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To evaluate strategies that test the safety and effectiveness of therapeutic approaches/regimens to reduce the probability of the emergence of antibiotic drug resistance by minimizing unnecessary drug exposure. Trial title: A Multi-Center, Randomized, Open-Label, Comparative Study to Assess the Safety and Efficacy of a Treatment Algorithm to Reduce the Use of Vancomycin in Adult Patients With Blood Stream Infections Due to Staphylococci.

Our goal is to scintigraphically detect, stage and ultimately treat ovarian carcinomas, using radiolabeled monoclonal antibodies preferentially reactive with epithelial ovarian carcinomas. Monoclonal antibodies raised against ovarian cancer, already shown capable of radiolocalizing human ovarian carcinoma xenografts in nude mice, will be further evaluated in that model. The effects of antibody dose, route of administration (intravenous as compared to intralymphatic or intraperitoneal), fragmentation, and labeling, will be evaluated to optimize antibody localization to tumor. Since ovarian carcinoma is generally a regional disease within the peritoneum and draining lymphatics, we expect this regional antibody delivery to significantly enhance tumor localization. Antibody localization will be evaluated by gamma camera scanning, tumor excision with gamma counting, immunoperoxidase staining, and microautoradiography. In parallel experiments, the specificity of existing and new monoclonals raised against ovarian cancer will be further evaluated by immunohistochemical techniques. The radiotherapeutic potential of the best monoclonals for imaging will then be evaluated in vitro using clonogenic assay techniques. The dose of radiolabeled antibody required to produce tumor kill of ovarian carcinoma lines in vitro will be determined. Based on these studies, the best antibodies will then be evaluated for radiotherapeutic potential in our nude mouse model of disseminated intraperitoneal human ovarian carcinomatosis. A variety of dosing schemes will be tested therapeutically with evaluation for the number of clonogenic ascites cells, mean survival, and cure as indices of the efficacy of radiotherapy. The monoclonal antibodies with the best localization to ovarian carcinoma xenografts in vivo, as well as showing the greatest in vitro specificity for ovarian cancer, will be evaluated in phase I imaging trials in patients with advanced ovarian cancer who are scheduled for a second debulking procedure. This will assure histologic, autoradiographic and gamma scan assessment of antibody localizaiton to tumor. This study will provide unique information regarding the optimal means of delivering radioantibody to ovarian tumors and about these tumor's sensitivity to radioantibody. This should result in a means of scintigraphically following residual ovarian carcinoma post-diagnosis and may eliminate the need for second-look diagnostic procedures. If adequate specific localization of radioantibody to ovarian cancer is achieved, then this approach may prove useful in the radioimmunotherapy of this common and lethal disease.

ABSTRACT Autism spectrum disorder (ASD) is a devastating neurodevelopmental disorder of undetermined etiology and without effective therapeutics. Recent advances in genomic approaches have led to the identification of over 65 ASD risk genes. Despite the genetic heterogeneity of ASD, several lines of evidence suggest that these genes share common molecular underpinnings. Therefore, we hypothesize that these genes will converge upon shared phenotypes when inhibited in model organisms and that these phenotypes will be the most central to the neuropathology of ASD. Identifying such convergent phenotypes requires a high-throughput system for modifying many ASD genes in parallel and assaying their effect(s) on embryonic brain development. Here we propose to leverage the diploid vertebrate tetrapod, Xenopus tropicalis, and the CRISPR/Cas9 system to identify convergent phenotypes among ASD genes during brain development. By injecting Cas9 protein and a single guide RNA (sgRNA) against an ASD gene at the two-cell stage, animals will be generated in which exactly half the body (separated by the midline) is mutant, allowing for direct comparison of mutant and control cells in the same animal. This will be performed for approximately 65 ASD risk genes, and the effects of ASD gene loss will be assayed by imaging neurons throughout embryogenesis using fluorescent reporters and by in situ RNA hybridization for neuronal cell fate specification markers. By employing fluorescence activated cell sorting (FACS), next generation RNA sequencing, and weighted gene co-expression network analysis (WGCNA), convergent transcriptional signatures of ASD gene loss will be characterized. The biological pathways indicated by these convergent signatures will be validated through targeted manipulation of key genes. Importantly, the validated biological pathways may provide clues about the observed convergent phenotypes. By combining the high-throughput capability of the CRISPR/Cas9 system and a tractable vertebrate model organism with a reliably-associated set of ASD genes, this study aims to understand the neuropathology of ASD at a critical developmental period, which should provide critical insights into the etiology of this disorder.

Chronobiology is the study of biological rhythms. There are a number of chronobiological effects which may have important clinical implications for antitumor therapies. For example, chronobiological effects on the replication of some types of tumor cells, the expression of certain drug toxicities, and the number of cytotoxic effector cells have been described. Interferon therapies are currently being evaluated in clinical trials for their potential as antitumor agents. This proposal focuses on possible chronobiological effects which may effect the potencies of various interferon therapies. We propose to evaluate the chronobiological effects on tumor growth of interferons administered (1) as single agent therapy; (2) as multiple interferon therapies (IFN-gamma plus IFN-alpha or IFN- beta); (3) as multiple agent therapies with hyperthermia; and, as multiple agent therapies with phenytoin. These studies will be performed both in vitro, using B-16 melanoma cells in cloning studies, and in vivo, using the B-16 melanoma/C57/B1/6 tumor system. Simultaneous treatments and various sequential treatments will be compared to identify the most potent treatment protocol. We also propose to evaluate the chronobiological effects on IFN-mediated bone marrow suppression of colony stimulating factor administration. These studies will be performed both in vitro using bone marrow colony growth assays and in vivo using total white blood cell counts as an indicator of bone marrow suppression.

The long-term goal of the laboratory is to gain a structural understanding of the molecular organization and function of biological macromolecules at the biological membrane interface. Our focus of this study is to understand physical mechanisms of ion selectivity, conductance regulation, channel gating of ion channel proteins, and the structural basis of molecular specificity for oligomerization by X-ray crystallography. The first specific goal is to determine the three-dimensional structure of the tetramerization domain of Shaker potassium channel. We have obtained three-dimensional crystals of the soluble domain of this channel that diffract X-ray beyond 2.0 Angstroms. Crystals belong to the space group I4 with one subunit molecule per asymmetric unit. We have also obtained three-dimensional crystals of the soluble, domain of a channel from a different subfamily of potassium channel Shaw that diffract X-ray to about 3.0 Angstroms. The second specific goal is to determine the atomic structure of an inwardly rectifying potassium channel ROMK1 from rat kidney. Towards this goal, the underlying hypothesis of our novel strategy is that the lipid- facing exterior of a channel protein can be mutated systematically without perturbing its channel function. Site-directed mutagenesis was used as a perturbation method in order to identify the lipid-facing residues. Based on this study, simultaneous changes have been introduced at sites in its transmembrane lipid-facing exterior of the channel, which do not alter the channel function. The surface of this exterior-modified channel has a sequence motif known to form a specific helix-to-helix interaction, through which the exterior-modified channel itself is solubilized by binding of synthetic amphipathic peptides to the channel exterior in the aqueous phase without the use of detergents. Understanding the structural basis for tetramerization will provide an essential knowledge on the physical nature of channel diversity. From the atomic model of the ROMK1 channel, we seek to establish the exterior- modification strategy that will be generally applicable to structural studies of a variety of integral membrane proteins such as ion channels and transmembrane receptors.

Neutrophil priming following trauma and sepsis is a key event implicated in causing Adult Respiratory Distress Syndrome (ARDS) and Multi-Organ Failure Syndrome (MOSF). Priming of the respiratory burst by cytokines following injury and sepsis results in excessive superoxide production by NADPH oxidase leading to auto-inflammatory tissue damage. Numerous studies have focused on details of how NADPH oxidase produces superoxide. A similar investigation into the mechanisms of priming has been relatively lacking. Our long-term goal is to develop a detailed molecular understanding of this priming phenomenon. Our previous studies and preliminary observations show that different signaling pathways activated by trauma and sepsis converge to phosphorylate two serine residues in a key protein of the NADPH oxidase, p47phox. We propose that through this phosphorylation, p47phox integrates upstream signaling events to control the level of NADPH oxidase activity by regulating protein-protein interactions within neutrophils. In the studies outlined in this proposal the consequences of priming-induced phosphorylation and the identity of proteins associating with NADPH oxidase components following priming will be investigated using neutrophil cell-biological and protein chemistry approaches. The results from these studies may assist in the development of novel therapies aimed at limiting the auto-inflammatory tissue damage patients suffer as a result of sepsis and trauma.

During this period, the collaborative team is working to validate and characterize previously identified hits from primary SENP screening. As a center, the NCGC has fostered and maintained over 130 active collaborations with both NIH and extramural investigators, facilitating drug discovery efforts across the entire spectrum of human disease. These efforts have led to dozens of high-throughput screens and a number of medicinal chemistry campaigns to further improve on screening hits, providing our collaborators and the general research community with publications and a variety of promising small molecule probes and leads. In addition, the NCGC has worked to advance a number of informatic initiatives to make better use of existing drug and disease target information and provide the general public with easily accessible resources, further catalyzing the development of new therapies for human disease.

OBJECTIVES: 1. To disseminate and coordinate sickle hemoglobin education in the community; 2. To counsel patients with sickle cell disease and their families and establish ongoing relationships with hospitals, clinics and other health agencies; 3. To develop educational materials on sickle hemoglobin to be used locally, nationally and internationally; 4. To make available to the medical community and general public a resource for the laboratory diagnosis of sickle and other hemoglobin variants; 5. To determine the molecular structure and intermolecular contact sites of hemoglobin S in sickle cell fibers; 6. To understand the mechanisms of translational and transcriptional control of the synthesis of specific hemoglobins; 7. To study the renal adaptation of patients with sickle cell anemia to overproduction of uric acid and the association of hyperuricemia and gout; 8. To determine monocyte bactericidal capacity in patients with sickle cell anemia and restore same in vitro by incubation with sickle serum cleared of red cell fragments; 9. To ascertain whether variations in severity of anemia among patients with sickle cell anemia are due primarily to differences in rate of red cell production or in red cell destruction and whether regulation of erythropoiesis is defective in such patients; 10. To study the role of prophylactic transfusion therapy in care of pregnant patients with sickle cell anemia.

Alveolar epithelial cells (AEC) cover the alveolar surface of the lung, which is the major acid-excreting organ in mammals. Because these cells are subject to an inconstant extracellular milieu with respect to pCO2, the regulation of intracellular pH (pHi) in alveolar epithelial cells is of particular significance. In previous work it has been established that alveolar pneumocyte pHi is regulated by several ion transport mechanisms that are capable of effecting acid/base entry or extrusion from the cell. These ion transport mechanisms are plasma membrane proteins asymmetrically distributed across polarized epithelial cell surfaces, thereby making transepithelial transport of acid/base equivalents possible. Their distribution with respect to membrane polarity across the alveolar epithelium, whose apical lining fluid pH is acidic (6.9), has not previously been established. We will investigate the mechanisms whereby acid/base transport by AEC is regulated by studying three hypotheses: (1) Acid/base transport by AEC occurs via specific ion transport mechanisms having a characteristic distribution with respect to membrane polarity across the cells, and which represent the activity of specific transport protein isoforms. (2) These ion transport mechanisms are both acutely and chronically regulable in AEC with respect to activity, expression, and polarity by specific extracellular stimuli that can thereby modulate acid/base transport under normal conditions and in disease states. (3) Regulation and localization of acid/base transport mechanisms in AEC influence alveolar fluid pH. The objectives of the studies proposed herein are therefore to further characterize the acid/base transport mechanisms in AEC, and in particular, to study regulation of their activity, expression, and polarity by specific exogenous factors and lung injury. We will use rat alveolar epithelial cell monolayers cultured on tissue culture-treated polycarbonate filters for many of these studies. This model is especially well suited for studies on alveolar epithelial polarity and acid/base transport. We will validate our findings using in situ studies on rat alveolar epithelium. The long term goals of this project are to define the importance of acid/base transport in intracellular homeostasis of alveolar epithelial cells, and to demonstrate the role of acid/base transport by alveolar epithelial cells in modulation of the extracellular alveolar milieu.

Abstract - Project 4: Cure rates for pediatric patients with relapsed or metastatic solid tumors remain unacceptably low. Cancer immunotherapies hold great promise, but scores of disappointing studies highlight our relative ignorance in understanding the immunosuppressive microenvironment within solid tumors. Because of their central role in mediating immunosuppression, tumor associated macrophages (TAMs), typically ?polarized? to a so-called M2- like immunosuppressive phenotype, and myeloid-derived suppressor cells (MDSC), are thought to be important therapeutic targets. We have found a clinically viable strategy that simultaneously reduces TAMs/MDSC (we dub ?myelolytic?) and polarizes the microenvironment (via oncolytic virus infection), resulting in significant antitumor efficacy. We hypothesize that targeting TAM and MDSC by combining ?myelolytic? therapies with pro- inflammatory therapies activates innate antitumor mechanisms that cause cancer regressions and reshapes the solid tumor microenvironment to be more permissive to cellular immunotherapies. In aim 1, we will determine the mechanism(s) by which combined myelolytic-virotherapy drives tumor regressions. We will use novel technologies such as fluorescent nanodiamonds to determine effects on innate immune cell phagocytosis of tumor cells. We will utilize the Genomics & Immune Monitoring Shared Resource Core B directed by Dr. Elaine Mardis to conduct flow cytometry with time-of-flight mass spectrometry and single cell transcriptomics to determine the effects on immune cell composition and polarization. We will also utilize gain- and loss-of-function approaches to determine if loss of MDSC are critical for enabling tumor regressions with myelolytic-virotherapy. We will also test combination therapies in xenograft and immunocompetent models of other cancer types to confirm its generalizability (osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, neuroblastoma). In aim 2, we will determine the effects of myelolytic-virotherapy on T cell-mediated immunotherapies. We will examine the effect of myelolysis alone and combined with virotherapy on the efficacy of antitumor T cells in a T cell exhaustion setting and with CAR-T cells (with Project 1 Leader Dean Lee and co- investigator Ruoning Wang, PI-DDN U01 member). We will work with Core B to examine the effects on T cell clonality using TCR sequencing. In aim 3, we will determine whether combined myelolytic-virotherapy enhances the efficacy of NK-based cellular therapies. We will work with Project 1 Leader Dean Lee and Project 2 Leader Mitch Cairo to study the effects on adoptive NK and CAR-NK cell therapy. Overall, with this project we will further elucidate, test and develop strategies to modulate the tumor microenvironment to facilitate innate immune cells as cancer therapy. Our findings may be applicable across a broad panel of pediatric cancer types and thus fits well into the aims of the Pediatric Immunotherapy Discovery and Development Network.

The pulmonary involvement in disorders of immune or inflammatory regulation can range from a primary manifestation such as with sarcoidosis or a relatively minor manifestations such as seen with the Mendelian Susceptibility to Mycobacterial Disease immune deficiencies. While both of these can be associated with granulomatous inflammation in the lung, the clinical pulmonary manifestations are quite different as are the management strategies. The Pulmonary Clinical Medicine Section (PCMS) has sought to capitalize on the close collaboration with the Laboratory of Clinical Infectious Diseases and other branches within the NIAID and NIAMS focused on these disorder to describe the pulmonary manifestations of known and emerging immune and inflammatory diseases. This has included characterizing the lung manifestations of newly described immune dysregulatory syndromes associated with mutations in such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and phosphoinositide 3-kinase (PI3K)and well known and characterized disorders such as chronic granulomatous disease. The PCMS has been actively involved in characterizing the protean lung manifestations of GATA Binding Protein 2 (GATA2) deficiency have provided unique insight into the role of alveolar macrophage dysfunction in development of pulmonary alveolar proteinosis and intracellular control of mycobacteria and fungi in the lung. Ongoing work in this area include characterization of lung manifestations of Autoimmune Lymphoproliferative Syndrome (ALPS) and Autoimmune Polyendocrinopathy, Candidiasis, and Ectodermal Dystrophy (APECED) Syndrome.

RESEARCH EDUCATION CORE SUMMARY The goal of the Research Education Core is to attract and catalyze a new generation of diverse research professionals and biomedical scientists who are committed to elucidating and addressing the cancer disparities outcomes faced by so many in South Carolina and the US. During the five-year U 5 4 project period, undergraduates at South Carolina State University (SCSU) and junior faculty at SCSU and the Medical University of South Carolina (MUSC-HCC) will be designated as South Carolina Cancer Disparities Research Center (SC CADRE) Scholars. Building from multiple ongoing efforts, the leaders of the SC CADRE are proposing a dual-level, integrated Research Education Core with an explicit focus on cancer health disparities. Expanded research education and training opportunities will be directed to both levels of Scholars. To realize the goal of the SC CADRE Research Education Core, the SCSU and MUSC-HCC Core Co-Leads plan to establish a new undergraduate SC CADRE Honors Program in Applied Oncology Sciences Research within the Department of Biological and Physical Sciences at SCSU to increase the pipeline of highly skilled, diverse undergraduates who pursue careers in biomedical research and cancer disparities. A new cancer research career development program at SCSU and MUSC-HCC will also be implemented for junior faculty who are interested in establishing independent careers in cancer disparities research. Within the Research Education Core, the SC CADRE undergraduate and junior faculty Scholars will interact as they participate in coursework and/or career development programming, weekly SC CADRE Research Club meetings, monthly seminar series, and an annual SC CADRE Cancer Health Equity Research Symposium. Through the Research Education Core, SCSU will hire two new junior faculty Scholars and MUSC-HCC will hire one new junior faculty Scholar, all of whom will be early-stage investigators (ESIs). Both levels of Scholars will actively engage in numerous SC CADRE enrichment activities and activities planned by the Community Outreach Core, which will work directly with Research Education Core leadership to s t r e s s the importance of community engagement in developing, conducting, and disseminating cancer disparities research.

Understanding how memory develops in the immune system is an important issue in basic science and in clinical medicine, both in terms of promoting an immune response and, conversely, in blocking the pathway that leads to an overactive state (allergy, autoimmunity). However, the mechanisms underlying the generation of memory lymphocytes are poorly understood. This proposal is aimed at answering the important question of whether the generation of memory T cells is associated with a stable alteration of cytokine expression. We focus here on the generation of mouse CD4+ memory T cells that help B cell antibody responses (memory helper T cells) in relation to attainment of the capacity to secrete IL- 4. We wish to distinguish whether there are pre-existing CD4+ subsets committed to this IL-4 potential, or instead whether such subsets are generated de novo by a maturation process following antigenic stimulation. We will examine TCR-V gene usage of CD4+ subsets by establishing T hybrids from a 2B4 TCR Vbeta (or Valpha) transgenic mouse to determine whether the cell subsets capable of rapid IL-4 secretion ("IL-4+") show restricted TCR V-gene usage, evidence for antigenic selection. Then we will attempt a direct demonstration of IL-4- naive cell transition to IL-4+ cells at the single cell level. Finally, we will examine whether memory T helper cells are already committed to a specific cytokine potential or not. If we can demonstrate that IL-4+ cells are produced during the process of memory T cell generation, then this provides an important concept of lymphocyte maturation; that is, specialization of effector function similar to memory B cell generation characterized by immunoglobulin isotype switch. These studies will provide greater understanding of the generation and function of memory cells in the immune system, both in normal and diseased states.

We established that neurons present in dorsal root ganglia (DRG), similar to leukocytes, express a wide variety of receptors for cytokines, chemokines, opioids, anandamide and other neuropeptides. We previously showed that prior exposure to chemokines such as MIP1αresults in PKC mediated desensitization of the chemotactic response to opioids by opioid receptors, and thus potentially enhances pain. This decrease in the analgesic effect of opioids was evident from the enhanced tail flick assay of rats administered MIP1αor RANTES prior to an analgesic opioid into the PAG of the CNS. We then extended these earlier studies by showing that prior administration of chemokines Asensitized and primed the calcium flux of capsaicin or anandamide stimulated vanilloid (TRPV1) algesic receptor on DRG neurons. This response also increased pain as shown by the enhancement of paw withdrawal in response to the intrathecal administration of the chemokine prior to capsaicin in vivo. This sensitization of the vanilloid receptor was also PKC dependent. Consequently, proinflammatory chemokines can increase pain both by suppressing opioid and enhancing vanilloid receptor responses. Based on these studies, we predicted that the anti-inflammatory effects of adenosine, which also interacts with GiPCR, might have effects on chemokine receptors. Indeed our studies showed that prior addition of adenosine results in suppressing the in vitro chemotactic response of leukocytes to a variety of chemokines. Furthermore, prior in vivo injection of adenosine inhibited the in vivo influx of leukocytes into a murine air pouch by about 90%. This cross-desensitization of chemokine receptors by adenosine A2a receptors was PKA dependent. The role of adenosine as an immunosuppressive effector molecule also has been reported to mediate the cell contact dependent effects of Tregs and to interfere with host resistance to tumors. Thus, studies of adenosine effects are relevant to tumor biology and immunology. These studies therefore reveal novel pathways of receptor mediated intercommunication of inflammatory as well as painful stimuli. Means of interfering with these PKC and PKA dependent signals and the pathophysiological relevance of this receptor cross-talk to inflammation and pain need to be further evaluated. Our current project focuses on neuroimmune interactions contributing to pain sensation in cancer patients funded by an INIP postdoctoral IRA financial grant from NIAID and NCI. As previously shown, chemokine receptor cross-talk suppresses analgesic opioid receptors, but enhances algesic transient receptor potential channel (TRP) receptors, thus resulting in painful inflammation. a) In collaboration with Dr. Jeffrey Cohen, NIAID we have investigated this in a cotton rat herpes virus infection model for chemotherapy induced Herpes Zoster. Herpes infection of dorsal root ganglia results in extremely painful inflammatory responses along nerve tracts. It has been reported that VZV infection produces TLR ligands and we have found that peripheral neurons present in dorsal root ganglia express TLR3, 7 and 9 which when stimulated express mRNA for many cytokines and chemokines. In addition, TLR ligand stimulation of neurons upregulate the expression of TLRs and TRPV1. Furthermore, preincubation of neurons for 16 hours with the TLR ligands, enhances the calcium flux induced by capsaicin stimulation of TRPV1. Consequently, products of the herpes virus interacting with these TLR's can either directly or indirectly, by inducing chemokines, enhance the response of TRPV1 pain receptors, providing one possible basis for herpes Zoster neuralgesia in immunosuppressed cancer patients. These studies in a pain model indicate that the peripheral pain reported by many cancer patients may be addressed by effective regulation of neuroimmune molecules.

DESCRIPTION: It is hypothesized that estrogen deficiency induces a decrease in the rate of fat oxidation which leads to accelerated gain of visceral fat mass and its associated insulin resistance. It is further hypothesized that viscera fat accumulation will be prevented by hormone replacement therapy through restoration of fat oxidation. To test this hypothesis, 100 women in early menopause will be metabolically characterized for substrate oxidation, fat distribution, and insulin sensitivity prior to being randomly assigned to receive either hormone replacement therapy or placebo for 2 years. Metabolic testing will be repeated at 2, 6, 12, 18 and 24 months.

Our knowledge about the organisation of cells, biological specimen structures, composition and properties in submicroscopic detail of biological samples is associated to a large extent to transmission electron microscopy (TEM). The Electron Microscopy Core (EM Core) Facility provides advice, technical services, training, equipment and facilities to NHLBI intramural scientists if their research requires electron microscopy (EM) to clarify questions. Using an electron microscope offers the advantage of increasing both the magnification of an object and the resolution over other imaging tools. These could be issues involving subcellular, supramolecular or macromolecular structure at a level of resolution below that obtained by a light microscope. The NHLBI EM Core Facility has supported projects using the following techniques in the past year: 1. Chemical fixation, embedding, ultra-thin sectioning and transmission EM digital imaging of tissues and cell culture. 2. EM immunocytochemistry, including immunogold, nanogold with silver enhancement and immunoperoxidase localisation of proteins and other antigens within and on the surface of tissues and cells by pre-embedding techniques. 3. Negative staining of large proteins, polymers and supramolecular structures as well as lipid and membrane vesicles for transmission EM digital imaging. 4. Rotary shadowing of large protein molecules, DNA and other macromolecules. 5. Preparation of platinum replicas of cytoskeletons, partially lysed cells and freeze-fractured/freeze- dried tissues and cells. 6. Chemical fixation, critical point drying, sputter-coating and scanning EM digital imaging of small organisms, organs, tissues and cells, as well as other materials such as artificial matrices. 7. Thawed cryosection immuno labelling technique (also called Tokuyasu method). 8. Immuno correlative light and electron microscopy (CLEM) on Tokuyasu cryosections. 9. Cryo-electron microscopy of vitreous sections (CEMOVIS) to observe biological samples in their most native, fully hydrated state. 10. High-pressure freezing (HPF) and freeze substitution (FS) allows improved morphological preservation compared to the conventional preparation. These techniques are available as a service to be performed by the EM Core Staff for the customer or they are available to be learned by users who will be trained to be independent users of the EM Core. In the past year, the NHLBI EM Core supported a total of 89 recorded projects for 37 investigators within the NIH. 62% of the services were requested by NHLBI research groups. The remaining 38% are divided as follows: NHGRI (8%), NCI (8%), NIA (1%), NIAID (2%), NIDCR (10%), NINDS (7%), NINR (1%) and OD (1%). We have also trained several postdoctoral fellows, students, and contractors in the use of the scanning and transmission electron microscopes and sample preparation techniques.

Dr. Philippe Szapary plans a career as a clinician investigator at the University of Pennsylvania School of Medicine, focusing his research on the critical evaluation of complementary and alternative medicine (CAM). His training will include formal course work towards a Master's of Science in Clinical Epidemiology with a specific focus on patient-oriented research (POR). To supplement his formal research training, he also plans to take courses in pharmacognosy and ethnobotany at the University of the Sciences in Philadelphia (USP). Using his formal training in POR and botanical pharmacology, Dr. Szapary plans to establish himself as an independent investigator focusing on new and existing cardiovascular CAM therapies. Environment: The University of Pennsylvania and nearby USP are uniquely suited to provide complementary training and resources for this award. At Penn, the Center for Clinical Epidemiology and Biostatistics will provide the formal POR research training. The Cardiovascular Risk Intervention Program and the Division of General Internal Medicine will provide study subjects. The General Clinical Research Center will provide ancillary support including nurses, a dietitian, a biostatistical programmer and laboratory services. At USP, the Department of Pharmacognosy will provide technical support in the analysis of study drugs. Research: In the last 10 years, patients have markedly increased their use of dietary supplements to treat and prevent chronic medical conditions like atherosclerotic cardiovascular disease. This widespread use continues despite little scientific evidence of benefit from randomized controlled trials (RCTs). Current estimates suggest that 30 percent of Americans are hypercholesterolemic. Serum cholesterol remains one of the strongest predictors of risk for coronary artery disease, the leading cause of death in Americans. Herbal therapies have multiple mechanisms of action making them attractive in the prevention of a multifactorial disease process like atherosclerosis. Two Ayurvedic herbals, gugulipid and curcuminoids, appear to have hypolipidemic, antioxidant and anti- inflammatory effects in humans. In this protocol, Dr. Szapary will primarily study the hypolipidemic effects of standardized extracts of curcuminoids and gugulipid in two RCTs. These trials will assess the safety and efficacy of these two agents when used alone and in combination, as done in Ayurvedic medicine, over a 3 to 6 month period. In addition, Dr. Szapary will examine their effects on state of the art biomarkers of oxidant stress and vascular micro-inflammation, both of which are important processes in the pathogenesis of atherosclerosis. These studies will help define the role of these herbal therapies in the prevention of atherosclerosis, and serve as a model for the critical evaluation of dietary supplements in cardiovascular disease.

Objectives: Sensorineural hearing loss (SNHL) is strongly associated with many aspects of military service including blast injury. The objectives of this proposal are to improve the prevention and treatment of SNHL in veterans. Research Design: To improve our understanding of cellular processes underlying hair cell (HC) damage and loss, the contributions of molecular isoforms within known HC damage and survival pathways will be evaluated by determining their expression, and by evaluating HC loss in gene knockout mice. To improve SNHL prevention, pharmaceutical agents that can protect cochlear HCs from damage will also be identified. To improve treatment for SNHL, transcription factors that enhance the ability of Atoh1 to induce the conversion of mammalian cochlear non-sensory cells into HCs will be identified. In addition, modifications to mechanisms of gene silencing will be studied to identify epigenetic limitations on the ability of nonsensory inner ear cells to adopt features of the HC phenotype. Methodology: Studies will be performed using in vitro cultures of inner ear sensory epithelia, and in vivo studies of noise damage to the cochlea. For identification of HC protectants and transcription factors, innovative high-throughput methods have been developed to permit rapid, but targeted, screening of pharmaceuticals and induced gene expression in mammalian cochlear epithelia. Progress over the past period of funding: Mechanisms of ototoxin entry into HCs have been identified; cellular patterns of reactive oxygen species accumulation into HCs have been determined and antioxidant treatments evaluated; two novel intracellular pathways of HC protection have been defined; three gene mutations that lead to deafness have been identified; a critical period of HC development in which transplantation is possible has been determined; two novel transcription factors that enhance the conversion of nonsensory cochlear cells into HCs have been identified, and epigenetic manipulation of the cochlear sensory epithelium to enhance gene expression induced by a viral vector has been performed. Clinical Relationship: The prevention and treatment of SNHL is of great importance to veterans and to the VA. The effects of SNHL on veterans' quality of life are substantial. SNHL and tinnitus also account for more disability compensation in the VA than any other disorder, and rehabilitation costs are high. The proposed research is targeted at developing new and improved therapies for prevention and treatment of this important health problem.

This research is directed toward synthesis and derivatization of novel DNA-binding peptides and detailed characterization of their DNA-binding and -condensing properties. A series of low molecular weight branched oligolysines and oligoarginines with a defined coupling site will be synthesized. We will determine the effect of number and positioning of positive charges on DNA condensation. Selected compounds will be derivatized with effectors such as receptor ligands, membrane-destabilizing peptides, carbohydrates and PEG. DNA binding properties of the various derivatives and the physical characteristics of resulting DNA complexes will be examined. Considering parameters like hydrophobicity and propensity of amino acids for particular secondary structures, we will introduce a limited number of rational mutations into a known membrane-destabilizing hexapeptide sequence and select peptides with highest membrane-destabilizing activities. The dependence of transfection efficiency in cell culture on DNA complex composition will be examined. Using the GCG package, membrane destabilizing peptides have been selected and compared for their primary, secondary and tertiary structure. This information and things like hydrophobicity indices etc. are essential in finding and designing new peptide sequences for the above-described project. The facilities provided by the Computer Graphics Laboratory are absolutely necessary for the continuation of this work. GL

Chronic cigarette smoking is very common in alcohol use disorders (AUD). Nevertheless, the effects of chronic smoking on in-vivo measures of brain injury in alcohol-dependent individuals have not been studied. Thus, it is unknown if the full extent of brain atrophy, cell membrane and microstructural injury, derangement of neurometabolism, low cerebral blood flow, and neurocognitive dysfunction described in alcohol dependence are solely attributable to chronic alcohol use, or if chronic smoking influences these measures. Preliminary results from our ongoing magnetic resonance (MR) and cognitive studies indicate that chronic smoking appears to exacerbate alcohol-induced abnormalities in brain morphology, neurochemistry, and blood flow, and may adversely affect recovery of surrogate markers of neuronal and cell membrane/myelin integrity as well as aspects of neurocognition during short-term abstinence from alcohol. The main goal of this competing continuation is therefore to test in treated alcohol dependent individuals (i) if chronic smoking compounds alcohol-induced neurobiological brain injury, (ii) if chronic smoking effects are functionally significant, and (iii) if chronic smoking influences recovery of alcohol-induced neurobiological and neurocognitive dysfunction during abstinence from alcohol. We will continue to apply an integrative approach of combining information from different MR modalities and neurocognitive testing in the same individual longitudinally to assess the neurobiological and functional consequences of chronic smoking in treated alcohol-dependent individuals. Specifically, we will continue our longitudinal 1.5 Tesla MR studies to quantitate regional brain structure, brain metabolites (reflecting neuronal and myelin viability), and regional cerebral blood flow as well as repeat comprehensive neurocognitive testing. In addition, new MR studies at high magnetic field (4T) will improve the quality of cerebral blood flow and diffusion data, and will measure cortical levels of glutamate and Y-aminobutyric acid, amino acids critically involved in the initiation and maintenance of substance dependence. Relationships of MR-derived and neurocognitive measures will determine the functional relevance of neurobiological measures and their changes over time and test the effects of chronic smoking on functional neurocircuitry in alcoholism. This application is responsive to PA-05-074 in that the proposed research will improve our understanding of the mechanisms underlying alcohol- and smoking induced brain injury and its potential reversibility with abstinence from alcohol. Chronic smoking may have hitherto unrecognized but significant contributions to these neurobiological processes and their cognitive and clinical consequences. The knowledge to be gained from this translational research can be used directly for public education, in new approaches to pharmacologic and behavioral interventions for AUD, and in monitoring treatment outcome.

A method for attaching fluorescent receptor groups at specific sites in large RNA molecules will be developed. The sensitivity of the labels to the solvent environment and to energy transfer from nucleic acid bases, proteins, and metal ions will be used as a way of investigating RNA secondary and tertiary structure. A small, functionally interesting region of the E. coli 16S ribosomal RNA (the S8/S15 binding region) will be isolated as a well-defined fragment and used to test the fluorescence labeling method. The fluorescent labels and other physical chemical methods will be used to confirm the predicted secondary structure of the region (seven potential helices) and to determine sites for tight Mg ion binding which associate with tertiary structure. The binding of two ribosomal proteins known to recognize this region, S8 and S15, will be examined in detail to determine what RNA recognition features are used by each and whether the proteins influence the structure of the RNA fragment.

The objectives of these projects are to: 1) develop equipment for use by investigators in Viral Oncology, 2) develop narrated, slide presentations for use by intra- and extra-mural members of oncogenic virus research on the hazards of laboratory procedures and equipment, 3) develop a training workshop for "Certification of Biological Safety Cabinets", 4) prepare monographs and safety notes on current hazards that occur within the laboratory, 5) assist in the development of standards for production and procurement of Class II biological safety cabinets, 6) provide consultation services on biohazards to Viral Oncology intra-and extra-mural personnel.

Puresyn, Inc. has developed purification processes for recombinant adeno-associated virus gene therapy vectors utilizing its proprietary chromatographic resin, PolyFlo. In Phase I, two tandem chromatography processes were developed: heparin affinity and PolyFlo and tandem PolyFlo in two different modes. Each process resulted in product of high purity and recovery that exceeds gradient centrifugation processes. We demonstrated each process removes significant amounts of host and viral contaminants. We are seeking Phase II funding to concentrate on AAV production to include large-scale purification up to 5 x 10e14 particles per run without compromising purity, yield or biological activity. We will refine and expand our current processes to assure they are applicable to AAV serotypes 1, 2 and 5. We will also address the significant problem of AAV aggregation. Finally, we will conduct an in vivo mouse study to determine the correlation between purity, biological activity and safety through the evaluation of immune and histopathological responses to the vector. Results of this research will have immediate impact for those engaged in the use of recombinant AAV vectors for gene delivery because it will allow the large-scale development of AAV vectors, It will have positive commercialization implications for PoIyFlo and Puresyn, Inc. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The common mold, Aspergillus fumigatus, is an opportunistic pathogen and the most frequently isolated causative agent of invasive aspergillosis. The disease is often contracted by immunocompromised hosts, including cancer patients, with reported mortality rates >80%. Our long-term goals are to develop a vaccine and to provide better means for the early diagnosis and monitoring of aspergillosis. Our specific hypotheses are: 1) certain aspergillus antigens have an immunoprotective effect, and 2) some antigens may serve as indicators of early stages of aspergillus infections. We base our hypotheses on the following observations. First, mice vaccinated with crude fungal protein extracts or viable conidia survive infection following immunosuppression (e.g., with corticosteroid treatment). Second, the fungus actively secretes a variety of specific proteins and peptides at different stages of its lifecycle. More than 60 antigens and allergens of Aspergillus have been described (1-4). However, only one of them, the polysaccharide galactomannan, is routinely assayed to diagnose Aspergillus infections. Based on these observations, our experimental focus is on the identification and structural and immunological characterization of A. fumigatus antigens. We plan to utilize and improve novel mass spectrometric techniques to detect and identify proteins and peptides of fungal origin from complex mixtures such as culture supernatants, bronchoalveolar lavage (BAL) fluid, blood and urine.

As a result of Phase I researches, a perfected design of a simple, reliable and economical phase modulation system has been achieved. Desig parameter perturbation and component selection has lead to this simplified and reliable design of a dual wavelength time sharing phase modulation spectrometer (dual wavelength PMS). The frequency has been optimized at 200 MHz; time-sharing of the oscillator frequency between two diode lasers at different wavelengths, laser diode driver circuits, fiber optic coupling, standardized detector heterodyne njection, amplitude and sensitive phase detection circuit, together with time sharing demodulation are engineered. This instrument had undergone rigorous electromechanical tests and has a remarkable stability corresponding to a draft of less than 0.04degree/hr of phase drift and a noise level of 0.9 mm or approximately 0.2degree of phase. Tissue signals are of the order of 4 cm in amplitude and thus signal to noise ratios of 5:1 with a 5 sec time constant at 3 cm input/output distance are feasible. The remarkable features of time and frequency domain spectroscopy in providing the optical path length for photon migration in tissues is well recognized. Phase I set forth to exploit design parameters of a simplified system suitable for biomedical research and for clinical studies. Phase II will focus on construction of dual wavelength phase modulation systems that will bring to the clinic the advantages of TRs in an economical and feasible device. We propose to pursue product development and testing under Phase II support leading to commercial exploitation under Phase III; that is, production prototype, FDA approval, marketing and sales.