. The Northwestern University Clinical and Translational Sciences Institute (NUCATS) was launched in 2007 to create a home supporting clinical and translational (C&T) science at Northwestern and beyond. Northwestern University (NU) is a leading national research university affiliated with nationally-renowned clinical partners. NUCATS is nationally recognized as an innovation leader in several key areas of C&T science, pioneering novel and scalable approaches in the areas of preclinical therapeutic development, health and biomedical informatics systems, research networking platforms and training programs to promote multi-disciplinary team science; and creation and leadership of a regional consortium of the three Chicago-area CTSAs to promote community engagement activities and practice-based research. In the present proposal, we describe our second generation institute, NUCATS 2.0. The Mission of NUCATS 2.0 is Speeding transformative research discoveries to patients and the population. Our Vision is to transform NUCATS into a national model of a highly integrated academic nexus that continually increases the quality, safety, efficiency and speed of innovative C&T research, by pursuing three Global Aims: 1) To speed translation and improve efficiency by integrating processes and programs that connect researchers with a continuum of resources, training, funding opportunities, and strategic partners; 2) To develop and implement innovative systems to identify, evaluate, facilitate and disseminate scientific breakthroughs, and improve the quality, safety and cost-effectiveness of C&T research; and 3) To promote Northwestern's culture of collaboration, innovation and translation through team-building, education, and training to empower the multi-disciplinary translational research teams of tomorrow. NUCATS 2.0 will be an innovative leader driving the rapid translation of life-saving and health promoting scientific discoveries to improv clinical practice and population health.

The two target populations have in common their potential for exposures to chemicals with resultant negative health outcomes. Both populations must have hazardous materials emergency response training as required under OSHA's Hazardous Waste Operations and Emergency Response standard (29 CFR 1910.120 or 40 CFR 311). In recent years. National Incident Management System (NIMS) mandates and development of the National Response Framework (NRF) have resulted in a nationwide approach to preparedness for all types of emergencies that reaffirms the critical role of first responders in dealing with these emergencies. Unfortunately, federal funds for preparedness assistance have not reached first responders in amounts sufficient to meet their needs for training. The populations targeted by this proposal typically have training budgets that are insufficient to meet the training needs, as indicated by the attached letters of support, particularly when travel is required to receive training. The current economic downturn has significantly eroded each population's ability to provide this much needed training and therefore makes the proposed cooperative agreement even more important.

Environmental toxicants with the ability to directly or indirectly modify the cellular thiols of lymphocytes can initiate immune dysfunctions resulting in increased incidence of infectious diseases, cancer and/or autoimmune diseases. The majority of our analyses will employ T cells, the regulators of the immune system; however, we will also compare the thiol characteristics and sensitivities of T and B cells; furthermore, we will evaluate the thiol-related biochemical differences between mouse and human lymphocytes. Preliminary data suggests that these species have differential thiol characteristics. The ability of exogenous and endogenous thiols, thiol-blockers, oxidants and anti-oxidants, as well as irradiation, to alter the subcellular mechanisms necessary for the activation and growth of lymphocytes will be assessed. In order to accomplish this objective we first must evaluate some thiol characteristics key to the biochemical activation of lymphocytes. The lymphoid subsets have different thiol sensitivities which we believe are due, in part, to variances in their exofacial thiols. Biochemical and cellular/molecular biology technics will be utilized to assess the influences of thiol modulation at various levels within the cell (exofacial, transmembranal, cytosolic, and nuclear). Novel thiol reactive chemicals and antibodies will be used to probe these cellular domains. Various cDNA probes, monoclonal antibodies, and anti-sense oligonucleotides will be used to assess the regulation of the mRNA expression, protein expression (and distribution) and need for thiol modulation of cellular cysteinyl proteins believed to be involved in lymphocyte activation. Some specific proteins to be assayed include protein kinase C, phospholipase C, calmodulin, and ornithine decarboxylase as well as some proto-oncogenes (c-myc; c-fos). The homogenous nature of cloned T cells, T cell hybridomas, and T cell tumor lines will aid in the analysis of the thiol-related sensitivities and activities of the subsets. Subcellular differences between the T cell subsets and cell lines will be compared. Evaluation of some biochemical changes involved in the stimulation of the different types of T cells will aid in the assessment of differences between non-proliferating and rapidly proliferating T cells and the processes involved in modulating their activation. The mechanism(s) of cell injury and modulation of activation and growth by toxicants with thiol reactivities will be further defined by our analyses. In addition, information may be obtained on numerous diseases which concomitantly have aberrant immune functions and redox states such as AIDS, Bloom's Syndrome, Ataxia Telangiectasia and Down's Syndrome.

The role of poxviruses, subviral particles isolated from virions or purified poxviral DNA in causing abnormal proliferation or neoplastic transformation of human and murine cells will be investigated. The structure and replication of ortho and paravaccinia DNA's will be studied in order to define: (a) the nature of the viral chromosomal structure and (b) the viral "replisome", in order to gain insight into the structure and replication of intrinsically more complex eukaryotic cell chromosomes. Alterations in cellular membranes following infection with various vaccinia strains will be compared and defined to: (a) determine how such alterations affect cell growth and cell-cell recognition; (b) function in providing "targets" recognized by the host's immune system. Restriction enzyme analysis and molecular hybridization techniques will be used to probe the genetic relatedness of poxviruses and their diversification in nature. This will provide the basis for future studies of the genetic information needed for determining host range specificity, pathogenecity and cell proliferation or killing after infection of cells with poxviruses.

The assembly and release of HIV-1 particles is quite complex, in that a number of viral and cellular RNAs and proteins participate, and while much has been learned about this process, much remains to be discovered. The Gag protein is central to the assembly and release of HIV-1 and all other retrovirus particles. A key function of the Gag protein that will be one focus of tis proposal is the recruitment and packaging of viral genomic RNA (gRNA). Reciprocally, gRNA may help to drive the recruitment of additional Gag molecules into a virion as assembly progresses. However, the full extent to which RNA participates in particle assembly in cells, and how Gag:RNA interactions change as assembly is initiated and completed remain to be defined. The experiments proposed in specific aim 1 will be the first to determine precisely how HIV-1 Gag and gRNA interact in cells. These studies could identify the very first as well as subsequent interactions between Gag protein and gRNA as well as between gRNA and antiviral cytidine deaminases. Following assembly, HIV-1 particles depart from the surface of the infected cell, to colonize neighboring or distal uninfected cells. During the previous funding period, we identified an IFN-induced membrane protein, termed tetherin, as a host defense molecule that inhibits HIV-1 particle release. Our work has demonstrated that tetherin acts directly, to retain nascent virions on the cell surface, but key mechanistic details of how tetherin functions, and what role t plays in preventing the dissemination of viral infection in cultured cells and in vivo remain to b determined. In specific aim 2, we will perform a series of in vitro and in vivo experiments to address these key questions.

Abstract The signals and processes by which diverse stimuli lead to keratinocyte cytokine production and cytotoxicity are presently not well understood. Moreover, chronic stimulation by agents such as ultraviolet radiation leads to malignancy. Recent studies have demonstrated that ultraviolet B radiation (UVB) damages human keratinocytes (HK) in part by inducing oxidative stress and cytokine production. Severe UVB damage to the keratinocyte can also result in apoptosis, or programmed cell death. Previous studies by our group and others have indicated that keratinocyte damage, especially via agents that result in oxidative stress results in the production of glycerophosphocholines (GPC) that can act as agonists for the Platelet-activating Factor (PAF) or the peroxisome proliferator activated receptor gamma (PPAR) systems. The long-term objective of this ongoing research proposal is to elucidate the role of these oxidized GPCs in the acute UVB-induced photoresponse. Three specific aims are designed to test the hypothesis that UVB-mediated oxidative damage to the keratinocyte results in the production of novel ox-GPCs that activate the PAF-R and PPAR systems. These novel lipids augment cytokine production, modulate apoptosis, and induce keratinocyte proliferation: 1). The effect of these ox-GPCs on acute UVB-mediated inflammation, cytokine production, DNA damage/apoptosis and proliferation will be assessed in mice deficient in PAF-R and epidermal PPAR; 2) Mass spectrometric methodologies will be used to structurally characterize ox-GPCs produced in response to UVB; and 3) The role of these ox-GPCs in the acute UVB photoresponse in humans will be determined. We anticipate that these studies using novel in vitro and in vivo model systems and novel methodologies will result in the characterization of ox-GPCs with PAF-R- and PPAR- agonistic effects in acute UVB-induced keratinocyte cytokine production, cytotoxicity, and proliferation. This information will aid in the understanding of the mechanism(s) by which oxidative stress can be involved in cutaneous pathophysiology. Since these mediators would be produced in response to oxidative stress in other organ systems, these studies should provide important insights into and tools to study the impact of ox-GPCs in human pathophysiology.

Aging and diabetes are associated with increased generation and deposition of Advanced Glycation Endproducts, or AGEs, the products of nonenzymatic glycation/oxidation of proteins/lipids. As human subjects age, the incidence of insulin resistance and, often, frank hyperglycemia rises. Long-lived proteins of the peripheral nervous system (PNS) are highly susceptible to AGE modification. AGEs, an heterogeneous class of molecules, may modify cellular function by an array of distinct mechanisms. One such mechanism is by their ligation of the signal transduction receptor, RAGE. RAGE also interacts with S100/calgranulins and amphoterin (or high mobility box group 1, HGMB1), which display diverse functions, such as neurite outgrowth and inflammation. Among the complications of diabetes, symmetrical neuropathy of the sensory and autonomic nervous systems affects significant numbers of diabetic subjects. Evidence accrued from the first five years of this Project has suggested an innate, survival role for RAGE in response to acute peripheral nerve injury. In euglycemia, transient and sharply-limited upregulation of RAGE (particularly in mononuclear phagocytes [MP] and neuronal/axonal structures) and its ligands is linked to RAGE-dependent inflammation and neurite outgrowth that contribute beneficially to regeneration. In diabetes, regeneration consequent to acute injury, such as crush, is significantly impaired. We hypothesize that in diabetes, chronic and sustained accumulation of AGEs and RAGE ligands, and upregulation of RAGE, perturbs homeostatic mechanisms in the peripheral nerve, leading to chronic dysfunction. Upon superimposed acute nerve injury, RAGE-dependent mechanisms sustain inflammation and neuronal dysfunction, and thwart regeneration. This proposal will address the fundamental question of whether RAGE/RAGE signaling globally, or specifically in cells of MP lineage or neurons, is protective or destructive to peripheral neurons in diabetes. We propose that in euglycemia, acute engagement of RAGE/RAGE signaling in both cell types is protective for peripheral neurons, but that chronic activation in diabetes promotes long-term neuronal dysfunction in the absence or presence of superimposed acute injury. This Project is critically linked to Project 1, as studies in chronic AB-enrichment in the CMSstrongly support deleterious and maladaptive roles for RAGE in neuronal stress. As antagonism of RAGE nears clinical trials in Alzheimer's Disease and diabetes, it is imperative to dissect the beneficial vs maladaptive impact of RAGE in both the CMS and PNS, and in chronic stress vs acute injury. Projects 1 and 2 are uniquely positioned to address these questions due to the long-time collaboration of the leaders of these Projects. Project 2 will employ Cores A and B through all five years of this Program.

PROJECT SUMMARY The International Consortium on Brain and Behavior Copy Number Variants (IBBC-CNVs) is a collaborative effort of 9 institutions with complementary experience and expertise in phenomics and genomics. The 22q11.2 and 16p11.2 loci are associated with significant risk for neuropsychiatric disorders across the lifespan. The clinical presentations are heterogeneous, manifesting in a range of developmental neuropsychiatric disorders, including Attention Deficit Hyperactivity, Anxiety, Autism Spectrum, and Psychosis Spectrum Disorders. Taking a `genetics first' approach of ascertaining patients based on known, homogeneous genetic etiologies will allow us to overcome barriers posed by the genetic and phenotypic complexity of idiopathic developmental neuropsychiatric disorders. We postulate that CNVs exert a large main effect on psychopathology, but the nature and degree of psychopathology observed in CNV carriers is multifactorial, with contributions from additional rare and common genetic variants, as well as environmental factors. Therefore, dissecting the effects of major CNV hits as well as additional rare and common variants on dimensional measures of psychopathology can elucidate the combined contribution of genetic mechanisms to psychiatric conditions and build models of risk prediction. Notably, the presentation and course of psychopathology in the CNVs resemble these features in idiopathic disorders. Therefore, beyond the specific genetic syndromes investigated, such a cross-CNV effort will identify convergent risk mechanisms for developmental neuropsychiatric disorders that are of relevance to the broader population. We propose to dissect dimensional measures of psychosis, social-emotional processing and neurocognition, and their genetic and environmental modifiers, to elucidate the architecture of risk for neuropsychiatric disorders in CNV carriers. Prospective evaluation with dimensional measures relevant to neuropsychiatric disorders will be applied to a cohort of 2000 individuals with 22q11.2 and 16p11.2 deletions and duplications (500 per group) and their relatives as feasible. In addition, categorical psychiatric diagnoses will be assessed in CNV carriers. Recruitment for prospective phenotyping will leverage existing large cohorts that carry these reciprocal CNVs, many of whom have already been ascertained and characterized with a range of phenotypic measures. New whole genome sequencing (WGS) will be performed in CNV carriers that have not yet been sequenced. We will also utilize existing genetic data from the largest available case-control samples diagnosed with SZ, ASD, and ADHD in the PGC to generate polygenic risk scores. Finally, we will examine family and environmental factors that contribute to the heterogeneity of presentation and developmental course in CNV carriers. This project will establish common phenomic and genomic resources. Our ability to conceive such a large-scale study capitalizes on our existing successful collaborations, complementary expertise, and institutional commitments to achieve these goals.

The aims of this competitive renewal application are to maintain and expand the present program for students participating in cancer research. The ultimate goal is to increase the number of health professionals, especially those from under-represented minorities, that will pursue clinical and/or research careers in cancer. The current grant award, Nutrition-Cancer Education Program (R25 CA47877) began September 1, 1988 and will end August 31, 1992. The grant award included two program components: Nutrition-Cancer Curriculum (Program I) and Short Research Experiences (Program II). This renewal application addresses the Short Research Experiences only, since Program I is not renewable. During years 01 through 04 of this award, the number of applications to the program have increased each year as has the quality of the students receiving the awards. Additional, there has been a higher percentage of minority student recipients when compared to the percentage of minority students enrolled in the Louisiana State University School of Medicine. In this application, the following activities are proposed: * continued funding for students from the health professions to participate in the Short Research Experiences in cancer/oncology; * expansion of the assistantships from 12 to 18 positions, including two positions for outstanding high school students; * new approaches for the recruitment of minority participation; * modification of the educational component of the Short Research Experiences; * use of newly-developed tools for evaluation of the Short Research Experiences; The above activities will be implemented in order to expand the educational and research opportunities of students in healthcare in Louisiana. It is anticipated that these opportunities will eventually contribute to cancer control in a state with excessive cancer rates, especially among the minorities.

Comprehensive examination of RNA structure and function can yield insights Impossible to giean from analysis of individual molecules. To aid understanding of the large and rapidly growing corpus of RNA structural data, we propose to create a structural classification of RNA (scaR). This database will organize features at four levels of complexity: three-dimensional motifs, tertiary interactions, structural domains, and whole molecules. Each of these may have multiple means of organization; for example, both by loop size and by base stacking. To initially define significant recurrent features, the classification will begin with manual inspection of all RNA structures. As the project progresses, observed characteristics will be formalized into algorithms that will be applied to perform rapid, reproducible, and accurate analysis and classification of RNA structures. [unreadable] [unreadable] The structural features will be mapped into a functional classification, and the associations between the two will be used to study general relationships between aspects of RNA structure and their function. To explore the evolutionary basis of RNA structure and the dependence of structure upon sequence, detailed links will be forged between classified RNAs of known structure and their evolutionarily related families. [unreadable] [unreadable] This classification will immediately elucidate the prevalence and variability of features in RNA structure. In addition, it is expected to have applications in RNA molecular modeling, molecular design and engineering, and RNA ligand and drug design. It will greatly enhance our understanding of RNA structure-function relations, the correlation between RNA structure and physical and biological properties, and evolutionary effects on RNA structure. The database will also provide a convenient and logical means for experts and neophytes alike to access and interpret the growing wealth of RNA sequence and structural data.

We have shown in our laboratory that rhesus monkeys with multiple inoculations of Brugia malayi L3 develop lymphatic pathology and immune responses to B.malayi adult worm antigen (BmA) that resemble, in many respects, those of human lymphatic filariasis patients. One similarity of the immune response between human and rhesus with brugian filariasis is that both exhibit T-cell unresponsiveness to BmA in vitro. In an attempt to overcome the cellular unresponsiveness to BmA, eight multiple-infected rhesus monkeys whose cells exhibited unresponsiveness to BmA and two uninfected controls were studied. PBMC were co-cultured with antigen and one of the following cytokines that have been reported to be effective in reconstituting T-cell proliferation interleukin (IL)-2, IL- 12, anti-IL- 10, anti-IL-4 and anti-IL- 13. We were able to overcome antigen-specific unresponsiveness in only a minority of the rhesus monkeys studied. Co-culture with either IL-2 or IL-12 were the only conditions which resulted in a slightly enhanced proliferation to BmA in these animals. Even high concentration of every cytokine was unable to render a significant antigen-specific proliferation. The results indicate that more than one distinct immunological mechanism may account for the antigen-unresponsiveness in rhesus monkeys infected with brugian filariasis.

This longitudinal study, now in its sixth year, assesses the effects of increased calcium intake on bone mass formation in females during puberty and early adolescence using double-blind, placebo-controlled trials of calcium supplements.

The objective of this program is to determine by a study of the nucleotide sequences in oncornavirus RNA whether the large 35S RNA subunits comprising the genome RNA are identical or unique polynucleotide chains. Regions of the RNA at the 5'-termini and adjacent to the poly(A) segments will be examined. In addition, the nature of the linkage of the 35S RNA subunits will be examined as well as the binding site(s) for the 4S RNA molecule which primes DNA synthesis by the viral RNA-dependent DNA polymerase.

This is a multi-center, randomized, open-label, parallel group trial to compare the efficacy of 9 ug interferon alfacon-1 administered sub-q three times per week for 72 weeks or 1 X day for 48 weeks to the same dose administered TiW for 48 wks in subjects with chronic HCV infection, previously untreated with interferon. Efficacy is defined by undetectable serum HCV RNa levels 24 wks following cessation of therapy.

The long term objectives of the proposed studies is to determine the regulation of UDP-glucoronosyl transferase (UDP-GT) inducer-mediated changes in thyroid follicular cell proliferation and apoptosis. This proposal describes a series of experiments that address how these chemicals alter rodent thyroid physiology, both in vivo and in vitro. Preliminary studies with other goitrogens suggest that UDP-GT inducers will effect thyroid follicular cell proliferation and apoptosis. Previous studies have emphasized feedback production of thyroid stimulating hormone (TSH) as the sole contributor to thyroid follicular cell proliferation, and ultimately, thyroid neoplasia. Recent findings such as, 1) while TSH inhibits apoptosis, propylthiouracil (PTU) treatment leads to an induction of apoptosis, and 2) insulin-like growth factor-1 (IGF-1) potently enhances TSH-mediated proliferation, strongly suggest that in addition to TSH that bother" factors are also important in regulating thyroid follicular cell proliferation and death. The findings from these studies are of importance to human health because there are several therapeutic and environmental agents that induce UDP- GT activity, decrease serum thyroid hormone, and potentially increase serum TSH concentrations. Yet, the relative importance of other factors is unknown. The studies outlined in this proposal will increase the knowledge base in the regulation of thyroid-follicular-cell proliferation and apoptosis, thus, enabling future studies with UDP-GT inducers to accurately assess which changes in thyroid physiology are impor nt.

DCCPS supports web-based, online, electronic and other communication and education efforts as part of its mission. These activities enable the organization to be more interactive with stakeholders by sharing important information about research portfolios and the resulting tools, products and scientific advances.

The long-range goals of this project are: (1) to evaluate the effects of prenatal exposure to environmental chemicals on the subsequent reproductive capacity of the offspring; (2) to investigate the mechanisms involved in the production of subfertility in mammals as a result of their in utero exposure to foreign chemicals; (3) to assess the transplacental carcinogenic potential of these compounds; (4) to study the physiologic disposition and metabolism of these compounds in the pregnant animal and fetus; (5) to study chemico-biological interactions of transplacental toxicants, with special emphasis on structure-activity relationships; (6) to determine if prenatal exposure to environmental agents can alter the cytoplasmic receptors for steroid hormones in reproductive tract tissues; (7) to develop and utilize organ culture systems to study the effects of environmental chemicals on the fetal ovary and reproductive tract in vitro; and (8) to evaluate the above animal models as predictors of human response. Special attention is given to diethylstilbestrol (DES).

Virtual product design and assessment has become a valuable tool in the development and evaluation of new buildings (and consumer products) as well as the interior design of automobiles. While these processes are beginning to work well for whole body designs targeted at accommodating population anthropometries, particularly in an effort to reduce injuries associated with awkward postures (Corlett et al. 1980, Chang et al. 2003, Chaffin 2005, Perez 2005), they have not been applied to hand tool or device design. It has been shown that repeated use of hand tools by individuals with hand dimensions significantly different than for which the tool was intended has resulted in an increased potential for hand-related injuries (Cobb et al. 1996, Meagher 1987, Kar et al. 2007, Markison 2007), particularly when significant force is required to operate the tool (Sancho-Bru et al. 2003, Molteni et al. 2008). Virtual hand models have been ineffective in addressing such anthropometric tool-fit issues. Current technological impediments related to available virtual hand models include (1) a lack of a solid geometry hand with accurate surface representation and accurate joint kinematics defined by centers of rotation, (2) a limited capacity in accounting for anthropometric scaling (e.g. the relationships between individual finger segment dimensions) (3) missing information on predicting how the hand grips objects and the forces applied during gripping, and (4) an inability to account for tissue compliance (particularly at the finger tips and palmar surface). The proposed project intends to address these impediments by developing a 3D geometric hand model, to be integrated into the current CAD design software products used by design engineers (e.g., SolidWorks, ProE) to evaluate the interaction between the hand and a new product. The proposed Phase 1 research will result in a virtual hand model that specifically targets the first two impediments (listed above) and will establish a draft framework (to be used to scope future research) for the necessary parameterization to address (3) and (4). At the conclusion of phase 1, a stand-alone software program with a scalable geometric hand representation with kinematically realistic articulating digits will be produced. The virtual hand model will be developed using the open-source SimTK core libraries (Sherman et al. 2005, Delp et al. 2007, Schmidt et al. 2008). The bones and joints of the hand will be modeled using rigid body structures to mathematically replicate laboratory recorded hand anthropometry, joint centers, and kinematics. The surface representation of the hand will be modeled using a similar method used by rigid body spring models (RBSM) (Kawai 1980). An integrated hand model, incorporating both the proposed surface-deformation/skeletal model and existing muscle models, would provide a powerful analysis tool for 1) understanding hand related injury mechanisms associated with grip posture and force and 2) optimizing tool design in-silico, prior to workplace deployment. PUBLIC HEALTH RELEVANCE: There currently exist no methods for design engineers to assess prospective design changes in a virtual environment as related to the overall tool-hand fit with respect to different population hand sizes. This has led to hand tool designs that are inappropriate for a large number of users and whose repetitive use will result in an increased potential for injury (Meagher 1987, Markison 2007). The overall goal of this project is to develop a scalable, virtual hand model that can be used to evaluate and determine appropriate hand-tool coupling interfaces, information that can be used to design hand tools to accommodate the hand sizes and hand shapes of end users.

We have found that the receptor FPRL1 recognizes Alzheimer's disease-associated amyloid beta peptides and promotes proinflammatory responses in the brain. We used mouse FPRL1 counterpart mFPR2 as a model for further understanding of the receptor in the progression of the disease. We found that mFPR2 in microglial cells, the macrophages in the brain, is upregulated by diverse proinflammatory stimulants, including agonists for the pattern recognition receptors and cytokines. Increased expression of mFPR2 on microglial cells enhances the capacity of the cells to endocytose amyloid beta peptide, therefore the receptor may profoundly affect the progression of the disease.

Modern biomedical imaging technologies allow for generation of high-resolution digital 3D images of many microscopic biological objects. However, effective strategies remain to be developed for automatic quantitative and statistical analysis of such microscopic structures' 3D morphologies. This proposal is aimed at developing algorithms for automatic morphometric analysis of neurons in intact brains. Quantitative and statistical characterization of individual neurons' spatial locations and their 3D projection patterns is not only essential for understanding brains' complexity, diversity, and plasticity with single-cell resolution, but also critical for elucidating subtle cellular pathological mechanisms underlying various neurological/mental/psychological disorders. New expertise will be explored to advance technologies in multiple areas of biomedical imaging, such as image computation and simulations of complex tissues. A GAL4-independent binary transcriptional system has been developed to label specifically the entire morphologies of the Drosophila olfactory learning and memory center, the mushroom bodies (MBs). In conjunction with MARCM (Mosaic Analysis with a Repressible Cell Marker) technologies, one can independently label various single MB neurons and the whole MBs in the same brains. Meanwhile, new algorithms have been developing to conduct automatic morphing (morphological deformation & matching) of irregular-shaped 3D objects. A virtual average MB will be constructed via statistical characterization of pair-wise morphing among multiple "standard" MBs. Morphometric analysis of distinct MBs and spatial mapping of individual MB neurons will then involve establishing point-to-point correspondence between the MBs of interest or the MBs, in which specific single MB neurons are differentially labeled, and the statistical model MB. Thus, one may be able to detect automatically and describe quantitatively any given MB's structural deviations and to identify individual MB neurons based on their 3D neuronal location/projection patterns.

The nicotinic acetylcholine receptor (AChR) is a pentarneric integral membrane protein with the subunit composition alpha2beta-gamma-delta . The five subunits form an ion channel at their center whose opening and closing is controlled by the binding of acetylcholine. The nicotinic AChR is muscle is the best-studied of a large family of neurotransmitter receptors that include the receptor for GABA glycine and the neural AChR. Glutamate receptors are formed according to a similar pattern, but are more distantly related. Our laboratory studies the structure, function, and assembly of the nicotinic AChR n muscle. We investigate both the properties of the receptors in muscle cells and in heterologous cells in which the AChR is expressed after transfection of receptor subunit cDNA. Because of the similarity of members of the family, findings that related to the AChR serve as a model for other receptors, which are targets for drugs that are relevant to the treatment of mental illness.

The goal of this research is to develop novel, biologically functional DNA nanostructures that dramatically enhance the reproducibility, sensitivity, and spatial density of chip-based DNA assays. These nanostructures will improve applications ranging from point-of-care diagnosis to genomic arrays used in basic research by enabling the development of next generation screening technologies that are faster, more sensitive, more reliable, and possibly more cost effective than those presently available in the life sciences market. To accomplish the stated goals, Nanolnk will develop a DNA patterning methodology based on Dip Pen Nanolithography (DPN) to generate sub-micron sized features of DNA on solid surfaces. This multidisciplinary effort will involve life and physical scientists at Nanolnk, MEMs and instrumentation engineers at our fabrication facility, in addition to support from outside experts in the fields of DNA microarrays and microfabrication. DPN, built upon the technique of Atomic Force Microscopy (AFM), allows one to deposit materials uniformly in a direct-write fashion on surfaces with nanoscale spatial precision. This strategy offers significant advantages over current microarray printing technologies that suffer from poor spot to spot reproducibility in terms of size, shape, and oligonucleotide density, as well as reproducibility across microarray slides. Preliminary work has demonstrated that the DPN technique can be used to deposit 12mer synthetic oligonucleotides on surfaces with extremely uniform sub-100 nm to several micron scale features. The DNA nanostructures formed robust films and exhibited selectivity in binding to complementary oligonucleotides. Thus, DPN can be used to generate uniform features of synthetic DNA far smaller than can be obtained with other spotting or photolithography techniques. In Phase I, Nanolnk will demonstrate feasibility of the DPN-based approach for generating sub-micron scale DNA nanostructures on glass surfaces. The resulting nanostructures will be analyzed using existing fluorescence probe technology to provide benchmarking standards for comparison to conventional microarray assays. In addition, for applications in life sciences and biomedicine, it is desirable and advantageous in terms of speed and throughput to extend the serial patterning capability of DPN to a parallel methodology. Thus, concurrent with ink development and patterning optimization, microfabricated parallel multipen arrays will be explored as a means for faster, simultaneous writing of multiple DNA inks.

Background: START, a Study of Trauma and Reduction of HIV Transmission, is a cross-sectional study that will examine whether traumatic stress contributes to potentially amplified transmission (PAT) risk behavior (the co-occurrence of detectable HIV viral load and HIV transmission risk) among stimulant-using men who have sex with men (MSM). Stimulant-using MSM are most heavily affected by HIV, despite the widespread availability of antiretroviral therapy (ART).1 Treatment as Prevention (TasP), defined as expanded ART access to achieve sustained viral suppression, is a promising biomedical prevention approach that will be utilized. While literature supports that early ART initiation decreases HIV transmission rates, stimulant-using MSM were excluded from these landmark trials. Stimulant-using MSM are known for low utilization and low engagement in HIV-related care thereby resulting in higher viral loads and high HIV transmission rates.2 The overarching objective of this proposed study is to identify modifiable risk factors associated with PAT to inform the development of innovative, theory-based interventions to optimize the effectiveness of TasP in stimulant-using MSM. Resulting information will inform the development of HIV/AIDS prevention approaches enhancing the effectiveness of TasP in stimulant-using MSM, thereby decreasing HIV transmission rates. Relation to Training: This proposal is designed to provide in-depth research training. Pathway analysis will be conducted to understand the role of psychosocial factors in the relationship between traumatic stress and PAT. The results will provide preliminary data for the preparation of a K99/R00 grant, which will be used to develop and pilot test a behavioral intervention to reduce traumatic stress symptoms and HIV transmission in stimulant- using MSM. This F31 proposal is a logical step in becoming an independent nurse investigator. Innovation: The study is innovative in three ways. First, PAT, a co-occurring outcome variable, will be utilized to model an outcome most closely linked with high HIV-viral transmission efficiency; second, the potential mediational role of psychosocial factors in the relationship between traumatic stress and PAT will be explored; and third, novel techniques will be utilized, including (a) the Balloon Analogue Risk Task (BART) measure of impulsivity while eliminating the limitation of a self-report measure requiring self-insight and awareness, and (b) Audio Computer Assisted Self Interviewing (ACASI), which is used in behavioral research to minimize recall and social desirability bias and enhance the veracity of self-report, thereby increasing internal and external validity.

There is an increase in the risk of bone fracture with aging and adult diabetes, and this increase cannot be solely explained by changes in bone mineral density (BMD). One barrier to new diagnostic tools and treatments is that the underlying cause for the disproportionate increase in fracture risk among diabetics and the elderly is currently unknown. Consequently, there is a need to identify the biophysical basis of the age- and diabetes-related changes in bone that decrease fracture resistance, not just bone strength. Addressing this, the proposal aims to determine whether increases in advanced glycation end-products (AGEs) explain the effect of diabetes and aging on the fracture resistance of bone (as characterized by fracture properties) and to determine whether an AGE inhibitor and/or an antioxidant can both improve the quality of bone structure and increase the fracture properties of bone. Aim 1 will determine the role of bone structure, BMD, and AGEs in the effect of Type 2 Diabetes (T2D) and aging on the fracture properties of bone. In the first experiment, bones will be collected from T2D rats and non-diabetic rats at 24 weeks and 32 weeks of age. In the second experiment, bones will be harvested from aging rats at 4 months (young), 12 months (adult), and 24 months (old) of age. All the bones will undergo extensive analysis in order to identify the relative contribution of compositional properties such as BMD and AGEs and structural properties such as moment of inertia to a set of biomechanical properties including traditional measurements of strength and new measurements of fracture toughness and fatigue life. Aim 2 will evaluate how exogenous glycation of collagen affects the fracture properties of bone. This aim investigates whether the direct accumulation of AGEs within the extracellular matrix of bone decreases the fracture resistance of bone and whether the AGE inhibitor pyridoxamine, a B6 vitamin, protects against such a change. With appropriate controls, both human cortical bone and rodent bone will be incubated in diabetic concentrations of glucose with and without the inclusion of pyridoxamine. After quantifying the concentration of pentosidine, a biomarker for AGEs, and BMD, each specimen will be subjected to a mechanical test. In the case of the human bone, tests will determine the effects of increasing AGE on bone strength, post-yield energy dissipation, fatigue life, and crack-initiation & crack-growth toughness. For the rodent bones, tests will determine the effect of increasing AGE on fatigue life and fracture toughness, the ability to resist crack propagation. Aim 3 will assess the efficacy of pyridoxamine and N-acetylcysteine to increase fracture resistance of bone in an aging rat model of T2D through changes in bone structure, BMD, and AGEs. In this translation aim, the role of oxidative stress and AGE accumulation in the aging and diabetic effects on bone will be investigated using these two compounds. Starting at 4 months of age, non-diabetic and T2D rats will drink water, water with pyridoxamine, or water with N-acetylcysteine, an antioxidant. After 4 months, 8 months, and 14 months (aging) of treatment, bones will be harvested for extensive analysis to determine the effects of treatment on the structural, compositional, and biomechanical properties of bone. Additionally, histological and cell culture assays will assess the biological effects of the compounds on oxidative stress and osteoblast differentiation. To achieve these aims, Micro-Computed Tomography will quantify volumetric BMD; high performance liquid chromatography will quantify the concentration of crosslinks and collagen content; and thermal gravimetric analysis will quantify the collagen and mineral fractions as well as water content. Statistical models will determine the relative contribution of the structural and the compositional properties to the fracture properties of bone. This will address the relevance of targeting oxidative stress and AGEs to improve the bone health of Veterans and prevent bone fractures. The long-term goal is to identify the factors affecting the important determinants of fracture resistance and developing accurate diagnostic assessments of fracture risk.

Although antiestrogens are known to be effective against many metastatic breast cancers and are presently undergoing extensive clinical trials, little is known of their mechanism of action beyond the fact that they mimic estradiol in binding cytoplasmic estrogen receptor and translocating it to the cell nucleus. I therefore propose to compare selected antiestrogens with active estrogens at four critical steps of estrogen action: 1) temperature-dependent estrogen-induced receptor transformation; 2) receptor translocation rate into cell nuclei; 3) receptor binding to specific nuclear acceptor sites; and 4) antiestrogen retention by nuclear receptor and its relation to clearance from the general circulation. Three model systems will be compared: the normal rat and mouse uterus, the dimethylbenzanthracene-induced rat mammary tumor system, and the MCF-7 human breast cancer cell line. The goal will be discovery of the critical differences between estrogen and antiestrogen action, particularly in mammary tumor cells and particularly as correlated with tumor responsiveness to antiestrogen therapy. The results should help improve the design of atiestrogen therapy alone or in combination with chemotherapy, should aid in the search for more effective antiestrogens, and should also contribute substantially to knowledge of normal estrogen action and the nature of hormone dependence in breast cancer.

Radiation-induced gastrointestinal syndrome (RIGS) results from a combination of direct cytocidal effects on intestinal crypt and endothelial cells and subsequent loss of the mucosal barrier, resulting in microbial infection, septic shock and systemic infiammatory response syndrome. Currentiy, there is no therapy for RIGS. Irradiation induces apoptosis of crypt endothelial cells, intestinal stem cells (ISC) and enterocytes within hours. We rationalized that the acute loss of cells in situ requires rapid compensation of their functions and this was best achieved with cell replacement therapies, e.g., blood transfusion for hemorrhage. The stroma of solid organs contains a variety of supporting cells, such as, mesenchymal and microvascular endothelial cells, macrophages and lymphocytes. These stromal cells provide the niche and could supply critical growth factor/signals for ISC regeneration. For example, upon intestinal mucosal disruption, resident macrophages in the intestinal submocosal layers are activated by pathogen-derived ligands for Toll-like receptors (TLR) and transmit regenerative signals to ISCs. We thereby propose intestinal regenerative therapy with a combination of systemic administration of growth factors and cell replacement therapy to salvage Gl function post-radiation exposure. In order to develop an stem cell-based therapeutic strategy for RIGS, we hypothesized that combinations of: a) intestinal stem cell growth factor, R-spondinl (R-spol), b) TLR ligands, and c) transplantation of bone marrow-derived endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) would restore the IR-damaged ISC niche, protect against IR-induced cell death and provide growth signals for host ISC regeneration, thus providing protection and mitigation from RIGS. Aim I Pathophysiologic Mechanisms, Discovery and Validation of Molecular Targets in RIGS Aim II will investigate whether acceleration of ISC regeneration could mitigate/protect RIGS by administration of a Wnt agonist, R-spondinl and a BMP antagonist. Aim III will examine whether repair of the ISC niche by TLR activation and/or bone marrow-derived adherent stromal cell-based therapies could mitigate RIGS in mice. The final goal is to identify radio-mitigating factors secreted by stromal cells.

This grant application requests five years of renewed support for the Arizona Alzheimer's Disease Core Center (ADCC). Scientifically, the ADCC is intended to optimize the development and use of its Cores, so as to capitalize on Arizona's scientific and organizational resources in the understanding, very early detection, and tracking of Alzheimer's disease (AD) and in the discovery of disease-slowing and prevention therapies. Organizationally, the ADCC is intended to establish a leading model of statewide collaboration in AD research. The Administrative Core provides the leadership and support needed to optimize the development, interaction, and use of its Cores. Working closely with researchers inside and outside Arizona, the National Alzheimer's Coordinating Center (NACC), and other AD Centers, it promotes the development and progress of AD-related studies and collaborations. It administers a program for the statewide solicitation, competitive review, and support of pilot studies. It helps solve the challenges and fulfill the opportunities associated with the ADCCs statewide collaborative model, and ensures the ADCC's accountability to the NIA. The Clinical Core maintains a large pool of clinically well characterized and annually assessed research subjects for the scientific study of AD and aging. The subjects include patients with AD and other dementias, patients with mild cognitive impairment (MCI), and normal controls, almost all of whom are enrolled in a brain donation program, and a growing pool of Latino and American Indian research subjects. This Core ensures the comparability and productive and appropriate use of subjects and data from its five clinical sites. It also promotes the productive and appropriate scientific use of longitudinally followed subjects, DNA, and data from three independently funded ancillary programs, yielding new information about the transition from cognitively normal aging to cognitive decline in persons at differential risk for AD. The Data Management and Statistics Core maintains the ADCC's database, helps ensure the quality of data and the protection of subject confidentiality, and provides statistical services in a manner that best serves the needs of the statewide ADCC. It works closely with researchers, NACC, and other AD Centers, sharing data in the most productive, timely, and appropriate way. The Neuropathology Core provides neuropathological diagnoses and extremely high-quality brain tissue from expired Clinical Core subjects to support research studies in Arizona and around the world. It also promotes the productive and appropriate use of biological materials from a large additional number of clinically and neuropathologically well characterized non-demented elderly subjects from its independently funded ancillary brain donation program, helping to address a critical need in the AD research community. The Education and Information Core provides training, innovative educational and outreach programs, and strategic partnerships to promote the development of AD-related researchers, address needs of professional and family caregivers, provide information about the ADCC, and address unmet needs of Arizona's American Indian and rapidly growing Latino communities.

Over the past five years, this Purdue University team has been collaborating with colleagues at Rutgers University to investigate the computational and experimental infrastructure for continuous manufacturing (CM) of solid oral dosage products. This work has built on a decade of research under the NSF supported Center for Structured Organic Particulate Systems. In parallel, the PI and Purdue co-workers have investigated CM approaches to small molecule API manufacture at micro and intermediate scales. This work has demonstrated the essential roles of process modeling, process analytical technology, active process control, intelligent process monitoring, material tracking and real time risk assessment. Moreover, current work is showing how these components must be linked through an integrated data management and informatics infrastructure in order to achieve the desired Industry 4.0 functionalities. While CM is an important development for the pharmaceutical industry, it is not a universal solution that meets all manufacturing needs, either technically or economically: often hybrid batch-continuous or fully batch modes can be advantageous. Moreover, given the major investment in existing batch facilities in the generic \manufacturing sector, there is an unmet need to bring Industry 4.0 functionalities to those manufacturers and thus significantly improve quality and reduce cost of medicines. The goal of this proposal is to expand the research on CM to develop and demonstrate a framework for the design and operation of batch and hybrid small molecule manufacturing systems. This will be achieved through five aims: Aim 1: Development of Pharmaceutical Model Library for small molecule and oral drug product manufacture Aim 2: General framework for the optimal synthesis of processes for small molecule-based API and product manufacture, encompassing the spectrum from batch to fully continuous processes Aim 3: Development of an Industry 4.0 real-time process management framework (RTPM). Aim 4: Demonstration of these technologies using several case-studies including high cost/low volume and high volume/low cost generic drugs at three different scales (lab, pilot and industrial) Aim 5: Development of instructional modules for conducting training programs for both FDA staff and industry as well as web-based access to tools and cases studies via pharmaHUB. This work will result in the development of the tool set necessary to implement Industry 4.0 across the pharmaceutical sector as well as the demonstration of the framework for systematic design and operation of processes for several specific drugs, including the effects of scale. The case studies will serve to inform and promote innovative manufacturing practices across the numerous batch and hybrid batch-continuous facilities existing worldwide. Moreover, by complementing the progress made in CM, it will enable the FDA to develop effective guidelines on the application of Industry 4.0 functionalities across the industry.

Glucose uptake by muscle and fat tissue is a primary mechanism for energy storage and represents a main control mechanism for glucose concentration in the circulation. A detailed understanding of the intracellular signaling system that mediates insulin-triggered translocation of the glucose transporter (GLUT4) will likely provide new findings for attacking diseases such as diabetes and obesity. The main goal of the proposal is to develop a kinetic screen to identify adipocyte regulatory proteins that control dynamic parameters for PIP3 signaling and for GLUT4 translocation and endocytosis. We will establish a wide-field TIRF system to measure the translocation and endocytosis rates of GLUT4 transporters, as well as the generation and degradation rate of PI3P lipids, in individual cells under 12 different perturbation conditions in parallel. We will create an RNAi library to target the approximately 800 adipocyte signaling/secretory proteins and a library of 800 expressed dominant negative and constitutively active expression constructs for complementary pertubation studies. In contrast to earlier approaches in which screens were based on end-point assays in fixed cells, the live-cell strategy proposed here focuses instead on the identification of adipocyte signaling and secretory proteins that control important kinetic parameters in insulin triggered PIP3 signaling as well as in GLUT4 translocation. In the second part, we will execute, analyze and interpret a screen of the insulin-PIP3-GLUT4 signaling network with this new approach and will also perform a screen of cross-talk between insulin and TNF-alpha signaling. These screens will be used to identify new players in the insulin-GLUT4 signaling network and to gain a better understanding of kinetic control mechanisms. The project will also provide a test case for a kinetic screening strategy and a test case for a combined screen using RNAi, as well as DN and CA expression constructs.

Complimentary and alternative medicines (CAM) are amongst the most commonly used therapies worldwide and in the US for chronic liver diseases. Silymarin, the active ingredient of the milk thistle, Silybum marianum, is one of the commonest used herbal medications but its efficacy in chronic liver disease is unproven. Liver disease secondary to both hepatitis C (HCV) and non-alcoholic steatohepatitis (NASH) represent 2 of the commonest liver diseases in the US and there is a need for the study of silymarin in these diseases in a controlled clinical trial. We are proposing three collaborative clinical trials to evaluate silymarin versus placebo in HCV patients who have never received treatment, in HCV patients who have failed treatment and in patients with NASH. These will be double blind, placebo controlled phase l/ll trials focusing on dose finding, safety and efficacy of silymarin. The endpoints of the trials will include histological improvement in liver disease and multiple secondary surrogate markers to understand both how silymarin works and to get a preliminary idea of the endpoints to be utilized in larger Phase 3 trials. We also intend to evaluate important secondary factors such as expectations of patients on CAM and effect of silymarin on quality of life. The goal of these studies is to determine whether there is a rationale for larger Phase 3 studies of silymarin in patients with chronic liver disease.

Dopamine (DA) signaling has received considerable attention for its role in reward-related processes, including the motivation to seek drugs and relapse in response to drug-associated cues (1-4). Destruction of DA cells in the ventral tegmental area (VTA), or DA terminals in regions such as the nucleus accumbens, disrupts drug self-administration (5-6) and administration of DA receptor antagonists attenuates the ability of drug-associated stimuli to promote reinstatement (7). In human addicts, striatal DA release in response to drug-associated stimuli is associated with increased drug craving and future relapse (8,9). Though DA has been generally implicated in drug taking and relapse, DA systems have thus far not been manipulated with the temporal precision and cell-type specificity required to isolate their role in specific aspects of those behaviors. Optogenetic tools have been applied to target DA neurons in transgenic mice, demonstrating that DA signaling supports behavioral conditioning and facilitates instrumental responding for food (20-21). More recently, a Th:Cre transgenic ratline was developed that allows for the selective targeting of DA neurons with optogenetic methods (22-23) in more complex behavioral paradigms optimized for use in rats. In this proposal, I will utilize Th:Cre rats, incorporating in vivo optogenetics and electrophysiology in combination with sophisticated behavioral analyses, to probe the causal contribution of DA signaling to different aspects of instrumental cocaine intake and relapse in response to Pavlovian cocaine stimuli. First, in Aim 1 I propose to test the sufficiency of VTA DA neuron activation to modulate cocaine self-administration and cocaine cue-induced reinstatement. Second, in Aim 2 I propose to test the necessity of DA signaling, via inhibition of VTA DA neurons, for cocaine intake and reinstatement. Additionally, the VTA contains a heterogeneous mixture of not only DA neurons, but also a substantial fraction of non-DA neurons that contribute to motivational processing (11-19), but little is known about how different populations of neurons in the VTA encode drug-related behaviors. Thus, in Aim 3 I propose to characterize the firing patterns optogenetically-identified VTA DA neurons (16) during self-administration and reinstatement.

The development of regulatory behaviors necessary for state, motor, and emotion control may have certain underlying physiological processes in the parasympathetic nervous system that play a role in their functioning. One specific psychophysiological correlate of regulatory behavior that has been extensively studied in the existing literature is that of heart rate variability, or vagal tone. Low heart rate variability in children has been associated with maladaptive outcomes (i.e., conduct disorder;Pine et al., 1996) and high heart rate variability has been associated with better social competence (Eisenberg et al., 1995). Thus, it is critical to understand the trajectory of vagal tone from infancy to childhood and how it both influences and is influenced by development. The objectives of the current application are 1) to examine the stability of baseline vagal tone and vagal regulation longitudinally in infants from 3 months to 3 years of age, 2) to examine the concordance between infants'behavioral and vagal responses to challenge tasks, 3) to examine the influence of factors endogenous (i.e., temperament) and exogenous (i.e., maternal sensitivity) to the infant on vagal trajectories over time, 4) to examine factors related to ethnicity and family income as predictors of vagal trajectories and 5) to examine baseline vagal tone and vagal withdrawal trajectories as predictors of socio-emotional functioning assessed by maternal report at 36, 48, and 60 months of age. The Durham Child Health and Development Study has demographic, maternal report, behavioral, and physiological data on 199 infants and their families at seven time points (3, 6, 12, 18, 24, 30, and 36 months). At each visit, infant vagal tone and behavioral response was measured before, during, and immediately following various challenge tasks meant to elicit negative reactivity and regulation from infants. Further, maternal behavior toward her infant during a free play and puzzle task was video recorded for behavioral coding. Our current understanding of vagal tone as a physiological index of regulatory functioning is limited. It is important to move beyond concurrent assessments of vagal response and identify patterns and trajectories that develop over time. The proposed analyses will allow us to examine the development of vagal tone over the first three years as a predictor of typical and atypical socio-emotional outcomes. Relevance to Public Health: The proposed study is designed to provide longitudinal analyses of the development of infant heart rate variability (i.e., vagal tone), and the regulation of vagal tone, across the first three years of life. This physiological system may underlie the processes of emotion regulation and executive functioning and has been associated with both positive and maladaptive behavioral outcomes in children. Understanding the various child and family characteristics that are related to its development will provide new insights into individual differences in such areas as self-regulation of emotion, responses to challenge such as the transition to school, and the acquisition of social competency in peer groups.

Chronic drug administration can produce allostasis, a maladaptive state related to drug tolerance. This proposal investigates nitrous oxide (N2O)-induced allostatic changes and the motivational consequences of being in an allostatic state. Allostasis refers to a disordered form of homeostatic regulation wherein a regulated variable, or one or more of its controlling determinants, persistently functions at levels significantly different from control values, potentially compromising an individual's health or viability. An allostatic model of drug addiction posits that biobehavioral control systems regulate variables relevant to drug taking behavior and that these control systems are vulnerable to drug-induced allostatic changes which promote the development of addiction. The proposed studies use a sophisticated experimental model that combines direct and indirect calorimetry so that core temperature and its determinants (metabolic heat production and heat release) can be simultaneously measured, enabling rigorous determination of allostatic dynamics during repeated N2O administrations. This thermoregulatory model system also provides a sensitive method for determining the motivational consequences of allostasis. Preliminary data indicate that adolescent rats are especially prone to develop drug-induced allostatic changes, suggesting that increased susceptibility to allostasis development may be a critical etiological factor for the heightened vulnerability to drug addiction during adolescence. Specific Aim (SA) 1 compares allostasis development in adolescent versus mature rats over a range of N2O concentrations, determines whether these allostatic processes stabilize, and explores how they can be extinguished. SA 2 compares the thermally motivated effects of a range of N2O concentrations in adolescent versus mature rats and assesses the motivational effects of an allostatic state during adolescence. In addition, the relationship between initial sensitivity, allostasis development and N2O self-administration behavior will be investigated. SA 3 examines whether factors measured in the allostatic state (N2O concentration, core temperature, heat loss, heat production) can be used as predictors of motivated behavior. This work has practical and theoretical importance for understanding the mechanisms underlying drug addiction. The proposed research has the added relevance of investigating an abusable inhalant during the adolescent period which NIH has identified as an important, yet understudied, research area. PUBLIC HEALTH RELEVANCE: A form of homeostatic dysregulation known as allostasis is suspected to play an etiologic role in the development of drug addiction. The proposed research uses an understudied inhalant to investigate drug-induced allostasis during a developmental period (adolescence) that is known for its heightened susceptibility to drug addiction. The findings of this research will contribute to our understanding of the pathogenesis and treatment of drug addiction.

Incontinence and urinary symptoms of frequency and urgency are prevalent in post-menopausal women. This project is designed to evaluate the effectiveness of topical estrogen in the behavioral treatment of urinary symptoms and incontinence.

The aim of this project is to clone and characterize the gene for a form of syndromic retinitis pigmentosa (RP), called. Hallervorden-Spatz syndrome (HSS) and characterized by abnormal electroretinogram, lipofuscin accumulation in the retinal pigment epithelium, and pigmentary retinopathy. Other features include dystonia, due to massive iron accumulation in the basal ganglia, and progressive deterioration leading to early death. Though lipid peroxidation is an hypothesized mechanism leading to the HSS phenotype, no knowledge exists of the molecular or biochemical defect in HSS. We have a unique opportunity to map the HSS gene using linkage analysis (homozygosity mapping) in a consanguineous Amish family with multiple affected members. Once the HSS gene has been mapped, we will search for mutations in candidate genes, as well as identify novel transcribed sequences that may contain the HSS gene. We will then characterize the gene and its protein product through homology studies to known sequences. Knowledge of the molecular basis of this disease will lead to a better understanding of the pathophysiologic process causing its pleiotropic effects. Understanding the etiology of a rare disease will often illuminate the mechanism at work in common, related diseases. Furthermore, by studying syndromic RP, we can use information about all of the syndrome manifestations (e.g. patterns of tissue expression, common metabolic or developmental pathways) to theorize a disease mechanism. Inference of a pathophysiologic process from a defective gene has proved frustrating for the forms of RP that are due to mutation in retina-specific genes. The HSS gene is not retina-specific, and a defect in it must account for rod photoreceptor degeneration as well as regional brain iron accumulation. Once the HSS gene is cloned and characterized, the other pathologic changes may provide a context for understanding the mechanism of pigmentary retinopathy. Since defects in this non-retina-specific process may cause other forms of syndromic and isolated RP and may be integral in disorders of lipofuscin accumulation, including aging macular degeneration, identification of the HSS gene may lead to greater understanding of RP as well as the macular dystrophies associated with sene ence. The HSS project forms the research core of Dr. Hayflick's training to become an independent biomedical investigator. Dr. Michael Litt, internationally recognized in the field of genetics, will he her primary sponsor with Dr. Richard Weleber, accomplished in the study of hereditary retinal diseases, as secondary sponsor. Unique strengths of her training program include a period of intense study in the Visiting Investigator Program through the National Center for Human Genome Research, which will provide her with expertise that will complement but not duplicate existing University research strengths, and the Oregon Health Sciences University and Department of Molecular and Medical Genetics research environments, which effectively foster collaboration with a diverse group of outstanding investigators.

Project Summary Antimicrobial resistance has evolved due to decades of inappropriate antibiotic prescribing by clinicians. In the emergency department (ED), nearly 50% of the 10 million antibiotic prescriptions written each year are inappropriate or unnecessary. However, antimicrobial stewardship programs (ASPs) for the ED have yet to be created. Failure to develop ED-based ASPs is a result of barriers unique to the ED, including lack of ED formatted guidelines, erratic workflow, rapid decision making, and diagnostic uncertainty. Electronic health record-based clinical decision support (EHR-CDS) can be customized to present ED- specific antibiotic recommendations at the point of care, in the usual clinical workflow. Therefore, EHR-CDS has great potential to overcome ED barriers and serve as platform for ED-based ASP. In this proposal, we build upon our preliminary single-center research to develop a multicenter EHR-CDS for antibiotic prescribing for two common pediatric ED infections, community acquired pneumonia (CAP) and urinary tract infection (UTI). National CAP and UTI antibiotic prescribing guidelines exist, but have not been adapted into ED context. We will use rigorous dissemination and implementation methods that this team has successfully used previously to create multicenter EHR-CDS. We also build upon our preliminary work to create a novel, multi- center EHR-CDS within the Pediatric Emergency Care Applied Research Network (PECARN). We will conduct workflow analyses and identify EHR triggers to determine the optimal timing for EHR-CDS activation, necessary to minimize alert fatigue. At each of the 3 participating sites, we will also develop an EHR-based mechanism to determine guideline adherent antibiotic prescribing for CAP and UTI. Through integration of the formatted ED antibiotic treatment guidelines for CAP and UTI, workflow analyses, and EHR activation triggers analyses, we will configure a prototype EHR-CDS for antibiotic prescribing. We will conduct heuristic review and subsequent scenario-based user testing at each site to produce a functional, scalable prototype EHR- CDS. Though we will initially build the EHR-CDS using a single her (with the largest market share), we will simultaneously develop a web-service version of the CDS for further scalability. Finally, as we have for prior studies, we will create automated clinician feedback reports using EHR data to provide antibiotic prescribing practices; these will complement the EHR-CDS as part of the overall ED-based ASP. The adaptable scalable EHR-CDS for CAP and UTI, and the provider feedback reports, will serve as the centerpieces for generalizable ED-based ASP. Upon completion of the work in this proposal, we will be poised to conduct a multicenter trial to test the effect of our EHR-CDS in pediatric and general ED settings.

The Quality Assurance Laboratory (QAL) in the Comparative Medicine Branch at the National Institute of Environmental Health Science (NIEHS)performs testing on the microbiological and/or chemical contamination of critical aspects of the overall NIEHS research program. Our primary task is monitoring the animal research program for microbiological and/or chemical contamination that may affect animal health and welfare, as well as, the physiological responses of animals used in research studies. Our research focuses on the physiological effects of natural or contaminating compounds in the micro-environment (e.g., animal feed, caging, bedding, water, etc.)of the research animals. Our goal is to assure that we minimize exposure to environmental compounds that may effect animal health and welfare or alter physiological responses of the animals resulting in unacceptable variability. Historically, QAL studies have focused on the potential for endocrine-disrupting compounds (EDCs) being present in the micro-environment of the research animals and the impact these compounds may have on study outcomes, especially reproductive development. For these studies, pre-pubertal CD-1 mice are weaned at post-natal day (PND) 15 and given test compounds orally (via diet, water or gavage) from PND 15 to PND 35 or until vaginal opening (VO) occurs which is a developmental milestone affected by hormones. We have shown that natural phytoestrogens (e.g., daidzein and genistein) present in commercially available rodent diets or added to diets free of these compounds can significantly (P less than 0.01) accelerate the time of VO in CD-1 mice. We have also shown that the total metabolizable energy (ME) in the diet can significantly affect these hormone-sensitive endpoints, although the predictability of this variable was less powerful than the phytoestrogen content. We have shown that the estrogenic mycotoxin zearalenone is ubiquitious in commercially available corn-cob bedding. 154 of 189 (84%) of the samples were naturally contaminated with zearalenone at levels ranging from 100 to 7,000 ppb (mean 500 ppb). We have shown that levels of 5-10 ppm can significantly advance the time of VO in immature CD-1 haired and SKH-1 hairless mice. We are currently performing studies looking at the effects of autoclave sterilization of rodent feed on the physical and chemical properties of the feed. Sterilization of rodent feed is important to prevent the introduction of microorganisms that may alter animal health or physiological response, and autoclave sterilization is a widely used method. Previous studies found that autoclaving rodent feed resulted in the production of acrylamide, as does any high starch containing food (e.g., potatoes, breads, etc.). These studies also determined that the levels of acrylamide produced by autoclaving the feed were high enough to cause measurable genotoxic effects (DNA adducts). Our study determined that increasing the sterilization temperature used resulted in an increase in the harness of the pelleted feed, as well as, the concentration of acrylamide. We are currently measuring the in-vivo effects of these levels of acrylamide.

The long-term goal of our laboratory is to identify the neural circuitry and neurochemical and neurophysiological mechanisms that underlie the expression and control of rage and aggressive behavior. The primary focus of the present grant application, which represents a major new direction in our research program, is to identify and characterize the roles of serotonin and cytokines in the medial hypothalamus in regulating these forms of aggression. This rationale for this application is based upon our most recent preliminary studies. They provide evidence that, in the medial hypothalamus, 5-HT1A and 5-HT2 receptors and cytokines, IL-lbeta and IL-2, powerfully modulate defensive rage behavior in the cat. The overarching hypothesis is that differential cytokine effects upon defensive rage and predatory attack are mediated principally through distinct neurotransmitter receptors of which serotonin and possibly GABA are primary candidates. Five experiments are proposed to test this hypothesis. The first will utilize immunocytochemical and neuroanatomical methods to characterize the pathway from the PAG to the medial hypothalamus mediating defensive rage and its relationship to serotonin axons and pre-terminals in this region. The second experiment will determine the effects of 5-HT1A and 5-HT2 receptors in the medial hypothalamus upon defensive rage. The third experiment will determine: the role of IL-1beta in the medial hypothalamus upon defensive rage, its relationship to 5-HT2 receptors, the distribution of IL-1R in this region and its relationship to serotonin axons and pre-terminals as well as to c-Fos labeled neurons following the expression of defensive rage. The fourth experiment will seek to determine the role of IL-2 in the medial hypothalamus upon defensive rage and their underlying neurotransmitter-receptor mechanism. The fifth experiment will identify the effects of activation of 5-HT and the above cytokines upon predatory attack behavior. The discovery that cytokines in the brain play a significant role in defensive rage represents a most significant observation. It has provided an entirely new direction of research - a direction in which the focus will address how cytokines and related substances in the brain may play critical roles in the expression and control of aggression and rage. By identifying the mechanisms underlying these effects, the proposed studies are of further significance because the strategies utilized here can now be applied for the study of how cytokines in the brain may regulate other behavioral processes.