Achieving long-term allograft survival in the absence of ongoing immunosuppressive treatment is an important goal in clinical organ transplantation. Homeostatic mechanisms that regulate T-lymphocyte survival during alloimmune responses are probably central to determining the fate of a transplanted organ. There is evidence to show that T-lymphocytes are susceptible to activation-induced apoptotic death following exposure to high antigen dose or persistent antigenic stimulation. The extent to which activation-induced T-cell death contributes to long-term allograft survival and transplantation tolerance is not known. The underlying thesis in this proposal is that activation-induced death of alloreactive T-cells is necessary but not sufficient for induction of long-term allograft survival and transplantation tolerance; suppression of factors that rescue T-cells from death is required to achieve these outcomes. Specific aim 1 is to study the role of activation-induced T-cell death in the induction of long-term allograft survival and transplantation tolerance to vascularized allografts. Heart transplant acceptance will be examined in gene-knockout mice deficient in membrane molecules or cytokines that mediate activation-induced cell death. Biochemical, histochemical, and flow cytometric techniques will be used to study apoptosis of alloreactive T-cells in vivo and in vitro. Specific aim 2 is to study the role of activated antigen presenting cells in rescuing T-lymphocytes from cell death, thus precluding long term-allograft survival and transplantation tolerance. In vivo and in vitro systems will be used to determine which antigen presenting cell cytokines or membrane molecules can rescue activated, alloreactive T-lymphocytes from apoptosis. An in vivo system consisting of scid mice transfused with T-cell receptor-transgenic lymphocytes will be utilized to study intragraft factors that prevent activation-induced T-cell death. The proposed studies will enhance our understanding of T-cell survival following allostimulation and refine our abilities to design and implement new therapies that induce tolerance to transplanted organs.


This proposal focuses on a brain area that is part of the central extended amygdala: the central nucleus of the amygdala (CEA). The CEA is involved in a number of responses to fear, stress and anxiety and contains distinct cell populations. However, it is not known whether the cell populations have distinct functions, and it is currently impossible to ascertain this in vivo with the tools available. This proposal aims to test the feasibility of developing a selective neurotoxic lesion of a specific neuronal population in the CEA using the neuropeptide B and W type 1 receptor (NPBW1) as a target. This receptor is very discretely expressed, specifically in the lateral CEA, but not in the surrounding areas. Furthermore, this receptor is expressed on cells of the lateral CEA that contain corticotropin releasing hormone (CRH) and dynorphin, but is not expressed on cells that contain enkephalin, or on cells of the medial CEA, a major source of amygdaloid output neurons. A selective ligand for this receptor (neuropeptide W-30) will be custom conjugated with saporin (by Advanced Targeting Systems). Aim 1 focuses on the development of an effective and selective neurotoxic lesion of the CEA. The ligand will bind to the NPBW1 receptor and when internalized will release saporin into the cell, causing the death of the cell. The utility of this approach has been shown for other receptor systems, including selective targeting of cells of the intercalated nuclei of the amygdala, using the mu opioid receptor as a target. The specificity of the localization of the NPBW1 receptor suggests that this lesioning method will selectively destroy cells containing CRH and dynorphin, but spare both enkephalin containing cells in the lateral CEA, and amygdaloid output cells within the medial CEA that are thought to be critical for modulation of several fear and anxiety responses (e.g. fear potentiated startle response). In Aim 2, the potential functions of CRH/dynorphin cells of the central extended amygdala will be assessed in adult male rats. These experiments will initially evaluate the effects of this neurotoxic lesion of the CEA on A) basal anxiety-like behaviors in the elevated plus maze and defensive withdrawal paradigm, and B) fear-potentiated acoustic startle reflexes. It is hoped that the development of a tool to selectively target a specific population of the CEA will help to define its functions, with an initial emphasis on the roles that the cells may play in fear and anxiety-like responses. It is hoped that with time, this work will ultimately lead to the development of neurotoxic lesions of several distinct cell types in the central extended amygdala, including the bed nucleus of the stria terminalis, and collectively, this will lead to a better understanding of the processing of negative emotions that are associated with the development and exacerbation of many fear and anxiety-related disorders. This proposal aims to selectively remove a specific neuronal population in the brain that is thought to be involved in stress, fear and/or anxiety responses. It is hoped that selectively targeting a specific population (rather than the more global approach currently available) will lead to a better understanding of how the brain processes these negative emotions. This in turn will be helpful in the design of treatment strategies for anxiety and fear-related disorders.


Resistance to chemotherapy is a major stumbling block in the treatment of cancer. Recently, a role has been defined for the overexpression of the oncogene bcl-2 in the production of resistance to chemotherapeutic-induced apoptosis in some tumors, including advanced beast cancer, acute myeloid leukemia, prostate cancer, and neural crest tumors such as neuroblastoma. It is therefore critical that new chemotherapeutic strategies be developed to overcome chemoresistance in tumors that overexpress bcl-2. Previous reports have documented that overexpression of bcl-2 is accompanied by an increase in the intracellular concentration of reduced glutathione (GSH). One possible way to overcome chemoresistance in bcl-2 - overexpressing cells would therefore be to use chemotherapeutic drugs whose activity is enhanced by reaction with GSH. We have performed extensive studies of one such group of agents, the anodynes. The anodynes are prodrugs that require reductive activation by thiols for their antineoplastic activity. As such, their activity both in vitro and in vivo and in vivo is directly proportional to the cellular concentration of thiols, including GSH. We have recently shown that, unlike other chemotherapeutic drugs, the enediyne neocarzinostatin (NCS) is a more potent inducer of apoptosis in tumor cells that overexpress bcl-2 than in those that do not. We propose now to determine the mechanisms for both the increase in GSH concentration and the increase in sensitivity to enediyne-induced apoptosis in bcl-2-overexpressing cells. We will then use our understanding of these phenomena and their mechanisms to design, implement, and optimize a chemotherapeutic strategy that exploits the unique molecular and biochemical characteristics bcl-2-overexpressing tumor cells to target them for drug-induced apoptosis. This approach has the benefit of maximizing efficacy in bc1-2-overexpressing tumor cells, minimizing toxicity to normal cells, and identifying those tumors for which this is likely to be an effective regimen. Furthermore, it will establish in a preclinical model the potential of this novel strategy for treatment of patients for whom these is otherwise no reasonable chance of survival.


This program project proposes to investigate the nephritogenic and transplantation activities of the streptococcal membrane and soluble fractions derived from it. Soluble fractions have been obtained from the streptococcal membrane which are on chemical analysis found to be low molecular weight glycoproteins which demonstrate several immunological and biological properties. These materials are immunologically cross- reactive with similar materials obtained from human glomerular basement membrane, induce homograft sensitivity in outbred mice to skin homografts and abrogate the cytotoxic activity of anti-HL-A antisera. These materials are being evaluated in in vitro correlates of cellular immunity in patients with rheumatic fever and post-streptococcal glomerulonephritis. Additionally, they are mitogenic for most human lymphocytes evaluated and may be involved in abrogating or altering MLC in humans. It is our intent to chemically characterize the effector molecules of the soluble fractions and to alter them so that they may be further explored in the above experimental parameters.


When presented with food or cocaine, dopamine (DA) in the nucleus accumbens (NAc) initially signals that the item is rewarding. When behaviors, such as drug taking, become compulsive we hypothesize that this reflects a shift in neural systems maintaining the behavior. There is enhanced DA release in the dorsolateral striatum (DLS) and attenuation of NAc DA release. Furthermore, within DLS, activity in the Direct Pathway from the striatum to the substantia nigra, that is important for initiation of appetitive behaviors is enhanced, while activity in the Indirect Pathway, from the striatum to the globus pallidus, that inhibits competing repetitive or stereotyped behaviors is attenuated. Females (women and laboratory rats) exhibit more rapid escalation of drug taking than do males and estradiol (E2) enhances acquisition of drug taking and motivation for drugs of abuse. Experiments from the Becker laboratory have demonstrated that E2 inhibits GABA release, down-regulates DA D2 receptors (D2DR), and enhances cocaine- or amphetamine-stimulated DA release in DLS but not NAc. The overarching hypotheses for this proposal are: 1) An attenuated cocaine-induced DA increase in NAc combined with an enhanced DA increase to cocaine in DLS is related to the propensity to develop a preference for cocaine over palatable food pellets in both males and females; 2) Estradiol's action in DLS of females enhances the cocaine-induced dopamine (DA) increase, inhibits GABA release, and inhibits D2 DA receptors. This combined effect enhances the rate of preference formation and motivation for cocaine over pellets in females; and 3) Decreased inhibition of the indirect pathway in DLS contributes to enhanced motivation for cocaine over pellets in both males and females. In females D2DRs are down-regulated in this pathway by E2, and this contributes to the more rapid preference formation in females and greater motivation for cocaine over pellets. Determining the mechanism(s) mediating formation of preference for cocaine over a highly palatable food reward, and how individual differences and sex differences contribute to this, are important for our understanding of and treatment of addiction in both men and women.


Stroke is a disabling and often fatal disease that affects all ages, but mostly in the elderly. Stroke incidence is expected to rise in the US as its population ages; therefore, its impact on public health and health resources utilization will continue to be significant. The Stroke Progress Review Group and NINDS have identified a need for a network of stroke clinical trials centers in order to prioritize and efficiently design nd conduct translational and exploratory early Phase I/II clinical trials and large Phase III clinical trials to identify and advance stroke treatments. The NINDS Stroke Trials Network is a multidisciplinary stroke research infrastructure that consists of a National Clinical Coordinating Center, a National Data Management Center (NDMC), and 25 Regional Coordinating Centers. The NDMC's role is to establish a collaborative relationship with all parties involved in the Network and provide efficient and standardized central data management that yields high quality data and statistical support in the planning and execution of the stroke trials. To this en, the Data Coordination Unit (DCU) at the Medical University of South Carolina has developed a web-based comprehensive integrated data and project management system, WebDCU, that enables distributed data entry from the participating clinical sites with extensive data quality control and also provides the necessary tools to efficiently manage operational activities for multiple trials, while ensuring compliance with the NINDS Common Data Elements and FDA regulations. Using the WebDCU? system, we will develop, implement and maintain a central database that streamlines and maximizes efficiency in the management of data collection, processing, and monitoring of clinical, biomarker and neuroimaging data. In addition, the WebDCU will incorporate trial management information system that will provide full support for all study operational activities in the Stroke Trials Network. The Neuroimaging Core of the NDMC will create neuroimaging repository for the stroke trials. In collaboration with the study Principal Investigator and the other parties of the Stroke Trials Network, the DCU biostatistics faculty members will: contribute to the innovative and efficient protocol development (including study design and case report forms development); statistically monitor study progress; generate reports for the DSMB and the study teams; conduct interim and final analysis and dissemination of study results via presentations and publications; and create public use datasets for data sharing. Finally, as the Statistics and Data Management Center for the Neurological Emergencies Treatment Trials (NETT) Network, the DCU offers unique advantage of seamless collaboration between the NETT and Stroke Trials Networks.


Psoriasis is a chronic inflammatory skin disease affecting 1-3% of the population worldwide. Several studies have shown a significant association between psoriasis and hyperlipidemia, a well-established risk factor for cardiovascular disease, suggesting these conditions may share common inflammatory pathways. While multiple immune cell types have been implicated in the pathogenesis of psoriasis, including conventional CD4+ and CD8+ T cells, the potential contributions of lipid autoreactive CD1-restricted T cells to psoriasis pathogenesis remain elusive. CD1 molecules bind and present lipid antigens to T cells. These antigens include mammalian self-lipids and foreign lipids derived from specific microorganisms. In humans, the CD1 family consists of group 1 CD1 molecules (CD1a, -b, and c) and the group 2 CD1 molecule CD1d. Mice lack group 1 CD1, but do express CD1d. The unique binding specificity of CD1 suggests a potential role for CD1 molecules in the presentation of modified lipids to autoreactive T cells in hyperlipidemic conditions. However, due to the lack of a suitable animal model, the role of autoreactive group 1 CD1-restricted T cells in hyperlipidemia-associated inflammatory diseases is unknown. To overcome this limitation, we have generated a double transgenic mouse model that expresses human group 1 CD1 molecules and a group 1 CD1-autoreactive T cell receptor. In this study, we crossed this novel transgenic mouse to the ApoE- deficient background to study the role of autoreactive group 1 CD1-restricted T cells in hyperlipidemia. Interestingly, the presence of group 1 CD1-autoreactive T cells under hyperlipidmic conditions resulted in the mice developing severe psoriasis-like skin inflammation. While this finding suggests that autoreactive group 1 CD1-restricted T cells contribute to the pathogenesis of hyperlipidemia-induced skin inflammation, when, where and how these T cells are activated is unclear. Therefore, in Aim 1, we propose to investigate the mechanisms by which group 1 CD1-restricted autoreactive T cells contribute to skin inflammation and the kinetics of activation and localizatio of lipid autoreactive T cells during the course of disease. In Aim 2, we propose to examine how these T cells are activated by deciphering the nature of the lipid antigens presented by group 1 CD1 molecules during disease progression and further examining whether hyperlipidemia affects DC function, thereby resulting in group 1 CD1-autoreactive T cell activation. Collectively, these studies will lead to a better understanding of how group 1 CD1-restricted autoreactive T cells contribute to hyperlipidemia-associated inflammatory diseases and provide the basis for manipulating these T cells to uncover new strategies for therapeutic intervention for psoriasis and other inflammatory disorders.


There is a very high incidence of treatment resistance in bipolar illness. Some two thirds of patients in academic centers remain highly symptomatic despite aggressive pharmacotherapy. Days depressed exceed days manic by a factor of three. This large pool of treatment-refractory patients has been the focus of study of the Branch both to better understand the pathophysiological mechanisms in recurrent unipolar and bipolar disorders and develop new therapeutic modalities. With the current availability of a series of treatment options, finding clinical and biological markers of which patients respond best to a given treatment, is a critical need for the field. For example, a double-blind, randomized trial of six weeks of treatment with an agent that enhances inhibitory GABAergic function (gabapentin, GPN), versus one that decreases excitatory glutamatergic function (lamotrigine, LTG), versus placebo, found significant benefit of LTG (20/39 or 51% response rate) over GPN (11/40 or 28%) and placebo (8/28 or 21%). Correlates of lamotrigine response included male gender, bipolarity, fewer prior clinical trials, and fewer prior hospitalizations for depression (Obrocea et al). Preliminary evidence suggests that a baseline pattern of hypo-perfusion on 0-15 PET scans was associated with clinical response. In relationship to the assessment of possible predictors of clinical response, depressed patients with global hypermetabolism on PET, especially in the left insula, are more likely to be responsive to carbamazepine (N=26), while those with the more classic pattern of frontal and left insula hypometabolism are more likely to be responsive to the dihydropyridine L-type calcium channel blocker nimodipine (Ketter et al). We have found that nimodipine increases somatostatin in cerebrospinal fluid (CSF) and those with lower CSF somatostatin levels at baseline are more likely to respond clinically to nimodipine (Frye et al). The Branch is now analyzing a series of studies of the use of repeated transcranial magnetic stimulation (rTMS) of the brain for the treatment of depression that we have helped pioneer. A double-blind, randomized, crossover trial showed significant antidepressant effects of active 20 Hz rTMS for two weeks compared with the sham (George et al). The next study assessed the differential responsivity to low-frequency (1 Hz) vs. higher frequency (20 Hz) rTMS vs. sham stimulation over left frontal cortex at 80% of motor threshold (MT). This study found differential clinical and metabolic responses within the same patient to these different frequencies. Moreover, those with a pattern of baseline hypometabolism tend to respond to the 20 Hz stimulation, while those with baseline patterns of hypermetabolism are more likely to respond to the 1 Hz stimulation (Kimbrell et al). Because the incidence and magnitude of clinical responsivity was not adequate for many patients, another rTMS study using higher intensities (100% of MT) was conducted. This study replicated the findings of differential responsivity within individual patients, and strikingly, revealed that 20Hz stimulation increased 0-15 blood flow in a long lasting fashion, while 1 Hz rTMS decreased it (Speer et al). The most recent rTMS study used higher intensities of rTMS stimulation (110% MT) for a longer time (3 weeks) in an attempt to increase the response rate. This study randomized patients to 20 Hz vs. 1Hz vs. sham stimulation without a crossover and confirmed the lasting differential effects of high vs. low frequency rTMS on blood flow. It also revealed superior antidepressant effects of both 20 Hz and 1Hz compared to sham. Thus, a number of promising and mechanistically novel treatment approaches have been pioneered in the Branch and the current effort is aimed at defining better optimal parameters for rTMS response and defining clinical and neurobiological markers of individual responsiveness.


Developmental exposure to bisphenol-A (BPA) at doses within the range of human exposure causes a complex array of adverse effects in animals. These outcomes are also known to be present in human populations and the rise in their occurrence coincides with the massive use of BPA and other endocrine disrupting chemicals in consumer goods. The main hormonal activity of BPA is as an estrogen mimic. Exposure to estrogens throughout a woman's life, including the period of fetal development, is considered a main risk factor for breast cancer. Developmental exposure to BPA altered mammary gland morphogenesis in rodents during the period of exposure and led to the development of pre-neoplastic and neoplastic lesions appearing in adulthood. The goal of this proposal is to identify the molecular, cellular and morphogenetic mechanisms underlying BPA-driven altered mammogenesis that predisposes to neoplastic transformation. To achieve this goal, we will use innovative tools such as a fetal mammary gland explant culture model that allows testing for direct effects of hormones and real-time observation of organogenesis. The Specific Aims of this proposal are to test three hypotheses, namely, 1: that the direct effect BPA on mammary gland development is mediated by ER1 and/or 2. 2: that BPA causes altered ductal morphogenesis i) by altering the composition and physical properties of the ECM and ii) by inducing adipogenesis. 3: that the different mammary gland phenotypes resulting from gestational and gestational plus lactational BPA exposure are due to alterations at the hypothalamic level.


Excessive alcohol drinking is an enormous public health burden in need of more radical therapy. Most health problems associated with excessive drinking are due to two types of drinking: (i) binge (BALs > 0.08 grams % in a 2-hour period) and (ii) relapse (sustained heavy drinking for at least 2 days following a single or multiple abstinence periods). Previously, we showed that alcohol- preferring (P) rats express elevated levels of the GABAA subunits for alpha1, alpha2 and toll-like receptor 4 (TLR4) innate immunity receptors, and using specific siRNA vectors infused into the central amygdala (CeA), demonstrated that regulation of binge drinking at this site is mediated by GABAA a2 regulated TLR4 (a2/TLR4 axis). While the alpha1 subunit was also shown to regulate binge drinking, it was associated with the ventral pallidum (VP) and was independent of TLR4 (Liu et al., PNAS, 2011). In the current grant, we propose to better elucidate the mechanism responsible for the TLR4 effect, focusing on the chemokine monocyte chemotactic protein-1 (MCP-1) and the dopamine rate- limiting enzyme tyrosine hydroxylase (TH)--implicated by data obtained after the application was last submitted--and on brain sites that regulate different domains of the alcohol addiction cycle. The working hypothesis is that neuronal TLR4 induces MCP-1 expression via activation of cell type- specific transcription factors and it, in turn, functions as a neurotransmitter to stimulate dopamine release and excitability (inferred by TH) in select reward loci. The specific aims are: Aim I. Define the expression of GABAA a2/a1, TLR4, MCP-1 and/or TH at alcohol reward loci from P vs NP rats. Aim II. Define the role played by the a2/TLR4 axis that encompasses MCP-1 and/TH at alcohol reward loci in impulsive binge drinking. Aim III. Define the contribution of a1, a2, TLR4 and its downstream targets (MCP-1 and/or TH) at alcohol reward loci in compulsive relapse drinking. Aim IV. Define the mechanism of TLR4-mediated regulation of binge drinking by focusing on downstream signals that upregulate MCP-1 and TH expression, through the use of the pHSVsiMCP-1 amplicon. Better understanding of the role of chemokines in neurotransmission and the regulation of distinct (viz. dopaminergic) neurons will help broaden current concepts of neuroimmune communication and their roles in excessive drinking. This highly innovative proposal will create a paradigm shift in understanding the relationship between innate immunity signals and neuronal responses that impact alcohol addiction. Use of non-toxic herpes simplex virus (HSV)- siRNA constructs to inhibit relevant genes at specific brain loci will define therapeutic gene targets and test the potential of gene therapy approaches for binge and relapse drinking.


It is the purpose of this project to characterize the polysaccharides isolated from certain species of parasitic protozoan hemoflagellates of the family Trypanosomatidae, including the human pathogen Trypanosoma cruzi. Topics under present and future investigation include: 1) the chemical nature of the polysaccharide and its possible linkage to other molecules; 2) subcellular localization of these molecules; 3) the antigenic and immunological properties of these molecules; and 4) possible relationship of these molecules to the stage of the parasite's life cycle. The characterization of the isolated carbohydrate indicates nonglycogen-like neutral polysaccharides in addition to negatively charged molecules. The antigenic nature of the polysaccharide has been shown by immunodiffusion and complement fixation techniques.


Recombinational repair is important for cell survival and stability of genetic material. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. Recent evidence suggests that, in all cells, every round of replication requires some form of replication fork repair. In this proposed study, we will address several important questions in prokaryotic and eukaryotic recombination that have relevance to replication fork repair in every organism. (1) How do the RecA paralog proteins facilitate recombination? Every organism appears to employ a RecA-/Rad51 orthologous strand exchange protein and at least one other RecA/Rad51 paralog protein. What are the paralogs doing? We will use a combined biochemical and genetic approach to address the role of the RecA paralog protein, RadA/Sms in genetic recombination of E. coli. (2) What branched DNA molecules are intermediates of recombination and which enzymes resolve them? Our knowledge of the enzymes that process branched DNA intermediates of recombination is incomplete. We will assay whether the RuvC-related protein of E. coli, YqgF, is involved in recombinational repair and can catalyze cleavage of branched molecules predicted by recombination mechanisms. (3) What factors mediate template-switch repair? This is a recombinational mechanism that leads to sister chromosome exchange without the requirement for a strand-exchange protein such as RecA. Our previous work identified the first two factors involved in template-switch repair of E. coli, the chaperone DnaK and the gamma/tau subunit of DNA polymerase III, DnaX. What other factors enable this reaction? Using genetic analysis, we will test the involvement of proteins of the replisome and seek factors presently unknown. Replication fork repair is important for cell survival and stability of genes. In humans, inefficiency of repair has been associated with cancer proneness, neurological and developmental defects and premature aging. This proposal seeks to understand the mechanisms of replication fork repair. [unreadable] [unreadable] [unreadable]


We will develop a novel approach to detecting transgene activity in tumors based on proton MRI of enzyme activated reporter molecules. Specifically, we will synthesize, evaluate, optimize, and apply novel agents to detect (3-galactosidase activity generated by the lacZ gene. Gene therapy holds great promise for treatment of cancer. However, major problems involve assessing delivery to target tissue, the uniformity (versus heterogeneity) of bio distribution and determining whether the genes are expressed. We propose a novel approach for evaluating gene expression using MRI. This research draws on foundations in optical detection of reporter genes, specifically P- galactosidase from the lacZ gene. Recently, 3,4-cyclohexenoesculetin-p-galactopyranoside, marketed commercially as S-Gal(tm), has been shown to generate an intense black color as a precipitate upon activation by p-gal in the presence of Fe3+ ions. This prompted us to consider the paramagnetic properties of the precipitate as a potential 1H MRI contrast agent. Preliminary studies have demonstrated feasibility and we now wish to translate and develop the concept to tumor cells for detection in vivo. Specific aim 1 will explore the utility of S-gal, with investigations to optimize and validate the technique. MRI parameters will be varied to maximize contrast with appropriate spatial and temporal resolution. Agent administration will be evaluated to maximize contrast and minimize toxicity. Specific aim 2 will focus on translation to tumors in living mice. MRI will be validated by comparison with optical methods in vivo and traditional methods such as histology and RT-PCR. Specific aim 3 focuses on the synthesis and development of alternate optimized substrates. We believe this approach can both become a valuable tool for detecting p-gal activity in vivo and serve as a proof of principle for a novel platform technology adaptable to other reporter genes and endogenous enzymes such as proteases. [unreadable] [unreadable] [unreadable]


This is application proposing to determine whether failure to undergo programmed cell death contributes to the pathogenesis of human neuroblastoma. Specifically, the investigator proposes to determine whether oncogene products which modulate programmed cell death (PCD) influence whether oncogene products which modulate programmed cell death (PCD) influence the malignant phenotype of neuroblastoma. The investigator previously determined that Bcl-2, which suppresses PCD, is present in pretreatment neuroblastoma tumore biopsies, and that, when deregulated, inhibits neuroblastoma tumor biopsies, and that, when deregulated, inhibits chemotherapy-induced PCD in neuroblastoma. Based on these prior findings, the investigator seeks to extend these observations in a system in which it is proposed that neuroblastoma is a disease resulting from failed apoptosis. The first aim will address the issue of oncogene cooperativity in the malignant phenotype of neuroblastoma,, specifically whether Bcl-2, in its capacity to inhibit apoptosis, cooperates with N-myc to transform cells. The second aim is proposed to determine whether Bcl-xs expression will lead to neuroblastoma cell death and enhanced response to chemotherapy (by pirating existing cell machinery). The third specific aim proposes to determine the expression of the death gene and survival gene in normal tissue from which neuroblastoma is thought to arise. The investigator feels these experiments will address the molecular link between the benign lesions and malignant neuroblastoma.


The Postdoctoral Training Program in Hepatology is an integral part of the robust liver-related research effort at UCSF. The purpose of the Program is to provide trainees a comprehensive background in liver biology and disease as well as the investigational skills to address new questions and contribute to new knowledge in hepatology. The Program is staffed by 13 faculty based within the Departments of Medicine, Surgery, Biochemistry and Microbiology & Immunology; collectively these individuals offer balanced expertise in basic and clinical hepatology and have a strong track record of working collaboratively with each other. A subgroup of 5 Program faculty comprise a Steering Committee charged with screening applicants, reviewing the training curriculum and monitoring the progress of active trainees. The top priority of the Program is to train physician- scientists who are admitted to UCSF as Gastroenterology fellows. MD applicants to the Program must have a prior record of research accomplishment and dedication to an independent investigative career in hepatology. PhD applicants are admitted largely from mentor laboratories; they must display similar academic promise and an orientation toward translational liver research. All trainees are placed through a core curriculum covering liver-related biology, liver-related research methods and general academic skills. They then undergo specialized research instruction under an individual mentor, with choices ranging from clinical epidemiology, health outcomes and genetics to cell biology, organogenesis, immunology, metabolism and fibrosis/carcinogenesis. Additional focused coursework is highly recommended for all trainees; for those pursuing clinical investigation, enrollment in a Master's Degree program is mandatory. Importantly, the Training Program benefits from numerous institutional resources including outstanding graduate programs in basic and clinical sciences as well as research support units such as the Liver Center and the Clinical and Translational Sciences Institute. Program faculty are strong figures in each of these units, and accordingly can guide trainees to utilize these resources to maximize the value of their postdoctoral experience. The ultimate goal of the Training Program is to provide sufficient group and individual mentorship to enable graduates to assume a faculty-level position and compete successfully for independent research funding in hepatology. PUBLIC HEALTH RELEVANCE: Postdoctoral Training Program in Hepatology is designed to provide MD and PhD scientists the skills to conduct independent research relevant to the liver. The Program fills a need for basic and clinical investigators who will direct new scientific knowledge to the prevention, diagnosis and treatment of liver-related ailments. The Training Program enables faculty to provide focused research instruction to 4 trainees per year. Importantly, it also enables trainees to enroll in didactic courses and obtain an advanced degree. The ultimate goal of the Training Program is for graduates to become independent investigators, meaningful contributors to the field of hepatology, and hopefully mentors themselves. The expectation is that the trainees' research successes will translate into improved outcomes for patients suffering from liver diseases.


There remains a clinical need for myocardial perfusion tracers that more accurately reflect cardiac blood flow over a wide range of flow rates while simultaneously demonstrating a reduced accumulation and/or rapid clearance from the tissues adjacent to the heart. Despite the excellent myocardial extraction and flow kinetics of thallium-201 for evaluating myocardial perfusion and ischemia, the energy spectrum of thallium- 201 makes it particularly prone to soft tissue attenuation artifacts. While there are two FDA-approved Tc-99m perfusion tracers, having more favorable energy spectra for imaging, Myoview(TM) (tetrofosmin) and Cardiolite(TM)(sestamibi), the first pass extraction fraction for both of these tracers is approximately 50% of TI-201, and they exhibit unfavorable abdominal artifacts. At very low flow rates sestamibi and tetrofosmin overestimate flow and at high flow rates (above 2 ml/min/g) underestimate myocardial blood flow. Because extraction of these Tc-99m tracers reaches a plateau at high flow rates, there is a concern that these tracers may not have adequate sensitivity to detect intermediate coronary artery disease during pharmacological stress, when blood flow is increased several times over baseline. Our plan is to exploit our versatile tridentate chelates by synthesizing a series of novel tridentate etherfunctionalized chelates (TECs) and synthesize the corresponding rhenium-TEC complexes, [Re(CO)3(L)3]+, for structural identification. The carrier-free Tc-99m TEC complexes will be prepared using [99mTc(CO)3(H2O)3]+ followed by determination of the in vivo distribution and pharmacokinetic properties of the 99mTc-TECs in rats. Comparisons with TI-201, 99mTc-sestamibi and 99mTc-Tetrofosmin will be performed as a benchmark for initial heart accumulation. Finally, in vitro partition studies of lead compounds with human blood components will be analyzed. This evaluation in normal resting animals will allow for the evaluation of the Tc-99m complexes as potential myocardial perfusion tracers. Successful completion of this project will enable a more rapid and accurate assessment of myocardial perfusion. Since heart disease is the major cause of death in the United States, accounting for nearly 40% of all deaths, as estimated by the American Heart Association, a more accurate agent to assess myocardial perfusion would warrant further development. The current market utilization is approximately 6 million procedures annually in the USA alone.


Hemophilia B is a severe bleeding disorder characterized by a functional deficiency in the blood coagulation protein factor IX (Christmas factor). Therapy for bleeding episodes in these patients requires the intravenous replacement of factor IX given as fresh frozen plasma or with commercially available concentrates of factor IX in which the protein is only partially purified. Although these commercial materials have proven efficacy in this disease, they contain many impurities, are often contaminated with virus, and their use is associated with a significant morbidity. The goal of this project is the development of a highly purified form of factor IX. The factor IX will be obtained by using a recently described invention which employs conformation-specific antibodies against human factor IX to purify this protein. This factor IX will be homogeneous and thus suitable for intravenous therapy. Published studies of factor IX purified by this methodology indicate that it is free of contaminants and infectious agents responsible for complications associated with the currently commercially available concentrates. In this Phase I study sufficient factor IX will be purified to accomplish studies of acute toxicity in rabbits and the ability to correct the bleeding deficiency in hemophiliac dogs.


Project Summary Autism Spectrum Disorder (ASD) disproportionately affects males (?) over females (?), possibly because of a Female Protective Effect (FPE). Characterizing the FPE may help us to understand and treat ASD in both sexes. Our Network has contributed to understanding sex differences in ASD at the levels of gene structure and expression, neural dynamics, brain function and connectivity. We have curated an unprecedented sex-balanced, age-, IQ- and severity-matched cohort of cognitively-able school-age ?, and ? with ASD, age- and IQ-matched typically developing (TD) children and unaffected siblings (US). At T1, we conducted behavioral phenotyping and measured key neural systems at the levels of brain structure, connectivity, function and temporal dynamics. Genotyping, whole-genome sequencing and gene expression analyses are underway. We now seek to pursue an extraordinary opportunity to assess our participants again (T2) as they make the transition through adolescence and into young adulthood. Our field has failed to generate a sufficient knowledge base to help optimize this transition for people living with ASD and their families. We will leverage the expertise of our Network to identify sex differences in ASD longitudinal brain development during this important transition. We will clarify both temporal and spatial characteristics of developing social perception, emotion regulation, reward and implicit language learning circuits, in addition to neural mechanisms for sensory habituation, creating dimensional, multi- level neural signatures of brain development. We will bridge DNA sequence and brain development and relate neural signatures to behavior and genetics to predict ?real-world? functioning in young adulthood. We will combine multiple levels of biology and endophenotypes?SNVs, CNVs, clinical measures, pubertal status, presence of seizures/epilepsy, sex hormones and multimodal, longitudinal measures of brain development?into one framework using an Integrated Weighted Gene Coexpression Network Analysis (iWGCNA). Finally, we will extend our T1 systems-biology approach through a collaboration*with ASD self-advocates/participants to evaluate the experiential validity of our findings. The proposed research marks the start of a new era in which advanced multimodal neuroimaging and genetic analyses will evolve into an integral part of a translational research chain. Novel behavioral treatment and pharmacotherapies for ASD may be further developed in adolescence and young adulthood with the tremendous benefit of directly and more precisely assessing impairment and change in neural circuits. By providing information about distinct, sex/gender-based developmental pathways in ASD, this study will identify if intervention/prevention strategies should include sex-based modifications.


DNA fragment size analysis by flow cytometry has been applied to the analysis of restriction enzyme digest of 3 bacterial gnomes E. coli, E. herbicola, and E. erwinia. Distinct restriction fragment fingerprints were observed and served as the basis for discriminating among the three species. Details are contained in a Research Highlight and a publication that is in press in Cytometry.


The objective consists of the development of methods for the controlled enzymatic synthesis of large oligoribonucleotides and oligodeoxyribonucleotides of defined sequence. The strategy involves the use of enzymes, which have the capacity to form the internucleotide bond, with substrates that contain specific reversible chemical modifications. The particular enzymes to be exploited in the study are polynucleotide phosphorylase, RNA ligase, and terminal deoxyribonucleotidyl transferase.


Work has continued to obtain information relative to the role of regional lymph nodes (RLN's) in patients with cancer so that a firmer biological basis for the management of such patients may be formulated. Interrelationships between a variety of histopathologic discriminants in mammary and colonic carcinomas as well as their correlation with lymphocyte transformation were determined. RLN's from patients with mammary carcinoma exhibited greater sinus histiocytosis and less lymph follicles than those from persons with colonic carcinoma. These latter tumors contained more lymphoid infiltrate and their component cells had higher nuclear grades than breast carcinomas. Differences in the relationship of these histopathologic parameters with lymphocyte transformation of RLNC's in these two tumor types further indicates the singularity of neoplasms of diverse origins. Lower nuclear grade and mild lymphoid infiltrate of mammary carcinomas were associated with nodal lymph follicle formation and the converse with the absence of such nodal structures. No significant difference in these histopathological discriminants were observed in patients with or without nodal metastases. Lymphocyte transformation of RLNC's from patients with mammary carcinoma was increased in those in whom their carcinomas were of low nuclear grade and contained only mild degree of lymphoid infiltrate. These findings suggest that nuclear grade and lymphoid infiltrates may be differently related to tumor host responses than previously contended. No functional or other relationships could be discerned for sinus histiocytosis. In a second study, RLNC's from patients with breast cancer were evaluated at the time of their removal (not after culture) relative to HT3 uptake. Such an investigation was carried out to determine whether such nodes displayed evidence of a response to the tumor, whether variations observed between nodes and between patients found following RLNC culture and PHA stimulation existed at the time of their removal. Another objective was to ascertain whether findings could be related to histopathologic discriminants in the tumor and nodes. In general, no correlation was obtained.


PROJECT SUMMARY/ABSTRACT The overall goal of this clinical research program is to provide infants with Down syndrome (DS) with the training and technology to independently explore their world to the same degree as their typically developing peers. Moving and mobility comprise up to 80% of a young child's waking hours. Such a high dose of sitting, standing and walking are a lofty gold standard for pediatric rehabilitation. Down syndrome (DS), which delays sitting, standing and walking by up to a year, significantly reduces infants' daily exploration with serious cognitive, language, and social consequences. This proposal specifically tests the feasibility and effects of a novel 'mobile therapy environment' that provides powered mobility, progressive exercise and functional skills training in one assistive technology device. Our modified ride on cars specifically combine the fun and exploration of powered mobility (such as a Segway) with the ability to advance balance, strength and coordination through therapeutic exercises while infants practice sitting, standing and walking. Over the last year, we have developed a series of electrical and mechanical modifications that allow a single car to be progressively driven in sitting, then safely driven as triggered by standing (Figure 1 upper) then triggered by over ground walking (Figure 1 lower). This proposal will allow us to determine the feasibility and effect of ride on car training on sitting, standing and walking delays (Aim 1) and on infants' broader cognitive, language and social-emotional development (Aim 2). This proposal, inspired by our mobile robotics work and supported by preliminary data, is scientifically principled, innovative yet feasible within the funding period. The data and devices from this 2 yr project will support more formal work on the clinical and commercial potential of a novel category of assistive technology: the 'mobile therapy environment'. Figure 1 Modified ride on toy cars provide inexpensive effective mobility and socialization as well as strengthening, balance, coordination during driving while standing (Upper) and driving while walking (Lower).


Studies of the physiological and pathophysiological characteristics of the stable hypertrophied myocardium will be continued. Attention will be focused on continuing studies of the regional distribution of coronary blood flow by hydrogen polarography and microspheres, on characteristics of thallium uptake and imaging by the hypertrophied heart, and on segmental function in the hypertrophied myocardium at rest, under stress, and with ischemia. Studies of preservation and protection of the hypertrophied ischemic myocardium during cardiopulmonary bypass will be continued, and the effects of coronary ischemia on the hypertrophied heart will be studied and characterized in terms of segmental changes and regional blood flow distribution.


Membrane proteins that function normally are vital to health;their defects are associated with many known disease states. Membrane proteins are the targets of many pharmacologically and toxicologically active substances and are responsible, in part, for the uptake, metabolism, and clearance of these substances. Despite the importance of membrane proteins, knowledge of their high-resolution structures and mechanisms of action has lagged far behind the knowledge of these properties of proteins in general. Theoretical modeling may help in deciphering the structure-function relationship of membrane proteins, however theoretical modeling of membrane proteins also lags behind the modeling of globular proteins. Our long-term goal in this proposed project is theoretical modeling of structure and function of membrane proteins to provide understanding of how the molecular and atomistic events during ion permeation and ligand binding lead to mesoscopic events of channel conductance and regulation. One problem in modeling ion channels is that characterizing their function, i.e. ion current - voltage relationships, requires modeling of processes on at least the microsecond time-scale, inaccessible for current atomistic simulations of proteins. The objective of this application is thus to create and apply reliable yet computationally efficient molecular-level models for ion permeation through open channels, which take into account channel protein molecular structure, polarizability and short time-scale flexibility, yet are capable of predicting observable ion currents (a slow process by Molecular Dynamics standards). In order to efficiently span a wide range of time-scales relevant to the ion permeation, the proposed models are of hierarchical nature. Our aims in this project are: to develop, test and apply hierarchical algorithms to model ion currents through open flexible channels of known or predicted 3D structures. In order to implement this hierarchical approach we will use several levels of resolution ("graining") of the system under consideration: from all-atom molecular modeling of the channel protein with its surrounding medium to coarse grained continuum approximate models, capable of spanning longer time-scales and mesoscopic sizes. Using this molecular/mesoscopic hierarchy of models we will study several systems of medical and medical engineering interest. We expect that the outcome of this proposed project will be significant for future direction of theoretical and computational approaches to study membrane proteins because once a functional model based on protein structure has been developed it will become possible to develop specific theoretical methodologies for designing drugs and drug delivery systems for membrane proteins via rational computer-aided design.


To determine the maximum total dose of paclitaxel when given with gallium nitrate and assess the objective response rate, duration of remission and overall survival in patients treated with paclitaxel plus gallium nitrate plus G-CSF.


As a federally qualified community health center, the applicant has as its core function to provide primary healthcare services to vulnerable populations. Access to health information is a basic healthcare need. The goal of this project is to contribute to the development of a free on-line multilingual multimedia digital library of health information. The focus of the work in this project is to build upon existing high quality multilingual written resources that have already been developed and put on-line by the NSW (New South Wales) Multicultural Health Communication Service (MHCS) of Australia. The project work will include the development of a community committee to identify the MHCS written materials that deal with topics and languages that are most critical to meet the health information needs of new refugees in the community. A development team will work with the local partners, MHCS and members of the target refugee community to create web-based multimedia versions of this written material. A minimum of four written documents will be developed into at least four languages for a total of at least sixteen individual multimedia software files. These will be created using Macromedia multimedia software. A website will be created that will link with the community health information website (Healthy Communities Without Borders - www.hcwb.org ) to assist end users in accessing the materials. Training for key staff at the performance sites will be provided to ensure that there is a clear awareness of the new materials and an understanding of how to access other quality multilingual on-line health information. Since this project is a key step to understanding how information technology can be better used to meet the health information needs of non-English speaking community members, a strong emphasis will be placed on evaluation activities. The evaluation will include an assessment of target users engagement in the software and whether any knowledge, skills or attitude changes occurred (Levels I and II of training evaluation). Evaluation of the implementation strategies and perceived usefulness at each of the four performance sites will also be carried out. The results of this project will provide guidance in future efforts to provide critical health information to our increasingly diverse population.


Many experimental vaccines have been developed that show promise in animal models of cutaneous or visceral forms of leishmaniasis, but an effective human vaccine still does not exist. In a series of phase III clinical trials in which killed Leishmania vaccines were tested in individuals exposed to infected sand fly bites in the field, no protection was observed. Remarkably, none of the candidate vaccines tested in animal models have been evaluated under experimental conditions using infected sand flies, and we addressed the possibility that vaccine failure in clinical trials may reflect the more stringent conditions of natural sand fly challenge. We compared needle and infected sand fly challenge to evaluate the efficacy of the only defined candidate vaccine currently in clinical trial, comprised of polyproteins (either KSAC or L110f), containing multiple antigenic epitopes from Leishmania delivered in a stable emulsion (SE) (water in oil) with glucopyranosyl lipid A (GLA), a TLR4 agonist suitable for use in people. Polyprotein-vaccinated mice had a 60-fold increase in CD4+ IFNg+ T cell numbers versus control animals at 2 wk postneedle inoculation of L. major, and this correlated with a 100-fold reduction in parasite load. By contrast, following challenge by infected sand fly bite, polyprotein-vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+IFNg+/IL-17+ ratios versus non-vaccinated controls. Importantly, mice with a healed primary infection were solidly protected against infected sand fly challenge that was associated with the speed with which effector cells appeared at a site of challenge, providing an immediate burst of effector cytokines that may be required to counteract the down-modulatory environment created by the highly localized, neutrophil-dominated, response to sand fly bite. We employed Ly6C, a T-bet regulated, GPI-anchored, surface glycoprotein expressed on T-bethi CD4+ T cells, to phenotype the rapidly recruited cells, and found that pre-existing, short-lived, CD44+CD62L- T-bet+Ly6C+ effector (TEFF) cells, not memory or memory-derived cells, mediate concomitant immunity in healed mice. Upon adoptive transfer and challenge, non-dividing Ly6C+ TEFF cells preferentially homed to the skin, released IFNg and conferred protection. Despite being short-lived, Ly6C+ TEFF cells were maintained at high frequencies in the mice with healed primary infection, presumably as a consequence of the persistent infection in these mice. The lack of effective vaccines against Leishmaniasis may be because protection against these infections requires concomitant immunity mediated by pre-existing TEFF cells, not memory cells, and is therefore not amenable to conventional, memory inducing, vaccination strategies. As indicated above, primary L. major infection typically produces cutaneous lesions that heal but that harbor persistent parasites. While the opposing roles of CD4+ T cell-derived IFNg and IL-10 in promoting parasite killing and persistence have been well established, how these responses develop from nave precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex II (pMHCII) tetramers to investigate the endogenous, parasite-specific primary CD4+ T cell response to L. major in mice resistant to infection, and applied this approach to enumerate the expansion, contraction, tissue distribution, and function of parasite-specific CD4+ T cells throughout the course of the infection. Maximal frequencies of IFNg+ CD4+ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL-10+CD4+ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type-1 (Th1) cells, not Foxp3+ regulatory T cells, were the chief producers of IL-10 and were not exhausted. Therefore, tracking antigen-specific CD4+ T cells revealed that IL-10 production by Th1 cells is not due to persistent T cell antigen receptor stimulation, but rather driven early and sustained locally by antigen encounter at the site of infection. In addition to studying the mechanisms underlying parasite persistence in healed mice, mouse models are also being used to study non-healing forms of disease. Infection of C57BL/6 mice with most L. major strains results in a healing lesion with minimal pathology at the site of inoculation in the skin. By contrast, using a strain of L. major (Lm Sd) isolated from a patient with chronic cutaneous lesions, C57BL/6 mice also fail to heal their dermal lesions or effectively control tissue parasite burden despite a strong and polarized Th1 response. In studies designed to identify the earliest cells and mediators that precede and promote the severe pathology, we detected elevated levels of IL-1b mRNA and IL-1b+ cells in the inoculation site 3-4 weeks post-infection, followed by a neutrophil infiltrate that persisted until the onset of the pathology. Whereas no phenotype was observed in IL-17 deficient mice, IL-1R deficient mice, as well as IL-1b, ASC, and caspase-1/11 deficient mice, each showed minimal pathology and healed their Lm Sd infection. These studies are unique in the innate immunity field in identifying inflammasome dependent IL-1b as preventing rather than promoting host defense against a microbial pathogen. The mechanisms underlying the failure to control the growth and systemic spread of Leishmania parasites in human visceral leishmaniasis (VL) are not well understood. A key immunological feature of VL is the inability of peripheral blood mononuclear cells (PBMCs) to proliferate or to produce IFNg in response to leishmanial antigens. IL-10 has been implicated in the suppression of antigen-specific T cell responses in human VL based on the elevated levels of IL-10 observed in plasma and lesional tissue, and its role in preventing clearance of L. donovani in murine models of VL. In unexpected findings from our recent studies we were able to show that in sharp contrast to assays employing PBMCs, a cytokine release assay involving L. donovani antigen-stimulated whole-blood cells was able to detect the secretion of IFNg by cells from the majority of patients with active VL. In 35 patients with active VL, 80% secreted high levels of IFNg as compared to 85% of cured VL patients, and 24% of EHCs with presumed subclinical infections. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the whole blood cells from patients with active VL also secreted IL-10, which better reflects the response that distinguishes individuals with active disease from cured or subclinically infected, immune individuals. More generally, the findings reveal that PBMCs may not accurately reflect the immune competency of peripheral blood cells.


PROJECT SUMMARY The natural history of high-risk, localized prostate cancer, accounting for a substantial proportion of the 29,000 prostate cancer?related deaths per year in the U.S., is highly variable: there are patients who are cured with surgery or radiation, while others develop metastases and succumb to their disease. To date, attempts to stratify patients within these subsets of high-risk, localized prostate cancer have proven inadequate. There is particular urgency within the African American population, where risk of lethal disease is highest. There is a tremendous need for biomarkers that can reliably distinguish the most aggressive forms of disease within high- risk groups. Specifically, we focus on the DNA damage repair (DDR) pathway, where certain variants appear to be associated with prostate cancer aggressiveness. In this proposal, we will address two important questions in the field: 1) Among patients with high-risk, localized prostate cancer, can we identify genetic aberrations in DDR pathway associated with the most aggressive forms of the disease? 2) Do DDR variants contribute to increased risk of aggressive prostate cancer among African American men? In the first aim, we focus on inherited germline variants in the DDR pathway. Using large retrospective and prospective cohorts of patients with high-risk, localized prostate cancer, we will perform sequencing in DNA derived from blood samples and examine associations with development of lethal prostate cancer over long- term follow-up. In our second aim, we will interrogate the somatic genome and matched germline using DNA derived from radical prostatectomy specimens and corresponding blood samples, and we will study associations with lethal disease. In the third aim, we will determine the prevalence of germline DDR in a larger cohort of African American prostate cancer patients and estimate the extent to which DDR mutations contribute to prostate cancer disparities. Completion of the experiments outlined in this proposal will provide an unparalleled look at the association between DDR and lethal forms of prostate cancer. We have the potential to i) discover specific germline and somatic biomarkers for lethal disease that could be used to determine treatment strategies at the time of diagnosis, perhaps using strategies such as PARP inhibition or platinum chemotherapy that target the DDR pathway; ii) discover germline biomarkers that could be developed as screening tools for the most aggressive forms of prostate cancer; and iii) define the extent to which mutations in in DDR pathway genes may explain racial disparities in prostate cancer. The genetic loci and target genes comprising this dataset together with the other projects will stimulate new targets for therapeutic intervention.


Family-based behavioral modification (FBM) is the "treatement of choice" for pediatric overweight. A comprehensive package, FBM includes a core set of intervention strategies (parents skills training; reduced sedentary behavior and increased physical activity; and reduced energy-dense and increased nutrient-dense foods). FBM also has strong theoretical foundations, which has allowed it to be rigorously evaluated. Despite these conclusions, the translation of FBM to primary care has received almost no investigation. FBM has been implemented almost exclusively in university-based clinics, by highly-trained clinical researchers, with motivated families having access to the clinic. This model does not reach the vast majority of overweight children in the popuation, especially those living in rural communities. Thus, there is a need for "translational" studies that adapt FBM for implementation by existing clinical staff in a cost-effective manner manner, that is well accepted by staff and patients. To this end, we propose a planning, pilot and feasibility intervention study that will be conducted within Geisinger Community Practice network. Geisinger is a comprehensive health care network that serves more than 2.5 million families across 38 counties of central Pennslyvania, many of whom reside in rural and underserved communities. The proposed pilot study, a 2-group randomized clinical trial, will be conducted out of a primary care clinic within the Geisinger network. Participants will be 60 oveweight or at-risk for overweight children, 4 to 8 years old, and a caregiver, who will be randomized to one of two conditions: Family-based Behavioral Modification (FBM; N= 30) or Minimal Nutrition Information (MNI; N=30). FBM will consist of streamlined parent- and child-group meetings, tailored for primary care and implimented by the staff nurse and pediatrician. MNI will consist of nutritional handouts and fliers. Primary outcomes measures will be 4-month changes in child weight status, diet and physical activity, and cardiovascular outcomes (for effect size estimation), and measures of treatment acceptability by families and primary care staff. This pilot study will provide Penn and Geisinger investigators the time, resources, and pilot data necessary to map out a larger- scale intervention study across the Geisinger primary care nework, using the R18 funding mechanism. [unreadable] [unreadable]


The aim of this research is to obtain basic information on mitochondrial chain enzyme systems enabling conclusions on sequence, action and mechanisms of electron and coupled energy transfer by the methods of sequential fragmentation, systematic reconstitution and related physiochemical techniques. This type of attack may open up new avenues leading to more advanced designs for experiments in the field of terminal electron transport systems. Indeed, some of the results obtained from the project in the last few years would not have been discovered by conventional methods, but were dramatically and unambiguously revealed by reconstitution experiments. This is a long term project which, of necessity, entails the preliminary isolation and characterization of respiratory components from intra-cellular organelles. BIBLIOGRAPHIC REFERENCES: Chiang, Y.L., Kaminsky, L.S., and King, T.E. A Complex of Cardiac Cytochrome c1 and Cytochrome c. J. Biol. Chem. 251, 29-36 (1976). Ohnishi, T., Salerno, J.C., Winter, D. B., Lim, J., Yu, C.A., Yu, L., and King, T.E. Thermodynamic and EPR Characteristics of Two Ferredoxin-Type Iron-Sulfur Centers in the Succinate-Ubiquinone Reductase Segment of the Respiratory Chain. J. Biol. Chem. 251, 2094-2104 (1976).


Recent studies suggest that reparative cells which contribute to healing in the lung following injury also possess regenerative capacity. Although the biology of post-natal lung regeneration is not well understood, mechanical pre-stress appears to be an absolute requirement for tissue regeneration to occur. Emphysema, a disease characterized by tissue destruction, is a potential target for cell-based therapy. This disease is associated with: 1) loss of resident reparative cells; 2) loss of the extracellular matrix that transmits pre-stress signaling; 3) and loss of pre-stress itself, the very signal required to trigger regenerative responses. Although these factors represent obstacles to the development of regenerative therapeutic strategies for emphysema, preliminary studies in our lab show that modulation of mesenchymal and epithelial cell proliferation using members of the fibroblast growth factor family complexed to carrier molecules in a biocompatible polymer can promote expansion of parenchymal tissues. The polymer scaffold, an air containing foam with mechanical properties similar to healthy lung tissue, effectively transmits stress to reparative/progenitor cells to promote proliferation and remodeling. Studies proposed here will test the hypothesis that 1) therapeutic post-natal lung tissue growth in emphysema can be achieved by augmenting the lung's innate healing response using growth factors to direct endogenous reparative lung cells following a localized mild injury; and that 2) the magnitude of this response can be modulated by altering pre-stress using concomitant bronchoscopic lung volume reduction therapy to increase transpulmonary pressures. We intend to advance this approach, known as pneumografting, into human trials under a physician-sponsored Investigation New Drug Application.


A Radiotherapy-Oncology Center has been established at the Tufts-New England Medical Center for the treatment of human malignancies, and to conduct research on the effects of irradiation on normal and malignant tissues. Education on clinical and biological aspects of cancer is carried out at all levels. Active efforts are sustained to cooperate with clinicians in affiliated hospitals in providing optimal care to cancer patients at the community level. Special efforts are directed toward the combined use of surgery, radiotherapy and chemotherapy in the management of various tumors. Research is ongoing in the application of the computer to all aspects of the practice of Therapeutic Radiology. Radiobiologic research centers on the role of the cell membrane in neoplastic conditions. BIBLIOGRAPHIC REFERENCES: Munzenrider, J.E. Fernando Bloedorn, M.D. -master radiotherapist. Tufts Health Science Review b:65-69, 1976. Pilepich, M.V., Bloedorn, F.G., Munzenrider, J.E., Rene, J.B., and Lowry, S.W. Split-course radiotherapy in advanced head and neck tumors. International Journal of Radiation Oncology, Biology, Physics Suppl. 1:31, 1976.


The precise inheritance of genetic material in eukaryotes requires that initiation at each of the hundreds to thousands of replication origins be subject to exquisite regulation so that the DNA is duplicated exactly once per cell cycle. When replication control mechanisms go awry, genomic instability is predicted to occur, but the precise molecular consequences of deregulated replication for genome integrity are completely unknown. Additionally, the full battery of regulatory strategies that govern replication has not been defined. Cyclin dependent kinases (CDKs) are key molecular regulators that both stimulate initiation and inhibit re- initiation of DNA replication. To understand the molecular basis of genome integrity, it is essential to develop a more sophisticated understanding of these CDK-dependent regulatory events. Additionally, it is critical to analyze how disrupting this regulation affects faithful inheritance of the genome. To this end, we have developed several innovative tools that allow us to study the genesis and consequences of re-replication in the budding yeast Saccharomyces cerevisiae. Our Specific Aims are as follows: (1) We have discovered that CDKs target polymerase alpha primase to block re-replication, which challenges the prevailing paradigm that the only strategy used by CDKs to inhibit re-replication is to prevent reassembly of a pre-replicative complex. Thus in this aim, we will characterize a novel replication control mechanism by investigating how CDKs inhibit polymerase alpha-primase to prevent re-replication. (2) We will continue to uncover new strategies for replication control by completing our ongoing screen to identify new CDK targets involved in either triggering initiation or preventing re-replication; this screen has already successfully identified five such targets, including polymerase alpha primase. (3) Using a robust copy number assay, we have obtained the first evidence that re-replication causes a heritable genetic change, namely a gene duplication event that represents an early step of gene amplification. We will exploit this unprecedented opportunity to examine the mechanisms by which re-replication promotes gene amplification. Because gene amplification is a primary means of activating oncogenes in cancer cells, these studies will shed light on the molecular triggers of tumorigenesis, and potentially identify targets of therapeutic significance. [unreadable] [unreadable] [unreadable]


Project Summary/Abstract A compact low cost digital microscope that combines brightfield microscopy and fluorescence is proposed. Innovations in key areas have enabled size and cost reduction, and the merging of brightfield and fluorescent microscopy. The microscope will fit into the CD bay of a personal computer and use a standard interface. An open software architecture will allow others to build applications, methods, and custom interfaces. Development will be complete by the end of Phase I. As proposed, the instrument will have broad applications in the advancement of healthcare technologies. Project Narrative Microscopy forms the underpinning for all life science discoveries. A low cost compact digital microscope will be built that enables wide spread use of this technology, including underserved communities.


Amphetamine and related drugs of abuse elicit species-specific motor responses characterized by repetitive or stereotyped patterns. Research on animals, typically rodents, as models of the human response, has implicated the striatum and related basal ganglia circuitry in the motor-activating effects of these drugs. Critical elements of this circuitry include both dopamine- and glutamate-containing fibers that contact neurons in dorsal striatum. During amphetamine-induced motor activation, these neurons establish a pattern of discharge activity mediated, at least in part, by a complex interaction between dopamine and glutamate inputs. Research in this application extends this line of work on behaving animals in two directions. One involves characterization of the neuronal response pattern to amphetamine in substantia nigra pars reticulata, a major target of striatal neurons and an important output nucleus of the basal ganglia. After basic neurobehavioral correlations are established, further studies will examine the extent to which amphetamine-induced changes in reticulata neurons are mediated by the striatum via descending GABA-containing projections. The aim is to determine how amphetamine-induced neuronal response patterns established in striatum are represented in reticulata neurons. A second focus of the proposed research is to examine at the single-neuron level how the major transmitters altered by amphetamine-- dopamine and glutamate--interact with each other and with GABA to influence the activity of striatal neurons in an intact, normally functioning animal. Dopamine, glutamate, and GABA will be applied directly by iontophoresis to electrophysiologically isolated single units in awake, unrestrained rats. Attention will center on the mechanisms by which synaptic dopamine modulates glutamate- and GABA- mediated responses. A major component of this work also involved iontophoresis of amphetamine and other indirect dopamine agonists in striatum to determine how local changes in dopamine transmission modulate striatal activity and to reveal the synaptic action of these drugs unaccompanied by concomitant activation of other neuronal pathways. Collectively, these lines of research will provide important new information on the neurochemical systems and processes by which amphetamine alters neuronal function and motor behavior.


In mice, many genomic regions contain variation that results in differences in adiposity (Reed 2003; 2006; 2007; 2008). The goal of this research program is to find the gene or genes on mouse chromosome 9 that account for the quantitative trait locus Adip5, which is associated with increased weight of the gonadal adipose depot. Although several lines of evidence suggest that a gene or genetic variant here has the ability to regulate adiposity, the exact gene or DNA sequence that causes this effect is not known. The QTL Adip5 has features that make it a practical target for a positional cloning approach: it is not particularly susceptible to maternal effects or epistatic interactions, and it is associated with a distinct phenotype (gonadal depot weight). Furthermore, the experimental plan is designed to identify Adip5 if the locus is imprinted (i.e., if there are parent-of-origin effects). Within the current Adip5 confidence interval, there are several credible candidate genes (Bbs4, Cpy19a1, Crabp1, Cplx3, Il18, Lipc, Nedd4), as well as dozens of genes and noncoding RNA of unknown function. Using a chromosome 9 substitution strain developed in our laboratory for this purpose (CSS-9), we will backcross these mice to the host strain (C57BL/6ByJ; B6) and conduct a genome scan to reduce the confidence interval of Adip5 (CSS-9 X B6 N2 genome scan; Aim 1). Based on the refined confidence interval provided by the genetic mapping information, we will parse this chromosome into small intervals through successive breeding cycles, and create microcongenic strains (<200 kb), one of which will contain the gene responsible for Adip5 (Aim 2). To identify the exact gene responsible for Adip5, we will genetically engineer one or more mouse strains with a segment of 129 DNA substituted into a B6 background by homologous recombination, and evaluate its effect on gonadal depot weight (Specific Aim 3). The long-range goal of this work is to develop an approach to systematically identify genes that contribute to normal variation in fatness among mice.


6. Project Summary/Abstract The myelination of CNS axons during development and the remyelination of demyelinated axons in adults require oligodendrocyte progenitor cells (OPCs) to migrate to their target axons where they mature into myelinating cells. Although a number of critical factors have been identified for these processes, our understanding of the molecular control of CNS myelination and remyelination remains incomplete. We have identified a zinc finger protein (Zfp191) that when mutated in mice results in the absence of CNS myelin despite the presence of normal numbers of mature, process-extending oligodendrocytes. Zfp191 mouse mutants express an array of myelin-related genes at significantly reduced levels, suggesting that this protein participates in the control of the CNS myelination program. Zfp191 belongs to a family of nuclear proteins whose members contain both DNA binding zinc finger domains and SCAN domains, which are responsible for protein-protein interactions. The goal of this proposal is to gain a better understanding of the role that Zfp191 plays in the myelination process. Zfp191 is expressed in all tissues and cell-types examined, including astrocytes and neurons, and the level of Zfp191 mRNA does not change as OPCs differentiate into mature, myelinating oligodendrocytes. Thus, a critical question that we will address in the studies outlined in this proposal is whether Zfp191 has a cell autonomous function in oligodendrocytes or whether other cell types contribute to the myelin abnormalities displayed by the Zfp191 mutants. Moreover, we will determine if the continued expression of this protein is required for the maintenance of the myelin sheath, and we will also assess if this protein has a similar essential function in the remyelination process. We will also explore the molecular mechanism by which ZFP191 controls the myelination program by determining its DNA and protein binding potential. Relevance: The studies described in this proposal will focus on the molecular control of the final stages of oligodendrocyte maturation, which result in the initiation of the myelination program. A better understanding of the factors that enhance oligodendrocyte maturation is essential in our effort to develop strategies to promote axonal remyelination in demyelinating neurological disorders (e.g. multiple scelrosis).


Our group will continue our studies directed to the understanding and control of latent human herpesvirus infections. We will focus on herpes simplex and human cytomegalovirus. For herpes simplex infection, we will study herpes genitalis in both humans and the guinea pig model of infection. We will continue to study gamma interferon as a correlate of disease containment in both systems as well as investigating the possibility of augmenting immunity conferred by isolated viral glycoproteins with immunomodulators such as interleukin-2. For human cytomegalovirus, we will continue to study the human immune response to two glycoproteins identified in the previous granting period. One glycoprotein is 86 kDa (p86) and the other is a complex of glycoproteins of 130 and 55 Da (p130/55). Our aim is to establish whether humoral and cellular immune responses to these specific glycoproteins correlate with protective immunity. We will also analyse the importance of gamma interferon in human cytomegalovirus infection to determine whether it correlates, as in herpes labialis, with containment of virus. We will examine whether it correlates, as in herpes labialis, with containment of virus. We will examine whether gamma interferon contributes to immunopathology of human cytomegalovirus infection in allograft recipients. Finally we will develop anti- idiotype monoclonal antibodies to the murine monoclonal antibodies with virus neutralizing activity described in our laboratory. We will use these anti-idiotype monoclonals as possible adjuvants for immunization with either the p86 or p130/55 as well as new probes for studying the regulation of the immune response following human cytomegalovirus infection. We will also use them to attempt to identify a cellular receptor for human cytomegalovirus.


PROJECT SUMMARY/ABSTRACT During dynamic exercise skeletal muscle blood flow increases rapidly and dramatically (exercise hyperemia) to meet the metabolic needs of the contracting tissue. Aging is associated with an attenuated hyperemic response during dynamic exercise. The mechanisms responsible for increasing blood flow at the onset of exercise as well as maintaining it over time in young adults involves a complex interaction between mechanical factors, the sympathetic nervous system and local metabolic and endothelial derived substances that influence vascular tone. The mechanisms responsible for the observed reductions in exercise blood flow in older humans are not completely clear. The applicant proposes two main goals: 1) to identify mechanisms contributing to the altered vasodilator responses to single muscle contractions and dynamic exercise in aging humans, and 2) to examine the effect of aging on the kinetics of skeletal muscle blood flow/vasodilation during exercise. During the K99/Mentored phase of the grant, the applicant will examine the mechanical, endothelial, and neural alterations in vascular function that occur with aging and determine how these changes relate to the attenuated rapid vasodilator response following a single muscle contraction. In the first portion of the R00/Independent phase of the grant, the applicant will examine the kinetics (rest to steady state transition) of vasodilation during rhythmic exercise and quantify the effects of aging on these responses. In the second portion of the R00 phase, the applicant evaluate whether the attenuated vasodilator response to single muscle contractions and slower kinetics of vasodilation during rhythmic exercise are similar in the upper and lower limbs of older subjects. Lastly, in the third portion of the R00 phase, the applicant will determine whether the changes in flow following single contractions and/or the kinetics of vasodilation in older humans is a result of physiological aging or related to training status. Collectively, the experiments outlined in this proposal focus on the mechanical, endothelial, and neural alterations that occur in the skeletal muscle vasculature with aging and how these changes impact blood flow in exercising muscle. Identifying the mechanisms by which blood flow to contracting muscles is altered with advancing age will help in understanding whether these changes are due to physiological age per se or a result of inactivity. During the K99/Mentored phase of the award the applicant will 1) continue to gain expertise in basic integrative physiology studies in conscious humans, and 2) continue to learn pharmacological and biochemical approaches to study the control of muscle blood flow from a mechanistic standpoint. Additionally, the candidate will gain new research skills and knowledge related to advanced cutting-edge ultrasound techniques and measures of arterial properties (specifically, Shearwave Dispersion Ultrasound Vibrometry; SDUV) under the mentorship of Dr. James Greenleaf (co-mentor). Training in an established and productive laboratory such as that of Dr. Michael Joyner along with the help of Dr. James Greenleaf at the Mayo Clinic will provide opportunities needed to achieve the goals listed above. Importantly, this training will facilitate the achievement of the applicant's long-term goal to develop an internationally-renowned independent research program in cardiovascular physiology.


Naltrexone, an opioid antagonist, is currently one of the most promising pharmacotherapies for alcohol dependence. In clinical trials, naltrexone has been shown to be more effective than placebo in reducing alcohol consumption and relapse rates. A number of critical questions remain, however, about the optimal strategies for using naltrexone in the treatment of alcohol dependence. The proposed research is designed to achieve four specific aims: (1) to evaluate whether a more intensive psychotherapeutic strategy provided with naltrexone enhances initial treatment outcomes in contrast to a less intensive intervention that may be used with naltrexone in primary care settings; (2) to evaluate whether long term treatment with naltrexone provides additional benefit in preventing relapse, alcohol-related impairments, and alcohol related psychiatric symptoms among those who respond to short term naltrexone treatment; (3) to explore predictors of response to the psychotherapeutic interventions and to naltrexone, including gender, craving, neuropsychological functioning, and severity of alcohol dependence; and (4) to evaluate the safety of long-term naltrexone treatment in an alcohol dependent sample. In order to address the question of treatment intensity, 192 subjects will be randomized to receive naltrexone and either weekly coping skills therapy or advice and clinical management for 10 weeks. The coping skills therapy is a treatment commonly used in specialty alcoholism treatment clinics and is designed to increase the patient's coping strategies in an effort to reduce the probability of relapse. Advice and clinical management is a less intensive treatment that is consistent with primary care treatment. Following completion of this study, two parallel discontinuation studies will be conducted in which treatment responders in each group are then randomized to receive naltrexone or placebo for six months. One study compares the outcomes of subjects maintained on naltrexone versus those on placebo among those initially treated using the advice and clinical management. The second study compares outcomes for naltrexone and placebo maintained subjects who were initially treated with coping skills therapy. Taken together, these studies should provide critical information about psychotherapeutic strategies to provide in conjunction with naltrexone and the optimal length of naltrexone treatment.


The Dutch Famine ('Hunger Winter') of 1944-1945 provides a unique opportunity to identify early life determinants of life course health and well-being. To date, studies have concentrated on health and mortality outcomes in ~45,000 army recruits who were examined at age 18 in the early 1960s. Famine exposure in these men can be inferred from place and date of birth. Funding is now requested to link a wide variety of socio-economic outcomes (employment, wages, and disability benefits) from government sources to our studied population. The study is collaboration between epidemiologists and demographers and economists (Dr. Heckman, University of Chicago). The newly linked data will be analyzed with state-of-the-art epidemiologic and econometric approaches to reliably identify, estimate, and interpret the effects of an early life shock throughout the lifecycle. This work will integrate currently separated research traditions from the medical and social sciences and lead to a better understanding of 'fetal programming' and its implications.


The Virginia Department of Mental Health, Mental Retardation and Substance Abuse Services proposes to enhance the data collection systems of the State's 40 community services boards. The goals of the project include implementation of the MHSIP recommended data sets and standards, improvement in the quality and reliability of data, and increased use of data and integration of data between community programs and inpatient facilities. The project will employ two full-time staff persons working with a Steering Committee of Community Services Board representatives. Activities will include modifying existing CSB data systems to meet MHSIP standards, developing a human resources data base, developing data audit guidelines and disseminating data analyses and reports.


We are studying regulation of sexuality in the male rat and are carrying out projects in the following areas: (1) Mechanism of action of androgens in the seminiferous tubules: Measurement of concentration and ratios of testosterone and 5 alpha-dihydrotestosterone in cytosol and nuclei, both bound and unbound, of seminiferous tubules at specied periods after hypophysectomy, and comparison with androgen concentrations in plasma, in effort to carrry out successful nuclear exchange assay and to assess role of androgen bindng in spermatogenesis. (2) Hormonal rhythms in the male laboratory rat: We are completing our second year of studying the pattern of matuation of plasma testosterone, LH and prolactin in rats from age of 30 through 150 days, in order to detect basic maturational pattern and influence of season and calendar year on this pattern. (3) Mating and the hormone neuroendocrine reflex: We are using the reproducible rise of plams testosterone, LH and prolactin at time of mating as a model for the study of its restoration in castrates by steroid replacement. (4) Peripheral regulation of plasma testosterone: The testicular-denervated, adrenal demedullated male rat is being tested both with stimulation of the preoptic nucleus and mating in order to evaluate CNS regulation of the denervated testis. BIBLIOGRAPHIC REFERENCES: Frankel, A.I., E.J. Mock, W.W. Wright and F. Kamel, 1975. Characterization and physiological validation of a radioimmunoassay for plasma testosterone in the male rat. Steroids 25:73-98. Mock, Edward J., Freja Kamel, William W.Wright and Arthur I. Frankel, 1975. Seasonal rhythm in plasma testosterone and luteinising hormone of the male laboratory rat. Nature 256:61-63.


The goal of the proposed research is to examine how exposure to parental violence operates within the contest of additional risk factors (low socioeconomic status, maternal depression, ;ack of moth-child relationships in the relation between exposure and children's social behavior. The aforementioned risk factors, aggressive expectations, severity and duration of violence witnessed, aggressive behavior problems, and social competencies of 150 children from violent homes will be compared to those of a community group of children where conflict ranges from verbal discord to low level aggression and does not include violence. Parents, shelter counselors, and teachers will complete several measures regarding family violence and their child's functioning, mothers will complete additional measures assessing their degree of depressive symptomatology and their perceptions of moth-child relationship positivity, and children will complete several measures assessing their aggressive expectations in peer relationships, mother-child relationship positivity, and their perceptions of warmth and conflict in their close peer relationship. This research has implications for improving models of the processes through which parental violence impacts on development and for informing prevention and intervention strategies to help child witnesses adjust to their experiences.


Project Summary/Abstract The overall goal of the Cancer Outreach Core (COC) is to reduce cancer health disparities among vulnerable migrant Pacific Islander populations living in Guam (GU) and Hawaii (HI). They are the Chamorro, Marshallese and Chuukese; the indigenous peoples of Guam, the Republic of the Marshall Islands (RMI), and Chuuk State in the Federated States of Micronesian (FSM), respectively. There are over 16,000 migrant Pacific Islanders from the RMI and from the FSM living in GU and over 17,000 living in HI. They have a disparate cancer health status compared to the rest of the populations in HI, GU and the Continental US. The cancer disparities of interest are preventable cancers which have a significant incidence in the Micronesian populations. As an example, the Pacific Islander women from the RMI and FSM have among the highest incidence rates of cervical cancer in the world: 79.7 per 100,000 and 42.4 per 100,000 (2007-2011, age-adjusted to the 2000 US Standard) in the RMI and in Pohnpei State, FSM, respectively, compared to 9.9 per 100,000 women in the United States (US) (1). The COC will build on the 2003-2015 University of Guam (UOG)/University of Hawaii Cancer Center (UHCC) Comprehensive Partnership efforts to prevent and control cancer in Pacific Islander communities. Through established community partnerships, the COC has three main tasks: (1) outreach to primary care physicians (PCP) to reduce preventable cancers in Chamorro, Marshallese and Chuukese peoples, (2) outreach to the Chuukese, Marshallese and other health disparity communities to increase HPV vaccination rates among adolescents, and (3) assist U54 investigators with recruitment of Pacific Islander populations in support of studies focusing on cancer health disparities. Community Health Educators (CHE) who have worked with the target communities will support the COC outreach strategies and the NCI National Outreach Network's coordinated agenda. The COC activities align with the overall goals of the Comprehensive Partnership to Advance Cancer Health Equity (CPACHE), and support hypothesis-driven research as proposed elsewhere in this application (e.g. Pilot Project I, Full Project II).


Recent events such as pandemic influenza A (H1N1)pdm09 have demonstrated how mutations in a viral genome can greatly impact disease spread and population health risk. Thus, there is now a greater need to merge viral genetics within state health agency surveillance practice. This is particularly relevant for zoonotic viruse that are transmittable between animals and humans such as influenza, rabies, and West Nile Virus. As an added complexity, there are many potential drivers of virus transmission that need to be considered including climate, population and travel, and ultimately, genetic polymorphisms in the virus itself. Zoonotic disease surveillance at the state level is most often performed using data that originates from passive case reporting by laboratories or clinicians rather than secondary data from resources such as GenBank. While these data are sufficient for federal reporting purposes and basic trend analysis, they only measure the number of suspected or confirmed cases and not the genetic characteristics of the virus. When states and federal agencies do use genotyping, it is often limited to certain pathogens (mostly bacteria) and only for samples that are reported through passive surveillance or during outbreak investigations. The omission of secondary viral genetic data limits the types of analysis by state health agencies. For example, current reportable disease data do not enable epidemiologists to determine the origin of a particular viral strain, trace how it has spread, or identify climate, population, and genetic factrs enabling it to propagate. In this study, we will develop and evaluate an integrated bioinformatics framework to supplement current zoonotic disease surveillance approaches at state health agencies. We hypothesize that a framework that properly merges viral genetic data with climate, population, and travel data can accurately predict the timing of initial peaks of seasonal epidemics caused by zoonotic viruses. Health agencies can then use these trends to prioritize control measures and reduce morbidity and mortality. In addition, we will address the barriers to health agency utilization of bioinformatics resources and secondary data by developing an online portal for accessing and querying of complex viral genetic models. We will measure the perceived usefulness of information from our framework as part of our long-term goal of utilization and adoption by health agencies. In Aim 1, we will develop an automated bioinformatics system that models virus diffusion while testing the significance of climate, population, and genetic predictors. As part of this effort, we will provide a publically available Web portal for health agencies and other users to access our results, and run their own models. In Aim 2, we will use our platform to identify significant climate, population, and genetic predictors of diffusion across different zoonotic viruses including influenza and WNV. In Aim 3, we will evaluate the accuracy of a bioinformatics system that uses statistically significant climate, population, and genetic predictors to identify seasona trends of zoonotic virus epidemics and communicate these findings to different health agencies.


The beta thalassemias are characterized by a deficiency of adult (beta) globin chains of adult hemoglobin (Hb), an excess of toxic, unmatched alpha globin chains, and intramedullary hemolysis. The resulting anemia develops only after fetal (gamma) globin synthesis and Hb F is suppressed in infancy. Induction of fetal (gamma) globin to levels which improve globin chain balance by even 10 percent can prolong red blood cell survival and diminish clinical morbidity. 5-Azacytidine has increased hemoglobin (Hb) levels by 1.8-3 gmd/d1 in thalassemia, but also causes general cytopenias and carries carcinogenicity risks. Fatty acids induce (gamma) globin experimentally. Arginine Butyrate, a prototype fatty acid, has been most effective when given intermittently or Pulsed, inducing Hb F to a mean level of 22 percent in 7/9 adults with sickle cell disease and increasing total hemoglobin by 3 gm/dl over baseline levels in 5/6 beta thalassemia patients. Two clinical pilot studies are proposed to test the hypotheses that therapy with Pulsed Butyrate, or rhu-EP0 + Pulsed Butyrate, will induce gamma globin chain synthesis sufficiently to improve non alpha: alpha globin chain balance and red blood cell survival, and increase total Hb in a significant proportion of patients with beta thalassemia intermedia. Baseline hematologic levels will be assayed four times over a two-month period. Butyrate will then be administered during an Induction Phase, to determine a patient's optimal dose, followed by a "Maintenance Phase" of therapy for 3 months. Pulsed Butyrate will also be tested with rhu-EPO. The proportions of patients on each study in whom the following endpoints are achieved, compared to baseline levels, will be analyzed: 1) an increase in total Hb of at least 2.0 grams/dl, 2) an increase in hematocrit of at least 5 percent, 3) a decrease in hemolysis, measured by LDH and bilirubin, 4) improvement in globin chain synthesis by 10 percent. Whether specific genotypes and in vitro response to Butyrate correlate with clinical responses will also be analyzed. These studies should determine the proportion and some genotypes of beta thalassemia patients which can benefit from Pulsed Butyrate plus/minus rhu-EP0 therapy.