The Metabolic Physiology Shared Resource (MPSR) is developed as an institutional core that will allow DRTC investigators to study metabolism using a range of animal models and human subjects. MPSR components include laboratories for study of mice, rats, canines, and human subjects. This resource is shared with two other centers. They are the Mouse Metabolic Phenotyping Center (MMPC) and General Clinical Research Center (GCRC). The MMPC provides comprehensive services for study of mice. The GCRC provides support for human studies in such areas as nutritional services, nursing, physical resources, biostatistics, and bioinformatics. The MMPC and GCRC both have NIH support independent of the DRTC. The DRTC will support laboratories for rat and canine experimentation and for the study of energy balance and nutrition in human subjects. The Rat Laboratory is an expansion of the DRTC efforts to centralize and develop techniques for studying in vivo metabolism (e.g. energy balance, insulin sensitivity, insulin secretion) in the conscious, unrestrained rat. While distinct from the MMPC, this component greatly benefits from and synergizes with the MMPC. The Canine Laboratory is a continuation of DRTC support of our novel approach to study in vivo metabolism (e.g. liver substrate balance, hormone action) using the conscious dog. The Human Energy Balance Laboratory is an essential complement of existing services available to DRTC investigators by way of the GCRC. It will support services for assessment of energy expenditure, body composition, and physical activity. The MPSR is directed by faculty (D. Wasserman, S. Davis) experienced in the range of experimental models supported by the resource and provides specialized technical assistance to investigators using the resource. The MPSR is structured to facilitate translational studies from genetically modified mice to humans and to allow investigators working in human subjects to explore more basic mechanisms in animal models. The MPSR represents the most efficient and scientifically sound approach to utilizing institutional resources that have common goals (i.e. study of metabolism in the whole organism). The MPSR effectively pools expertise and in some cases physical resources, allowing DRTC investigators the ability to study metabolism in scope that is unprecedented.


The Postmenopausal Estrogen/Progestins Intervention (PEPI-1) trial of 875 participants followed at 7 centers for three years is the largest and longest randomized double-blind placebo-controlled clinical trial of hormone treatment in postmenopausal women -- and the only long-term trial of estrogen-progestin regimens. It is the only long-term trial designed to compare the effects of Estrogen Replacement Therapy (ERT) and combined (estrogen-progestin) hormone replacement therapy (HRT) on heart disease risk factors. It is also unique for the number of women who began ERT or HRT more than 5 years after the menopause. Details of the study design, hormone replacement regimens and study population are given in Project I, as are the major analyses of PEPI-1 data proposed for completion during the extended follow-up period. Project II, Studies of Repository Samples, was planned to utilize plasma, urine and other samples already collected but not analyzed by the close of PEPI-1. Both Project I and Project II, submitted by the Coordinating Center to reflect the centralized activity necessary for a multicenter project, will be conducted with the Clinical Investigators as a cooperative project. Project III, submitted in parallel from each of the 7 PEPI Clinical Centers, describes a three year prospective observational follow-up study designed to: ensure PEPI women's safety and learn more about possible delayed adverse hormone effects; study treatment choices, compliance and reasons related to these decisions; and monitor long-term effects on heart disease risk factors and bone density. All participants will be invited to an annual clinic visit, (conducted according to the established PEPI-1 protocol) which includes a medical history and limited examination, mammogram, endometrial biopsy, and standardized questions about quality of life, sexuality, symptomatology, and medication use. We will measure primary PEPI endpoints (HDL-cholesterol, blood pressure, insulin and fibrinogen) and their covariates, and bone density at the hip and spine.


The proposed research is designed as an analytical and manipulative investigation of mechanisms critical to cell migration in the patterning of the developing nervous system. The studies will focus on two developing cranial nerve nuclei in the chick embryo: The oculomotor ventromedial subnucleus and the trigeminal motor nucleus. In studying the pattern of migration and possible mechanisms important for the proper execution of these patterns, a combination of approaches will be used, including interspecific embryonic transplantation, sequential electron microscopic analysis of intercellular interactions during disparate migration phases, analysis of cellular-substrate interactions, and dual embryonic microsurgical-autoradiographic study. Within the oculomotor ventromedial subnucleus, the experiments will determine the pattern of migration whereby the primordial cells attain their definitive position, the nature and sequence of intercellular contacts formed during migration, (both within and between populations), and will examine pinocytosis as a possible mechanism for mediation of guided cell movement. Within the trigeminal motor nucleus, the proposed studies will examine the nature and sequence of intercellular contacts formed prior to, during and after migration and will experimentally analyze the importance of extracellular influences (i.e., ingrowing ganglionic afferents) to migration of the nucleus.


Drug-resistant tuberculosis is a serious public health threat. Diagnosis of drug-resistant strains presents a particularly urgent challenge, as such strains are responsible for an increasing burden of morbidity and mortality worldwide. Genome-wide scans that have revealed multiple genetic variants that are associated with drug resistance. The goal of this project is to identify and validate which ofthe resistance-associated mutations are causally connected to either high-level drug resistance or an increased risk of developing resistance. In addition, for selected mutations and agents, we plan to further explore the spectrum of potential mutations that either have not yet been found in clinical isolates or whose association with drug resistance is unclear. Specifically, we will construct isogenic strains containing resistance-associated mutations, producing strains with single point mutations in an otherwise wild type genetic background. We will determine the drug resistance phenotype of the resistance-associated mutations, and we will also predict mutations associated with resistance not yet seen in clinical isolates. Taken together these studies will lead to validated SNPs that will serve as the basis for much needed sequence-based diagnostic tests for tuberculosis.


BIOLIBRARY & PATHOLOGY SHARED RESOURCE Directors: Devon Kelly, M.S. and George Thomas, M.D ABSTRACT/PROJECT SUMMARY The Knight Cancer Institute (Knight) sponsored BioLibrary & Pathology Shared Resource brings together tissue collection, processing, clinical annotation, and histopathology services into a single core. The unit provides members with: 1) a repository of well-annotated human tissue, blood and saliva, 2) a database of annotated clinical variables including patient demographics, treatments and clinical outcomes; research and clinical trial participation; genetic profiles, research analyses and specimen imaging, 3) tailored data in the form of client-specified case reports, 4) pathology services for studies on human and animal tissues, including cryostat sectioning, paraffin-embedding, microtome-sectioning, histochemical staining, immunohistochemistry, immunofluorescence and fluorescent in situ hybridization, 5) support in producing high quality RNA and DNA samples from both frozen and fixed specimens, and 6) pre-award consulting for preparation of grant and other funding applications. The BioLibrary & Pathology Shared Resource is accredited through the College of American Pathologists (CAP) and is Clinical Laboratory Improvement Amendments (CLIA) certified. It is staffed with members who have decades of combined experience in handling samples from small animals and human to ensure that tissues are properly prepared to meet the various needs of the research community, both for immediate analyses as well as long-term storage and use.


Description: The International Union of Operating Engineers (IUOE) has developed a training plan to provide hazardous materials health and safety training to its membership, in particular the Hoisting and Portable (H&P) engineers and Stationary Engineers. They plan to expand and administer their on-going training effort to ensure that their membership who could work in hazardous waste cleanup have the skills to recognize and control hazards. The IUOE is a current grantee in the NIEHS program. They claim to be the largest provider of hazardous waste worker training in the United States since the inception of their training program and grant funding in 1988. Under their current NIEHS cooperative agreement they trained 7,439 workers in the 40-hour worker course and 22,803 workers in the 8 hour refresher. This training program is one part of the IUOE s long tradition of providing worker training programs, including a formal apprenticeship program and Job Corps Program.


Cell signaling mediated by the Hedgehog (Hh) family of secreted proteins plays crucial roles in animal development and human diseases. The Hh pathway is operating in a similar way among organisms ranging from insects to human. Drosophila has been a powerful model organism to study Hh signaling mechanisms, as sophisticated genetic, molecular, and biochemical tools are available to dissect this important pathway in whole organisms as well as in cultured cells. The long-term goal of my laboratory is to delineate the complex regulatory network that governs Hh signal transduction in order to understand how graded Hh signal is transduced to generate multiple developmental outputs. Hh exerts its biological influence through a conserved signaling cascade that culminates in controlling the balance between the activator and repressor forms of the transcription factor Ci/Gli (CiA/GliA and CiR/GliR). The goal of this research is to investigate the multifaceted regulatory mechanisms that control Ci activity. Our recent study has uncovered a dual role of the Ser/Thr kinase Fused (Fu) in the regulation of both the production of CiR and the activity of CIA, and revealed that Fu is activated through dimerization-mediated phosphorylation of its activation loop. These findings provide a critical inroad into a mechanistic dissection of Ci activation. We will explore the mechanism by which Fu promotes the maturation of Ci into CiA and investigate how the Hh gradient is translated into a Ci activity gradient (Aim 1). In a genetic modifier screen, we have discovered that the SUMO pathway can modulate Hh signaling activity and identified Ci as a SUMO substrate. We will further characterize this new post-translational modification of Ci to explore its role and mechanism of action in Hh signaling (Aim 2). The molecular mechanism by which Sufu inhibits Ci is still poorly understood. We have uncovered a previously unidentified nuclear localization signal (NLS) that overlaps with the Sufu binding domain in Ci. We will further study the function of this NLS and its regulation (Aim 3). Finally, how Ci functions in the nucleus to control Hh target gene expression has not been fully explored. We have identified multiple domains required for CiR-mediated repression. Identifying cofactors that interact with these domains and investigating their roles in Hh signaling should shed important lights into how Ci regulates its target gene expression. Therefore, we will carry out protein- protein interaction screen and in vivo RNAi screen to identify Ci co-repressors (Aim 4). The proposed study should provide a much deeper understanding of the Hh signal transduction mechanism and shed new light into how graded Hh signals are translated into different developmental outcomes. PUBLIC HEALTH RELEVANCE: The Hh pathway is a major signaling pathway that controls embryonic development and adult tissue homeostasis. Deregulation of Hh signaling has been attributed to many human disorders including birth defects and cancers. Investigation of the multifaceted and conserved mechanisms that regulate Hh signaling activity will not only provide insights into fundamental developmental problems such as how cells interpret different levels of spatial signals but may also provide new avenues for developing diagnostic tools and therapeutic treatments for cancers caused by deregulation of Hh signaling activity.


This application requests the competing renewal of UNC's MSTP award. Our primary goal is to train an outstanding group of young men and women who are committed to become physician-scientists, fully capable of bridging the gap between basic science and clinical medicine. These individuals will go on to become the next generation of leaders in biomedical science, thereby making significant contributions to and improvements in human health. At the same time, they will become teachers and scholars not only at many of the best medical schools, but also at major research institutes and leading organizations in the biomedical and pharmaceutical industry. We hope to achieve our goal by identifying and then recruiting to UNC candidates from diverse backgrounds who bring with them a great variety of academic and research interests. Once here, they find a strong education in clinical medicine that is well integrated with superb research opportunities. Since the time of our last competing renewal, we have expanded our Leadership Team to include: Kim Rathmell, MD-PhD, the Director for Translational Science and Mohanish Deshmukh, PhD, the Director for Basic Science. In addition, based on the feedback that we received at the time of the last competing renewal of this MSTP grant, we have implemented several new initiatives designed to link the graduate school-based research activities of each student with relevant clinical experiences. Examples include: a) a Longitudinal Clinical Clerkship: this provides each student with clinical experiences that are related to and complement his/her research project; b) a monthly Clinical Case Conference: this is organized and directed by our senior MD-PhD students working in conjunction with Chief Residents from Medicine and Pediatrics; and c) a focus on the Clinical Relevance of Doctoral Dissertation: we are asking that each thesis committee include one clinician- scholar (often a physician-scientist), whose role is to ensure that the clinical relevance of the research is always at the forefront of the student's thinking. W believe that these additions have addressed the concerns of the last review panel and at the same time served to integrate the two phases of our training program. Over the past five years, our program has grown from 64 to 76 students, drawn to UNC from many of the best colleges and universities all across the USA. Their academic credentials are superior: this year's incoming class, for example, had a mean GPA of 3.75 and a mean MCAT of 37.6. Once here, our students are performing at a high level in both the classroom and the laboratory. They are pursuing their graduate training in 20 individual departments and curricula representing the Schools of Pharmacy, Public Health, Medicine, and the College of Arts and Sciences. They are receiving a high percentage of honors, publishing on average four manuscripts, successfully competing for a variety of awards and independent funding (e.g., 26 of our students hold F30 awards from the NIH), and completing the dual degree program in eight years. After completing their post-doctoral training, many of our graduates are becoming faculty members at many excellent institutions.


The photometabolism of bilirubin is to be studied by broad band fluorescent radiation of jaundiced Gunn rats fitted with a bile duct cannula. The photoproducts of bilirubin are to be isolated from the bile by thin layer and high pressure liquid chromatography and identified as either photo-isomers, photo-oxygenations and/or photo-adducts of bilirubin. Similar studies are to be performed using as the light source monochromatic light from an argon tuneable lasar. The latter will permit the determination of the in-vivo action spectrum of bilirubin. The diarrhea that is associated with the clinical use of phototherapy for neonatal hyperbilirubinemia is being studied in experimental rodents by small bowel perfusions with bile pigments. The mechanism of the enterocyte secretion of sodium and water that is induced by non-conjugated bilirubin is being compared with other classic secretogogues. Reversal of the bilirubin-induced secretions is also being investigated by blocking the enterohepatic circulation of bilirubin to prevent enterocyte exposure to non-conjugated bile pigment. The microsomal enzymatic abnormalities for the conjugation of bilirubin in the Gunn strain of rats is to be further defined by comparison with the other microsomal deficiencies in this strain of animal. Ohter microsomal activities and lipid composition are to be examined. Novel amino acid conjugates of bilirubin have been qualitatively found in the meconium of some infants, in bile of patients with hemolytic disorders and in the Crigler-Najjar syndrome Type I. Attempts to induce synthesis of these conjugates in experimental animals will be made.


Disruptive behavior disorders (DBDs; conduct disorder and oppositional-defiant disorder) with severe aggression constitute a public health problem for which evidence-based treatment options are limited. Increasing numbers of youth with aggression are being treated with atypical antipsychotics without good evidence of safety or incremental advantage over safer stimulants. This double-blind, placebo-controlled, parallel groups study will compare the effectiveness of (a) parent training in behavior management (PMT) + placebo (PBO), (b) PMT + d-methylphenidate (d-MPH), and (c) PMT + d-MPH + risperidone (RIS) in children with severe aggression, primary DBDs, and comorbid ADHD. Participants must exhibit a clear history and current pattern of serious physical aggression (i.e., moderate or higher scores on the Modified Overt Aggression Scale (OAS-M), a Clinical Global Impressions (CGI) Scale Severity score of 4 or higher for aggression), and high scores on the on the Disruptive-Total of the Nisonger Child Behavior Rating Form (NCBRF) . The primary aims are to determine (a) if PMT + d-MPH are superior to PMT+ PBO and (b) if PMT + d-MPH + RIS are superior to PMT + d-MPH and to PMT + PBO. Secondary aims include determining whether type of aggression (reactive vs. proactive) moderates treatment response. Design: Two hundred sixteen children across 4 sites (Case Western Reserve, Ohio State, Pittsburgh, & Stony Brook) will be randomized to 9 weeks double-blind of PMT + PBO (n=72), PMT + MPH (n=72), or PMT + MPH + RIS (n=72). All groups will receive a 12-session course of carefully monitored, empirically-based PMT. Responders will be followed on their assigned treatments in a 12-week Extension, and all participants will be assessed at one year after baseline. Clinical change will be measured by (a) parent ratings on the NCBRF & ADHD Symptom Checklist (CL) ; (b) teacher ratings on the ADHD Symptom CL; (c) clinician interview of the child with OAS-M; (d) clinician CGI-Improvement score (CGI-I); and direct observations of child-parent behavior. The primary outcome measure is the NCBRF Disruptive Total score; secondary outcomes are CGI-I, response rate (NCBRF reduction of at least 25%, plus CGI-I score of 1 or 2), other NCBRF and ADHD Symptom Checklist subscales, and cognitive tests. Baseline score on the Antisocial Behavior Scale will assess type of aggression (reactive or proactive) as a potential moderator. AEs and tolerability will also be assessed. This study will assess the use of placebo, d-methylphenidate (Focalin), and d-methylphenidate plus risperidone (an atypical antipsychotic drug; brand name Risperdal) against the back-drop of behavior therapy which will be taught to the parents of participants. The participants will be children ages 6 to 12 years, inclusive, who have been diagnosed with a disruptive behavior disorder plus ADHD and who display significant aggressive behavior. Given the increasing rates and severity of violence in our society, this investigation will help to determine if drug therapy, combined with parent-provided behavior therapy, can reduce child aggression. [unreadable] [unreadable] [unreadable]


The Emergency Responder Health Monitoring and Surveillance (ERHMS) Technical Assistance Document (TAD) aimed at National Response Teams highlights the need for a technology platform for collecting data, before, during and after disasters, to assess the effect training has on response-related illness and injury for emergency responders. This SBIR grant application is aimed at developing a software platform that will not only provide the means to efficiently collect data for the assessment of training impact; but also will provide the means to efficiently deliver pre-deployment and site specific training (SST), against which the assessment of training impact on responders' illness and injury can be evaluated. If successfully implemented, the software platform will be a useful tool both for delivering just-in-time training and for supporting environmental health research relating to responders' exposure to potentially harmful environmental contaminants during and after disaster events. This research project proposes an innovative system for generation and distribution of incident-related knowledge (including manuals, general and site-specific trainings) with integrated monitoring and reporting functionality to address the needs described above. The system will allow the incident command official to generate training materials and data collection tools by simple and straightforward import from existing materials, as well as to customize previously generated tools to better-fit the current situation. All the materials will be delivered to emergency responders in the form of instantly generated smartphone (iOS, Android) and web applications, which will significantly reduce costs of logistics and latency of materials delivery. Materials encapsulation into mobile apps will address the prevalence of mobile devices (smartphones, tablets) at the scene and will ensure that emergency responders can access the content of any situation and circumstances anywhere in the field in the most convenient form, independently of what kind of device they have. Phase I will concentrate on assessing, validating and prioritizing the most demanded tools together with a representative set of key stakeholders; developing a system prototype for digitization and modularization of training and data collection tools, and instant distribution via rapidly created mobile application; prototyping specific interfaces for emergency responders and incident command personnel; testing and finally, evaluating the first prototype. Phase II will concentrate on capabilities of electronic collection and distribution of health status and surveillance data, on integration with an external IT system for two-way communication of hazard data outside incident command, and on generation and delivery of additional materials in response to changed environmental conditions.


Project Summary Oxidation reactions are among the most important reactions in organic chemistry and play a crucial role in the synthesis of pharmaceuticals, natural products, and other bioactive compounds. Advances in catalytic oxidation reactions have potential for major impact in the discovery and production of pharmaceuticals. The majority of existing catalytic oxidation methods face challenges in their efficiency and selectivity, including chemo-, regio- and stereoselectivity, limiting their use in small- and large-scale applications. The proposed research will develop new oxidation and oxidative coupling methods that form carbon-carbon and carbon-heteroatom bonds, including C(sp3)?H functionalization reactions. Some of the resulting methods will streamline the discovery of new bioactive molecules with diverse three-dimensional architectures, addressing key challenges in medicinal chemistry and drug discovery, while others will provide the basis for streamlined process-scale synthesis of pharmaceuticals. Three complementary project directions are outlined in this proposal. The first focuses on the development of oxidase-type aerobic oxidation catalysts that feature a transition metal and a redox active organic co-catalyst. New bioinspired catalyst systems will be explored that exhibit second-order biomimicry, whereby simple organic precursors undergo oxidative self-processing to create the essential co-catalysts. This process resembles the post-translational modification of amino acid side chains to generate reactive cofactors in Nature. The second project will pursue new electrochemical oxidation methods for the synthesis of organic molecules that are difficult to access via classical synthetic methods. These efforts target the identification of versatile mediators and electrocatalysts that permit the reactions to proceed at low electrode potentials, thereby tolerating diverse functional groups and enabling broad scope and utility. Finally, we will develop radical relay methods for benzylic C?H oxidation and oxidative coupling to afford new C(sp3) C?O, C?N, C?X, and C?C bonds. These efforts will be applied to pharmaceutical building-block diversification, core-modification, and late-stage functionalization. In each of these project areas, empirical reaction discovery efforts will be complemented by mechanistic studies of the catalytic reactions. Close interactions and collaborations with pharmaceutical companies in all phases of this project will play an important role in ensuring the broadest possible impact of our efforts.


The specific goals of this study are: a) to investigate whether the cellular tropic and cytopathic characteristics of distinct HIV-1 isolates allows for infection and death of hematopoietic stem/progenitor cells; and b) to investigate whether HIV-1 infection reduces the "stem cell capacity" of the putative CD34++CD38+/- stem cells engrafted NOD/SCID mice transplanted with a human thymus.


Annual Report 2001 This project investigates signal transduction pathways that regulate survival, growth, and differentiation in the lens and corneal epithelium. Recent studies have focused on the signaling pathway linking EGF receptor activation with c-fos transcription in the lens and on the functions of Cdk5, a member of the cyclin dependent kinase family, in both lens and corneal epithelium. Our previous studies of lens epithelial cells have shown that 12(S)HETE, a lipoxygenase metabolite of arachidonic acid, is required for EGF-dependent activation of PKCalpha and PKCbeta.. Further studies of this pathway have now demonstrated that exogenous 12(S)HETE alone leads to activation of these two PKC isoforms and of PKCgamma, an isoform that is not activated in the presence of EGF. We are now investigating the possibility that EGF-dependent signals may prevent 12(S)HETE-dependent activation of PKCgamma. Since we have also shown that 12(S)HETE-dependent activation of PKCalpha and PKCbeta is necessary but not sufficient for c-fos transcription in lens epithelial cells, we are investigating sequences in the c-fos gene that mediate the response to PKC. Our studies of Cdk5 suggest that this enzyme may regulate cell adhesion, migration, and survival in a variety of non-neuronal cells, including lens and corneal epithelial cells. To investigate the possible mechanism of these effects in the lens, cDNA library has been constructed from E18 embryonic rat lenses for yeast two-hybrid screening using Cdk5, p35, and p39 as 'baits'. Interacting clones are being characterized. To extend our previous studies of Cdk5 and p35 in corneal epithelial cells, we have stably transfected a mouse corneal epithelial cell line, A(6)1, with Cdk5 or Cdk5T33 and have used these lines for in vitro studies of cell adhesion and migration. Results indicate that Cdk5 promotes cell adhesion to a fibronectin or collagen matrix and inhibits cell migration, as measured by an in vitro scratch assay. Similarly, we have found that overexpression of Cdk5 in the corneal epithelium of transgenic mice retards corneal wound healing. However, this inhibitory effect on cell migration seems to be relatively specific for corneal epithelial cells. In several other cell types that we have examined, overexpression of Cdk5 increases, rather than decreases, the rate of cell migration. Possible explanations for this cell-type specific difference are being explored. Finally, we have found that Cdk5 overexpression induces apoptosis in COS1 cells and in certain other cell types. This effect is also produced by the Cdk5 activator, p39, but not by the alternative activator, p35, or by the kinase deficient form of Cdk5, Cdk5T33. Cdk5-transfected COS1 cells were found to release a substance to the medium that causes apoptosis even in non-transfected COS1 cells. The nature of this substance is being examined.


Project Summary: Coronary artery disease can lead to myocardial infarction (MI), which is a major cause of morbidity and mortality in the US. Blockage of the coronary arteries prevents blood from reaching downstream target tissue, causing cardiomyocyte necrosis. This results in infarct expansion, dilation of the heart, scar formation, and depletion of cardiac pump function. Current therapies to restore blood flow to the affected tissue causes ischemia reperfusion (IR) injury which increases cardiomyocyte death and can lead to heart failure and mortality. Our lab has recently discovered a novel stem cell in the bone stroma, which we have called cortical bone stem cells (CBSCs). In both murine and porcine MI models, CBSCs were demonstrated to reduce scar size, promote cardioprotection of cardiomyocytes, induce new myocyte formation, and improve cardiac pump function post MI. Additionally, cardiomyocyte mass was shown to increase following CBSC treatment in pig MI/IR models at 3 months post MI. However, it is unclear if this increase in viable mass mainly results from protection of cardiomyocytes from death or the induction of myocyte growth from preexisting cardiomyocytes, rather than CBSCs. We will determine if CBSC secreted exosomes (CBSC-exo) are responsible for improving cardiac structure and function post MI/IR as seen with CBSC therapy. We will also define how CBSCs and/or their exosomes (CBSC-exo) directly protect adult cardiomyocytes from cell death, induce cardiomyocytes to reenter the cell cycle, and promote angiogenesis. For experimental studies, mice will undergo MI/IR surgery followed by injection of GFP+CBSCs, CBSC-exo, or saline. Cardiac repair will be assessed by echocardiography, invasive hemodynamics, histology and molecular studies to evaluate cardiac function, structural remodeling, cardiomyocyte density, and new blood vessel formation. To study the direct effects of CBSCs and their paracrine factors on myocytes and endothelial cells, adult feline cardiomyocytes or human endothelial cells will be subject to hypoxic damage and then treated with CBSCs, CBSC-exo, or CBSC cultured media. This proposed research should provide insight on how stem cells mediate cardiac repair through paracrine signaling. Results from these studies will determine whether exosomes can be used as a novel cell-free therapy to better repair the heart after MI/IR.


Activities in this area are focused on the development of agents with novel mechanisms of action, and the development of novel combinations. A range of compounds are under study. Currently, phase II studies being conducted in ovarian cancer include: 9-AC, a topoisomerase I inhibitor; MGI-114, a novel DNA damaging agent; and, CAI, an anti-angiogenesis agent. A phase I study is beginning, using SU5416 with carboplatin in recurrent ovarian cancer. SU5416 is an anti-VEGF agent with molecular properties that inhibit angiogenesis, and may impair platinum-DNA adduct repair. Preclinically, gene therapy approaches in ovarian cancer are being explored, using dominant negative constructs to AP1 that have been developed by Dr. Charles Vinson of DBS. Also, proteasome inhibition is being explored on the preclinical level in human ovarian cancer cells. In prostate cancer, a range of agents have also been studied. Such agents include gallium nitrate (a heavy metal with a nubmer of activities), high dose tamoxifen (PKC inhibition), thalidomide (anti-angiogenesis) and thalidomide combinations, CAI (anti-angiogenesis), and ketoconazole (anti-androgenic agent) and ketoconazole combinations. - angiogenesis, ovarian cancer, pharmacology, platinum compounds, prostate cancer, - Human Subjects & Human Tissues, Fluids, Cells, etc.


Genomes can be altered in a variety of ways including single base pair changes, deletions and duplications of sequences, and chromosome rearrangements (translocations and inversions). The rates at which these events occur affect 2 quite different processes. Genetic changes are required for evolution and the rate of these changes, therefore, can in principle affect the rate of evolution. Second, many cancers are associated with elevated rates of chromosome rearrangements, and this type of instability appears to be strongly associated with metastatic tumors. When mammalian cells are treated with aphidicolin (a DNA polymerase inhibitor), the chromosomes are broken at specific sites (fragile sites) that are related to translocation breakpoints observed in tumor cells. High levels of chromosome breaks and other aberrations are also observed in mammalian cells with mutations in the ATM and ATR genes. Our general goal is to mimic this type of genetic instability in the yeast Saccharomyces cerevisae, and to characterize the mechanisms responsible for this instability. Our specific aims are: 1) to determine the DNA sequence properties that characterize yeast fragile sites and to characterize the chromosome rearrangements that are associated with DNA breaks at fragile sites, 2) to characterize chromosome aberrations in yeast strains with mutations in genes affecting DNA damage checkpoints, telomere length regulation, recombination, and/or chromatin assembly; for example, strains with mec1 and/or tel1 genes (yeast homologues of the mammalian ATR and ATM genes, respectively) will be examined, and 3) to understand the genetic instability induced by ionizing radiation. These studies will emphasize techniques that allow examination of the entire yeast genome, including DNA microarray analysis (to detect changes in gene dosage and to map translocation breakpoints), and gel technologies that allow separation of intact chromosomal DNA molecules. [unreadable] [unreadable] [unreadable]


The olfactory system is able to recognize thousands of odorants thus maintaining food consumption in humans. The ability to identify odors by humans is dependent on odorant receptors within olfactory neurons that line the nasal epithelium. This epithelium is under continuous cell division by a basal stem cell population that produces immature olfactory neurons, which then become mature olfactory neurons. Odorant receptors (ORs) are clonally expressed in immature olfactory neurons. The mammalian genome consists of ~1500 OR genes; these genes belong to the seven-transmembrane receptor superfamily. However, each allele of each gene is treated as a separate entity leading to ~3000 OR alleles to be expressed in a clonal (singular) manner by olfactory neurons. The ability for this singular OR allele expression is a multi-step manner. A critical step is the production of a functional OR protein. Olfactory neurons are found in domains within the epithelium such that a given olfactory neuron chooses (in general) between ~100 OR genes (~200 alleles). The selection process occurs in two-steps: one of ~200 alleles is chosen for expression (1st choice) within an immature neuron, followed by OR protein production, which is tested for functionality. Failure to produce a seven-transmembrane protein allows for the singular choice mechanism to choose another OR allele in that same immature olfactory neuron (2nd choice). If the chosen seven-transmembrane OR is unable to couple with the signal transduction machinery, then that olfactory neuron will fail to mature and die. The critical step in the expression of an OR gene is the initial choice of one OR allele within an immature neuron (1st choice). To date, it is completely unknown how this singular choice process occurs at a molecular level. Analysis of very small OR promoter regions (~300bp) have lead to the identification of two transcription factors that may play a role: an O/E-1 type binding protein and a homeodomain type biding protein. The identification of more candidate DNA binding proteins and their interactions is necessary to understand the singular choice process. This project fully exploits the small regulatory regions used to produce OR singular choice in immature olfactory neurons. In Aim 1, the most minimal OR control region will be defined in order to identify other candidate transcription factors. There are at least 10-15 different olfactory neuronal cell types each fated to express one of ~200 OR alleles. In Aim 2, two minimal OR promoters normally expressed in different olfactory cell types will be used to identify additional transcription factors. This will allow the understanding of how specific patterns within the olfactory epithelium are obtained. In Aim 3, a candidate homeodomain protein will be deleted within immature and/or mature neurons in order to determine its role on OR gene expression. In Aim 4, I will make every attempt to promote this work into publications and greater funding.


The proposed research can be divided into two broad sections: 1) A. -Special histological studies will be continued in cases demonstrating alterations in fluid pathophysiology of the inner ear. This would include study of the endolymphatic sac and cochlear aqueduct systems together with their respective vascular channels, as well as the vestibular organs and cochlea. B. -Detailed study of cochlear otosclerosis, with and without sensorineural deafness, will be performed, relating possible etiological factors to certain specific tissue changes in some cases of cochlear otosclerosis. 2) The laboratory has well over 500 temporal bones, received either as UCLA autopsy specimens or from the National Temporal Bone Banks Program. Histological examination of these temporal bones creates interest in detailed studies of particular middle and inner ear diseases. Many specimens demonstrate unexpected or unusual changes, and one cannot precisely predict in which direction such findings may lead.


To identify alcoholism risk genes, we are collecting and testing for linkage and pattern of genetic transmission families from three different American Indian populations which are relatively homogeneous and in which alcoholism is highly prevalent. This study also addresses the genetic epidemiology and psychiatry comorbidity of alcoholism in American Indians using structured psychiatric diagnostic interviews of subjects in large families. Data sets of Cheyenne Indian and Pima Indian subjects from large families have been collected. A large panel of random polymorphic probes is being typed in Pima Indian and Cheyenne Indian alcoholics and controls and genetic linkage and transmission analyses are in progress for these groups. An analysis of psychiatric comorbidity in Pima Indian alcoholics and nonalcoholics has been completed.


An estimated 23 million Americans, including adolescents and the elderly, suffer from circadian rhythm sleep disorders, such as delayed sleep phase disorder, advanced sleep phase disorder and winter depression. These conditions are characterized by persistent insomnia and/or excessive daytime sleepiness, impaired performance, reduced well being and lower quality of life. The negative symptoms result from a misalignment between the timing of sleep and the internal circadian clock. Clinical research has demonstrated that circadian rhythm sleep disorders are most effectively diagnosed (differentiated from other causes of insomnia) and treated if each individual patient's circadian phase is known. The timing of the master internal circadian clock is most reliably measured from the onset of the endogenous circadian rhythm of melatonin, a neuroendocrine hormone, as measured in dim light (dim light melatonin onset, or "DLMO"). However to date the reliable and valid assessment of the DLMO is limited to the research laboratory setting. This application answers PAR-08-212 "Methodology and Measurement in the Behavioral and Social Sciences" which calls for research to "improve and develop methodology and measurement in the behavioral sciences through innovations in data collection techniques and measurement...approaches that integrate behavioral science research with biological science research are particularly encouraged...developing and validating research instruments that fill a gap in research needs...improve measurement in the real-world setting." Our goal is to develop a streamlined procedure for the accurate assessment of circadian phase (DLMO) outside of the laboratory that will provide clinicians and researchers with a novel diagnostic and research tool. In this way the underlying neurobiological cause of a patient's insomnia and/or circadian rhythm disorder can more readily be diagnosed and treated. Specific Aim 1 is to validate procedures for at-home circadian phase assessment in a large sample of healthy people (n=120). Validation will occur by (1) objectively measuring compliance to the at-home procedures and (2) comparing DLMOs collected at home to DLMOs collected in the laboratory, in a within-subjects counterbalanced design. We have pilot data to support the validity of our at home procedures. Specific Aim 2 is to validate the same at home procedures in patients with delayed sleep phase disorder (n=30). Specific Aim 3 is to conduct rigorous analyses to inform future users which subject characteristics and light levels predict (1) compliance to the at home procedures and (2) valid at-home DLMOs. The results of this 3 year study will have substantial implications for the translation of basic and clinical research to the community: (1) the diagnosis and treatment of insomnia and circadian rhythm sleep disorders will be significantly enhanced, thus improving public health and safety, mood and quality of life, (2) community participation in research will be improved, particularly in vulnerable and under represented populations, thus increasing scientific knowledge and (3) research and clinical costs will be substantially reduced. PUBLIC HEALTH RELEVANCE: This research will test a new method to measure the timing of the body clock at home, instead of in the research laboratory. The results will facilitate the diagnosis and treatment of circadian rhythm sleep disorders such as advanced sleep phase disorder, delayed sleep phase disorder and also winter depression, thereby improving public health and safety, well-being, mood, mental function, and quality of life.


The major aim of this project is to examine which subjects are likely to succeed in a behavioral obesity treatment. The first objective is to empirically derive predictors of successful response to a behavior modification approach. Predictors which will be examined are weight loss history (prior dieting), severity of overweight, extent of binging, and attitudes about dieting (perfectionistic goal setting). The second objective is to cross-validate these predictors on another sample of subjects. Moderately overweight females, from 150-200 lbs, at the time of their application and ranging in age from 25-45 will be studied. Treatment will be conducted in groups led by experienced behavior therapists.


The first aim of this research is to investigate the properties of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) whose constituent fatty acyl chains are highly asymmetric in length. There are many important membrane components, such as the ceramide, sulfatide, ganglioside, and sphingomyelin molecules of nervous tissue, which contain two hydrocarbon chains of inequivalent length. The results of the research proposed in this project will help lead to an understanding of the effect of chain length asymmetry on the properties of lipids in natural membranes. It has recently been demonstrated that the asymmetric-chain phospholipid, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, possesses important physiological functions as a platelet activating factor (PAF) and as an antihypertensive agent. A long term research project is being initiated in my laboratory with the objective of studing the physical and physiological properties of this important phospholipid. Thus, a second aim of this proposal is to begin this project by studying the physical properties of synthetic PAF and to attempt to isolate the receptor for PAF that has recently been shown to exist in human platelet membranes. Specifically, we propose to (1) investigate the properties of saturated mixed-chain PEs, C(18):C(18)PE through C(18):C(2)PE. This work will complement the studies that we have already carried out on the properties of saturated mixed-chain PCs. (2) to investigate the properties of binary mixtures of mixed-chain PCs and PEs with symmetric-chain phospholipids with particular emphasis on the mixing properties of the two lipid types as well as the possible existence of nonlamellar phases in the binary combinations; (3) to study the properties of synthetic PAF in comparison to those of C(18):C(2) PC. As C(18):C(2)PC is physiologically inactive, a comparison of the properties of these two lipids may serve to elucidate some of the important structural properties of PAF necessary for its physiological function; (4) to attempt to isolate the membrane receptor for PAF that has been shown to exist in human platelets by employing a combination of detergent solubilization and affinity chromatography. Principal techniques to be used are differential scanning calorimetry and densitometry, 31P-NMR, electron microscopy, quasi-elastic light scattering, Raman spectroscopy, detergent solubilization, and affinity chromatography.


Intravascular administration of cell-free hemoglobin appears to be strongly correlated with a rise in pulmonary and systemic blood pressure. This hypertensive response has been attributed to the reaction between hemoglobin and the endothelium-derived relaxing factor (EDRF), which is now believed to be nitric oxide (NO). We explored the interactions between NO and the oxidized forms of a number of chemically modified hemoglobins. These hemoglobins are blood substitute candidates due to their lower oxygen affinity and greater stability of their tetrameric structures. Using fast reaction techniques, the stopped flow apparatus, we documented that there are differences in the rate of the initial NO binding to the ferric forms of these proteins and in the subsequent NO-driven reduction step. Since NO oxidizes oxyhemoglobin to ferrihemoglobin (metHb) extremely rapidly in vivo, we hypothesized that a redox cycle between NO and metHb could be partially responsible for the depletion of NO as a biological transducer, leading to altered vascular tone with variable effects observed for different hemoglobin preparations. This and other related work was presented at a recent international meeting on blood substitutes and recently appeared in full in Arch. Biochem. Biophys. Work is in progress to explore the mechanism(s) of interactions of NO with other hemoproteins to fully understand the degree to which heme pocket chemistry and other structural features determine the binding of NO to hemoproteins. This will ultimately contribute to the design of hemoglobin that, along with desirable oxygen binding characteristics, exhibits a suppressed ability to react with NO.


Summary: With the potential threat of smallpox as a bioterrorism agent, a major effort should be invested in two main areas: a) development of new effective preventive vaccines, and b) production of high titer vaccinia IGG (VIG) that can be used as a first line of defense in exposed and/or high risk populations. The current stocks of VIG are limited and outdated. Scientists at CBER and elsewhere (MPH) were encouraged to produce new lots of VIG and subject them to careful titration in validated neutralization assays. In addition, experiments are underway to compare the neutralization capacity of different VIG subclasses, in order to optimize viral neutralization in future production of VIG.The current neutralization assay for vaccinia (plaque-reduction), is labor intensive, slow (up to 7 days), and depends on scoring plaques by eye (i.e., subjective). This assay is difficult to validate and to transfer to sponsor-based laboratories. As an alternative approach, our laboratory initiated late last year the development of an alternative vaccinia neutralization assay, which is fast, quantitative, and easy to perform and transfer to other laboratories.This assay is based on the use of recombinant vaccinia virus expressing a bacterial gene coding for the enzyme b-Gal under the control of the vaccinia late promoter. Several recombinant viruses were tested and two of them, vSC8 and vSC56 are currently under investigation in our laboratory. We also tested several cell substrates for the assay: BSC1, Vero, and HeLa. Results: - We have established and standardized a novel assay to measure vaccinia neutralization. It is based on the expression of a reporter gene coding for the bacterial b-galactosidase enzyme (b-Gal) under the control of a synthetic E/L promoter. We demonstrate that the new assay is rapid (24 hr), of equal sensitivity to PRNT assays, reproducible, objective, and easy to transfer. In addition, we describe preliminary results with a second reporter gene assay based on a vaccinia-EGFP recombinant virus. The expression of the GFP is induced by IPTG. The two assays provide high throughput capabilities and may be established in clinical laboratories for the evaluation of multiple samples from clinical trials. - Thus far, it was found that the MPH VIG is somewhat better than the Baxter (4oC) VIG with ID(50) values of 10-15 ug/ml and 25 ug/ml, respectively. - We also tested Ig subclasses derived from the MPH VIG and found that the neutralization efficiency of the IgG1 subclass is superior to all other subclasses and to the total (unseparated) VIG preparation. - We tested multiple lots of IVIG from 5 different manufacturers. Several lots were found to have significant anti-vaccinia neutralization titers.


This revision of U24 l\/lH068457-08, "NIMH Center for Collaborative Studies on Mental Disorders," (NIMHC) requests funding for a "Repository Supporting Stem Cell Research Relevant to Mental Disorders" in response to NOT-MH-10-024. Since late 1998, the NIMHC, whose biorepository is at Rutgers University and whose data repository is at Washington University School of Medicine, has established a robust infrastructure for the receipt of blood samples and externally produced cell cultures. DNA and RNA has been extracted from blood and over 75,000 lymphoblastoid cell lines have been produced in-house for the NIMHC, with 99.8% success on the first attempt. In the past 5 years, the NIMHC has distributed -450,000 DNA, ~15,000 RNA and ~8,000 cell lines to researchers throughout the world. Based on serving the needs of NIMH Genetics Initiative grantees, the NIMHC maintains secure clinical (phenotypic) and genomic databases and a password protected, secure website for NIMH-approved researchers to access. The NIMHC has also developed and made available web-based bioinformatics resources for the design, analysis and interpretation of studies in psychiatric genetics. We propose to utilize the facilities, expertise and experience of the NIMHC to serve researchers wishing to use human patient and control primary cells, adult stem cells and induced pluripotent stem cells (iPSCs) for investigation of the cellular bases of mental disorders. The NIMHC will receive primary tissue samples for the establishment and banking of primary cell cultures or adult stem cell lines. The NIMHC will also receive cultures of primary cells, adult stem cells and iPSCs for characterization and banking. These cell lines, along with subject clinical and genomic data and molecular and phenotypic data describing the cell lines, will be distributed to NIMH-approved researchers. The NIMHC will also provide consultation regarding the nature and use of these stem cell lines. To a lesser extent, the NIMHC will also produce, characterize and bank iPSCs made from primary cells. Freely sharing primary and stem cells within the research community is likely to greatly accelerate progress in understanding the cellular bases of mental disorders. Public Health Relevance: Mental disorders are a common, widespread and heterogeneous group of diseases whose biological bases are poorly understood. Elucidation of the underlying genetic and physiological bases for these disorders is necessary for the creation of effective therapeutics. Nerve cells or other cells derived from adult stem cells offer the possibility of modeling these disorders in cell culture and for preliminary screening of pharmaceuticals.


This study will enroll 10 people currently participating in the extension phase of ACTG 306 plus an additional 60 people not participating in ACTG 306. The study will last six months. The purpose of the study is 1) to compare the effects of two ZDV-containing drug combinations to see how many people ahve an undetectable viral load at both weeks 20 and 24; 2) to compare the safety of each of the three drug combinations.


Project Summary Problems frequently encountered social and behavioral science researchers concern heterogeneity of the: 1) individuals needing treatment, 2) interventions responding to individuals'specific needs, and 3) array of potential outcomes resulting from treatment (Goldenberg, 1978;Gordon, Powell, Rockwood, 1999;Rockwood, Joyce, &Stolee, 1997). A common response to these problems is to identify various outcome measures that encompass the variability in patients, treatments, and outcomes (Kazdin, 2005). In these studies, the patients treated are often assessed on all outcomes. Analyses often involve multivariate approaches or latent class models. A problem with this design and analysis strategy is that when patients are assessed on all potential measures, many are assessed on outcomes that their specific treatment plan was never designed to address. The project is designed to expand the research base on analytic methods to assess the statistical significance associated with interventions targeted at heterogeneous populations. Specifically, the project will continue the study of the maximum individualized change (MIC) procedure developed by Boothroyd, Banks, Evans, Greenbaum, and Brown (2004). The method was initially developed as an analytic alternative to the more traditional multivariate and latent model approaches often used in studies examining individually-tailored interventions. Our initial developmental work on this approach suggests that it offers a number of significant advantages over traditional statistical approaches in studies where a number of measures are used to assess potential treatment outcomes. These advantages include increased statistical power to detect smaller program effects and few problems associated with missing data. Despite the MIC's early promise, more basic work is necessary to determine whether this approach should become a standard analytic tool in future studies. The study will consist of two phases. First, an expanded simulation study of the MIC procedure will be conducted (Boothroyd et al. 2004) broadening the initial set of assumptions. Specifically, the impact of seven variables will be systematically examined. The variables include varying the: 1) level of correlation among outcome measures, 2) number of outcome measures/domains being assessed, 3) distribution of response effects, 4) number of outcome measures on which a person improves, 5) sample size, 6) weighting the outcome measures based on qualitative assessment regarding the likelihood of change, and 7) amount of missing data. In phase two, the MIC procedure will be used in the re-analysis of data collected as part of SAMHSA-funded multi-site national study examining the impact of managed care. In a recently published article summarizing the study findings, the authors concluded, "Managed care and fee-for-service Medicaid programs did not differ on most measures;however, a lack of sufficient power was evident for many measures" (Leff, et al., 2005).


This is the first submission of an application to investigate the mechanism of Zn2+ toxicity in the CNS. Recent findings indicate that this cation is elevated during stroke, traumatic brain injury, and oxidative stress, and that elevated intracellular Zn2+ is associated with the death of cultured neurons. Elevated Zn2+ is also associated with mitochondrial dysfunction, and with the death of cells by necrosis and apoptosis, although the mechanism of these effects is unknown. The investigator and his associates have recent findings that seem to bear upon the mechanism of Zn2+ toxicity at the cellular level. They have found that submicromolar concentrations of Zn2+ inhibit the mitochondrial alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes, and that Zn2+ stimulates the mitochondrial permeability transition. They also note that oxidative stress can release Zn2+ from metallothioneins and that the metallothionein expressed in brain is associated with the mitochondrial outer membrane. Putting all of this together, the investigator hypothesizes that cell injury caused by oxidative stress arises, at least in part, because Zn2+ is released from metallothionein, inhibits the TCA cycle at the level of one or both of the dehydrogenases, and thereby, or through other mechanisms, provokes the permeability transition and subsequent cell death. To begin evaluating this hypothesis, Dr. Brown will investigate the mechanism by which Zn2+ inhibits the dehydrogenases using enzyme kinetics and the study of partial reactions. Mechanisms transporting Zn2+ in mitochondria will be determined, and respiration studies will be conducted to ascertain the relative importance of Zn2+ action on the dehydrogenases, compared to the electron transport chain, in producing mitochondrial dysfunction. Finally, the mechanism by which Zn2+ influences the permeability transition will be determined. In several cases data obtained from brain, heart, and liver mitochondria will be compared, to ascertain the generality of the findings, and to allow a cross comparisons of literature, which is developed to different extents for mitochondria from these various organs.


Mechanisms and Importance of Opioid and ORL-1 Receptor Coupling to Ca2+ Channel Subtypes 1. What are the molecular identities of opioid and orphanin FQ-sensitive Ca2+ channel subtypes? a. Do ORL- 1 receptors couple like opioid receptors to L-type Ca2+ channels when expressed in GH3 cells? b. Which different L-type Ca2+ channel a1 subunit transcripts are expressed by GH3 cells? c. Do antisense oligonucleotides to the a1 subunit abolish the opioid- and orphanin-sensitive Ca2+ current component? d. Do opioid and ORL-1 receptors expressed in HEK293 cells couple to Ca2+ channels formed by a10 subunits? 2. What G protein mechanisms couple opioid and ORL-1 receptors to CA2+ channels? a. Is there a consensus sequence at which bg binds to all opioid and orphanin FQ regulated Ca2+ channels? b. Does a peptide mimicking this sequence prevent receptor coupling to L- type Ca2+ channels in GH3 cells? c. Can a similar approach uncouple opioid receptors from a1a, a1b and a1d Ca2+ channels in HEK293 cells? 3. What is the contribution of L-type Ca2+ channels, relative to other effectors, in the modulation of vesicular release by opioids and orphanin FQ? a. Other than Ca2+ channels, what effectors are regulated by opioids and orphanin FQ in GH3 cells? b. Do opioids inhibit pro[actin release from GH3 cells through G protein bg subunits? c. What is the contribution of L-type Ca2+ channel inhibition in the opioid regulation of prolactin release from GH3 cells? 4. Do specific Ca2+ channel subtypes have a role in the central actions of opioids and orphanin FQ? a. Are a10 subunits (or other opioid and orphanin FQ sensitive subunits) coexpressed with opioid and/or ORL-1 receptors in central neurons? b. Do specific Ca2+ channel subtypes participate in the opioid and/or orphanin FQ regulation of dopamine release in the reward pathway?


The mission of the University of Toronto MHSc in Bioethics International Stream is to strengthen individual and institutional leadership capacity in bioethics, with particular emphasis on research ethics, in low- and middle-income countries. We work in partnership with existing and emerging programs in strong institutions in the developing world to identify leaders who will make those programs successful and sustainable. Building upon the success of the program over the past 8 years, (32 graduates as of June 2008, program experience rated highly by graduates), the reach of the program will refocus on West Africa (especially Ghana and Nigeria), and continue to focus on South Asia (extending beyond India and Pakistan to Bangladesh). Key emerging issues of global ethics (such as genomics/biotechnology and public health ethics) will be incorporated into the curriculum. We will build on continuous professional development program for graduates. The University of Toronto Component consists of 10 months of course work covering breadth and depth areas in bioethics, and resulting in the MHSc degree. The Home Country Component consists of 14 months of mentored research, educational, and leadership activity in bioethics in the trainee's home country culminating in a workshop in Toronto to facilitate intertrainee exchange of experiences, ideas, successes, and challenges. The program draws upon an internationally-distinguished faculty from a variety of disciplines. Over 4 years, the program will train at least 12 highly-qualified individuals nominated by home institutions and carefully selected for potential impact in their home country. We believe our targeted and strategic approach, intellectually rich environment for bioethics scholarship, legitimizing effect and discipline of the graduate degree, emphasis on adult learners and career development, continuous evaluation and improvement of our programs, and long-standing and trusting partnerships with institutions in developing countries, are the key success factors of our program. PUBLIC HEALTH RELEVANCE: As clinical research expands globally it is imperative to build capacity in the developing world for the ethical assessment and regulation of research in the contexts where the research will be conducted. The University of Toronto, Masters of Health Sciences in Bioethics (MHSc), International Stream is devoted to contributing to capacity building efforts in international research ethics. Over the past 8 years, working with established organizations in the developing world, the program has graduated 32 fellows with MHSc degrees who are leaders in scholarship and practice in ethics and regulation of human research. In this renewal we intend to continue our efforts in capacity building with a specific focus on Nigeria, India, Pakistan and Bangladesh, four of the most populous nations in the world, with pressing health needs. [unreadable] [unreadable] [unreadable]


PROJECT ABSTRACT Most neurodegenerative disorders exhibit highly heterogeneous genetic underpinnings. For example, with Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Parkinson's disease (PD), mutations in different genes are causal in distinct subsets of families. In addition to this genetic heterogeneity among inherited cases, the majority of individuals exhibit sporadic forms of these disorders in which there are no known causal mutations. In part because of this sporadic onset, it is widely accepted that (largely unknown) environmental forces are at play in the initiation and/or progression of each of the above disorders. This proposal will use a systems approach in Drosophila to identify forces that drive initiation, as well as common cellular responses that may modulate progression. This will be accomplished by three scientific aims. First, we will test a series of cellular stressors, behavioral stressors, and models of injury/inflammation. The effects of these manipulations will be assayed by following 7 different neurodegenerative phenotypes and biomarkers, including a novel assay of endogenous retrovirus replication. Second, we will use a relatively new approach to purify the population of cells that are most impacted, and profile active transcription within the nuclei. This experiment will identify common downstream cellular responses. Finally, we take advantage of high throughput genetic approaches in Drosophila to systematically test for functional impact of identified gene targets.


The overall goal of our grant proposal is to evaluate the possibility that natural HIV viral env sequences that emerge during the course of HIV infection, can be used as immunogens to elicit broadly-reactive anti-HIV neutralizing antibodies. These env sequences will be derived from HIV infected subjects which during a very short period of time (2-3 years) developed broad and potent anti-HIV cross-neutralizing antibody responses. In Project 1, we will monitor HIV infected subjects to identify those that develop broad cross-neutralizing antibody responses shortly following infection and we will characterize in detail these responses. We will amplify viral env from longitudinal samples from these patients and in conjunction with Project 2 we will examine what role the CD4+ T cell helper responses have in the development and maintenance of broad anti-HIV neutralizing antibody responses. The Envs identified in this Project 1 will be used as immunogens in Project 3 to test the hypothesis that they can elicit broad cross-neutralizing antibody responses in animals. RELEVANCE (See instructions): Our proposed studies will explore a novel concept in the development of an immunization protocol that will lead to the elicitation of broad cross-neutralizing antibodies against HIV. As such, the proposed studies are highly relevant to the development of an effective anti-HIV vaccine.


Please reference the abstract RELEVANCE (See instructions):


The investigators objective is to determine the physical and molecular basis for the cooperativity found between the troponin-tropomyosin complexes which make up the regulatory strand of the thin filament. To this end, methods have been developed or are under development to manipulate the regulatory proteins of chemically skinned mammalian muscle fibers. Troponin C, I, T and tropomyosin will be extracted or replaced either singly or in various combinations with structurally different isomers or with chemically modified ones purified from various muscles. When extraction without replacement is possible, the effect of extraction on cooperativity during thin filament activation by Ca2+ (slope of the pCa/tension relationship) and by rigor crossbridges (slope of the pS/tension relationship) will be examined. Through replacement techniques, fluorescent probes or other structural modifications will be introduced into the regulatory strand of skinned fast and slow muscle fibers. Fluorescence changes in single fibers will be used to measure Ca2+ binding, crossbirdge binding and the state of the regulatory strand simultaneously with tension. The apparatus developed for these studies automates solution mixing, protocol execution, and the collection of fluorescence and tension data; similar apparatus without the fluorescence measurement capability is used to develop better methods of exchanging the regulatory proteins of the thin filament. The cooperative regulation of thin filaments will be modeled with the classical formalism for allosteric proteins and with an induced shift formalism under development. By comparing pCa/tension and pS/tension data from control, extracted, and reconstituted fibers with the model expectations, experimental tests of proposed cooperative mechanisms are developed and new insights into the relationship between molecular form and thin filament physiology are realized. The cooperativity found between the regulatory complexes of the thin filament is more extensive than any molecular interactions hitherto described; it integrates the actions of at least 75 regulatory protein molecules and unifies control over hundreds of actin molecules. To explain it in physical terms may require the development of new ways to envisage molecular interactions in biological structures. Through this contribution to basic knowledge, the proposed work will contribute to the capacity of biomedical researchers to develop treatments for disease.


Guanosine triphosphate-binding proteins regulate numerous processes in all cells. The types of process include synthesis, signal transduction, growth regulation and intracellular transport, and several others. These proteins vary in size and structure, but they share regions of conserved protein sequence, in their GTP-binding domains. Moreover the GTP-binding domains of two of these proteins, ras p21 and elongation factor Tu, possess nearly identical tertiary structures. The similarity of these structures suggests that GTP and GDP similarly alter the structures of these proteins and indeed those of all GTP regulatory proteins. This conclusion warrants the use of reasoning by analogy to define the functions of these proteins. The peptide chain elongation factors Tu and Ts comprise a GTP-regulatory system whose interaction with a variety of cellular components make it an informative subject both for the study of GTP-promoted processes and for the identification of protein-protein and protein-nucleic acid interactions. EF-Tu presents a useful model for these processes because it is an abundant, soluble protein of tractable size whose tertiary structure has been largely solved. EF-Tu engages in three separate but interdependent processes-GTP-mediated aatRNA-binding, ribosome-mediated GTP hydrolysis, and EF-Ts-mediated GDP exchange. Other GTP-regulatory proteins engage in analogous processes. To understand how these processes occur, we are determining how single amino acid replacements alter each of the functions. The structural consequences of these changes are determined by reference to the EF-Tu structure, and the effect of the analogous change on other GTP-regulatory proteins is predicted. The reliability of this approach can be assessed by comparing the effects of analogous changes on the functions of EF-Tu and ras p21, whose structure is also known.


This Phase II SBIR will develop Continuous Real Time (CRT) ShuntCheck, the first portable, non- invasive device for real time, continuous monitoring of changes in flow in CSF shunts. This device will result in improved clinical management of hydrocephalus by providing a non-invasive method for monitoring and researching shunt function. Hydrocephalus, a common condition in which CSF accumulates in the brain ventricles, is corrected by placing a VP shunt that drains excess CSF to the abdomen. Shunts frequently malfunction, usually by obstruction, but the symptoms of shunt failure are unspecific - headache, nausea. Diagnosis of shunt malfunction is expensive and presents risks (exposure to radiation from CT Scans, risk of infection from radionuclide testing). Additionally, ongoing clinical management of shunted patients is complex (due to a lack of tools for investigating CSF over drainage, for assessing the performance of specific shunt valves and siphon control devices and for streamlining the adjustment of programmable shunt valves). NeuroDx's existing device, ShuntCheck-Micro-Pumper, is a shunt obstruction detector and addresses the need for a non-invasive test for shunt malfunction. While this makes it a valuable tool for the Emergency Dept, the short duration of the test limits its utility for shunt valve adjustment, investigating suspected shunt over drainage, etc. A non-invasive, non-radiologic device which can track changes in CSF flow rate would address many ongoing clinical management needs and become a valuable tool for the neurosurgery clinic. In our Phase I studies, we developed a laboratory prototype CRT based upon a breakthrough innovation in our thermal dilution technology and validated its safety and accuracy in bench and animal studies. CRT can reliably differentiate between no, low and robust shunt flow and can track changes in shunt flow rates over extended time periods. The goal of this Phase II project is to refine CRT ShuntCheck from a laboratory prototype to a production-ready device, validate its safety and accuracy in bench and animal testing, and complete a 510k submission for FDA clearance. Post-Phase II clinical studies will demonstrate the clinical utility and cost effectiveness of CRT ShuntCheck for streamlining valve adjustment in pediatric and in adult NPH patients. NeuroDx's business model for this product involves the generation of revenue primarily from the ongoing sale of single-use, disposable sensors for these tests. Shunt management testing constitutes approximately 105,000 shunt flow tests annually in the United States alone. The need for new diagnostic tools for managing hydrocephalus patients is highlighted by the NIH announcement Advanced Tools and Technologies for Cerebrospinal Fluid Shunts (PA-09-206), to which this application is responding. Our application directly responds to the request for Diagnostic tools for use in a hospital or outpatient setting that work in real-time to quantitatively determine shun function.


Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase that is a critical component of signaling pathways regulating cell function. The heterodimeric core of PP2A associates with a variety of regulatory subunits that contribute to the specificity and subcellular localization of the enzyme. PP2A also interacts with a number of proteins that are not considered to be conventional regulatory subunits of the enzyme. The investigators anticipate that identification of additional interacting partner for PP2A will provide new insights into the cellular organization of the enzyme and that such organization dictates specificity of enzyme action. One sub-population of PP2A is associated with microtubules in neurons. Tau and MAP2 are neuronal microtubule-associated proteins (MAPs) that are highly phosphorylated in vivo and are targets for numerous protein kinases in vitro. The phosphorylation state of tau and MAP2 determines their abilities to bind to and thus stabilize microtubules. Results from investigators' laboratory indicate that PP2A dephosphorylates tau and MAP2 in vivo, suggesting the enzyme regulates the neuronal cytoskeleton. An important aspect of this work is that one of the pathological hallmarks of Alzheimer's Disease includes hyperphosphorylation of tau and disruption of axonal microtubules. The proposal has three specific aims. 1) The first aim will test the hypothesis that an anchoring protein that promotes targeting of PP2A to microtubules is required for efficient dephosphorylation of tau and MAP2. The molecular basis of PP2A interaction with the microtubule cytoskeleton will be explored. Putative anchoring protein(s) will be purified, characterized, and cDNAs cloned. The role of anchoring in regulating PP2A will be examined with the assumption that direct protein-protein interactions define the specificity of PP2A toward neuronal MAPs. 2) The underlying hypothesis of aim 2 is that suppression of PP2A in neurons of intact animals will lead to increased phosphorylation of tau and neuronal degeneration. SV40 small-t antigen, a specific inhibitor of PP2A, will be expressed in transgenic mice using neuron-specific promoters. The effects of reduced PP2A activity will be assessed by measuring tau phosphorylation and neuronal morphology. 3) The hypothesis to be tested in this aim is that two novel PP2A- interacting protein influence the activity and function of PP2A. Characterization of p31 and p17, two PP2A-interacting proteins identified in a two-hybrid screen, will be pursued with high priority. The effects of these proteins on activity, subcellular localization, and in vivo function of PP2A will be determined. Additional candidate interacting proteins, identified in the two-hybrid screen will also be characterized.


Autologous skin grafts are the preferred method for covering large burn surfaces. The healing of autologous skin grafts, and the re-harvesting of skin donor sites for autologous skin grafts, is often slowed by the altered metabolic state of burn victims. Accelerating the healing of the skin graft donor site would increase the number of sites that could be used and decrease the re-harvest time between grafts. One of the single most important factors in healing a skin graft donor site is the neovascularization and reestablishment of oxygen tension and nutrient levels in the wound. Ultimately the oxygen, and some of the nutrients, are used to produce adenosine triphosphate (ATP). ATP is used for nearly every aspect of the wound healing process, including protein synthesis, growth factor production, and mitosis. We have developed a technique for the rapid and controlled delivery of ATP using fusogenic lipid vesicles (VitaSol). VitaSol, when applied topically, provides wounds with ATP during hypoxic periods and accelerates the healing process of acutely wounded skin. The long-term objective of this proposal is to develop a safe and effective technique to accelerate the healing of partial-thickness wounds in swine using VitaSol, and prepare the compound for a Phase I clinical trial for autologous skin grafts. The specific aims of phase I project are: 1) Determine the mechanism by which VitaSol accelerates partial-thickness wound healing in swine. We hypothesize that the beneficial effects of increasing wound ATP levels are related to increased angiogenesis, increased angiogenic growth factor synthesis, and accelerated mitosis of keratinocytes and fibroblasts; 2) Optimize the formulation of VitaSol for maximal acceleration of partialthickness wound healing in swine. Once the mechanism of how VitaSol accelerates wound healing is known, the formulation will be mixed with an excipient to create a hydrogel which can be easily applied to wounds, and helps maintain ATP levels during a 24 hour period; and 3) Stabilize the optimized VitaSol formulation and examine efficacy in swine. The optimized VitaSol formulation will be prepared as freezedried powder and examined for stability and efficacy in partial-thickness wound healing. The success of this project is likely to have a major impact on medicine. It will enhance our understanding of how ATP delivery to wounds affects the wound closure rate and time to complete wound closure. Our paradigm shift in wound healing will provide a totally new therapeutic approach that could significantly increase number and frequency with which skin grafts can be harvested and used clinically. [unreadable] [unreadable]


The lateral geniculate nucleus (LGN) is a major relay station in the visual system. Relay cells in the LGN summate direct excitatory inputs from retinal ganglion cells. Also, these ganglion cell inputs provide inhibitory inputs to the relay cells, via LGN interneurons. This project will attempt to characterize the way in which all of the retinal ganglion cells that overlap the general region of an LGN neuron's receptive field contribute to, or influence that LGN unit's firing. This will be done in the cat by recording simultaneously from individual ganglion cells and from individual LGN neurons. At both the level of the retina and the LGN, attention will be paid to the W/X/Y classification scheme, as well as to the familiar ON and OFF-center types of receptive fields. It is known that in terms of direct excitation, the anatomically based W/X/Y pathways are kept relatively separated all the way from the retina to the visual cortex. At the LGN, interneurons are used to provide inhibitory influences that modify the direct excitatory signals that pass along these channels. By recording from ganglion cells and LGN neurons simultaneously we will ask what, if any, is the specific organization of this inhibition? That is, to what extent are various pathways segregated in inhibitory terms as they are known to be in excitatory terms? Inhibitory influences from both eyes onto any particular relay cell will be studied. In a related series of experiments, we will record independently from two neighboring ganglion cells and an LGN relay cell to ask the basic question, to what extent is simultaneous firing of these ganglion cells important as an influence on the relay cell's firing? Various levels of background illumination, including complete darkness, will be used.


This research examines the effects of variations in serum testosterone levels on GH secretion and basal metabolic rate in normal men. Subjects will be studied 3 times, in random order, under conditions of low, normal, and elevated testosterone. Both stimulated and non-stimulated GH secretion, BMR, and lipids will be measured during each admission. CORE LAB ONLY.


DESCRIPTION: The main goal of the project is to study the mammalian metabolism of the anticancer agent betulinic acid using microorganisms as in vitro models of mammalian metabolism. Microorganisms, particularly fungi, have been utilized successfully in studying the mammalian biotransformations of drugs and other xenobiotics. The use of microorganisms as in vitro models to mimic and predict mammalian biotransformations for initial metabolism studies has many advantages including the ability to produce large quantities of metabolites for full structural characterization and biological evaluation, substantially reduced cost over in vivo studies, and lessened animal demand. Befulinic acid, a pentacyclic lupane-type triterpene, is a relatively common natural product that was recently reported as a melanoma-specific cytotoxic agent following both in vitro and in vivo studies. Because of its high antitumor activity and lack of toxicity, the drug is currently undergoing preclinical development for the treatment or prevention of malignant melanoma. An essential part of preclinical development of a drug is to elucidate its mammalian biotransformations and evaluate the biological activity and/or toxicity of its mammalian metabolites. Since there have been no reports on the mammalian metabolism of betulinic acid, it is the main objective of this study to utilize microbial cultures as model systems to predict and prepare potential mammalian metabolites of betulinic acid. In addition, the generated metabolites of betulinic acid will be evaluated for antitumor activity and/or toxicity.


Our ability to analyze hazardous material in toxic waste sites has improved dramatically in recent years. However, we are very limited in our ability to trace the movement of hazardous materials from Superfund sites through various media or to prioritize and mitigate the hazards involved. Our ability to predict exposure, much less susceptibility or effect, of these materials on humans and their environment is still more limited. This Program consists of eight integrated projects, three research cores, a training core and an administrative and outreach core to address these problems. We will determine the fate and transport of hazardous materials in ground water, surface water, and air as they move from toxic waste sites. Concurrently we will develop sensitive systems for evaluating the exposure and effect of populations to these materials. These biological markers will be based upon immunochemical and other detection systems and based on a fundamental understanding of the toxicological processes involved. The project will emphasize pulmonary, dermal, and reproductive systems in mammals as well as microbial and fish systems in the environment. We also will explore new technologies for thermal and bioremediation of toxic waste and address possible health risks associated with these technologies. Rapid immunochemical analysis will supplement classical technologies for the evaluation of sites, validating models of transport from these sites, as well as determining human susceptibility, exposure and effect. The biomarkers developed in this project will serve as biological dosimeters in epidemiological and ecological studies in this and sister projects. The technologies developed in the project will be tested at field sites and transferred to end users.


This project will target the enzyme, fatty acid synthase (FAS), for the treatment of lung cancer. Our preliminary studies have found that the vast majority of non-small cell lung cancers express high levels of this enzyme compared to normal tissues. This increased expression of FAS is significant because inhibition of this enzyme in cancer cells leads to a metabolic imbalance and cellular apoptosis. In a series of in vivo experiments, we found that treatment of orthotopic xenografts of human mesothelioma cells with an agent that inhibits FAS essentially abolished the growth of established tumors. Furthermore, in preliminary experiments, we found a promising anti-tumoral response of lung cancer orthotopic xenografts (in nude rats) treated with an FAS inhibitory compound. Importantly, these treatments did not result in any recognizable damage to normal tissue but did lead to dose-limiting anorexia. The proposed studies will further develop the use of FAS inhibitory therapy for lung cancer treatment. In the first phase of our preclinical studies, we will compare several novel FAS inhibitory agents, using in vivo and in vivo experimental systems, to identify a lead compound with high level of activity against lung cancer cells and tolerable levels of toxicity/anorexia. In the second phase of the preclinical studies, we will optimize dosing protocols for the treatment of lung cancer orthotopic xenografts using this compound. This optimization of treatment protocols could be useful for designing treatment protocols to be applied in the clinical setting. The third pre-clinical aim of this project is to examine the effects of the FAS inhibitory compound when used in combination with other agents, such as those currently used to treat lung cancer or being evaluated for treatment of lung cancer. Because the FAS target represents a pathway distinct from those targeted by other compounds, there is a significant potential that such combinations could have synergistic antineoplastic activity, thus allowing reduction of doses of the respective agents. The final aim of this project is to initiate a phase I clinical trial for a compound identified by the preclinical studies to have the best potential for lung cancer treatment. Successful completion of this phase I trial and the preclinical modeling studies will provide a framework for further evaluation of an FAS inhibitory compound in the treatment of lung cancer.


People with HIV/AIDS survive to older ages, but we still do not know if or how HIV-associated neurocognitive disorders (HAND) is exacerbated in the aging brain. We will evaluate the hypotheses that brain inflammation drives an interaction between HAND and brain aging by modifying the ubiquitin proteasome system (UPS). The UPS is the main route of neuronal protein turnover;it is required for normal minute-to-minute physiological changes in synaptic potentiation, learning and memory formation. The UPS plays a key role in preventing the accumulation of misfolded neuronal proteins in pathological neurodegeneration during brain aging. We established that the UPS is abnormal in brain cortex of people with HAND: Immunoproteasomes (IPS) are induced in response to interferons. IPS induction was linked solidly with HIV-associated neurocognitive disorders (HAND). It was relevant pathologically because it was prevalent in subjects with HIV encephalitis (HIVE) and/or replicating HIV concentration in the brain. It was anatomically pervasive in critical neuronal compartments including synapses, axons and neuronal nuclei, perikarya, and isolated synaptosomes. The proposal will test the hypothesis that IPS induction perturbs the synaptic protein economy by diverting the UPS substrate repertoire pathologically, which alters the substrate repertoire, synaptic protein turnover, and synaptic plasticity in HAND and brain aging. The first aim will use human brain specimens to determine how IPS induction affects macromolecular assembly and enzymatic functions of the UPS, including the dynamic interaction with synaptic proteins. The second aim will use interferon-stimulated mixed brain cell cultures to determine how IPS induction influences synaptic protein turnover and holoenzyme kinetics, and will determine whether inhibiting the IPS reverses these changes in the synaptic protein economy. A final aim will use autopsy specimens to determine if the age-of-onset or rate-of-accumulation of misfolded proteins during pathological brain aging is perturbed in association with IPS induction. The program overall addresses a highly novel mechanism for synaptic dysfunction in HAND and has important implications for the mechanism of interaction between pathological brain aging and HIV/AIDS. PUBLIC HEALTH RELEVANCE: "The project is important because it will clearly define a novel basic mechanism regarding how HIV infection in the brain disturbs learning and memory, and how HIV worsens brain degeneration during aging. The studies will determine if changes in brain proteins that occur can be blocked, in order to stop dementia and brain aging in HIV infected people"


This project is the synthetic organic chemistry component of this program project. Our tasks is the synthesis of research quantities of lead compounds. These compounds are supplied from other projects for biological testing. Amounts ranging from 100 mg to 1 g are sufficient for confirmation of initial in vitro activity and testing in secondary systems, such as mouse mammary organ culture (MMOC). If the results are promising, short-term animal testing can also be performed with these quantities of test materials, as can mechanistic evaluations. In this context, this project is also responsible for derivatization and synthetic modification of lead compounds for structure-activity relationships (SARs), which will be used for lead optimization. In addition, project 5 will perform scale-up syntheses of lead compounds for use in full-term animal studies for the assessment of chemopreventive activity. Scale-up synthesis will involve multi-step processes and possibly stereoselective transformations for quantities ranging from 500 g to 2 kg. The goal of project 5 is the efficient and timely synthesis of compounds from the discovery part of the program to the stage of complete biological evaluation. To accomplish this goal, a flexible, competent, and responsive synthetic group has been assembled. In ongoing studies, this project will focus on large scale syntheses of sulforamate, oxomate, sulforaphane, brassinin, cyclobrassinin, 4'-bromoflavone, E-resveratrol and 4'-bromorot- 2'-enonic acid for inclusion in animal carcinogenesis models (project 3). We will also continue analog syntheses of resveratrol, rotenoids and isoflavones. Thus, in a highly coordinated manner, project 5 will contribute to the further development of lead compounds that have or will emerge from this program project.


To advance the work already performed in our laboratory with rats, fetal substantia nigra or adrenal medulla was grafted to the denervated caudate of the rhesus monkey in our continuing research on brain tissue transplantation. Although only moderately successful thus far, the experiments have demonstrated for the first time that peripheral tissue autografts can survive transplantation into nonhuman primate central nervous system.


Variola (smallpox) remains a bioterrorism threat in the 21st century. The Dryvax vaccine is too toxic for many persons. Little is known about the specific T cell response induced by intradermal Dryvax. We propose detailed studies of the epitopes and antigens recognized by vaccinia-specific CD8 and CD4 T-cells in humans. The planned antigen discovery method, expression cloning, interrogates the viral proteome with libraries of cloned vaccinia genomic DNA. We have successfully used this method with HSV-2, which has a complex genome similar to that of vaccinia. The responses of vaccinia-specific human T-cells lines and clones will be validated in quantitative measurements of immune responses using direct ex vivo assays. In Aim 1, we will derive CDS CTL lines and clones that are specific for vaccinia, and determine which vaccinia open reading frames and peptide epitopes they recognize. Epitopes will be validated in both CTL and IFN- gamma assays and dose-response curves done to document high-avidity recognition. We will then measure the diversity of the CDS response and the evolution of the response over a one year period in a subset of primary vaccinees. We predict that the immunodominant CDS antigens will be non-structural early proteins, and that immunodominance will also be controlled by HLA type and will be stable over time. CD4 T-cells provide important help for antibody and CDS responses. Therefore, in Aim 2, we will detemine the fine specificity of vaccinia-specific CD4 T-cell lines and clones using our proven molecular library method. Selected vaccinia proteins that stimulate CD4 T-cell responses will be expressed as full-length proteins in baculovirus to study CD4 T-cell immunodominance in a population of vaccinated subjects. We predict that structural proteins will be dominant and that the CD4 response will include reactivity with vaccinia proteins that are the targets of neutralizing antibodies. Several such proteins are already available as baculovirus constructs. The data will be medically useful in evaluating the immunogenicity of candidate smallpox vaccines that are being evaluated for widespread use in the case of possible variola exposure. Poxviruses expressing heterologous microbial or cancer proteins are being modified for increased immunogenicity, with the goal of prevention and therapy of other infections and malignancies. Our data will assist in evaluating the immunogenicity of these constructs.


Reactivation of BK virus (BKV) following renal transplantation (KTx) is common and can lead to BK virus- associated nephropathy (BKVN), a serious and increasing problem. Current therapeutic options are limited to tapering of immunosuppression and experimental use of cidofovir. Available evidence suggests that BKV- specific T-cell responses are important in controlling infection, and we hypothesize that viral reactivation in KTx drives the expansion of functional BK-specific cells which control BKV in the majority of patients, but in a minority, this immune response is absent or dysfunctional, which may predispose to progression to BKVN. If true, functional BKV-specific T-cells could represent correlates of protection against BKVN. We will test this hypothesis by using state-of-the-art flow cytometry methodologies to track and characterize BKV-specific T-cell responses in two cohorts of KTx patients at UCLA Medical Center. The first cohort will comprise 40 Ktx recipients who will be prospectively followed beginning 1 month and until 1 year post- transplant. A second cohort will consist of KTx patients with clinically confirmed BKVN and enrolled at the time of diagnosis. The patients will be monitored by plasma PCR for BKV viral load. Global analyses of immunological responses to BKV will consist of flow-based intracellular cytokine assays employing as antigens overlapping peptide libraries spanning the majority of the viral proteome. These will be supplemented by more sensitive immunological probes to a panel of immunodominant BKV T-cell epitopes we have previously identified. The functionality of these BKV-specific CD4 and CDS T-cells in terms of cytolytic potential and production of different cytokines will be characterized and correlated with viral load and clinical status to see if these cells represent markers for BKV reactivation and/or protection against BKVN. Immunological probes to detect these cells may form the basis for tests to guide clinical management. Relevance: With the increasing problem of Type II diabetes, the number of kidney transplants being performed within the US is rising. Although BK virus is ubiquitous in human populations and establishes a life-long infection, it is only life-threatening in the context of a minority of immunocompromised KTx recipients. Information from the proposed study may allow identification of patients at risk of progression from BKV reactivation to clinical BKV disease and guide therapeutic interventions. [unreadable] [unreadable] [unreadable]