The astroglial transporter GLT-1/EAAT2 is responsible for the largest percentage of glutamate transport in forebrain. Abnormal expression/function of EAAT2 is common to sporadic ALS, to transgenic models of the disease, as well as other neurological injuries. The mechanism that underlies transporter deregulation is not fully understood, but may involve altered promoter activation. We hypothesize that regulating expression of these proteins could provide powerful therapies to retard disease progression. Initial studies provide exciting evidence that increasing EAAT2 protein/activity can retard neurodegeneration in ALS animal models;diminish seizures associated with epilepsy, and retard brain tumor growth. In this proposal we will use recently generated GLT1 and GLAST-promoter reporter mice to evaluate the regulation of transporters. These rodents will be used to study the dysregulation of transporters in neurodegenerative disease. We propose to alter transporter gene activation as a novel means to delay neurodegenerative disease. The completion of these studies will provide a comprehensive understanding of transporter regulation in normal and abnormal CNS and their potential as neuroprotectants. Specifically we propose: 1) To understand the normal regional and temporal regulation of astroglial glutamate transporter gene expression, thru analysis of GLAST-BAC and GLT1-BAC promoter reporter expressing transgenic mice;2) To test the hypothesis that altered glutamate transporter expression in neurological injury is the result of promoter deregulation;3) To determine if altered cellular expression of astroglial glutamate transporters can prevent neuronal degeneration. Using drugs capable of increasing GLT1 and GLAST promoter expression we will determine if increased expression of the astroglial transporter(s) can prevent neural injury in vivo and we will determine if cell specific expression of glutamate transport alters chronic neurodegeneration using an animal model of amyotrophic lateral sclerosis. We hypothesize that over expression of GLT1 or GLAST will be neuroprotective in disease models. Over all Significance: In this proposal, we will develop a basic understanding of expression of GLAST and GLT I, discover reagents that can alter glutamate transporter activity, and determine if these approaches are useful for chronic neurological insults.


This program tests molecular imaging variables, how they predict response to therapy, and how they change over the course of different treatments. We propose 5 interactive projects: 1. Proliferation studies to validate how well FLT compares to TdR in patients with a variety of tumors and explores ways to simplify the PET procedure and still distinguish flux from transport. Our hypothesis is that FLT with appropriate data analysis can be as accurate as TdR for reporting tumor proliferation. We will test the hypothesis that FLT PET plus MRI can distinguish between tumor progression and pseudoprogression in patients with GBM that finish initial therapy with external beam RT plus concurrent TMZ. 2. Tumors have variable levels of multi-drug resistance that reduces effectiveness of drug treatments. We will quantify transporter activity of P-glycoprotein as a suspected cause of treatment resistance and failure by analyzing [11C]-verapamil kinetics. We will test the hypothesis that pts who up-regulate their tumor Pgp activity after neoadjuvant chemotherapy will have shorter survival and time to progression. 3. Hypoxia is an important resistance factor that is imaged using [18F]-FMISO. In pts with brain tumors we will test the hypothesis that FMISO images following initial surgery define an appropriate target for focal boost RT that will improve time to tumor progression and survival. In pts with ER-neg metastatic breast cancer, we will compare the extent of hypoxia to response to systemic therapy and progression-free survival. Other experiments will compare FMISO and [64Cu]-ATSM and we will measure temporal changes using an FMISO test-retest protocol and BOLD-MRI to look for short term changes in regional oxygenation of tumors. 4. This project will test the use of 18F-FES PET to choose therapy in pts with a history of ER+ breast cancer who have failed prior regimens and are being considered for salvage endocrine therapy and use FES PET to measure regional ER as a pharmacodynamic response to endocrine therapy and FDG PET to localize active tumor and identify early effects of drug therapy, including HER2 and ER. The project will also image androgen receptor function in prostate cancer to identify heterogeneous AR expression. 5. Preclinical radiopharmaceutical development and research. New methods will be developed to image important aspects of the tumor phenotype. Some are new molecules;some are new applications for old molecules;all to develop promising imaging agents for characterizing the tumor phenotype. These include ligands for HER2 as a factor in treating breast cancer, new agents for imaging of androgen biology, a monoamine oxidase A ligand to select patients for a new clinical trial for prostate adenocarcinoma and studies comparing Cu-ATSM and FMISO for different mechanisms of uptake.


The purpose of this investigation is to improve understanding of the effects of children's direct exposure to 9/11 and future mental health disorders, substance use or abuse and other risky behaviors as they now enter into adolescence and emerging adulthood. Based on the behavioral development literature, our city-wide assessment of children 6 months after 9/11 and the numerous findings of post- 9/11 consequences amidst exposed adults, there is strong evidence to suggest that exposure to 9/11 early in life would increase directly exposed children's vulnerability within the domains of interet in this investigation. Unfortunately, to date, the science-base of post-disaster behavioral consequences for youth, including those who were 9/11 exposed as children, lacks a longitudinal assessment, that includes in-depth measurement of the multiple, complex relevant domains, from type and severity of exposure to well-defined behavioral outcomes and intrapersonal, familial, academic and environmental factors. The proposed study will assess 1,000 children directly exposed to 9/11 at ages 0-12 and currently ages 12- 24, drawn from the World Trade Center Health Registry (WTCHR) Pediatric sample, who will be compared to unexposed Controls (N=500). The study involves a synergistic collaboration between the Child Psychiatric Epidemiology Group (CPEG) at Columbia University-Research Foundation for Mental Hygiene and the WTCHR. The WTCHR has longitudinal data on this population since 2002-2003, including excellent participant tracking information. CPEG has conducted many large-scale assessments of vulnerable youth, including 9/11 exposed children and their parents, addressing themes of high


This project is concerned with 8 major areas of neuropsychopharmacologic drug investigation: 1) early rapid-screening activity studies of new short-acting and long-acting psychoparmacologic compounds in severely ill and chronically hospitalized schizophrenic patients (phase I and II); 2) intensive double-blind studies in this same population; 3) improvement and development of blood extraction procedures, e.g., chromatographic analyses, to correlate those blood concentrations; red blood cell concentrations as well as plasma and serum levels of psychopharmacologic agents required to produce specific therapeutic changes in man (since recent measures of plasma levels show no correlation with therapeutic response--see supporting data concerning long-acting haloperidol); 4) attempts to correlate these blood concentrations required to produce CNS side effects in man; 4) six month evaluations of those long-acting oral and intramuscular compounds that appear to be especially promising therapeutic agents for evaluation of toxicity as well as ability to show sustained antipsychotic activity in the above patient groups; 6) controlled inpatient studies in adolescent schizophrenic patients as well as in acute adult schizophrenic subjects; 7) early rapid screening studies of new antidepressants (including comparison studies with lithium) and antianxiety compounds in nonpsychotic alcoholic inpatents in order to determine if one or more specific psychopharmacologic-responsive subgroups exist within this population; 8) double-blind studies of new active antianxiety and antidepressant agents in the same patient population (see supporting data fo drug-placebo differences in this population).


SUMMARY Intestinal failure is caused by a variety of enteropathies ranging from short bowel syndrome due to conditions such as necrotizing enterocolitis to congenital intestinal disorders. The mainstay therapy for patients with intestinal failure is parenteral nutrition (PN) but there are significant clinical complications associated with PN including infections, hepatic dysfunction and sepsis. Thus, a major challenge for intestinal research is to successfully develop new concepts and therapeutic approaches for the treatment of patients with intestinal failure that are dependent on PN. Advances in intestinal stem cell (ISC) biology, obtained through analysis of genetically-modified mice and cell lineage tracing studies, have garnered much interest in ISCs as a therapeutic target. Our long-term goal is to understand the extracellular signaling pathways that regulate human ISC niche, and how these pathways can be utilized for regenerative medicine or modulated in disease states such as intestinal failure. The recent development of directed differentiation of human pluripotent stem cells into intestinal tissue has afforded new opportunities to study human gastrointestinal development, tissue homeostasis and disease processes in vitro. Human intestinal organoids (HIOs) provide an in vitro model to study the direct interactions of epithelial and mesenchymal signals involved in maintenance of the ISC niche. Significantly, for patients with severe enteropathies where access to intestinal tissue is restricted, iPSC-derived HIOs provide a novel approach to study the intestinal biology of these patients. ADAM17 (TNF? converting enzyme) is a ubiquitously expressed sheddase responsible for shedding and activation of several growth factor/cytokine receptor families. In the mouse intestine, ADAM17 is implicated in complex intercellular communication involving EGFR/ErbB and TNFR signaling within multiple cell types of the ISC niche to maintain intestinal homeostasis, especially when faced with intestinal injury and inflammation. Recently, several patients from two different families were identified with distinct homozygous autosomal recessive ADAM17 null mutations defining a new rare congenital intestinal disorder termed neonatal inflammation skin and bowel syndrome. Our central hypothesis is that ADAM17 regulates signaling cross-talk between epithelium and mesenchyme of the human ISC niche. We will address this hypothesis in 3 aims: 1. generation of ADAM17 mutant and ADAM17 mutation corrected iPSC lines from dermal fibrobalsts obtained from an ADAM17 null patient. 2. Analysis of intestinal programming from ADAM17 mutant and ADAM17 mutation corrected iPSCs using HIOs and HIO xenograft transplantation and 3. Study the contribution of ADAM17 signaling from epithelial and mesenchymal layers from these HIOs. At the conclusion of this project, we should have gained detailed understanding of the role for ADAM17 signaling in maintaining the human ISC niche and defined ADAM17 or its substrates as potential new molecular targets, that may result in new approaches for stem cell therapies in regenerative medicine and for the treatment of intestinal failure.


We are interested in how high frequency (30+ Hz) brains oscillations are generated using the mouse olfactory bulb as our model system. These oscillations are a prominent feature of neural activity in many brain areas including the olfactory system. The mechanisms that underlie these oscillations, as well as the functional role they play are poorly understood, but they have recently been implicated in many aspects of brain function and disease. In the previous funding period, we have described a novel mechanism, called stochastic synchrony, by which correlated fast fluctuating inputs can generate synchronous gamma-frequency (30-80Hz) oscillations in populations of neurons. While much of our previous work used olfactory bulb neurons as a model system for analysis of this novel mechanism of synchronization, this prior work ignored many details of olfactory bulb neurons and circuits in order to describe the general features of this phenomenon. In this application we propose to extend our previous work by considering how key features of olfactory bulb circuitry and physiology modulate stochastic synchrony. We are particularly interested in whether the observed heterogeneity of neural properties is a useful feature of neural networks or is a "bug" that results from the intrinsic imprecision of biological systems. Homogeneity should enhance synchrony but recent data suggests that heterogeneity across neurons of the same type may provide certain functional advantages. We are also interested in how the synaptic connectivity of the olfactory bulb may facilitate or disrupt synchrony. Exploring these mechanisms will improve our understanding of the function and disorders of synchrony, especially in the context of the vertebrate olfactory system PUBLIC HEALTH RELEVANCE: Activity of neurons in the olfactory system represents information about sensory stimuli in the environment. One of our long term goals is to understand how to interpret features of neuronal activity as representing features of olfactory stimuli. To accomplish this goal we believe that it is critical to develop our understanding of the dynamics of neuronal activity through the use of experiments and computational models. In this proposal we describe an integrated experimental and computational approach to analysis of the mechanisms of patterns of periodic synchronized activity in the olfactory system. In particular we want to understand how the detailed and diverse biophysical and synaptic properties of olfactory bulb neurons contribute to the generation of patterned activity.


The broad, long-term objective of this proposal is to understand which tissues and genes respond to caloric restriction, and to explain how such changes affect the overall physiology, health and longevity of animals, including humans. The hypotheses to be tested are: (1) An H1 linker histone, HIS-41, is a direct target of DAF-16 and is one key transcriptional output of scenarios that increase longevity. (2) HIS-41 is necessary, but not sufficient, for the metabolic shift and increased longevity conferred by reduced insulin-like signaling or caloric restriction. (3) Distinct longevity mutants induce different HIS-41 accumulation patterns appropriate for the affected target tissue. (4) Caloric restriction extends life span by activating two synergistic processes: HIS-41 dependent silencing that reduces metabolic rate and reactive oxygen species levels; and increased production of factors that are co-regulated with HIS-41, such as superoxide dismutase. The specific aims are: (1) Identify the sequence elements required for DAF-l6- dependent induction of his-41 in vivo (hypothesis 1). (2) Assay fat accumulation and life span in calorie restricted his-41 mutants or his-41- gerontogene double mutants; and determine whether forced HIS-41 expression is sufficient to phenocopy longevity mutants (hypothesis 2). (3) Identify genes that regulate HIS-41 levels and genes that are coordinately regulated with his-41 (hypotheses 1,2). (4) Determine the cell type specificity of HIS-41 accumulation in calorie restricted vs. single and double mutant combinations of distinct longevity pathways (hypothesis 3). (5) Measure the life span of transgenic animals that constitutively express HIS-41, SOD-3, and genes that are co-regulated with HIS-41 (hypothesis 4). A central view of this proposal is that widespread genomic silencing, mediated by an H1 linker histone, is a common feature of many genetic pathways that extend life span and increase stress resistance. Environmental factors, including caloric restriction, increase longevity by turning on the H1- mediated repression of genes for rapid growth and glucose-based metabolism, thereby reducing the overall rate of metabolism and the production of reactive oxygen species. The activation of the H1 repression system is done by the same transcription factor (DAF-l6/forkhead protein) known to up regulate enzymes for fat storage and utilization, as well as enhanced life maintenance (e.g. superoxide dismutase).


Susan Steck-Scott, PhD, MPH, RD is a research assistant professor in the Department of Nutrition at the University of North Carolina at Chapel Hill. Through this Career Development Award, she is seeking training in molecular epidemiology and cancer survivorship to complement her strong background in the nutritional sciences and cancer prevention. Dr. Steck-Scott's primary mentor for this proposal, Dr. Marilie Gammon, is a renowned breast cancer epidemiologist with extensive experience in mentoring young investigators. In addition, Drs. Lisa Carey, Robert Millikan, Michael Schell and Fred Wright are well established investigators in their fields and have agreed to be co-mentors on this project. Training will include coursework and workshops in ethics, molecular epidemiology, carcinogenesis, pharmacology, and biostatistics, as well as a laboratory experience to learn the techniques of genotyping. This training will help the candidate to develop a unique niche in the cancer research community and establish an independent research program in the area of gene-nutrient interactions in cancer prevention and control. The primary focus of the award is a research project to examine the interaction between cruciferous vegetable intake and polymorphisms in genes encoding metabolizing enzymes in breast cancer prevention, recurrence and survival. The proposed research capitalizes on dietary data, urine and blood samples available from an already conducted population-based, case-control study, the Long Island Breast Cancer Study Project of which Dr. Gammon is the Principal Investigator. Breast cancer subjects are currently being followed up for recurrence and survival status. The specific aims of the research are 1) to examine the associations between cruciferous vegetable intake, urinary isothiocyanate (ITC) excretion, and polymorphisms in GSTM1, GSTT1 and GSTP1 in control subjects to determine whether having the non-null or active genotype for GSTM1, GSTT1 and/or GSTP 1 results in increased urinary excretion of ITC; and 2) to examine the interaction between cruciferous vegetable consumption and polymorphisms in GSTM 1, GSTT 1, GSTP 1 and CYP 1A 1 in relation to breast cancer risk in a large population-based case-control study; and 3) to examine the interaction in relation to breast cancer recurrence and survival in a follow-up study of the cases. This study represents a unique opportunity to examine whether cruciferous vegetables are modifying the effect of genetic polymorphisms in breast cancer prevention, recurrence and survival. By identifying those individuals who may be genetically more susceptible to the benefits of cruciferous vegetable intake, future and ongoing interventions may become more effective by being targeted to those people who will receive the most benefit.


The goal of the University of Pittsburgh Global Health Research and Research Training eCapacity Initiative is to enhance evidence-based, data-driven decision-making in India through innovations in information and communication technology using modeling and simulation to assess the health and economic impacts of alternative disease control strategies. To globalize the advantages of methods and curricula developed in the Public Health Dynamics Laboratory (PHDL) at the University of Pittsburgh, we believe it is essential to have local public health experts take the lead in developing, running and analyzing the models. Our objective is to enhance the capacity of researchers to use computational modeling, not only at our Fogarty training site, SHARE INIDA (D43 TW009078), but also at multiple Fogarty training sites in India. In the introductory Phase 1, a two-day symposium (35 participants), SHARE INDIA, Hyderabad, will introduce the concept of the integrated decision support pathway, and basic modeling software and tools. This symposium will showcase research that enables data-driven decision making using cutting-edge methods including mathematical, network, and agent-based modeling. Five Indian investigators plus one modeling support staff will receive in-depth, hands- on training in computational modeling during a week-long workshop at the University of Pittsburgh PHDL. This workshop on complex agent-based modeling will use our open-source modeling platform, FRED (Framework for Reconstructing Epidemic Dynamics). This experience will foster establishment of a new collaborative network for modeling studies among Indian researchers and with PHDL researchers. Two small modeling developmental grants will be awarded. Phase 2 will focus on sustainability. Two small research grants will help sustain newly developed, collaborative modeling projects. Two PHDL modelers will return to India to visit the recipients of the small grants and solidify collaboratios. Two week-long short courses on computational modeling in public health, incorporating educational materials and free software from the Pittsburgh Workshop, will be presented by Indian faculty at CR Rao Institute targeting 25 participants each. A second introductory symposium for 35 new participants, led by newly trained Indian modelers, will stimulate a new wave of interest in modeling. Our aims address an unmet need in Indiai.e. building capacity of researchers to use cutting-edge, yet appropriate technology to address policy-relevant questions. The education program will result in increased knowledge among Fogarty-affiliated researchers in India of the kinds of data needed to inform models and basic methods in modeling. Trainees who attend the workshops in Pittsburgh and sustain their use of modeling through small research grants are expected to continue to apply computational modeling methods to address Public Health questions of importance to India. These trainees are also expected to lead computational modeling collaborations within India and with PHDL faculty. In the future, the program is expected to result in funded research that aids data-driven decision-making in Public Health in India.


The goal of the proposed research is to characterize the amino acid regulatory interactions which occur in Saccharomyces cerevisiae. Growth of this organism in medium supplemented with a variety of amino acids results in a decrease in the intracellular arginine pool, but concomitant induction of arginine catabolic enzymes and repression of arginine anabolic enzymes. The specificity and magnitude of these interactions will be determined. The contribution of changes in enzyme levels to the observed decline in the arginine pool will be investigated. The kinetics of enzyme induction and repression will be compared to that observed when arginine is added to the growth medium. The role of the intracellular arginine pool in enzyme induction and repression will be determined. The physiological significance of these interactions will be examined by characterizing the behavior of mutants impaired in: (1) specific arginine metabolic regulation; (2) cross pathway regulation; and (3) nitrogen catabolite repression. The role of amino acid compartmentation in the regulatory interactions will be determined by measuring the distribution of arginine between vacuole and cytosol in cells growing in supplemented and unsupplemented medium.


To assess long-term symptomatic and objective relief in patients who underwent successful coronary angioplasty performed in 1980, 1981, and 1982.


A. Summary. We have preliminary data showing that in addition to its role in promoting osteoblast (OB) function and bone formation, fibroblast growth factor 2 (FGF2) is a negative regulator of mesenchymal stem cell differentiation into mature adipocytes (AD). We hypothesize that loss of FGF2 expression results in a shift of stromal mesenchymal progenitors from OB differentiation towards adipogenesis. The proposed studies will increase our understanding of the molecular mechanism (s) by which FGF2 affects aging bone as well as the role of FGF2 in the osteogenic and antiadipogenic effects of PTH in bone. Specific Aim 1: Determine how FGF2 modulates the adipocyte phenotype using Fgf2+/+ and Fgf2-/- mice in Col3.6-GFP or aP2-GFP genetic backgrounds. We will test the hypothesis that in the absence of FGF2, marrow progenitors have a reduced ability to choose the osteogenic pathway. To assess the age-dependence of the phenotype, we will examine young adult mice at 6-8 weeks of age and compare them to 4-5 month old adult mice that already exhibit reduced bone mass. Aim 1A: i) determine the temporal and quantitative onset of GFP expression in primary bone marrow stromal cultures (BMSC) from Fgf2+/+ and Fgf2-/- mice harboring transgene reporters for the OB (Col3.6-GFP) or AD (aP2-GFP or -Cyan) lineage; ii) characterize the AD and OB potential of GFP positive and GFP negative cells isolated via FACS analysis and then cultured in the absence and presence of exogenous FGF2 and PTH; and iii) examine changes in gene and protein expression. Aim 1B: Define the function of FGF2 during osteogenic versus adipogenic differentiation in vivo. Using mice developed in Aim 1A, we will i) assess whether there is a correlation of changes in bone mineral density and whole body and bone fat content. ii) examine the expression of key adipogenic and osteoblast signaling molecules from whole bones and from freshly isolated marrow; and iii) assess the effects of PTH, administered to mice alone or in combination with FGF2 on adipogenesis in ex vivo BMSC cultures. Specific AIM 2: Determine whether FGF2 is a necessary factor for PTH-mediated pro-osteogenic and anti-adipogeneic effect on mesenchymal progenitor cells. We hypothesize that FGF2 inhibits adipogenesis through modulation of Wnt 10b and PPARg in mesenchymal progenitors. We also hypothesize that in the absence of FGF2, PTH is unable to inhibit mesenchymal progenitor cell differentiation towards adipogenesis and this is mediated through regulation of PPARg by Runx2 and Wnt 10b downstream effects. Aim 2A: Examine the mechanisms by which FGF2 deficiency modulates the development of the OB or AD phenotype. We will determine whether FGF2 modulates Wnt 10b and PPARg2 activity and what signaling pathways mediate this in CFU-OB and CFU-AD from young and adult mice in vitro. Aim 2B: Define the transcriptional mechanisms underlying PPARg regulation by PTH and FGF2 signaling. We will test the hypothesis that one possible mechanism through which FGF2 and PTH crosstalk may regulate adipogenesis is through Runx2 and Lef-1/-catenin mediated control of the PPARg 2 promoter.


This is the second and final revision of a research grant to characterize nicotine withdrawal in pregnant smokers. Maternal cigarette smoking is the leading preventable cause of poor pregnancy outcomes and pediatric morbidity and mortality in the U.S. While 80% of pregnant cigarette smokers report seriously thinking about quitting while pregnant, most are unable to quit for even one week. Efficacious interventions have been developed for smoking cessation during pregnancy, but cessation rates are low, typically under 20%. One factor thought to play a substantial role in early smoking relapse and extensively investigated in non- pregnant smokers is nicotine withdrawal. To our knowledge, there are only two reports on nicotine withdrawal during pregnancy: a retrospective study in pregnant adolescents by Albrecht and colleagues (1999) and our group's recent prospective pilot study (Heil et al., 2006). Given nearly 30 years of research on smoking cessation during pregnancy, this represents a striking gap in this literature. Our initial pilot study resulted in two seminal findings. First, we observed that pregnant smokers report substantially elevated levels of withdrawal symptomatology prior to the cessation attempt. Second, even with this pre-cessation elevation, we were able to validate a number of withdrawal symptoms in pregnant smokers who sustained abstinence for five days and establish their incidence, magnitude, and acute time course. While a significant step forward, our pilot study raised additional questions about nicotine withdrawal symptomatology in pregnant smokers. The two aims that make up the proposed study are designed to follow up and extend the literature in this area. First, we propose performing a thorough examination of pre- cessation elevated withdrawal symptomatology, comparing levels in pregnant smokers to those in pregnant non-smokers and non-pregnant female smokers in an effort to elucidate the source of the increased symptomatology. Second, we propose performing a more comprehensive and rigorous characterization of nicotine withdrawal during pregnancy, increasing the sample size and lengthening the time course from our pilot study. The proposed study will partner with an ongoing clinical trial testing the efficacy of voucher-based incentives to promote smoking cessation during pregnancy. We will analyze nicotine withdrawal data collected from pregnant women who abstain as part of this trial and compare results to those of abstinent non-pregnant female smokers and pregnant non-smokers. PUBLIC HEALTH RELEVANCE: The proposed study has the potential to contribute important new information about nicotine withdrawal among pregnant women. Such new knowledge may contribute to the development of more effective treatments for one of our nation's most daunting drug abuse problems.


The Prevention & Control Research Core has been developed with Prevention & Control and Clinical Researchers of the DRTC to provide a range of consultation and direct services to support behavioral, patient centered/clinical, educational, and prevention research. Seven core services are: (1) assistance in recruitment of clinical and non-patient samples and recruitment of research settings for community oriented research; (2) assistance regarding clinical aspects of diabetes and assessment of clinical status and diabetes risk; (3) assistance regarding epidemiological aspects of research and analyses and accessing administrative databases; (4) assistance in planning, developing and evaluating health education interventions, including addressing cultural and psychological factors, diet and nutrition; (5) measuring psychological factors and quality of life among those with diabetes; (6) data gathering, administration of population-based and clinicbased surveys, and data management; and (7) statistical analysis of data, including biostatistical consultation and analysis using geographic information systems. Because the core is newly developed following NIDDK's changed DRTC guidelines, plans are in place to monitor the use and utility of the varied core services, eliminate underutilized services, add promising services, and reallocate funds accordingly. Not including research support for projects not using the Prevention & Control Core, users include six investigators with independent NIDDK funding (Brownson, Klein, Lustman, Racette, Sinacore, White), five investigators with other NIH funding for diabetes-related research (Haire-Joshu, Holloszy, Mueller, Newcomer, Wilfley), three investigators with non-NIH, externally funded diabetes-related projects (Fisher, McGill, Polonsky), and five investigators with prior or current Pilot & Feasibility funding and/or diabetesrelated projects planned (Auslander, Hams, Hershey, Perkinson, Schootman).


We have run two subjects in the past 12-month period. The co-investigator on the grant, Dr. Thomas Schlaepfer, has temporarily returned to Switzerland in order to oversee the running of his own Neuroimaging Laboratory at the University of Bern. Pilot results so far are encouraging, and show activation in expected regions of basal ganglia, frontal lobe, and mesial temporal areas, which we hypothesized would be involved in cocaine's cerebral activity. Dr. Schlaepfer is returning to the United States for a visiting academic appointment in the 1999-2000 period and more subjects will be run in the study during this time.


The objective of this project is to develop a package of brief, safe and reliable measures of exercise tolerance that, in total, is broadly applicable and highly discriminating in population studies of older persons. The first phase of testing and evaluation has been completed at the Baltimore Veteran's Administration Medical Center through an interagency agreement (Y01-AG-4-0260). The following tests were administered on two occasions 7 to 10 days apart to a volunteer sample of 50 men and women age 54-80 years, 24 of whom had peripheral arterial disease: (1) timed usual and fast pace 4-meter walks; (2) timed usual and fast pace 20-meter walks; (3) 6-minute usual pace corridor walk; (4) seated step-test; and (5) treadmill walk. Heart rate at work, recovery heart rate, and blood pressure were measured. Two questionnaires were also piloted; one is a refinement of Taylor's Leisure Time Physical Activity questionnaire and the other measures physical function including ease of performance and level of fatigue as well as difficulty in performing higher order functional tasks. Preliminary results on test-retest reliability and associations among the walking and seated step tests were presented at GSA, November 1995. Analyses of the reliability and validity of questionnaires and final analyses of the exercise tolerance measures is proceeding. The original scope of work was completed in March 1996, approximately $15,000 under budget. A second phase of testing has been planned and will commence September 1996. To increase the measurement ceiling and improve the reproducibility of the 6-minute corridor walk, several consultants have suggested the walk be performed as fast as possible for the entire 6-minutes or for at least some extended portion. None of the recommended modifications have been tested against the more accepted treadmill walk protocol nor is there data or test-retest reliability. This will be accomplished in the second phase of the pilot study in which we will recruit 20 to 25 older healthy adults and test them twice 7 to 10 days apart. The following tests will be administered: (1) 400m corridor walk, with the first 100m serving as a warmup and the last 300m performed as fast as possible with encouragement and (2) treadmill walk. Heart rate will be continuously monitored and the Borg perceived exertion scale will be presented every 50m.


Little is known of the transduction mechanisms used by human taste cells. It is the purpose of this application to enhance techniques for taste cell isolation from fungiform papillae of human volunteers and to then investigate the physiological characteristics of these cells. Successful completion of this feasibility study will open new important routes for studying normal human taste response and give impetus to determining the course of human chemosensory disease. The three aims of this study are: to develop protocols to maximize viability of isolated human taste cells; to characterize stimulus-regulated changes in intracellular calcium ion activities and membrane potential in these cells; and to perform loose patch analyses on these cells to identify stimulus-regulated currents in these isolated cells. Procedures developed to date by the investigators on this proposal for isolated human taste cells have yet to be optimized, resulting in unacceptable variations among preparations. In order to allow characterization studies to proceed efficiently, isolation protocols need to be better defined. Experimentation with enzyme type/amount, trituration, media and holding conditions will be performed. Imaging studies of intracellular calcium and membrane potential using the dyes Fura 2 & di-8-ANEPPS will permit basic characterization studies to be performed. These will emphasize characterization of ion channels and normal responses of taste cells to pharmacological challenges. Responses to primarily sweet and bitter stimuli will be evaluated. Loose patch studies will be used to characterize individual cells as to taste modality and chemical specificity. Human taste cells have not been the subject of study for physiological experiments. The unique specificities of human taste can be used to test individual cells. Volunteers can be taste tested prior to have fungiform papillae removed to determine their response to particular stimuli. This research is of high impact since it deals directly with the receptor cells of humans, thereby strengthening the likelihood that therapy regimens can be developed to treat chemosensory disorders. Successful completion of this study will allow correlations to be made between psychophysical measures of taste and biophysical characteristics of the receptor cells. A more complete understanding of peripheral versus central processes of taste will be a likely outcome.


Acetylcholine (ACh) is a neurotransmitter widely distributed throughout the mammalian central and peripheral nervous systems. In the central nervous system the predominant physiological effect of ACh is a slow depolarization mediated by muscarinic receptors (slow m-ACh potential). Slow postsynaptic potentials (psps) may underlie some of the long-term changes in the nervous system. Long-lasting synaptic effects such as facilitation or potentiation may account for the synaptic plasticity that underlies the more complex psychobiological phenomena of learning and memory. Thus, better insight into mechanisms of specific slow psps may lead to better general understanding of synaptic plasticity and neuronal integration. Cholinergic synapses, in particular, are thought to be of prime importance in cortical memory processes, and disruption of cortical cholinergic pathways is involved in the development of certain dementias. A prevailing hypothesis of the pathogenesis of Alzheimer's disease is that the primary pathology is degeneration of a discreet group of cholinergic neurons in the basal forebrain. Because these neurons are the sole source of cholinergic afferents to the entire cortex, their loss leads to widespread cholinergic denervation of the cortex. The precise ionic mechanisms involved in the slow m-ACh potential in mammalian neurons are not known. Furthermore, the role of second messengers in mediating the m-ACh response has not been defined. Thus, compared with the detailed knowledge of nicotinic ACh receptors, the present state of knowledge of neuronal muscarinic ACh receptors lags far behind. These studies will use an in vitro system, sympathetic neurons in tissue culture. Voltage-clamp and patch-clamp techniques will be used to gain insight into the ionic mechanisms and the metabolic determinants that underlie the slow m-ACh response. Experiments will focus on specific ion conductance mechanisms and how they are controlled by biochemical second messengers. The specific aims are: 1) to establish the ionic mechanisms underlying the slow m-ACh depolarization in rat sympathetic neurons in tissue culture; 2) to determine the pharmacological sensitivity of the m-ACh response to various muscarinic agonists and antagonists; and 3) to ascertain if biochemical second messengers mediate the slow m-ACh potential.


Despite the significant reduction in HIV burden associated with HAART, reservoirs of latent virus remain in treated individuals. These reservoirs serve to provide a source for new virus when treatment is interrupted, and a site for creating drug resistant variants. In some individuals, discontinuation of HAART stimulates virus-specific T cell responses that appear to control virus. This phenomenon suggests that immunization with an effective vaccine during therapy may augment the effect of HAART. Two criteria are critical in the design of such a strategy: 1. Identification of the reservoir(s) so that immunization can be specifically targeted to it, and 2. The vaccine must induce responses that clear virus infected cells in the reservoir. We hypothesize that the intestinal lamina propria serve as a major reservoir of virus during HAART. Thus a mucosal vaccine is needed. An additional advantage is served by this approach because the GALT serves as an extrathymic site for T cell maturation, therapy overcoming the thymic destruction induced by chronic HIV infection. We propose to use a novel vaccine utilizing skin immunization with viral DNA co-delivered with vectors expressing genes for potent mucosal adjuvants, bacterial enterotoxin genes (CT or LT). Using the SIV: macaque model for AIDS this project will implement in vivo primate studies designed to compare the immunogenicity of SIV DNA with or Without vectors encoding CT or LT (project 1). Test vaccines encoding DC-specific chemokines and chemokine receptors (projects 3 and 4), and evaluate the ability of the optimized vaccine adjuvant formulation to serve as an effective adjunct to HAART in monkeys treated with PMPA (projects 1-4). Monkeys will be immunized, infected with titered stocks of SIV/DeltaB670 to determine the efficacy of treatment. The role of the GALT as a reservoir, and the ability of DNA immunization+/- PMPA to stimulate recovery (HAART +/- vaccine therapy) of immune responses in the GALT will be determined by analysis of antibody in mucosal secretions, function of CD4+ and CD8+ T cells, and quantitation of SIV-specific CD8+ cells by tetramer staining. T cells from the GALT will also be phenotyped to determine changes in lymphocyte subpopulations, the source of repopulating cells (naive versus memory subsets) and functional capability. The presence of SIV tetramer- binding CD8+ cells expressing alpha4beta7, the receptor mechanism whereby priming in the skin can induce effect responses in the mucosa, an ddetermine the relative magnitude of these responsres induced in the GALT by the different vaccine adjuvant formulations. The demonstration of this approach in identify the induction of mucosal immune response in the periphery of monkeys immunized during therapy will encourage similar studies in the analogous human trials described in project 5.


Biochemical, immunological, genetic and transgenetic experiments all indicate that the prion protein (PrP) is central to prion replication and disease susceptibility. Infectious prions are composed of a disease-specific PrP isoform designated PrPsc, and allelic forms of the PrP gene (Prnp) determine scrapie incubation time in mice. Mutant alleles of the human PrP gene (PRNP) are linked to familial prion diseases, and mice homozygous for a null allele of Prnp are resistant to prion disease and fail to support prion replication. However, additional factors impinge on prion replication and prion disease. Studies exploiting chimeric PrP transgenes and Prnp null mice suggest that efficient prion replication involves at least one auxiliary molecule, designated protein X. Both scrapie incubation time and the areas of the brain that are affected can differ dramatically among inbred strains of mice that express identical PrP molecules following infection with a single strain of scrapie. Treating prion incubation time as a quantitative trait led to the identification of at least two modifier genes. To identify the genes underlying these quantitative trait loci (QTLs), the QTL intervals will be isolated in congenic strains and the interval narrowed though high resolution mapping. The congenic strains also will be used to characterize the effects of each QTL on incubation time, pathology, distribution of PrPsc in the brain, PrP biochemistry, and concentration of PrPsc at onset of illness. Collaborative efforts with Dr. Hood (Project II) will take full advantage of advances in the Human Genome Project for candidate gene prioritization using oligonucleotide arrays and other technologies. BAC transgenesis and gene targeting will be used for identification of these and other prion disease modifiers that will be identified. QTL analysis is dependent on naturally occurring polymorphisms between mouse strains and thus samples only a small subset of potential modifier genes. Therefore, a screen will be established for chemically induced mutations that alter prion incubation time in transgenic mice so that all genes potentially are targeted. Finally, in collaboration with Drs. Hood (Project 11) and Prusiner (Project III), we will identify networks that are perturbed by misexpression of the PrP-related protein Dpl and screen for modifiers of Dpl-induced phenotypes. The combination of approaches will help define the genetic bases for susceptibility to prion disease and apply this new genetic knowledge to understand the underlying molecular processes.


During this period, emphasis of the work shifted from head restraint methods to methods of patient repositioning and to methods for reducing head motion not entirely eliminated by the thermoplastic mask restraint system now adopted as "standard" across the PET program. Testing revealed that the thermoplastic mask cannot, itself, be reliably used to reposition a subject from one imaging session to the next even though the mask is custom-made to each individual and can be attached to the imaging table in only one way. As a result, we used the spatial tracking system described in previous reports to measure (in normal volunteers) the repositioning accuracy of a method based on wall-mounted laser line projectors. Testing of this widely used technique revealed that although portions of the same tomographic plane can usually be replaced in the same imaging slice and within a spatial resolution element, exact repositioning of the entire slice is an uncommon event. Given this pair of results, work on the interactive repositioning system scheme using the spatial tracking device was resumed with testing ex- in the near future. Measurements have revealed that even with aggressive head restraint, residual motions within the restraint continue to compromise image quality. Independent knowledge of head position during the scan procedure can be used to correct these image data for patient motion. Feasibility studies of two such correction schemes have been carried out for both PET and conventional scintigraphic imaging studies with favorable results in both cases. Methods for efficient implementation of these computationally intensive techniques are now under development.


Graves? disease (GD) is the most common cause of hyperthyroidism, accounting for 60 to 80 percent of more than 250 million cases of hyperthyroidism globally. It is an autoimmune disorder caused by thyroid stimulating antibodies (TSAbs), which bind to the thyrotropin (or thyroid-stimulating hormone (TSH)) receptor on the surface of a thyroid cell. This leads to stimulation of the thyroid gland and subsequent synthesis and secretion of excess thyroid hormone. One crucial diagnostic tool is the use of assays for the quantification of TSAb, which can provide important guidance for the diagnosis and management of patients with both thyroidal GD and extrathyroidal manifestation. In addition, detection of TSAb is critical in predicting relapse or remission after initial anti-thyroid drug treatment or disease progression. Although many assays have been developed to detect autoantibodies against TSHR (TRAbs), it has been shown that among all commercially available assays, only cell-based bioassays can differentiate between stimulatory and inhibitory activity (TBAb) of circulating TRAb. However, cell-based bioassays are time-consuming, laborious, and relatively expensive. Therefore, there is a need for the development of simple, cost effective, and sensitive immunoassays that can differentiate between TSAb and TBAb in patient blood samples. Mediomics has developed a novel assay platform (PINCER assays) that allows simple homogenous mix-and-read detection of a variety of targets. These rapid-result assays can reach high specificity while remaining easy to use and at a low cost. In addition, our long-term collaborator, Dr. Tomasz Heyduk?s laboratory, developed a novel screening platform by combining both ribosome display and Next Generation Sequencing (NGS) technologies to quickly identify linear and conformational epitopes of antibodies from patient samples. Here, we propose to take advantage of Mediomics? innovative assay technology and the novel screening platform to develop highly specific immunoassays to detect stimulating and blocking autoantibodies (TSAbs and TBAbs) against the thyrotropin receptor. We have two specific aims in our Phase I project. Aim 1. Explore the entire protein sequence space of TSHR to identify peptide ligands specifically recognizing isolated human TSAbs or TBAbs. Aim 2. Prepare, optimize, and evaluate the performance of peptide-based immunoassays. If this project is successful, we would have validated the commercial potential of our assay, and will thus lay a foundation for transition to Phase II, where we will embark on the development of a commercialization-ready product and its clinical validation with well-defined clinical samples.


One important question about effects of media violence on anti-social behavior is whether exposure is likely to produce delayed or long term effects (Liebert & Baron, 1972). The proposed research is designed to assess whether "memory" variables influencing delayed recall of previous aggressive stimulation will strengthen delayed aggressive responses. Previous research has focused on the short term effects of exposure to "external" aggressive stimulation, but delayed responses to aggressive stimuli may occur if recalled from earlier exposures (internally-generated stimuli). The present interactive memory model predicts that an individual is likely to be aggressive if he is aroused and uninhibited when he recalls aggressive stimuli (e.g., media violence). Thus, aroused but uninhibited subjects who recall aggressive words are more likely to produce aggressive reactions than subjects who do not recall aggressive words. Three experiments are proposed to test the hypothesized interaction of recall, arousal, and inhibitions. These experiments are designed to manipulate accessibility (likelihood of recall); arousal; aggressive connotation of words; and evaluation apprehension. Subjects will serve in the "Buss procedure" (Buss, 1961), and shock intensity will be the measure of aggression. If the experiments support the proposed model, then it might be extended to important research questions about delayed effects of exposure to mass media violence.


The intestinal epithelium (IEC) with its luminal microflora serves as an ideal experimental and medical model that merges innate immune recognition in the context of a tissue regeneration environment. Genetic predispositions that cause imbalanced microbe-host interaction may contribute to the pathogenesis of inflammatory bowel disease (IBD). Various susceptibility loci for Crohn's disease and ulcerative colitis have been identified; however, their contributions to the disease mechanism remain poorly defined. A Crohn's disease susceptibility locus at chromosome 15q22 is immediately linked to RAB11A. This gene encodes a small guanosine triphosphatase (GTPase) that regulates the recycling endosome function. The applicant's laboratory has genetically targeted the mouse Rab11a, and analyzed IEC-specific Rab11a knockout mice. Preliminary data suggested that Rab11a controls epithelial-cell-intrinsic inflammatory cytokine response to enteric microbiota. Enterocytes deficient in Rab11a in Drosophila and mouse intestines overproduced proinflammatory cytokines, and caused early-onset enteritis, higher susceptibility to inflammatory neoplasia, and premature mortality. The long- term goal is to understand how the endosomal sorting of microbial receptors in IECs influences innate mucosal immunity and microbe-host homeostasis. The objective of this proposal is to establish the molecular mechanisms, by which Rab11a endosome-mediated sorting of Toll-like receptors (TLRs), in particular TLR9, support intestinal mucosal tolerance to microbiota. The central hypothesis is that Rab11a sequesters TLR9 to an inactive endosomeal compartment and dampens unwanted immune response to enteric microbiota at steady state conditions. Information learned about IEC-mediated modulation of inflammatory cytokine production and immune response may help reduce the adverse inflammatory response in IBD. This hypothesis will be tested with 2 specific aims: (1) to establish Rab11a endosomal compartment as an epithelial-cell-intrinsic modulator of cytokine response to microbiota; and (2) to determine the mechanism of Rab11a-controlled TLR9 transport and activation in response to microbial agonists. The experiments will use the novel Rab11a conditional mouse model that facilitates inducible Rab11a deletion, IEC-specific Drosophila Rab11 RNA interference (RNAi) lines, RAB11A-depleted human colon epithelial cell lines, and in vivo perfusion of microbial agonists. The combination of these genetic models with in vivo physiologic analyses in the study of endocytic control of microbe-host homeostasis is innovative and a major step toward understanding IBD.


The Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a devastating consequence of systemic inflammatory conditions such as sepsis that afflicts almost 200,000 people a year in the US with 75,000 deaths. The hallmark of ALI is inflammation-induced disruption of the endothelial cell (EC) barrier that lines the pulmonary vasculature, resulting in leakage of fluid, protein, and cells into the airspaces of the lung. Our laboratory has extensively studied the mechanisms involved in maintaining and enhancing EC barrier function as a tool for identifying possible therapeutic targets. The current paradigm of EC barrier regulation suggests a balance exists between barrier-disrupting cellular contractile forces and barrier-protective cell-cell and cell-matrix tethering forces. Both competing forces in this model are intimately linked to the actin-based endothelial cytoskeleton by a variety of actin-binding proteins. Our work has defined an essential role for the actin-binding protein, cortactin, in the resolution phase of vascular permeability with this critical function occurring via EC cytoskeletal rearrangement. Very little is known about the mechanisms governing recovery of EC barrier function following injury. Thus, cortactin is an attractive molecular target for novel therapies and warrants the intense structure/function studies we propose in this application. With this background, the PI proposes to investigate the hypothesis that cortactin regulates EC cytoskeletal rearrangements that result in altered barrier function during ALI syndromes. In SA#1 we will mechanistically characterize the key portions of the cortactin molecule involved in barrier regulation through the use of molecular biology and proteomic techniques utilizing in vitro models of barrier disruption (e.g., thrombin, TGFpl) to focus on cortactin's role during the barrier recovery phase. Transgenic animal models of ALI will extend these studies in vivo. In SA#2 we will examine the role of cortactin in cortical actin and junctional protein rearrangements that regulate pulmonary endothelial barrier function using novel atomic force microscopy (AFM) and other techniques to functionally characterize cortactin's role in peripheral cytoskeletal rearrangements involved in barrier recovery, focusing on cortical actin structures, junctional complex formation, and lipid raft signaling. In SA#3 we will characterize the functional consequences of an ALI- associated coding single nucleotide polymorphism (SNP) we have identified in the cortactin gene through a combination of molecular biology and proteomic techniques. This aim will determine the mechanistic effects of this ALI-associated SNP on cortactin function as it pertains to endothelial permeability and barrier recovery using the in vitro and transgenic animal techniques described above. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]


PROJECT SUMMARY\ABSTRACT Predisposition to develop Rheumatoid arthritis (RA) is associated with the presence of certain HLA alleles while others offer resistance. HLA-DQ8 and DRB1*0401 molecules render humans and mice susceptible to develop arthritis while DQ6/DR2 and DRB1*0402 provide protection. Collagen-induced arthritis (CIA) susceptible HLA transgenic mice produce rheumatoid factor and anti-cyclic citrullinated peptide (CCP) antibodies similar to that in RA patients. Similar to humans, the HLA-DR4 transgenic mice develop CIA more often in females than males. Our hypothesis is that DR4 has a role in gender-bias of arthritis, where DR4 expression, presentation of antigen by various APCs and ensuing responses are different in males and females. Female sex hormone, estrogen, is involved in pathogenesis by enhancing production of peptidyl arginine deiminase (PAD) enzyme that leads to citrullination of synovial proteins. These citrullinated proteins are presented with greater efficiency by HLA-DR4 leading to an autoreactive T and B cell response and disease. Smoking and aging can result in higher amounts of citrullination. On the other hand, DQ8 can present native and de-amidated peptides thus generating a robust immune response to an antigen. In this proposal we will investigate the mechanism of association of DR/DQ with arthritis by studying its interaction with receptors involved in innate and adaptive immunity via 1) binding to calreticulin, an established innate immunity receptor, and triggering signaling, 2) by selection of regulatory cells and 3) by selection/ deletion of autoreactive T cells. In addition, we will use DRB1*0401 and DRB1*0402 mice to determine the mechanism of protection to understand how 3 amino acids difference in both molecules lead to protection by the latter. In aim1, we will define the mechanism of predisposition by using mice expressing RA susceptible and resistant HLA class II molecules. In aim 2 we will test role of shared epitope in protection versus susceptibility by studying interaction of *0401 and *0402 with calreticulin, and role of T regulatory cells and Th17 cytokines. As a corollary, we will study mechanism of remission during pregnancy by studying function of T regulatory cells and antigen presenting cells. Recent literature has shown a protective/ susceptible effect of non-inherited maternal antigen (NIMA) on the development of arthritis. In aim3, we will use various matings to generate mice that have been exposed in utero to one NIMA. These mice will be evaluated for in vivo arthritis, autoantibodies and function of T and B cells. The proposed experiments will test several new, original and innovative theories on the role of MHC genes in predisposition, onset, progression, severity and modulation in arthritis. The findings would shed light on the role of immune system in health and disease and may identify new therapeutic approaches for arthritis.


DESCRIPTION: Environmental exposure to polychlorinated biphenyls (PCBs) occurs worldwide, and most individuals in developed nations have measurable levels of organochlorines in adipose tissues. This project proposes to estimate the risks associated with perinatal exposure to PCBs for developmental deficits or delays on various measures of neurologic, neurobehavioral, and intellectual outcomes. The investigators, with support of the European Commission, have established a birth cohort of 200 children recruited consecuttively since March, 1994 from a relatively homogenous population in the Fazroe Islands. The northeast Atlantic Ocean where the Faroes reside is known to be one of the largest environmental reservoirs of PCBs. Due to consumption of pilot whale blubber, the average PCB exposure in this cohort is believed to be substantially higher than established averages in Northern Europe and is expected to vary 10 to 20 fold. Developmental exposure to PCBs will be determined prenatally by maternal and cord serum and breast mile PCB concentrations. This cohort has already been evaluated at 10 to 15 days of age with the Touwen neonatal neurologic scale. Currently, the revised Bayley Scales of Infant Development, the Fagan Test, and measures of infant linguistic performance are being administered at 7 months of age. Follow-up assessments include at 18 months the Bayley-II, and a neurologic examination at 30 months, with more extensive test batteries at ages 42, 54, and 66 months.


We have found that T4 tail fibers have a myosin-like activity at their proximal half and a rare cross-beta folded protein structure at their distal half. We propose to clone the T4 tail fiber genes and sequence their primary structures. We will then look at the mechanism of their folding and dimerization into mature tail fiber halves by conventional methods as well as in clones containing plasmids with genes expressing only the tail fiber genes. We have developed rapid and sensitive methods for selecting these clones and assaying the immature and mature tail fiber parts. Our overall goal is to explain in molecular terms the rigid structure of the tail fiber parts, the springlike angle in the middle and the mechanism of action of both termini with respect to attachment to the cell surface (LPS) and the phage baseplate.


When challenged with inhibitors of steroidogenesis, the cultured vascular smooth muscle cell showed depressed synthesis of cholesterol and of dolichyl(pyro)phosphate and an accompanying loss of cellular cholesterol. It was deduced that a regulatory site for the synthesis of both substances might occur at the HMG-CoA reductase. As a consequence of this inhibition cellular glycoprotein synthesis was diminished, and it was concluded that the availability of dolichyl(pyro)phosphates which mediate glycoprotein synthesis may also contribute to the regulation of their assembly. Cellular response to these changes may involve redistribution of subcellular membrane systems. This redistribution, perhaps necessary as a defense response, may present the cell with a new set of problems. Such redistribution will be assessed in a variety of conditions using subcellular fractionation schemes and marker enzymes assays. It is now clear that loss of cellular cholesterol may also contribute to defective glycoprotein assembly and membrane biogenesis. Such an effect points to the importance of a metabolically active cholesterol pool which the cell synthesizes to make certain that proper assembly is maintained. In view of this, contribution of exogenous cholesterol, supplied in a variety of forms, to such a pool will also be investigated.


Neurotransmitter release is a key step in synaptic transmission. However, the molecular events that trigger fusion of synaptic vesicles and neurotransmitter release are unknown. One attractive hypothesis is that Ca2+-activated channels in the vesicle membrane control fusion of vesicles. If true, such a channel would provide a simple molecular link between Ca2+ entry and vesicle fusion. Previous experiments have lead to the identification of several kinds of ion channels that are credited to the synaptic vesicle membrane. The objective of this project is to study these ion channels to determine: 1) which, if any, are from the synaptic vesicle membrane; 2) the biophysical properties of these channels; and 3) regulators of the channels (such as voltage and Ca2+). These goals are directed toward elucidation of the mechanism(s) of fusion of synaptic vesicles to the nerve terminal membrane and the possible role of vesicular ion channels in this process. Plan: Planar bilayer experiments will be used to identify and characterize ion channels from synaptic vesicles purified from the electric organ of the electric fish, Torpedo californica. Vesicle membrane will be induced to fuse with bilayers by using a new technique that both enhances fusion of all vesicles and makes it possible to estimate the prevalence of any observed channels. That is, it will be possible to determine if a particular channel is representative of the population of synaptic vesicles or if it is from a minor contaminant. Preliminary Results: Synaptic vesicles from Torpedo californica have been isolated and treated so that they fuse with planar bilayers. Two kinds of ion channels have been detected from these vesicles having estimated single channel conductances of 180 pS and 14 pS.


Development of a miniature orally inserted blower with embedded sensing for the diagnosis and treatment of sleep disordered breathing is proposed. This Oral Positive Airway Pressure (OPAP) device represents a potentially radical change in the interface, machine, and patient adherence of sleep disordered breathing treatment. The ergonomic design of the device will facilitate oral and nasal breathing during sleep, eliminate external machines and tubing, and will be retained in the oral cavity during sleep. Inter-individual differences in airway response to sleep disordered breathing will be automatically accounted for by innovative real-time sensing of airway collapse and by the closed-loop coupling between respiratory effort and machine delivery. Ease of use and improved adherence to therapy is anticipated. This research will utilize monitoring technology embedded in the device to objectively document adherence. Information obtained will determine how the nightly duration of use and long term adherence to OPAP can affect morbidity, mortality, healthcare utilization, and quality of life issues. Phase I of this project investigates the feasibility of several candidate OPAP device designs, and evaluates their therapeutic (including air blowing and ergonomics) and diagnostic capacities in airway simulation models and oral cast models. Phase II of the project will obtain clinical trial data of this device and sensor in a clinical trial of sleep disordered breathing patients with the subsequent intention to market and sell this product. Phase II research will also support the investigation of cardiovascular outcomes for those adherent and non-adherent to OPAP treatment for OSA. [unreadable] [unreadable] [unreadable]


The overall objective of this proposal is to understand the mechanism and regulation of transplacental calcium transport during mammalian embryonic development. Based on our previous identification of specific calcium-binding proteins (CaBP) in the human and mouse placentae, experiments are proposed here that directly examine the functional involvement of these proteins in the placental calcium transport process. Specifically, these projects aim to study how the CaBPs interac with Ca++, where they are located within the placental cells, how they participate in transmembrane calcium transport, and what are the cellular and molecular steps regulating their specific, temporal expression during development. These projects will be approached by first preparing purified placental CaBPs which may be used to raise specific antibodies. Detailed physicochemical analyses will be carried out to reveal conformational changes that may accompany Ca++ CaBP interactions. The specific antibodies will be used to localize the ultrastructural distribution of the CaBPs and will also be used to probe the nature of their functional involvement in placental calcium transport using cellular and subcellular methodologies. Finally, the developmental regulation of CaBP expression will be explored by 1) identifying putative regulatory factors and 2) characterizing the molecular processes leading to gene expression. The integrated approach taken here should lead to a fuller understanding of how a developing embryo obtains its calcium supply and futhermore provide general insights to the regulation of embryonic development.


This research proposal constitutes an attempt to develop biological-biophysical data about specific patterns of ribonucleic acid biosynthesis and those enzyme systems involved in their processing, in particular the transfer RNA-methylases, as they relate to the leukemogenic process. It is intended that this information will afford an expanded level of knowledge concerning the biochemical responsiveness of leukemia cells found to be sensitive or resistant to various antineoplastic agents (e.g. vincristine, cystosine arabinoside, cytoxan, methotrexate) administered alone or in combinations. In this connection, information will also be sought as to how such agents can modulate the biosynthesis of particular species of RNA in leukemic cells. The basic techniquesto be employd entail the isolation and purification of mouse ascites tumor cells (e.g. L1210, P383, L5178Y) and certain resistant sublines as well as human peripheral blood leukocytes, cell-culturing where appropriate and variable-interval pulsing with radioactive precursors of ribonucleic acid (uridine, L-methionine). In vitro evaluation of tRNA-methylase activities in various leukemic cells will be conducted using either a homologous or heterologous assay system in the presence of unfractionated tRNA plus partially purified enzyme preparations. Special techniques to be used for characterizing individual RNA species and ribonucleic acid components (nucleoside level) include thermal-detergent extractions, sucrose-gradient centrifugation, polyacrylamide gel electrophoresis and computer analyses of "RNA-fingerprint" data.


Elucidating childhood precursors of osteoporosis may lead to interventions that prevent or mitigate osteoporosis later in life. The central focus of this application is to investigate bone mass as measured by DEXA, including bone mineral content (BMC), bone area (BA) and bone mineral density (BMD) in relation to a wide range of putative precursors of osteoporosis. We will study a large prospective twin cohort (-2,000 twin pairs) of children and adolescents, enrolled previously in 1998-2000 in Anqing, China, with an age range of 6-21 years. The twins are currently followed by a NIH funded study to identify precursors of metabolic syndrome (MS). The specific aims are: (1) To measure biomarkers that are known or suspected to affect bone health in 1,500 twin pairs at baseline and follow-up study, including bone modeling and remodeling markers: osteoprotegerin (OPG), soluble receptor activator of NFKB ligand (sRANKL), parathyroid hormone (PTH), osteocalcin (OC), tartrate-resistant acid phosphatase 5b (TRAP-5b); nutritional markers: magnesium, vitamin K1, and 25(OH) vitamin D; and also examine other relevant biomarkers covered by the funded MS study, including steroid hormones: androgen, estrogen, testosterons, LH, and FSH; metabolic markers: fasting and 2-hr post OGTT insulin, glucose, HOMA-IR, fasting lipids; growth hormones: insulin-like growth factor (IGF-I); adipocyte markers: leptin, adiponectin; and inflammatory markers: C-reactive protein (CRP), lnterleukin-6 (IL-6), and tumor necrosis factor (TNF); (2) To conduct cross-sectional and longitudinal co-twin analyses to test the following hypotheses: (a) BMC, BA and BMD attained levels are associated with biomarker levels at the baseline and follow-up survey, respectively; (b) BMC, BA, and BMD changes between the baseline and follow-up survey are associated with biomarker levels at the baseline survey; (c) BMC, BA, and BMD changes are associated with biomarker changes between the baseline and follow-up surveys. We will further examine if the above relationships are independent of other important covariates, including age, gender, pubertal stage, body weight and composition, nutritional intake, and physical activity, and if there are interactions between the biomarkers and the covariates. The identification of potential important precursors of osteoporosis in children and adolescents would represent a huge step forward in our understanding of the biological basis of bone mass and should have important implications for the detection of individuals at high risk for osteoporosis. [unreadable] [unreadable] [unreadable] [unreadable]


PROJECT 1: ENCODING AND RETRIEVAL PROCESSES IN DECLARATIVE MEMORY In the prior funding period, patients with and at risk for schizophrenia were found to show a differential deficit in recollection that scaled with phase of illness, while familiarity was intact. This deficit was correlated with reduced hippocampal and parahippocampal activity at retrieval on items for which associative memory was evident. There are two (not mutually exclusive) possible sources ofthese deficits: 1) disrupted structural connectivity, resulting from aberrant developmental changes in gray and/or white matter during peri-adolescence;and 2) failure of patients to disengage default mode network (DMN) activity during memory encoding and/or retrieval. The first hypothesis is suggested by our preliminary work indicating a steeper rate of gray matter reduction in prefrontal regions and a lack of normal age-related increase in medial temporal white matter tracks in FE and prodromal patients who convert to psychosis. The second hypothesis is based on recent evidence of reduced suppression of DMN activity and less task-related activity and functional connectivity during a working memory task in schizophrenia patients and their siblings. Increased DMN activation appears to accompany attention to internal thoughts and self-referential thinking;reduced suppression ofthe DMN may thus interfere with patients'ability to activate the episodic and associative memory circuitry at encoding and/or retrieval. In the next phase of the study we will pursue these two hypotheses in cross-sectional and short-term longitudinal studies of prodromal, FE and chronic patients and controls, using MRI and DTI to index structural connectivity and fMRI during an associative memory task and resting scans to index functional connectivity between memory-related and DMN regions. The findings will help to clarify the nature and neural underpinnings of declarative memory deficits in schizophrenia, whether behavioral and physiologic deficits predict the onset of schizophrenia over and above behavioral indicators of risk and are differentially predictive of poor functional outcome. Findings maybe used in prodromal research to identify those at greatest risk for schizophrenia.


Initially, this Phase II study investigated the effects of daily administration of DMP 754, a IIb/IIIa platelet inhibitor, versus aspirin on the inhibition of platelet aggregation, the incidenceof bleeding and thrombocytopenia in patients with coronary artery disease over a 6 month period of time. The study was amended in March 1998 to include combination therapy arms of DMP 754 and aspirin. Preliminary data suggested increased risk of bleeding so the protocol was amended in July 1998 to remove the 2.0 mg DMP 754 + Aspirin arm and its comparitor (2.0mg DMP 754 alone). We enrolled 8 patients between May and October 1998. Of those eight patients, two were discontinued, one due to decrease in platelet count and the other because of a positive hemoccult. DuPont closed enrollent December 1998 after meeting enrollment goals. The six-month study follow-up will conclude in May 1999. Clinical endpoints include death, progression to myocardial infarction, CABG, PTCA for recurrent ischemia, hospitalization for ischemia or unstable angina. We expect to generate an abstract for presentation, on which Mayo investigators will be co-authors, in late 1999 followed by a manuscript.


Pancreatic cancer is one of the most lethal malignancies in the US. The primary curative treatment is surgery and is only appropriate for approx. 20% of presenting patients because most will present with locally invasive or metastatic disease. Although Gemcitabine has been proven superior to 5-Fluoracil in advanced pancreatic cancer, long term survival is rarely seen. Other agents are clearly needed to treat this refractory disease. Strong evidence points to the importance of raf kinases in the development and maintenance of human cancers. For example, raf proteins have been demonstrated to be direct downstream effectors of ras protein functions within the MAP kinase signaling pathway. Since 75% of patients with pancreatic cancer have ras mutations, novel therapy directed against raf kinases are particularly exciting in this malignancy. ISIS 5232 is a phosphorothioate oligonucleoide antisense inhibitor of human c-raf kinase or are in an expression in-vivo and in-vitro. This is a limited institution ECOG phase II trial which will look at the response rates and toxicity of ISIS 5132 administered by continuous infusion via portable pump (provided free of charge). Numerous bug samples for pharmocokinetics and measuring of downstream effects will be needed during the course of this trial. This novel therapy has numerous avenues for future therapeutic directions, including adjuvant therapy or perhaps combination therapy with other standard chemotheraputic agents.


This proposal requests funding to assist in upgrading of laboratory animal facilities at The Rockefeller University, with the goal of providing research scientists with pathogen free rodent colonies. In a previously funded 1987 facilities improvement grant from the NIH we upgraded equipment, investigator safety, sanitation, surgery, water and environment components of The Rockefeller University Laboratory Animal Research Center and Field Research Center. These improvements have helped us remain a regional center for training and excellence in laboratory animal science. Matching funds are now being requested for a modification in our basic rodent husbandry care system which will enable our scientists to maintain and work with mice and rats free of common pathogens. We propose, with the assistance of this grant, to expand a limited 1200 sq ft of room with rodent microisolator housing by 2800 sq ft of additional pathogen free experimental animals. our existing limited pathogen free rodent housing was established by Howard Hughes Institute and NIH funding in addition to supplemental Rockefeller University funding. This proposal will enable us to maintain virtually all rodents which originate as pathogen free strains or stocks as pathogen free experimental animals, thus greatly enhancing the biomedical research of this institution. Approximately $45,000,000 in federal research funding is utilized at this university. Nearly two thirds of the laboratories conducting this research require laboratory animals for their studies and utilize the facilities of the Laboratory Animal Research Center.


Pseudomonas aeruginosa is an important cause of keratitis. The results of antibiotic treatment are unsatisfactory. We have shown that physiologic concentrations of calcium in vitro antagonize the effects of polymyxin B, colistin, and gentamicin on P. aeruginosa. These antibiotics may have less activity in vivo than one would predict on the basis of in vitro tests. We have also found that chelation of calcium significantly enhances the activity of these antibiotics in vitro. The combination of antibiotic and chelating agent may also have greater activity in vivo than the antibiotic alone. We propose to determine the relative potency of polymyxin B, gentamicin, and carbenicillin, with and without chelating agents, in the treatment of experimental Pseudomonas keratitis. Chelation may sharply increase the efficacy of treatment of Pseudomonas keratitis. Recent studies suggest that collagenase may play an important role in the pathogenesis of corneal ulcer and that chelation may block collagenolytic activity. We also propose to clarify the relationship between cations, chelation, and corneal ulcer.


Low and variable efficiency is a major problem in targeted gene alteration, which is used as a primary tool in gene therapy and animal model studies. We tested several types of constructs, alone, or in combination with other factors, to introduce a point mutation into the alphaB-crystallin gene in one-celled mouse embryos. We found that co-injection of single stranded DNA (ssDNA) along with antibodies against Ku70/86, or supplementing the system with hRad51/hRad54, increases efficiency of targeted mutagenesis. These findings suggest that proteins in the homologous recombination DNA repair pathway contribute, and that proteins involved in the alternative non-homologous end joining pathway inhibit, ssDNA-mediated targeted mutagenesis. This is the first successful demonstration of targeted mutation in early mouse embryos. This novel methodology of supplying protein factors to stimulate gene modification in the nucleus has not been reported previously. For this initial study we have been using an extremely time consuming PCR-based restriction fragment length polymorphism (RFLP) assay to detect point mutations introduced into genomic DNA. To improve the detection method, we have developed transgenic mouse lines in which correction of a mutation in a fluorescent protein gives an instant read-out, and many embryos can be screened simultaneously. We engineered a cassette encoding a bicistronic mRNA (mutated red fluorescent protein DsRed / an IRES element / green fluorescent protein). All mouse embryos expressing the transgene should fluoresce green (endogenous control for transgene expression), but only embryos in which the point mutation in DsRed has been corrected should also fluoresce red. We have used several enhancer/promoter elements, which have been used successfully in preimplantation stage mouse embryos or mouse ES cells, to drive expression of the cassette. A successful reporter line would exhibit easily detectable levels of fluorescent protein expression at the earliest stages of embryo development. All promoter/fluorescent protein transgenes were constructed and tested for expression of EGFP and intact DsRed in both transient assays in mouse embryos and in F1 transgenic mouse embryos. Several promoters/enhancers were used in attempts attain early embryonic gene expression. A construct, whose expression is driven by the EF1alpha promoter, was able to generate green fluorescent protein in eight cell stage embryos (48 hr pf) and red fluorescent protein at 72 hr pf. A line of transgenic mice with the EF1alpha promoter / mutated red fluorescent protein / an IRES element / green fluorescent protein construct appears to be extremely promising at this time. Four transgenic mice lines with mutated DsRed protein were established. All four lines show expression of EGFP at 48 hr pf. Sequence analysis of a PCR-amplified region of the DsRed gene, from mouse genomic DNA, confirmed presence of the nonsense mutation in codon 15 of DsRed. RT-PCR analysis shows that three transgenic mouse lines have low copy number of the construct (2 copy) and one has a high copy number (26 copies of the construct). One line of mice (with 2 gene copies) is being bred for further experiments. In addition to our work with ssDNA as a targeting vector, we are also working, in single celled mouse embryos, on another type of gene targeting which we have named Forced Homologous Recombination (FHR). In this case, we use a significantly larger targeting vector, similar to those currently used in ES cell targeting procedures, with regions of homology to a selected gene or chromosomal locus at both 5'and 3'ends. Co-microinjection of the targeting construct with key factors involved in homologous recombination (for example Rad51/Rad54 proteins, which has been shown be able to form a filament-complex with nucleotides in vitro) may increase gene targeting efficiency. We are planning to use a nucleoprotein filament complex instead of the naked DNA vector to further increase alteration efficiency. The presence of long homology arms on both ends should increase specificity of gene modification and also dramatically decrease any random integration of the targeting vector, compared to our ssDNA strategies. Two genes were selected for in vivo testing of this FHR system. The first being the alphaB-crystallin gene. This gene had previously been knocked out, however another neighboring gene, Hspb2, was also knocked out in that mouse line. If successful, the FHR system will enable elimination of only one gene with precise accuracy. The second gene is caspase-6, a knock-out of which was also developed earlier, by eliminating the first exon. However our preliminary data suggests positive identification of caspase-6 by western blot in some tissues, which could possibly arise by alternative splicing or an alternative translation start site. We are working on a construct for FHR study that should eliminate the active center and dimerization domain from caspase-6 with precise accuracy. Generation of these two vectors is presently underway.


This is a request for an ADAMHA Research Scientist Award. Sleep changes in late-life depression may be permanent or may recover only slowly. Hence, to clarify the relation of persistent sleep abnormalities, pathogenesis, and illness course, we propose three related longitudinal studies of: Study 1 - 200 elderly (60-80 yo) patients with recurrent unipolar depression participating in an already funded maintenance therapies research protocol (MH43832-01, C.F. Reynolds, P.I.); Study 2 - 80 elderly subjects (60-80 yo) with spousal bereavement (n=40) and bereavement-related depression (n=40); and Study 3 - 60 healthy elderly (60-80yo) non- depressed, non-bereaved controls. Participants in all three studies will have repeated (four to seven) sleep EEG assessments over a two to three year period to determine if, as hypothesized: 1a) abnormal pretreatment (T1) measures of REM sleep cyclicity/density predict elevated risk of relapse and recurrence of major depression; 1b) EEG sleep abnormalities persisting into continuation therapy (T2) and into maintenance therapy (T3-T7) predict elevated risk of relapse and recurrence, respectively; 2a) depressed bereaved differ from non-depressed bereaved elderly in baseline (T1) and followup (T2-T5) measures of REM sleep cyclicity/density; 2b) baseline (T1) and followup (T2-T5) abnormalities of REM sleep in bereavement predict a more protracted course (and need for psychiatric intervention); and 3) longitudinal changes in sleep/sleep quality (T1-T4) of healthy elderly controls are related to subclinical depressed mood. Multiple logistic regression and survival analyses will model the hypothesized relation of REM sleep abnormalities (independent variable) to outcome: recurrence/non-recurrence of depression (Study 1); or presence/absence of RDC-defined major depression at 1 and 2 years after spousal bereavement. A repeated-measures MANOVA will contrasts the long- term evolution of selected sleep variables (sensitive to aging, depressed mood, or both) in recurrent depression, bereavement, bereavement-related depression and controls. Exploratory data analyses will assess possible interrelations among biological controls. Exploratory data analyses will assess possible interrelations among biological REM sleep abnormalities, severity of psychopathology, and disruption in social rhythms.


The goal of this training program is to prepare physicians and basic scientists for careers focused on the development and use of radionucleotides in the diagnosis and treatment of cancer. Support is requested for five trainees, who may be either physicians or basic scientists. Physician trainees will have completed the major portion of requirements for board certification in nuclear medicine. Basic science trainees will hold the Ph.D. degree in chemistry, physics, biology, or a related science.. Each will be aiming for a career in research in a academic setting. The training program will be two to three years in duration. A broad based didactic curriculum will cover fully the field of nuclear medicine as applied to cancer. Each trainee will give focused concentration in selected tracks coordinated by a senior faculty mentor. Specific research tracks are: (1) Tumor antibodies/radiopharmaceutical, (2) Endocrine tumors, (3) Brain tumors, (4) Radiopharmaceutical therapy, (5) Quantitative radionuclide imaging, and (6) Radiopharmaceutical development. Research in one or a combination of these tracks will involve at least 50-75% of trainee time. The program will be conducted in the Division of Nuclear Medicine and Comprehensive Cancer Center facilities. Training will be supervised by members of the large, multi- disciplinary nuclear medicine faculty. This group publishes over 90 papers per year and receives over 6 million dollars per year in research grant support. Nuclear Medicine facilities include 90,000 square feet of clinic space, 17,000 square feet of laboratory space, a dedicated cyclotron, and 3 PET scanners. This program is integrated with University of Michigan Comprehensive Cancer Center activities. The combination of faculty motivation, structured curricula, excellent facilities, and superb intellectual environment is expected to prepare individuals to be tomorrow's leaders in cancer research.


Arthritis has reached epidemic proportions in the United States. It is the leading cause of disability, affecting about one-third of adults. Osteoarthritis (OA) is the most common form of arthritis with a prevalence conservatively estimated at 21 million Americans, or 12.1% of the adult population. We recently showed that a 5% weight loss in obese adults with knee OA had a modest effect on clinical outcomes; however, these participants remained obese at the conclusion of the intervention, so their risk of disease progression was not altered. Indeed, the nature of the relationships between weight loss and the mechanisms associated with disease progression are undetermined. We hypothesize that a more intensive weight loss (2 to 3 times greater than previously achieved in this population) would provide the stimulus necessary to reduce inflammation and joint loads and alter disease progression. The study population will consist of 450 overweight and obese, older (age > 60 yrs) adults with tibiofemoral osteoarthritis of one or both knees. Participants will be randomized to 1 of 3, 24-month interventions: intensive dietary restriction-plus-exercise; exercise-only; or intensive dietary restriction-only. The primary aim is to compare the effects of these interventions on inflammatory biomarkers and knee joint loads in overweight and obese adults with knee OA. Secondary aims are: to compare the effects of these interventions on self-reported function and pain, and mobility; to determine the dose response to weight loss on disease progression and the mechanisms associated with the OA disease pathway; to determine if inflammatory biomarkers and knee joint loads are mediators of the interventions, with significant effects on function, pain, and disease progression; and to determine the association between quadriceps strength and disease progression as a function of knee alignment. Weight loss is the most modifiable risk factor for knee OA; however, no study has determined the extent of weight loss that is beneficial for this population. This study could make intensive weight loss the standard-of- care for overweight and obese adults with knee OA, as it enhances our understanding of the OA disease process and weight-loss recommendations for older people generally. [unreadable] [unreadable] [unreadable] [unreadable]


With the recognition that cancer is a genetic disease, many cancer researchers, including scientists in the Mayo Clinic Cancer Center (MCCC), require access to routine and advanced cytogenetics services. Such services can aid in the identification of genetic regions important for the development of specific malignancies. Such services can also be used to characterize the clonality of tumor specimens and determine the origin (e.g., mouse vs. human) of a specific tumor culture. Thus, the Cytogenetics Shared Resource will provide cytogenetic and molecular cytogenetic services to MCCC members. In addition, this Shared Resource will prepare EBV-transformed B-lymphocyte cell lines for MCCC members.


Recognizing objects is a fundamental cognitive ability, e.g., for reading or social communication, and its loss or impairment is associated with a number of neural disorders. For example, many studies have shown that individuals with Autism Spectrum Disorder (ASD) are impaired in face recognition, possibly contributing to their difficulty in social interaction. Differences in social cognition are one of the defining features of autism. However, the precise nature of the differences in the neural mechanisms of face processing seen in autism has been difficult to study and its relationship to social behavior is unknown. While our understanding of the network of brain areas underlying human face perception has advanced considerably, current models are still qualitative and treat processing within specific brain areas as "black boxes". To understand the graded (rather than all-or-none) recognition deficits and to identify targets for therapeutic intervention, a quantitative and mechanistic model of the neural computations underlying face perception is required that rigorously links visual stimuli to neural activation and behavior. The purpose of the proposed project is to explore the neural mechanisms of face processing in typically developing and autistic individuals using an integrated approach based on a tight interaction of computational modeling of neural processing in the brain areas underlying human face recognition and of model-driven behavioral and fMRI experiments. In particular, the model motivates experiments to probe the face representation in typically developing and ASD subjects, and to mechanistically link differences in behavior and fMRI to differences at the level of neural processing -providing insight into the neural causes of the observed face recognition deficits along with an opportunity to evaluate current theories of neural processing differences in ASD. In a second part of the study, the model will drive experiments to study high-level plasticity and perceptual learning in typically developing and autistic individuals. This will not only leverage the insights from face perception to explore more general differences between the two subject groups, but is also expected to provide a foundation for remediation efforts. The relevance of this research to public health lies in its direct application of the results of this research program to achieve a better understanding of impairments found within and outside the autism spectrum.


The objective of this protocol is to measure the effect of six months treatment with human growth hormone at a physiologic dose on the muscle strength and endurance and on the functional capabilities of subjects with post poliomyelitis syndrome.


An unexplained decrease in muscle strength occurs with increasing age. Persons over seventy years old average about 50 percent of the strength of those less than 35 years old. As strength declines with increasing age, mobility, functional capacity and independence are lost, and the risk of injury as a result of falling increases. Decreased muscle strength is also a well-documented symptom of vitamin D deficiency and particularly of 1,25 D deficiency. The myopathy associated with 1,25 D deficiency, like that seen with aging, is characterized by proximal muscle atrophy and a selective loss of type II muscle fibers. Administration of 1,25 D to patients with 1,25 D deficiency restores muscle strength. Serum concentration of 1,25 D decreases with increasing age. Serum 1,25 D levels in persons over 70 years old average about 55 percent of those in persons in their mid thirties. In uncontrolled studies, administration of 1,25 D to elderly subjects has improved muscle strength. In a pilot study, we showed a 27.5 percent increase in muscle strength in healthy elderly men after one month of oral treatment with 1,25 D, and a 35 percent improvement in those over 70 years old. In the pilot study the percent increase in strength was strongly correlated with the percent increase in serum 1,25 D level (correlation coefficient = .9). Based on these observations and biochemical evidence linking 1,25 D to muscle function, we hypothesize that the weakness associated with aging is, in part, due to inadequate serum concentrations of 1,25 D in the elderly. To test this hypothesis, we proposed to perform a randomized, controlled, double-blinded trial of the effects of oral treatment with 1,25 D in 100 persons over 70 years old using the isokinetic dynamometer as a precise measure of strength. Our study will have a 90 percent power to show a 20 percent difference between the treated and placebo groups. A positive study will establish a metabolic etiology for the weakness presently considered a "normal" part of the aging process and open a new area for biochemical research in aging. If effective, treatment with vitamin D may make it possible to improve mobility, reduce functional dependence and decrease the risk of falling in the elderly.