This proposal has been targeted at two areas of catalytic antibody research: The obtainment of new antibody catalysts and the development of new strategies for obtaining improved antibody catalysts, including antibodies with altered specificities and chemical reactivities. The development of new antibody catalysts will center around a hapten design strategy which we introduced and termed "bait and switch" catalysis. We will apply this strategy for our investigation of antibodies which can catalyze cationic cyclization processes. We will explore two types of cationic cyclization reactions. The first involves antibody catalyzed olefin cyclization processes and thus the formation of carbon-carbon bonds. To augment these studies, we intend to also explore the possibility of utilizing antibody catalyzed cationic cyclization reactions to form carbon-heteroatom bonds. A long-term goal of these two projects is to channel this type of antibody catalysis for the formation of unique steroid and unusual heterocyclic molecules. An equally important reaction in the formation of carbon-carbon bonds is the Aldol condensation. Using our "bait and switch" approach, we plan to investigate bimolecular antibody catalysis for an intermolecular Aldol reaction. We will engage a multisubstrate "bait and switch" tact for the induction of amino acid residues in an antibody's combining site to act both as a Lewis acid catalyst and, to provide additional binding forces to overcome entropic loss which is seen when two substrates are brought together. An advantage of such catalytic antibodies would be the avoidance of environmentally damaging Lewis acids which are commonly employed in Aldol reactions. Hybridoma methodology has been used exclusively in the obtainment of catalytic antibodies. However, this process of isolating catalytic antibodies suffers from some limitations. Of special concern is the ability to generate a large enough array of monoclonal antibodies to screen for the desired activity. We intend to apply antibody phage display semisynthetic combinatorial libraries in conjunction with panning methodologies which are designed to select for antibody chemical catalysis. To complement these studies, we will also utilize antibody phage display methodologies to examine the potential of altering a catalytic antibody's specificity and chemical reactivity. This arsenal of antibody phage display studies which we have selected to investigate should provide us with a means to access more sophisticated modes of antibody catalysis.


The inflammatory bowel diseases (IBD), ulcerative colitis and Crohn's disease, are histologically characterized by an accumulation of lymphocytes and plasma cells in the lamina propria and foci of polymorphonuclear lymphocytes. An immune mechanism has long been suspected in the pathogenesis of IBD but, the etiology remains unknown. It is also not clear whether alterations in lymphocyte populations or their function mediate the mucosal injury or are epiphenomena of inflammation. Current research efforts investigating the functional properties of intestinal mucosal lymphocyte subpopulations, especially the intraepithelial lymphocytes (IEL), have been hampered by the inability to specifically identify IEL in cell suspension after isolation from intestinal specimens. The major goal of this project is to define possible phenotypic and functional differences in the mucosal lymphocyte subpopulations in IBD and normal intestine. This will be accomplished by using recently developed monoclonal antibodies that are primarily restricted to mucosal lymphocytes and IEL in particular. Monoclonal antibodies that recognize the T cell receptor gamma/delta will also be used to determine the intestinal distribution of these cells in IBD and their functional characteristics. A difference in the distribution or function of lymphocyte subpopulations in the lamina propria or epithelial compartments, may play a role in the pathogenesis of IBD.


The Duke Department of Pediatrics'training program is based on developing pediatric Junior Faculty into physician-scientists who are skilled in cutting-edge methods of laboratory research and who will pursue independent academic careers investigating important issues related to childhood diseases. Our program is based on our pool of outstanding candidates, a strong curriculum of didactic courses, experienced mentors who perform state-of-the-art research, and an excellent research environment. Our commitment to develop future academic pediatricians is evidenced by the academic success of our Junior Faculty. Our Principal Investigator is Dr. Joseph St. Geme, III, Chairman of the Department who is assisted by Dr. Page Anderson, Program Director, Dr. Delbert Wigfall, Minority Recruitment Advisor, and an Internal and External Advisory Committee. Four young scholars will be supported each year. They will be drawn primarily from our fellows and Junior Faculty. Recruitment of underrepresented minorities is a focus of the program. Our faculty, which includes many mentors from other Departments, has strong track records in research, funding, and mentoring. Our research training centers are in the areas of Developmental Biology, Cell Biology and Cell Signaling, Microbiology and Immunology, and Genetics, Genomics, and Proteomics. Didactic courses, including a multi-lecture course on writing, will complement the laboratory research experiences, enabling the trainees to write and submit grant applications to support their career. The trainees will be required to take a five lecture course in Responsible Conduct of Research. The young Scholars will have access to all the research resources of the NCI-funded Comprehensive Cancer Center, the shared facilities at Duke University, the Institute for Genome Sciences and Policy, and the Center for Human Genetics.


Our short-term aim is the development of algorithms for locating, enhancing, and recognizing selected classes of objects in radiographic images. Our long-term aim is the development of a man-machine technology capable of storing, analyzing and retrieving radiographic pictures in greater numbers, with greater accuracy and reliability, and at lower costs than ever before. We also wish to establish a research environment where medical and engineering researchers can work effectively together on the automation of image analysis. To achieve these objectives we will build upon the results of our research over the past several years on computer-aided image processing, pattern recognition, and trainable classifiers. Our plan of research consists of three concurrent branches all three of which are major forces for the success of the project: 1) basic theories and techniques, 2) medical applications, with special emphasis on radiology, and 3) the picture-processing computer facility. It is expected that the techniques and insights developed in this project will be useful not only in radiology -- which receives the major emphasis here -- but also in a wide class of clinical and research activities involving the analysis of images.


It is the hypothesis of this grant application that the pathogenesis of rheumatoid arthritis (RA) depends not upon the absolute numbers of T cells in the blood or the joints, but rather upon the relative percentages of auto-antigen specific T cells that produce and respond to pro-inflammatory and anti-inflammatory signals, including cytokines and chemokines. These T cell populations may be the target for therapeutic intervention. In preliminary experiments that prompted this grant application, we have shown that: i) peptides of bacterial origin (E. coli dnaJ heat shock protein) are strong immunogens in RA patients. dnaJ shares with HLA DRB1*0401 the susceptibility sequence to RA (shared epitope); ii) T cells in the blood RA patients that react with the shared epitope can be identified and isolated, using a novel technical approach; iii) T cell receptors usage and patterns of cytokine production by these shared epitope specific T cells can be analyzed. Based upon these initial results, we now need to test our hypothesis in a larger cohort of RA patients with different disease courses, and responses to treatment. Thus, the aims of this project are: 1. To characterize frequencies, function and phenotypes of T cells in RA patients reactive with the shared epitope. Parameters to be studied will include cytokine production, T cell receptor usage and membrane markers of activation and memory. Chemokine receptors will also be studied. 2. To determine the effects of disease activity and severity, and of slow acting anti rheumatic drugs, on function and phenotype of the shared epitope specific and bystander T cells. 3. To develop methods to modulate the Th1-type phenotype of shared epitope specific T cells, using in vitro immune manipulation with altered peptides. The long term objectives of this project are: i) to contribute to a better understanding of T cell-mediated events in the pathogenesis of rheumatoid arthritis; (ii) to develop paradigms for the prediction of disease outcome, and for evaluation rapidly new models of therapy.


Peripheral neuromodulation has been pursued to control conditions such as chronic pain and dysfunctions of pelvic organs. The expansion of such therapies is challenged by incomplete understanding of peripheral neural circuits, which comprise complex networks of peripheral ganglia and nerves that innervate multiple organs and carry axons of afferent as well as efferent peripheral neurons. Methods for manipulation of neural activity through genetically engineered receptors and channels such as channelrhodopsins and DREADDs (Designer Receptor Exclusively Activated by Designed Drug) create an opportunity for the development of tools for targeted neuromodulation through viral vector-mediated cell-specific gene transfer. The use of cell-specific promoters and combinatorial vector systems for targeted expression of optogenetic or pharmacogenetic transgenes presents a potential solution to the neuroanatomical challenges of peripheral neuromodulation. The long-term objective of this research program is to develop strategies for peripheral neuromodulation through cell-specific delivery of neuromodulatory transgenes to peripheral ganglia using adeno-associated viral (AAV) vectors. Based on our work on the biodistribution of AAV vectors, the objective of this application is to provide proof-of-concept fo site-specific targeting of transgene expression to afferent and efferent systems. We will address the central hypothesis that cell-specific targeting of a neuromodulatory transgene to peripheral neurons by AAV vectors enables organ-specific functional interventions. To address the Specific Aims of the project, we will develop AAV vectors for cell-specific targeting of DREADD, validate neuroanatomically their restricted biodistribution, and demonstrate the neuromodulatory function of the transgenes using behavioral, in vitro and in vivo neurophysiological, and in vivo imaging analyses. Aim 1: Test the feasibility of a strategy for organ-specific neuromodulation of afferent systems through AAV-mediated gene transfer. We will test the hypothesis that combinatorial AAV vector targeting of colon-innervating sensory neurons, using a Cre- dependent vector that carries the gene for the inhibitory DREADD (hM4Di) and an AAV-Cre vector, will enable organ-specific control of afferent activity. Aim 2: Test the feasibility of a strategy for organ-specific neuromodulation of efferent autonomic systems through AAV-mediated gene transfer. We will test the hypothesis that cell-specific AAV vector targeting to parasympathetic bladder post-ganglionic neurons, using a vector that carries the gene for the excitatory DREADD (hM3Dq) under the control of the cholineacetyl transferase (ChaT) promoter, will promote bladder emptying in a rodent model of spinal cord injury. The proposed strategies and vector tools will enable functional dissection of peripheral neural circuits, applicable to multiple organs and across species and animal models. These approaches can be extended to optogenetic neuromodulation, to integration with closed-loop platforms, and potentially to development of next generation neuromodulation therapies as the clinical translation of AAV-mediated gene transfer advances.


The Administrative Core will facilitate and coordinate communications between the members of the Consortium themselves and between them and the 4 members of the Scientific Advisory Board (SAB), all of whom will be Consultants to the Consortium. The major thrust of the proposals made in this application is to investigate and hopefully resolve the problems associated with coagulation dysregulation following organ xenotransplantation in pig-to-primate models. Frequent communication, most likely in the form of conference calls, is likely to be required between members of the Consortium and Consultants, all of whom are experts in the field of xenotransplantation and/or coagulation. The Core will help with travel arrangements and hotel accommodation for Consultants visiting Pittsburgh for the annual workshops at which the results of the Consortium's efforts will be discussed and plans made for the future 12-month period. The Core will also arrange travel and hotels for the members of the Consortium who will be participating in the annual meeting with other scientists funded through this RFA. The immunologic and coagulation assays to be used in the studies in Projects 1 and 2 will, as far as possible, be standardized between the two centers, and this will require exchange of samples, tissues, and reagents. This will be organized and coordinated through the Administrative Core. Data from the various experiments and assays will be collected and collated in the Administrative Core. The Core will also be responsible for assisting with the preparation of manuscripts reporting the scientific work of the consortium to be submitted for publication. The Core will do what it can to ensure timely reporting in peer-reviewed journals of the results obtained by the Consortium. It is the Consortium's plan to invite other scientists who are working in this field to the annual workshops that will take place in Pittsburgh, even if those scientists are neither Consultants to the Consortium nor funded by the RFA (although these scientists will have to fund their own travel expenses). The members of the Consortium believe that it is only through full collaboration and exchange of ideas and information that the problems of xenotransplantation will be resolved in a timely manner. The Administrative Core will provide assistance to visiting scientists with regard to reserving hotel accommodation, etc.


DESCRIPTION: (Applicant's abstract) The recently-determined sequence of the genome of bakers' yeast (S. cerevisiae) revealed that almost two-thirds of the genes of this well-studied organism had escaped detection. Thus, we know almost nothing about the function of most of the genes that comprise this simple eukaryotic cell. To catalyze approaching the ultimate goal of a comprehensive understanding of the function of all the proteins that constitute a eukaryotic cell, we propose to construct quickly the complete set of mutants deleted for each of the approximately 6000 genes of this important model organism. It seems certain that wide distribution of this collection of mutants will greatly accelerate the pace of discovery of gene function. This goal will be achieved by a consortium of 5 laboratories, each disrupting 300 genes in each of two year, yielding 3000 mutants. The other 3000 genes of the organism will be disrupted by our European and Canadian colleagues. The mutants will be screened for a few standard phenotypes to provide necessary information for the users of the mutant collection, then distributed widely to all interested scientists for more detailed and complete analysis. We propose to replace the coding sequence of each gene with the KanMx gene encoding resistance to the drug gentamycin. In addition, a novel sequence tag will be introduced into each of the mutants to uniquely identify them. Non-essential genes will be disrupted in haploids of both mating types; essential and nearly essential genes will be disrupted in diploids. Gene disruption will be accomplished using a facile and popular PCR-based technique that will enable construction of the complete set of mutants in two years. In the final year of the project, we will enhance the value of the mutant collection by constructing conditionally-expressed versions of essential and nearly essential genes, constructing approximately 750 double mutants deleted for both members of a duplicated gene pair, and disrupting small genes as they are revealed.


Abstract A key problem in chronic infections is progressive dysfunction of the infected organ. Importantly, patients with similar risk factors often exhibit marked variation in the rate of disease progression, as do individual patients at different times. The chronic Pseudomonas aeruginosa (Pa) lung infections in people with cystic fibrosis (CF) are a prime example. Pronounced variability in the rate of lung function decline is seen across patients even when host CFTR genotype and airway microbiology are similar. While both bacterial and host factors likely contribute to disease variation, several new findings lead us to focus on genetic variation that evolves in infecting Pa. First, CF Pa strains have been found to genetically diversify in vivo, producing clonally-related variants that differ markedly in virulence, and these variants can co-exist simultaneously inside lungs. Second, our data show that the distribution of genetic variants changes over time and the emergence of highly virulent genetic variants can be temporally associated with changes in disease. Finally, many groups have investigated host genetic and environmental factors that affect disease, but the effects of bacterial genetic variants on disease progression are relatively unexplored Here we exploit unique clinical and technical resources, and our knowledge of Pa pathogenesis to test the hypothesis that some gene variants that evolve in the Pa populations infecting CF patients increase lung injury. We will test this hypothesis in three steps. First, will we measure Pa gene variants in sputum collected at specific time points based on the lung disease phenotypes. Second, we will perform genetic association analysis to delineate which variants/variant groups are most strongly associated with the phenotypes. Third, we will investigate the functional effects of gene variants on Pa virulence, host injury, and inflammation. This work could identify potential cause and effect relationships and provide proof of principle for a disease progression mechanism that may operate in many chronic infections.


The goal of this research is to understand how the nervous system encodes information about odors. The sense of smell presents unique problems to the nervous system in terms of stimulus detection, neural encoding and recognition of complex stimuli; understanding how the brain solves these problems will likely lead to new general insights into how the brain processes information. The initial code for odors consists of patterns of activity across olfactory receptor neurons, which project to glomeruli in the olfactory bulb, the first stage of synaptic processing of olfactory information. Here, patterns of activity across thousands of receptor neurons are transformed into spatially organized maps of glomerular activation - these glomerular patterns are unique for a given odor and odor concentration. The research in this continuing project uses optical imaging methods to visualize these patterns and to investigate how they represent olfactory stimuli. Previous work built on the observation that spatial maps of receptor input to glomeruli are temporally dynamic, and asked how these dynamics participate in odor coding and shape the initial stages of olfactory processing. A key finding was that much of the temporal dynamics of odor maps are organized around the respiratory cycle, which is heavily modulated in the awake, behaving animal and is integral to the act of smelling. The experiments proposed here will investigate, for the first time, how odor sampling behavior (i.e. - `sniffing') shapes early olfactory coding at the level of the olfactory bulb and the transformation of receptor inputs into patterns of postsynaptic activity. The experiments will image receptor input to olfactory bulb glomeruli while an animal is awake and actively performing odor-guided tasks, and relate these patterns to the animal's sniffing behavior. Different patterns of sniffing will also be played back in the anesthetized animal in order to separately evaluate effects of sampling behavior and effects of behavioral state-dependent modulation of receptor inputs. The experiments will also ask how sniffing shapes the transformation of odor representations by the olfactory bulb, using electrophysiological recordings from individual olfactory bulb projection neurons during activation by odorants sampled with different sniffing patterns. In addition to testing, for the first time, several longstanding hypotheses about the role of sampling behavior in shaping odor codes, this work will be important in understanding how olfactory information is encoded and processed in the awake, behaving animal. This work has the potential to lead to new treatments for olfactory deficits or therapeutic approaches to improving odor or flavor perception in individuals with impaired nasal function. PUBLIC HEALTH RELEVANCE: The sense of smell presents unique problems to the nervous system in terms of stimulus detection, neural encoding and recognition of complex stimuli; understanding how the brain solves these problems will likely lead to new general insights into how the brain processes information. This project in particular focuses on the importance of respiratory behavior in determining how the brain represents and processes olfactory information. Understanding this relationship could lead to new treatments for olfactory deficits or therapeutic approaches to improving odor or flavor perception in individuals with impaired nasal function.


PRAM-1 is a phase-II, randomized, open-label, multicenter, multi-arm protocol. HIV-infected, clinically stable children, 24 months to 17 years of age, treated with the same antiretroviral therapy for >16 weeks, will be randomized to one of the three following treatment arms: 1) Zidovudine (ZDV) plus Lamivudine (3TC); or 2) Stavudine (d4T) plus Ritonavir; or 3) ZDV plus 3TC plus Ritonavir. Subjects will be stratified by CD4% <15% or >15%. The first eight subjects randomized to the d4T+Ritonavir and ZDV+3TC+Ritonavir arms will participate in a real-time Phase-I pharmacokinetics study (16 subjects in total). The three arms will be evaluated primarily with respect to change in plasma HIV-1 RNA copy number from baseline to 48 weeks and with respect to safety and tolerance.


Dementia is the most common neurological disease in the elderiy. Experimental, epidemiological, and neuropathological evidence suggest associations between vascular factors and cognitive decline and dementia regardless of subtype, with vascular pathology present in half of all dementia cases. However, the mechanisms which underiy these associations are a topic of debate. While large vessel cerebrovascular disease and cortical stroke have been linked to cognitive decline and dementia, the specific role of cerebral small vessel disease is unclear. Few population based studies have assessed cerebral microvascular structure and function in relation to cognitive decline and dementia, due in part to the difficulty of applying specialized and costly imaging techniques in large population based samples. We propose to apply two non[unreadable][unreadable] invasive, relatively low cost techniques for assessing microvascular status (with retinal phootography) and function (transcranisal Doppler ultrasound (TCD) with C02 challenge) to test the hypothesis that cerebral hypoperfusion and microvascular disease precede the onset of dementia and contribute to cognitive decline. Because retinal and cerebral arterioles have similar anatomy, physiology and embryology, retinal characteristics assessed by standardized grading of fundus photographs provides a surrogate marker for cerebral microvascular status. Similarly, TCD with C02 challenge is a safe non-invasive technique for assessing cerebral vascular function. Recently, these techniques have been studied in relation to dementia, although the majority of studies have been limited by cross-sectional design, restriction to clinical AD cases, or limited neuropsychological data. Little is known of their associations to eariy cognitive decline and preclinical dementia. Applying these techniques in the EAS will allow us to assess whether retinal microvascular status and cerebrovascular function predict cognitive decline and dementia, with a focus on eariy cognitive changes. To elucidate mechanisms that may underly these associations, we will evaluate whether they mediate associations of vascular risk factors with cognition and will assess the relation of retinal and TCD measures to neuroimaging measures of structual brain changes. RELEVANCE (See instructions): Much is known regarding the prevention and treatment of vascular disease. Better understanding the role of vascular status and function in dementia will direct the application of these strategies toward the prevention of this prevalent condition.


The purposes of this study are to collect data on severe headache in order to measure the prevalence and to describe the demographic characteristics of the major types of headache. To this end a survey of the general population has been designed. A survey questionnaire, which includes sections on demography, descriptive headache features, medical information, and history, has been developed. The data will also be used to identify and assess the etiological and environmental factors associated with the major idiopathic headache types. The study was designed in two parts: a feasibility study and an area survey. The feasibility study has been completed. Telephone interviews have been conducted with the patients from four headache clinics. The questionnaire data have been processed together with information abstracted from the physician records about the headaches. The planning and design of the area survey has been completed. The area survey will not be funded and this study is thereby completed.


: The Children's Computerized Physical Activity Reporter: Reliability and Validity, PI: Patricia Flannery Pearce, MPH, PhD, RN, APRN, FNP Assistant Professor University of Utah, College of Nursing Salt Lake City, Utah Purpose: The purpose of this descriptive, comparative study will be to: (a) evaluate the test- retest reliability and (b) concurrent validity of an innovative, computer-based self-report questionnaire, the Children's Computerized Physical Activity Reporter (C-CPAR), compared with a commonly used paper-based questionnaire and objective criterion of accelerometry. Background: Physical inactivity is linked to obesity, metabolic syndrome, and early cardiovascular risk. There are global directives to better understand children's physical activity, but physical activity measurement is difficult, especially with self-report. Currently available self-report questionnaires provide adequate reliability, but less than robust validity. The inconsistent validity of currently available physical activity questionnaires may be due the lack of attention to matching the questionnaire content and format to children's understandings of the underlying constructs, or to the form and structure that children need for precise recall. The C-CPAR was developed with children, for children, in a study aimed at identifying children's understanding of physical activity, their needs for reporting, and their perspectives on currently available questionnaires. Usability and feasibility of the C-CPAR were excellent in the original study. In a pilot feasibility study (N=31), with the National Youth Sports Program (NYSP), comparison of MET minutes of NYSP camp activity reported for two 1-day reports demonstrated strong reliability (r=0.64, p<.0001). Average MET level for scheduled NYSP activities and C-CPAR reported activities for the NYSP time period, correlations were significant, ranging between r=0.66 (p<.01 Day 1) and r=0.37 (p<.01 Day 2). Comparisons of total NYSP activity reports to crude pedometer counts were not significant (r=0.33).77 Time to completion for the C-CPAR was significantly faster on Day 2 of the study. A Spanish language alternative is being developed. However, C-CPAR reliability and validity must be assessed further, to determine the relevance of the C-CPAR for clinical or research venues. Participants: Participants will be 180 junior-high school students from a single, ethnically-diverse public junior high school using, selected with stratified random sampling (grade and gender). Methods: Children will be randomly assigned to two groups in a crossover design to control for questionnaire sequencing effect. Participants will wear an Actical(r) accelerometer for three days (Friday to Monday), complete one self-report (C-CPAR or PD-PAR) on each of the two following consecutive days, followed by crossover to the alternate report daily for two subsequent days. Analyses will include Pearson correlations for test-retest reliability and validity, Bland Altman plots to evaluate associations between measures, and differences in correlations for test of accuracy. Differences in age, gender, and ethnicity will be evaluated. Implications: The C-CPAR may provide the format and content that matches children's understanding and needs for accurately reporting their activities. If reliability and validity are found adequate, the C-CPAR potentially can be used to assess children's activities in clinical and research venues. Improved measurement will translate to improved understanding and provide a stronger foundation for intervention. The long-term goal of this program of research is to develop intervention studies, specifically contributing to knowledge of physical activity and its behavioral components, while maximizing the use of technology. Future research will focus on the utility of the C-CPAR for therapeutic application in research and practice. The World Health Organization, Healthy People 2010, and the Surgeon General encourage the accumulation of at least 30-60 minutes of moderate activity daily for all age groups. Promotion of physical activity and counseling about its importance should be a priority for all health care professionals. Yet there is an increasing trend of inactivity well documented. To understand physical activity in children, measurement is essential. Many of the measures of activity for children are modifications of instruments originally developed for adults, but the study of physical activity of children is complicated by developmental differences, vocabulary, and unique activities in a rapidly evolving technologic environment. Currently available self-report questionnaires are inadequate for children's reporting. The C-CPAR was designed with children, for children, to support children's reporting. The current proposal is designed to examine the reliability and validity of the C-CPAR, and expand knowledge regarding children's physical activity and its self-report measurement. A valid self-report tool will aid researchers and clinicians in understanding physical activity. More in-depth understanding will serve as a strong foundation for interventions. The long- term goal of this program of research is to develop intervention studies, contributing to knowledge of physical activity and its behavioral components, while maximizing the use of technology. Future research will focus on the utility of the C-CPAR for therapeutic application in research and practice. [unreadable] [unreadable] [unreadable]


The ribonucleases of human pancreas have been less completely characterized than similar enzymes of other organisms. This project is concerned with the isolation and biochemical characterization of human pancreatic ribonucleases, and with the definition of the relationship between the human pancreatic enzyme and ribonucleases found in other human tissues, in serum, and in urine. In addition to enzyme characterization, a radioimmunoassay procedure will be developed to quantitate the pancreatic protein in various tissues, and to estimate cross-reactivity of structurally related RNases in other organs and organisms. Radioimmunoassay procedures will also be used to quantitate nuclease of pancreatic origin in serum of individuals suffering from a variety of diseases, particularly pancreatic carcinoma; this could permit development of a technique for early diagnosis of this malignancy.


Latina women living in the U.S. are twice as likely to be diagnosed with cervical cancer and 40% more likely to die from the disease than non-Latina whites. Lower utilization of Pap smear contributes to greater cervical cancer mortality among Latinas, with recently-immigrated, uninsured women from Mexico at greatest risk of inadequate Pap smear utilization. To reduce this disparity, we propose to: 1. Increase routine Pap smear screening rates among uninsured Latina women living in the southeast using an empowerment-based, communication skills-building intervention. 2. Explore changes in knowledge, barriers, self-efficacy, and shared-decision making after the intervention and evaluate the impact of these factors on Pap smear screening rates. This pilot study uses a quasi-experimental community trial design to address these aims. The proposed community-based intervention uses a lay health advisor model to educate women about cervical cancer etiology and preventive strategies and to empower them with communication skills that will help them negotiate the medical encounter. Our research has several advantages beyond what has been documented to date: (1) The research is targeted to women in need of a Pap smear and who are at highest risk of not having one ; (2) The intervention combines education AND empowerment, a considerable advantage over previous research that has focused exclusively on increasing knowledge. (3) Empowerment, through skills- building, is grounded in theory and is culturally-appropriate; (4) The quasi-experimental community-trial design allows us to move beyond feasibility and actually test the effect of the intervention at the community- level; (5) The intervention will be easily replicated and disseminated given the size and scope of the project; (6) To date, no such interventions have been rigorously tested among newly arrived uninsured Latinas in the southeast. Given that the southeast has the fastest growing Mexican immigrant population in the U.S. and health systems are just beginning to respond to their health needs, this research study is strongly justified. Our research aims address NCI's 2006 strategic investment cancer research to "overcome the unequal burden of cancer experienced by various population groups by ....developing effective interventions to reduce those disparities, and facilitating intervention delivery." [unreadable] [unreadable] [unreadable]


The repair of extensive bone injuries remains an unmet clinical challenge. By developing two new and complementary models of large-scale bone regeneration in zebrafish and mouse, we aim to understand the role of the cartilage callus in generating large segments of full thickness bone. While the periosteum generates osteoblasts during homeostasis, the specific subpopulation that builds the repair callus remains poorly defined. Using transgenic lineage tracing, we provide compelling preliminary evidence that a rare bi-potent Sox9+/Runx2+ periosteal population generates new cartilage and bone during repair. This newfound ability to label, manipulate, and isolate a specific stem cell population allows us to test whether the remarkable regenerative capacity of the rib is due to the unique properties of its periosteal stem cells. In Aim 1, we team up with an orthopaedic surgeon to test that Sox9+ cells from the rib periosteum can be expanded in culture and used to heal a critical-sized femoral defect. The formation of a cartilage callus is a common feature in bone repair, yet how the periosteum generates cartilage only during repair remains a mystery. In preliminary data, we find that the cartilage callus is severely compromised when either the Ihha ligand is deleted in zebrafish or the Hh receptor Smo is deleted from Sox9+ cells in mice. In Aim 2, we test that this reflects a repair-specific role for Ihh, which is markedly different from its developmental role in osteoblast differentiation and chondrocyte proliferation. Further, our preliminary data suggest that these Ihh-induced repair chondrocytes differ in important ways from those in the growth plate since repair chondrocytes co-express osteoblast genes even at pre-hypertrophic stages. This increase in osteogenic character subsequently correlates with a conversion of chondrocytes into osteocytes. In Aim 3, we test whether repair and developmental chondrocytes represent distinct cell types by comparing global gene expression at different stages of maturation. We also use lineage tracing to quantitate the extent to which cartilage-derived osteocytes preferentially contribute to full thickness bone during repair. Lastly, we use powerful new chromatin accessibility assays to test that as Sox9+ periosteal cells become cartilage, their greater osteogenic character results from the maintenance of poised osteoblast enhancers. Our findings will reveal how a rare population of periosteal stem cells can be induced to make cartilage during injury, and how this specialized repair cartilage can be used to regenerate full thickness bone. A better understanding of these important stem cells will form the basis of future pre- clinical trials aimed at healing large-scale skeletal lesions in other parts of the body.


Chronic ethanol consumption results in microsome proliferation and an increase in the level of a cytochrome P-450 (P-450alc). The relative of this pathway and the alcohol dehydrogenase (ADH) pathway in ethanol metabolism in the liver is subject to debate. The ADH-negative and ADH-positive deermouse stocks have suggested that a substantial amount of ethanol metabolism may occur by non-ADH mediated pathways. The P-450alc has been purified from the deermouse and used to raise antibodies which are monospecific as judged by Western blotting. Antibodies to P-450alc inhibit ethanol metabolism in vivo. These antibodies will be used to identify cloned cDNA for P-450alc in a Lambdagtll expression lkibrary. The P-450alc cDNA will be sequenced. The mechanism of induction of P-450alc will be studied by measuring P450-alc synthesis rates in control and ethanol-fed animals using radiolabeling and specific immunoprecipitation techniques. Levels of P-450alc mRNA in control and ethanol-fed ADH-positive and ADH-negative deermice will be measured. An analysis of P-450alc mRNA levels in baboons and humans will be started. Investigation of possible genetic variation in P-450alc induction in many representative inbred mice will begin as will a survey of P-450alc gene structure in inbred mice. With P-450alc cloned DNA, the possibility exists to study variation in this gene and surrounding sequences in human alcoholic and non-alcoholic populations to identify possible genetic variation which may be of significance in controlling the expression or structure of this physiologically important protein. An analysis of DNA sequences upstream, where interesting regulatory sequences reside, from the structural gene will require the isolation of a genomic clone. This should be made readily feasible by the availability of the cDNA for P-450alc.


The expression of a region of the bacteriophage T4 genome is being studied as a model for examining developmental gene regulation. Throughout infection, T4 uses the host transcriptional apparatus to transcribe its DNA, but with time different regions of the T4 genome are transcribed. To determine signals and factors that regulate this transcription, the expression of the T4 genes uvsX (recombination protein), 40 (stimulates head formation), and 41 (primase-helicase component) have been examined by nuclease Sl protection experiments and Northern analyses. Changes in the transcription pattern of the uvsX-40-41 region are programmed with phage development. Early in infection a heterogeneous population of transcripts is observed, having major 5' starts about 900 and 200 bases upstream of uvsX. The bulk of these RNAs continue through 40 and into 41, but a portion (about 1/4) stop just downstream of uvsX (within gene 40). Later in infection a 5' start closer to uvsX (about 50 bases upstream) is observed, in addition to the ends seen earlier, and a significant fraction of the uvsX RNA ends at the stop. Thus, as infection proceeds, the level of 40, 41 mRNA decreases relative to that of uvsX. Analysis of uvsX-40-41 transcripts expressed by a plasmid with this region reveals different 5' ends from those after T4 infection, but significant utilization of the RNA stop downstream of uvsX. Thus, T4 infection is needed for the 5' starts, while the host can produce the RNA stop. To help determine what host factor(s) are needed for this stop, transcripts were examined after infection of the E. coli transcription termination (rho) mutant, rho026. Previous work has suggested that in rho026, transcription termination may be enhanced, resulting in less expression of regions downstream of a rho-dependent termination site (Stitt et al. (1980) J. Virol. 35, 775). My transcription mapping indicates that infection of rho026 results in several-fold less 40,41 mRNA than infection of the rho+ parent, because of a 2.5-fold greater use of the RNA stop and less transcription overall. Thus, rho factor either acts to terminate transcription at the stop or influences other factors needed for the regulation of this site.


Summary Hypercholesterinemia-mediated cardiovascular diseases can be treated by statins. However, there are many instances of unresponsiveness and intolerance. Thus, there is urgent need for new approaches to lower plasma LDL, preferably that act synergistically with statins. Blocking liver VLDL, the precursor of LDL, secretion has long been recognized as an effective alternative to lower LDL in the circulation. However, this can have unwanted consequences, such as lipid accumulation in the liver. Therefore, targeting VLDL secretion without causing lipid accumulation is challenging but has great promise. Preliminary studies done for this proposal have revealed an important role of liver phospholipid transfer protein (PLTP) in regulating VLDL secretion, and thereby plasma VLDL/LDL levels. However, the mechanism involved is still not quite clear. The establishment of liver-specific PLTP expression (not overexpression)/apoB100-only mice gives us a unique opportunity to evaluate whether liver-generated PLTP plays a critical role in human-like VLDL production. Our working hypotheses are 1) PLTP plays a major role in intracellular VLDL lipidation, transport, and post-translational degradation, thus promotin VLDL production; 2). Cellular PLTP activity can be tightly regulated by some factors, furin (PCSK3) is one of them. We have two specific aims. Aim 1: To investigate the effect of liver-specific PLTP activity on human-like VLDL production. We will attempt to answer the following questions: 1) Is PLTP activity required for the VLDL lipidation in hepatocytes? 2) Is PLTP activity required for VLDL transport vesicle (VTV) formation? And 3) Does PLTP activity prevent VLDL degradation in hepatocytes. Aim 2: To investigate the impact of furin-mediated PLTP regulation on VLDL production. We will characterize the interaction between PLTP and furin, in particular its pro-segment (profurin) in vivo, and determine the effect of such interaction on live VLDL production. We will also investigate why profurin/PLTP-mediated VLDL less production does not cause lipid accumulation in the liver. We anticipate that this project will broaden our understanding of both PLTP and furin biology and provide evidence for a novel strategy for lowering plasma VLDL/LDL levels that will be effective in humans.


Retrospective dietary information of high validity and reproducibility is critical for use in case control studies of the relationship of diet to cancer, cardiovascular and other chronic disease and disability. A cohort of Tecumseh residents completed interviews on frequency of food use and 24-hour dietary recall in 1967-69 as part of a general epidemiologic survey. Approximately 1700 members of the cohort aged 35-64 will be re-interviewed in 1981-82 about their current diet, recollections of past diet and perceptions of dietary change, and the results will be compared to past reports. Within-person comparisons will eliminate the component of variance between persons present in the comparison of matched controls. Characteristics of respondents which influence reproducibilty of historical dietary reports will be investigated. Sensitivity and specificity of the retrospective categorization according to food usage, nutrient intake and dietary practices of interest in the etiology of chronic disease and disability will be determined. Multivariate statistical methods will be used to evaluate quantitative changes, changes in dietary patterns, and reproducibility of retrospective dietary reports within the individual. Recommendations will be made for the design and use of a new form of retrospective dietary instrument which will be based on findings validated by measurements in the same individuals over 13 years.


The present proposal outlines a number of specific projects designed to elucidate significant unanswered questions in the shikimic acid biosynthetic pathway. Synthetic approaches to shikimate-3-phosphate, 5-enolpyruvylshikimate-3-phosphate, chorismic acid, isochorismic acid and bacilysin are described. Mechanistic possibilities from some of the enzymatic transformations of the above metabolites are presented.


Innovative and culturally appropriate multilevel health communications interventions are desperately needed to address the chronic disease epidemic in high-risk populations, such as low-income urban African Americans (AA). However, the vast majority of communications strategies have focused on educating individual consumers about healthy food choices, while in poor urban settings the lower availability of affordable healthy food choices greatly limits the impact of these messages. We will work with 3 wholesalers and 24 small retail food stores to develop and test novel strategies in Baltimore, Maryland, including: 1) multilevel health communications alone directed at wholesalers, retailers and low-income AA consumers intended to enhance willingness to stock and/or purchase healthy foods; 2) pricing strategies (performance based allowances) directed at wholesalers and retailers to increase their stocking of healthy foods at reduced prices; and 3) combined health communications and pricing strategies. Intervention strategies will be tailored to meet the needs of the target populations based on formative research and stakeholder input. Our proposed work is based on significant field experience in this setting, including the development of evaluation tools to assess change in stocking and pricing of key foods (at the store level), and psychosocial factors, dietary intake, and food purchasing behaviors (at the consumer level). Our study has the following aims: 1) Conduct formative research with representatives of multiple levels of the Baltimore food environment (i.e., local wholesalers and retail food store owners) in order to select key foods for promotion, determine appropriate communication strategies (e.g., messages, channels, materials) for each level, and select the most appropriate pricing approach (i.e., performance based allowance structure and stipulations). 2) Pilot the multi-level program with three wholesalers and 24 food stores (6 control, 6 health communications only, 6 pricing only, 6 combined), and assess program implementation through detailed process evaluation. 3) Assess impact of the pilot program on a) the stocking, pricing, marketing, and sales volume of promoted foods at wholesale and retail levels, and b) food purchasing behaviors and associated psychosocial variables (i.e., self-efficacy, intentions, perceived cost) at the consumer level (final sample n=12 consumers/store, 288 total). The proposed research directly addresses the objectives of the PA by seeking to develop effective, multilevel communication strategies to improve diet and reduce risk for diet-related chronic diseases. We anticipate this design will demonstrate the value of a multipronged and multilevel health communications approach to obesity and chronic disease prevention, and will lead to a large-scale trial and informed policies designed to improve food availability and affordability in low-income urban settings.


Despite the detailed understanding of signals 1, 2, and 3 and the critical function of T cells and dendritic cells (DC), clinical transplantation tolerance is almost never achieved. In transplantation, models of immune function, and interventions designed to promote immune regulation, have been based on simplified receptor-ligand and cell-cell interaction models. There has been a lack of studies that place immunological recognition within anatomic contexts and evaluate the critical role of anatomic microdomains in the regulation of the immune response. Our preliminary data now demonstrate that during tolerization there is alloantigen specific clustering of T cells and plasmacytoid DC (pDC) in the T cell areas of the lymph node (LN) near the abluminal surface of the high endothelial venules (HEV). Within these clusters T cells undergo either priming or development into de novo CD4+CD25+ regulatory T cells (Treg). Importantly, B cells presenting specific alloantigen are present in these cell clusters and contribute to Treg development. We hypothesize these multicellular clustered interactions in the LN are key to the induction and maintenance of tolerance. Specifically, we hypothesize that the precise interaction of T-APC (pDC, B cells) determines the outcome of T cell migration, positioning, proliferation, and maturation, and ultimately whether rejection or tolerance are induced. This hypothesis integrates many of the known receptor-ligand and cell-cell interactions, and places these interactions in the context of secondary lymphoid organ structure. To investigate the role of these cellular and structural elements in LN clustered interactions, we propose the following specific aims: Specific Aim 1. What are the T-APC-LN interactions that are important for tolerance? Using a transplant model that allows the tracking of alloantigen specific T cells, specific alloantigen presenting APC, and the positioning of the cells with respect to HEV, we will characterize specific receptor-ligand interactions between T-HEV and pDC-HEV that regulate tolerance, and define the interactions between T-pDC and other DC that determine the generation of Treg and inhibition of effector T cells within the LN. Specific Aim 2. How is the priming of effector T cells altered in the LN during tolerance? This aim will study the specific interaction between effector T cells and Treg in the LN clusters during tolerization. We will determine how Treg-T effector interactions regulate effector T cell migration, proliferation, and differentiation. We will characterize specific receptor-ligand interactions between Treg and T effector cells that determine whether T cell priming and rejection versus suppression and tolerance predominate in the immune response. Specific Aim 3. Determine the role of B cells in tolerance in the LN We will investigate the roles of B cells in the LN during the T-APC clustered interaction that results in tolerization. Analyses will focus on B cell APC function, chemokine production, and immunoglobulin production. PUBLIC HEALTH RELEVANCE: The research will investigate and define the cellular and molecular interactions that are important for generating regulatory suppressor T cells and antigen specific tolerance. The ability to define these interactions and achieve tolerance is important for achieving good graft survival and patient survival for transplant recipients.


This project has continued to focus upon the potential mechanism(s) underlying the selective brain tumor (CNS) cell line cytotoxicity of certain quaternized ellipticine derivatives. Sensitivity of these glial derived human brain tumor cells to N2-methyl-9-methoxyellipticinium acetate (MMEA) correlates with peak cellular concentration of the drug (r=0.976, p<0.0001). Peak cellular concentration of MMEA by sensitive CNS cell lines is achieved 10-15 hours following initiation of drug exposure and represents intracellular drug concentrations several hundred-fold in excess of that found in the extracellular medium. Cellular accumulation is mediated by a high-affinity, sodium-independent transport system which is competitively inhibited by the plant alkaloid reserpine, secondary and tertiary tricyclic antidepressant drugs and, with much lower affinity by catecholamine neuro-transmitters and serotonin.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A series of three studies were conducted in African green monkeys (AGM) to test a proprietary compound, TMC 353121, formulated by Tibotec Pharmaceuticals against RSV in nonhuman primates. In previous studies, the compound, an RSV fusion inhibitor was shown to have efficacy against RSV in vivo in smaller animals. These studies were performed in collaboration with Bioqual, Inc. with animals and veterinary personnel at their facility. My laboratory was to provide not only virus challenge stock but performed all virological testing of samples taken from the animals during each of the three studies. We performed serological testing of 48 AGMs to prescreen and identify 36 RSV seronegative animals to be used for the studies. The first study, NC282, was an infection study and utilized 6 AGM. We prepared 37, 1ml vials of RSV strain at each of two concentrations of 1x10e3pfu/ml and 1x10e4pfu/ml and cryopreserved them for the entire study. The animals were divided into two groups with each group receiving either 10e3pfu/ml (Gp.1) by intranasal and intratrachael administration and the other receiving 10e4pfu/ml (Gp.2) by similar administration. Animals were monitored for clinical signs of RSV infection with nose and throat samples taken daily and bronchoalveolar lavage samples taken every two days and tested for virus loads by plaque assay. Peak viral loads were reached in both groups by day 6 in the throat with 3.7e2 pfu/ml and 1.4 e3pfu/ml and in the BAL with 2e2 pfu/ml and 3.7e2 pfu/ml and for Gps1 and 2 respectively. These results confirmed the model and the decision to utilize the 10e4pfu/ml inoculation dose for the two subsequent efficacy studies was made. Two efficacy studies with the continuous intravenous infusion of the compound were performed. Study 2 had 15 animals divided into three groups with Gps. 1 and 2, therapeutic and prophylactic arms with TMC 353121 was administered at a plasma level of 50ng/ml and Gp 3 the vehicle control arms;and Study 3 had 12 animals divided into three groups with Gps 1 and 2 therapeutic arms only with TMC 353121 administered at plasma levels of either 5ng/ml or 500ng/ml and Gp 3 a vehical control group. Animals were preconditioned to wear the jacket and tethering system, underwent surgery for implanting the catheter, and infusions begun. The animal studies have recently been completed, however, we are still conducting plaque and antibody assays and compiling data.


The transfer of phospholipids between the ER and mitochondria is critical for mitochondrial biogenesis. Mitochondria cannot synthesize many of the lipids they require for membrane biogenesis and yet there is little or no vesicular trafficking to mitochondria. It is thought that lipids are transferred from the ER to mitochondria by a poorly understood nonvesicular mechanism. This transport has been proposed to occur at regions where the ER and mitochondria are closely apposed. We have found that lipid synthesis at contacts between the ER and mitochondria (and other contact sites) promotes lipid exchange and are working to discover the mechanism. In a second project, we have identified a protein that facilitates contacts between the Golgi and the ER and facilitates ceramide transport. Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. We identified a novel mechanism by which cells prevent the toxic accumulation of ceramides; they promote nonvesicular ceramide transfer from the ER to the Golgi complex, where ceramides are converted to complex sphingolipids. We found that the yeast protein Nvj2p is an ER-Golgi tether that generates close contacts between these compartments and promotes the nonvesicular transfer of ceramides to the Golgi complex. The protein normally resides at contacts between the ER and other organelles but during ER stress it relocalizes to and increases ER-Golgi contacts. ER-Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER-Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the build up of toxic amounts of ceramides. A third project focuses on sterol transport to the vacuole. We have found that sterol-enriched domains form on the vacuole membrane in response to various cellular stresses. The domains regulate the stress response. We are working to understand how sterol transport is mediated and have found that conserved class of lipid transport proteins is required. The mammalian homologues of these proteins are seem to perform the same function. A fourth project focuses on the biogenesis of lipid droplets in the ER. We have found that lipid droplet emergence from the ER is regulated by a conserved family of proteins called FIT proteins. We think these proteins modulate the lipid composition of the ER and we have evidence that lipid droplet emergence from the ER can be driven the lipid composition of the ER.


The antitumor agents, 6-thioguanine and 6-mercaptopurine, have been reported to produce cellular toxicity as a result of incorporation of either agent into DNA as 6-thioguanine nucleotide. However, there is no data which demonstrates directly that a DNA polymer containing 6-thioguanine has altered functional properties. The purpose of this proposed project will be to ascertain the functional potential of a DNA containing 6-thioguanine in internucleotide linkage, thereby establishing methodology for future studies on the mechanism of action of other purine and pyrimidine analogs. The three methods of approach will be as follows. A. Allow vaccinia virus to incorporate 6-thioguanine into its DNA, and after harvesting viral particles, determine the ability of the genomes to express their information, namely: 1) ability to induce virus-specific mRNA synthesis and 2) ability to induce the synthesis of virus-specific proteins, i.e., thymidine kinase, DNA polymerase and deoxyribonuclease. The study will be controlled by monitoring the extent of 6-thioguanine incorporation into the viral DNA and relating this level to that found to be toxic to mammalian cells grown in tissue culture. B. The significance of incorporation of 6-thioguanine into cellular DNA on induced-protein synthesis will be evaluated as follows. Cultured cells will be treated with 6-thioguanine and washed free of non-incorporated drug. The cells will then be stimulated with an interferon-inducer or with interferon, and the production of induced interferon and induced antiviral activity by or in the cells will be quantitively determined. C. The significance of 6-thioguanine metabolites on DNA and protein synthesis in the cell cytoplasm will be determined by pretreating cells with 6-thioguanine. The ability of the pretreated cells to permit vaccina virus replication will then be assessed. This DNA virus replicates in the cytoplasm independently of the host cell genome.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We will use the recently completed rhesus macaque genome map, to identify approximately 83,333 single nucleotide polymorphisms (SNPs) randomly distributed throughout the rhesus macaque (Macaca mulatta) genome. At least ten copies of each DNA fragments in reduced representation libraries (RRLs) constructed from pooled (n=25)DNA samples from 7 different regional populations will be sequenced, then filtered, parsed and compared to the rhesus macaque draft sequence and to each other using bioinformatics tools we previously developed to identify the locations, flanking sequences and minor allele frequencies (MAFs) of 83,333 SNPs. Genotyping assays on lliumina iSelect chips will be constructed for the 7,600 SNPs whose frequencies differ most between Indian and Chinese rhesus macaques and genotyped in geographically representative samples of 192 Indian rhesus macaques, and192 Chinese rhesus macaques and in 678 Indian/Chines rhesus macaques with varying proportions of Indian and Chinese ancestry. Genotypes for the 7,600 SNPs will be used to 1) confirm Mendelian segregation of SNPs in multigeneration families, 2) assess the ability of the SNPs to accurately estimate proportions of Indian and Chinese ancestry in Indian/Chinese hybrid, pedigreed animals and 3) identify ancestry informative markers for admixture mapping of QTLs and to conduct admixture mapping of a QTL for "temperament.". Our SNP map will be made publically available through several outlets including dbSNP. The 384 most potentially informative of the 83,333 SNPs for longtail macaques will be genotyped in 96 samples from six different longtail macaque populations to identify minimum panels of 96 SNPs each for identifying country of origin and for parentage analysis and estiinating parameters useful for genetic management. Assays will be constructed of these two panels and made available to NIH investigators, providing cost recovery. The genotypes of SNPs described above will be used for genomic and population structure analyses of regional populations of rhesus and longtail macaques. PUBLIC HEALTH RELEVANCE (provided by applicant): Rhesus and longtail macaques are the most and second most used models for the biomedical study of human diseases. Genetic resources developed for rhesus macaques are not available for longtail macaques. Mapping disease phenotypes and QTLs is possible with the recent sequencing of the rhesus genome but requires the identification of many known polymorphic loci (e.g., SNPs) throughout the macaque genome. The most practical method for conducting whole genome association studies is admixture mapping.


1. OBJECTIVES/METHODS: Bleed vaccines annually; test serum for rabies- neutralizing antibody by the Rapid Fluorescent Focus Inhibition Test (RFFIT); boost those with <1.0 International Unit rabies-neutralizing antibody/ml serum with HDCS Rabies Vaccine (Institut Merieux). 2. RESULTS: Distribution of human serum quantified for rabies-neutralizing antibody in IU/ml: Employing Agency Number of People Number of RFFIT (serum source) Sampled Tests CBER-FDA 12 24 NIH 90 180 TOTAL HUMAN SERUM TESTS 102 204


Cell surface receptors coupled to the heterotrimeric GTP-binding proteins are universally responsible for the transmembrane transmission of extracellular messengers such as hormones, neurotransmitters and a variety of sensory stimuli. Because of this direct involvement in the regulation of the most crucial cellular functions, G-protein coupled receptors (GPCRs) are among the most important targets of therapeutic intervention. It's estimated that about 50% of drugs in use act on GPCRs. Thus, understanding of the receptor and the G- protein functions at the molecular level is among the highest priorities of public health research. Several competing models aim at describing the universal mechanism of G- protein activation by GPCRs, but none has presented compelling and conclusive experimental evidence so far. HYPOTHESIS: G-protein 23-subunit complex is a key molecular switch at the center of the gear-shift model of G-protein activation. We will test this hypothesis using the prototypical GPCR rhodopsin (R) and the G-protein transducin (Gt) responsible for phototransduction in retinal rod cells as a model system. Three interconnected Specific Aims will test various aspects of the hypothesis, such as questions of the molecular organization of the receptor- G-protein complex, the high-resolution picture of the receptor-G-protein interface, the mechanism of signal transfer from the receptor, and the roles of individual G- protein subunits, especially the G23 subunit complex, in this dynamic process. This project aims at understanding the universal principles underlying cell- to-cell communications and cellular responses to a variety of sensory stimuli. Several competing theories describing these basic molecular mechanisms will be tested to gain insights into the inner workings of cell surface receptor proteins and specific protein-protein interactions. Because almost half of all therapeutics on the market today target these signaling pathways, knowledge obtained as a result of these studies will be essential in new drug design and fighting a wide range of diseases such as heart problems, asthma and vision disorders.


Abstract Ischemic stroke is a leading cause of death and long-term disability. Current drug treatment is limited to tPA, which has a low success rate and potentially severe side effects. Acidosis is a common feature of neurological disorders such as brain ischemia, and it has been shown to play a critical role in stroke. The mechanisms, however, remained elusive. The discovery that protons activate a distinct family of cation channels, the acid-sensing ion channels (ASICs), has shed new light on acid-signaling and acidosis-mediated brain injury. The studies in our laboratories in the past 10 years have provided convincing evidence suggesting that activation of ASIC1a contributes markedly to acidosis-mediated ischemic brain injury. Following our initial report, others have demonstrated an important role for ASIC1a activation in spinal cord injury, traumatic brain injury, and axon degeneration. Thus, ASIC1a represents a novel therapeutic target. Despite its well-established role in neurological disorders, the detailed mechanisms underlying ASIC1a-mediated neuronal injury in stroke remain unclear. We now have strong evidence suggesting that, besides the well-documented Ca2+ toxicity, a combination of increased ASIC1a surface expression, Zn2+ toxicity, and an ion conducting independent cell death pathway participate in ASIC-mediated neuronal injury in ischemia. The objective of this application is to investigate the detailed molecular mechanisms and pathways underlying ASIC-mediated neuronal injury. Given the limitations of currently available pharmacological inhibitors that target these channels, e.g. the non-specificity of amiloride and large molecule nature of PcTX1, the proposed studies may disclose novel and alternative therapeutic strategies for ischemic brain injury.


This application for an R34 award proposes to refine and conduct a pilot RCT of a peer-led cognitive-behavioral intervention to determine the feasibility, acceptability, and outcomes for preventing depression and other negative outcomes among out-of-school inner-city adolescents and young adults in a community-based employment training program. The specific aims of this study are: (1) to refine the intervention protocol for the peer-led cognitive behavioral intervention, develop an intervention fidelity checklist, develop a training manual for peer leaders who deliver the intervention, and train peer leaders to deliver the intervention;(2) to conduct a pilot RCT of 142 adolescents and young adults, aged 16-22, that compares outcomes for youth involved with our peer-led cognitive-behavioral intervention with outcomes from youth participating in an attention control condition;and (3) to obtain the information and experiences required for designing and implementing a larger randomized trial. To achieve Specific Aim 1, will refine our peer-led intervention, develop an intervention fidelity checklist, develop a training manual for peer interventionists, and train peer interventionists. To achieve Specific Aim 2, we will conduct a pilot RCT of our peer-led cognitive behavioral intervention. 142 adolescents and young adults with elevated depressive symptoms but without a current depressive episode will be randomized to our 9-session intervention or a 9-week life skills attention control. Assessments post- intervention, 6 months, and 12 months post-intervention will measure depressive symptoms, depressive episodes, anxiety and PTSD symptoms, cognitive appraisals, engaged coping strategies, quality of life, and education and employment milestones. To achieve Specific Aim 3, we will use findings from our pilot RCT (e.g., implementation fidelity, recruitment, attendance) to develop a R01 application to be submitted during the final year of our R34 project. The public health significance of this project is substantial. Given consistent evidence that depression is a debilitating and chronic condition, interventions must attempt to prevent initial depressive episodes from occurring. Adolescence and young adulthood is an ideal time for preventive interventions to occur given the high depression incidence rate in this age group. The innovation of this project is also notable, as it brings mental health services to employment training programs that are being promulgated with new federal funding initiatives. These employment training programs serve large numbers of adolescents and young adults who are not reached by existing mental health interventions. This project is consistent with the goal of the R34 mechanism to encourage research on the development and/or pilot testing of new or adapted interventions and with two high priority areas of NIMH'S Division of Services and Intervention Research to develop innovative interventions and personalize them for optimal use in diverse populations and to employ strategic partnerships and community participation to enhance research capacity and infrastructure. PUBLIC HEALTH RELEVANCE: Depression is a significant public health problem, with adolescents and young adults among the most affected. This project tests a peer-led intervention that aims to prevent the onset and worsening of depression in adolescents and young adults (16-22) who are out-of-school and not in the workforce and enrolled in an employment training program-a group that has not been the focus of previous mental health research. Our intervention may have great benefits to society, as preventing depression among this population has the potential to increase the employability, and ultimately, economic self-sufficiency of participants.


Size regulation requires both extrinsic and intrinsic mechanisms that define growth limits. Extrinsic mechanisms sense nutrient availability and control growth systemically. In contrast, intrinsic mechanisms regulate growth in individual organs and tissues in an autonomous manner. It has been postulated that one way intrinsic size regulation of organs is achieved is through organ compartmentalization. The aim of this proposal is to investigate how developmental compartments contribute to organ growth and size. Expression of dMyc in the Posterior (P) compartment of the imaginal disc results in a proportionately normal size wing. We have found that cell death is increased within the P compartment, and also non-autonomously, in the Anterior (A) compartment in this case which might account for the proper size regulation. I propose studies to examine the role of cell death in regulation of the size of each compartment, and of the entire developing wing. Our results point to a possible asymmetry in growth regulation imposed by compartments. I propose experiments to address whether asymmetry exists and the possible mechanisms involved in this potential asymmetry. [unreadable] [unreadable] [unreadable]


The goals of this project are to develop, evaluate, and disseminate an Internet (web) application, Moderate Drinking (MD). MD will use a behavioral self-control training (BSCT) protocol that helps drinkers learn skills to either moderate their drinking or to abstain. The target population is members of the self-help organization, Moderation Management (MM) and to visitors to their web site. MM (www.moderation.org) describes itself as an organization that can be helpful for problem drinkers wanting to either moderate their drinking or to abstain. Its members tend to be less severe problem drinkers with few signs of dependence. MM offers written materials describing a program of behavioral change, an online mutual-help community, and face-to-face meetings. Their behavioral change program is a version of a current generation BSCT protocol that will be used in the web application. BSCT is an intervention with strong empirical support. The interactive program will be unique on the Internet. The program will help users choose a goal (moderation or abstinence) then learn cognitive and behavioral skills necessary to achieve and maintain their goal. Modules include goal setting, self-monitoring, "doing a 30,"increasing motivation and self-efficacy, identifying and managing triggers, developing alternatives, coping with lapses/relapses, and a segue to abstinence if the user is having difficulty achieving and maintaining moderation. Because moderate drinking is an option, the program may to appeal to less severely dependent drinkers who might otherwise avoid a program that is purely abstinence based. This group of less severely dependent drinkers is an under served but growing population. Phase II goals are to complete the development of the program, pilot test it, and conduct a randomized controlled trial with a 12 month follow-up to assess its effectiveness. The experimental group will receive training via the MD web site and will participate in MM's online mutual help community. The control group, will only participate in the online mutual help community.


Beneficial mutations can arise within individuals neutral to the mutation's utility; variants demonstrating the change can then be selectively sorted from the progenitor population by the environment. This observation alone does not exclude the possibility that selective forces can also influence either the generation of genetic alterations, beneficial or otherwise, or the retention of alterations. Inheritable changes can be of at least two types: those that alter the genome by changing its DNA sequence (genetic), and those that establish a self- perpetuating physiological state (epigenetic) in individuals within an otherwise isogeneic population. Both genetic and epigenetic changes that favor a cell's survival may occur in organisms after exposure to an environment. I have demonstrated the existence of mechanisms that allow eukaryotes to adapt to growth-halting environments after exposure of the organism to the environment. The mechanisms result in the change of genomic DNA sequence at specific (selected) loci nonrandomly. These changes are the result of recombination and require only some of the components of the homologous recombination machinery. Environmentally induced changes in microbes could compromise the efficacy of vaccine- based therapies.


Three transparent gels participate successively in the visual process: The cornea, the lens and the vitreous. The molecular composition of these gels are known but to a large extent the way these molecules form aggregates and superstructures in the tissues are not fully elucidated. Optico-mechanical studies on the gels themselves are advantageous because they do not destroy the tissues, they don't introduce any artifacts and furthermore they can be utilized to study the gels under dynamic conditions (either unidirectional or cyclic vibrational motions). The same optico-mechanical techniques such as light scattering, birefringence, dielectric studies, and dichroism can be used to study the gels and solutions of single macro-molecular components of the gels of the eye. In addition, hydration and fluorescence studies will be employed on single components as well as electron diffraction, if microcrystals of macromolecules can be obtained. The same techniques will apply to study 2, 3 and higher number component mixture of macromolecules. Thus, the description of gel state will be done both frm synthetic (components and sums of components) and from the analytical (the whole tissue) point of view.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Maternal smoking is the major preventable cause of intrauterine growth retardation and prematurity. Recent evidence shows that developing lung is also highly sensitive to maternal smoking and that smoking during pregnancy leads to decreased lung function, increased respiratory diseases and increased incidence of sudden infant death syndrome (SIDS) in the offspring. Given that every year more than 400,000 infants in the US alone are born to women who smoked during pregnancy, it is of major importance to find ways to prevent those changes. Our data shows that nicotine is one of the factors responsible for the changes in pulmonary function present in children born to smoking mothers. In this project, rhesus monkeys are used to characterize the effects of chronic exposure to low levels of nicotine throughout pregnancy on lung development and function. The purposes of this project are to 1) characterize the molecular basis for nicotine's actions by determining how nicotine effects the functioning of nicotinic receptors in fetal monkey lung;2) characterize the effect of fetal nicotine exposure on lung development by functional, morphometric, immunohistochemical and molecular analysis;and 3) develop ways to block the effects of nicotine on lung development that can lead to clinical interventions that can be combined with vigorous smoking cessation programs in pregnant smokers in order to help the offspring of smoking mothers.


The Vaccine Pilot Plant (VPP) is a multiuse facility designed to manufacture vaccines for human clinical trials according to current Good Manufacturing Practices (cGMPs) required by the US Food and Drug Administration (FDA). The facility is composed of a warehouse to receive the raw materials, central process support facility to manufacture buffers and media components, manufacturing suites to produce active vaccine ingredients, a filling suite to fill the product into vials for delivery to the clinic, Quality Control laboratories to test the vaccine product, and supporting office and utility space for staff and equipment. These areas all work in concert to take raw materials and turn them into vaccine product that can be evaluated in humans.


Dengue virus and other flaviviruses including West Nile virus, tick-borne encephalitis virus, and Yellow Fever virus, are widespread, mosquito- or tick-borne human pathogens. About 40% of the world's population lives in areas with substantial risk of dengue transmission, and as many as 100 million people may be infected annually, experiencing dengue fever and potentially lethal severe dengue. Development of a vaccine effective against all four dengue serotypes has been difficult because infection with one ofthe four serotypes does not lead to protective immunity against any of the other three and may lead to enhanced risk of severe, life-threatening illness, especially in children. Antivirals against dengue are of interest due to their potential to reduce the severity of dengue-associated disease as well as to reduce transmission. This proposal will contribute to efforts to develop dengue antivirals by developing mechanistically well-characterized small molecule inhibitors of dengue entry and using these to validate the dengue E protein as an antiviral target in vitro and in vivo. We have previously identified three structurally distinct lead compound series and inhibitors that exhibit a spectrum of activities against dengue virus entry. In the proposed work, we will use a combination of x-ray crystallography, medicinal chemistry, direct protein-small molecule affinity measurements, single-virion fusion and live-cell imaging assays, and resistance studies to optimize the pharmacological activity of our compounds and to elucidate the biochemical and structural mechanisms of action that correspond to potent, pan-serotype inhibition of dengue. Key to these efforts are quantitative assays, analogous to enzymatic activity measurements, for studying the biochemical functions of the viral envelope protein and that allow us to link the biochemical mechanism of small-molecule inhibitors with the step(s) in the cell-entry pathway at which they block infection. Optimized dengue entry inhibitors will be tested in a murine model of dengue infection and pathogenesis. Collectively, our efforts are aimed at producing compounds that can be advanced as preclinical candidates for dengue drug discovery efforts. More broadly, this work establishes the template for a rational approach to the development of small molecule inhibitors of other enveloped viruses as potential antivirals


We propose to conduct the first pharmacotherapy relapse prevention study in body dysmorphic disorder (BDD). BDD, an often-delusional preoccupation with an imagined or slight defect in appearance, is a distressing, impairing, and relatively common body image disorder. It is associated with high rates of functional impairment and markedly poor quality of life. It appears that SRIs are often--and selectively--efficacious for BDD and that many BDD patients receive SRIs. It also appears that most patients discontinue an efficacious SSRI at some point, as the alternative is life-long treatment. However, no relapse prevention studies have been done. Such a study is important from a clinical and public health perspective, because BDD appears to often be chronic and require long-term treatment. It is therefore critically important to investigate the risk of relapse with SSRI discontinuation, and whether continuation SSRI treatment decreases relapse risk. 128 subjects will be enrolled and first treated openly for 14 weeks with escitalopram; 58 escitalopram responders will then be randomized to double-blind continuation treatment with escitalopram or placebo for 6 additional months. Our primary aim is to compare time to relapse and relapse rates in responders to acute escitalopram who are then randomized to placebo versus continuation treatment with escitalopram. Secondary/exploratory aims will explore 1) Whether subjects who receive continuation escitalopram perform better on secondary outcome measures (e.g., quality of life) than those on placebo; 2) Whether subjects taking continuation escitalopram have further improvement in BDD symptoms during the continuation phase; 3) Predictors of relapse after escitalopram discontinuation; and 4) Acute treatment response. Because this study will offer a unique opportunity to investigate the genetic basis of BDD, we will explore BDD's genetic basis and the relationship of selected candidate genes to treatment outcome and side effects. In summary, this study will be the first relapse prevention study in BDD and the first study of continuation pharmacotherapy in BDD. It will provide critically important information on relapse with continuation versus discontinuation of an SRI, whether continuation treatment protects against relapse, and whether patients further improve with continuation treatment. It will explore previously unstudied questions about BDD's pharmacogenetics, which may ultimately guide and enhance medication treatment. This study will yield unique and clinically important data, and will fill gaps in knowledge about this relatively common, severe, and understudied illness.


Myeloid lineage osteoclasts are the sole effective bone-resorbing cells. Osteoclasts are required for the resorptive phase of physiological bone remodeling that maintains musculoskeletal integrity and regulates bone mass. Under physiological conditions the generation and function of osteoclasts is tightly regulated and coupled to the function of bone-forming osteoblast lineage cells. Many pathological conditions associated with excessive bone resorption and bone loss are characterized by loss of normal regulation/coupling, and excessive osteoclastogenesis. The long term goals of this project are to elucidate new mechanisms that suppress osteoclastogenesis, with the associated goal of using this information to develop new therapeutic approaches to suppress pathological bone resorption. Inflammation is an important driver of pathological bone loss. Inflammation decreases bone mass by suppressing osteoblast-mediated bone formation, and concomitantly strongly promoting bone resorption by increasing the differentiation and bone-resorbing function of osteoclasts. Thus, inflammation induces local bone erosion/osteolysis at inflammatory sites in diseases such as rheumatoid arthritis, periodontitis, infections, and orthopedic implant loosening. Recent work, including contributions from our laboratory, has made clear that potent inflammatory factors, such as the inflammatory cytokines IFN-? and GM-CSF and ligands for Toll-like receptors (TLRs) that sense microbial products and tissue damage, also induce feedback inhibition mechanisms that restrain osteoclastogenesis and thus limit the amount of bone resorption that occurs in inflammatory settings. Little is known about feedback inhibitory mechanisms induced by inflammatory factors to limit osteoclastogenesis. Based on our overarching hypothesis that augmenting feedback mechanisms represents an attractive alternative therapeutic approach to suppress pathologic bone resorption, we have investigated mechanisms by which inflammatory signaling restrains osteoclastogenesis. We have discovered two novel and complementary mechanisms by which inflammatory factors, including ligands for Toll like receptors and ITAM-associated receptors, suppress differentiation of osteoclast precursors. These are: 1. Modulation of a proteolytic pathway that generates a biologically active intracellular fragment of the Fms receptor for M-CSF. 2. Induction of the BCL6 transcriptional repressor to inhibit expression of the 'master regulator' of osteoclastogenesis NFATc1. These novel inhibitory mechanisms target two major nonredundant proteins required for osteoclast differentiation, Fms and NFATc1, and effectively inhibit osteoclastogenesis. We will characterize these inhibitory mechanisms to obtain knowledge that can be used to develop new approaches to suppress osteoclastogenesis and pathologic bone resorption by augmenting these mechanisms therapeutically.


Mosquitoes are insect vectors responsible for the transmission of many infectious diseases to hundreds of millions of people worldwide. Females of most mosquito species require blood from vertebrate animals for their egg development. Multiple bloodfeedings enable mosquitoes to transmit disease pathogens, including malaria parasites and dengue virus, from one person to another. Our long term goal of this project is to elucidate the molecular mechanisms that regulate mosquito egg production and identify target molecules that can be utilized for mosquito control. Mosquito egg development is governed by alternating peaks of two major insect hormones - juvenile hormone (JH) and 20-hydroxyecdysone (20E). Deprivation of JH in newly emerged adult female mosquitoes will halt egg maturation. On the other hand, topical application of JH to mosquitoes shortly after blood feeding interferes with the normal responses to 20E and impairs egg production. We have recently demonstrated that the mosquito Methoprene-tolerant (AaMet) protein is a key player in the juvenile hormone signaling pathway in the newly emerged adult female mosquitoes. AaMet protein binds to JH and forms a complex with AaFISC protein, a coactivator of the 20E receptor. AaMet and AaFISC are found to be associated with the promoters of JH target genes and activate their expression. In addition, our preliminary studies imply that AaMet mediates the inhibitory effects of JH on 20E-induced gene expression. Taken together, the results suggest that AaMet and AaFISC are components of a juvenile hormone receptor. The objective of this project is to elucidate the molecular details of how AaMet functions in juvenile hormone signaling that regulates egg maturation in mosquitoes. In Aim 1, we will perform structure-function studies of the juvenile hormone binding domain in AaMet and define the structural determinants required for high affinity binding to JH. In Aim 2, we will investigate how AaMet and AaFISC proteins are recruited to juvenile hormone response elements in the JH target genes. In Aim 3, we will test the hypothesis that AaMet is involved in the crosstalk between juvenile hormone and 20E signals in blood-fed female mosquitoes. The study will significantly advance our understanding of the molecular action of juvenile hormone in mosquitoes, and provide a structural basis for designing new pesticides that specifically target the mosquito JH signaling pathway.


Premitec, Inc., in collaboration with the Doheny Eye Institute at the University of Southern California has developed novel, 3-dimensional wide field microelectrode arrays (3D-WFA) for retinal prosthesis. These devices can double the visual field for retinal prostheses versus the devices currently being tested in clinical trials. The electrode arrays have a 10-mm diameter area of contact with the retina, thus enabling a wide field of vision. This work is funded by the National Institute of Neurological Disorder and Stroke (NINDS) at NIH. A conclusion of our results so far is that the large area polyimide array, although it has a three dimensional curvature to match an "average" eye, nonetheless has the tendency sometimes to separate from the retina, particularly at the edges, due to difficulty of the implant procedure, the nature of the polyimide array edge and variation in eye curvature. In addition use of a single retinal tack to affix the prosthesis to the retina not only damages the retina at the insertion site but also leads to greater non-uniformity in distance to the retina across the array. To address these shortcoming we propose to develop a novel hybrid silicone/polyimide wide-field array and eliminate the tack altogether. Recently we have demonstrated that the silicone surface can be modified to bind an adhesive protein which also binds strongly to the inner limiting membrane of the retina, and more generally to brain and other tissues. Replacing the tack with an adhesive will virtually eliminate the distance between the electrode and the retina, and thus reduces the stimulus threshold. By incorporating adhesive-silicone technology into the design of the 3D-WFA, we can address two important issues: increase implant safety by reducing the likelihood of retina damage and improve implant efficacy by reducing the distance between the array and the retina. PUBLIC HEALTH RELEVANCE: Advancements in microfabrication and materials have made possible the development of flexible neural interfaces. These medical devices are targeted at treating incurable neurological disorders and solve difficult technical problems that are limiting the capability of neural implant devices. Such diseases are widespread in the population as a whole and their impact on individual health is profound. The progress made by us so far with support from NINDS through Phase II SBIR demonstrates that our technology has the potential to lead to a successful commercialization of complex and challenging neural interfaces like the retinal stimulator and make a real impact on public health by increasing the capability of implantable neural prostheses.


The Mid-America Cancer Center Program is continuing to develop its strengths in research, education and patient care in order to bring the increasing cancer expertise at four participating institutions to bear on the cancer problems in the portion of the Midwest centered in Kansas City. The Center brings together scientists at The University of Kansas Medical Center, The University of Kansas-Lawrence, Kansas State University and Midwest Research Institute in program of mutual interest in the cancer field. The research strengths shared include drug development from basic design to clinical trials, virology, biology, immunology, carcinogenesis, epidemiology and radiobology. Based at the major tertiary care facility in the region, the opportunity exists to combine these research strengths of the Cancer Center with a rapidly improving clinical capability to bring outstanding care to the cancer patients of the region. The transmission of this knowledge to the medial profession as a whole remains a major goal of the educational and outreach program of the MACCP. At the same time, the MACCP is increasing its total information efforts to insure that all facets of cancer are understood by not only the professionals but by the public through cooperative programs with the Amerian Cancer Society divisions in the area. The Cancer Data Service has become a computerized regional registry providing the broad statistical base necessary for effective evaluation of the efforts of all participating elements of the program and for the conduct of epidemiological studies.


The 2007 UNAIDS report estimated that for each person treated with potent antiretroviral therapy, 4-6 new individuals became infected. Without a foreseeable cure for HIV, prevention measures must control the HIV epidemic and structural/barrier/behavioral methods simply have limited efficacy or applicability. There is a critical need, for coitally-independent dosing strategies which women can initiate and are imperceptible to their partners. It is increasingly clear that antiretroviral pharmacokinetics predict efficacy: using samples from the CAPRISA 004 study, our laboratory demonstrated that subjects who maintained the highest tenofovir concentrations in cervicovaginal fluid (and thus in genital tract tissues) were the least likely to become infected with HIV-1 and HSV2. Although topical formulations such as gels and vaginal rings are being investigated, oral antiretroviral drugs also hold significant promise for prevention. Currently, antiretroviral doses used in prevention studies are those that are FDA-approved for treatment of HIV infection. However, there are no data to confirm that exposures with these standard treatment doses will protect mucosal cells from HIV acquisition, or to inform alternative dosing strategies. We propose a highly significant plan to develop a model for oral antiretroviral prevention strategies. In healthy women volunteers, we will determine the ability of 3 doses of 4 antiretroviral drugs to concentrate in 3 at-risk mucosal surfaces. In explant tissue cultures, the concentration of these drugs, alone and in combination, required to protect tissues exposed to multiple infectious molecular clones will be identified. A new approach to normalizing tissue responses to cell numbers and composition will also be implemented. Once the in vivo tissue pharmacokinetic results and ex vivo target concentration results are known, a mathematical model will be developed to predict the antiretroviral doses/regimen maximally protective at all mucosal surfaces. Finally, a second proof-of-concept study is proposed to dose women with the final antiretroviral regimen and challenge tissue biopsies with HIV to determine risk of infection.