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Posterior tibial tendon dysfunction (PTTD) results in a debilitating flatfoot deformity which is increasingly recognized as a major problem in the treatment of foot and ankle disorders. In fact, this disorder is felt to be the number one foot and ankle problem requiring intensive scientific investigation. The deformity is caused, at least in part, by the loss of the tendon function with subsequent muscle imbalance. Bony procedures without major fusions are being used in an effort to treat deformity when possible. However, with a tendon transfer of one third the muscle mass and a similarly smaller tendon, the original problem of muscle imbalance remains. If the problem of muscle imbalance and increased strain could be resolved, patients could be treated early to eliminate those factors that promote deformity and decreased function. This would make for a successful long-term result. A cadaveric model has been developed for the posterior tibial tendon deficient foot. Physiologic tendon, bone and ground reaction loads can be applied and monitored, and the resulting strain in the arch of the foot can be measured. Using this model, the following fundamental questions in the treatment of PTTD will be addressed: 1) does loss of the posterior tibial tendon increase strain in the arch and thereby promote deformity?; 2) how well does the present flexor digitorum tendon transfer replace the posterior tibial tendon muscle unit and does it leave muscle imbalance and increased strain in the arch?; 3) does a bony procedure, like a medial calcaneal displacement osteotomy, adequately assist the flexor digitorum tendon transfer?; and, most importantly, 4) if the flexor digitorum longus tendon transfer is inadequate (as would be expected based on clinical results), are there procedures that can adequately normalize strain and joint position?

This is a renewal application, and would provide funding to administer the Neurosurgeon Research Career Development Program (NRCDP) for another five years. The purpose of this award is to provide the basis for a continued national effort to support, train, and mentor junior neurosurgical faculty members at appropriate institutions nationwide. This application describes all key aspects for the mechanism of recruiting applicants and the developing them into surgeon-scientists through continuing the NRCDP. The proposal addresses topics such as the rationale for the program, the relevant history of the PI and Institution, and the ongoing need for the NRCDP. The proposal also addresses the rationale for the selection of a National Advisory Committee (NAC). It also details a well-developed system for the recruitment of applicants. In addition, there is a description of the existing and robust system for selection of scholar-applicants based on their potential for success, the track record of their mentor, and the support of their Chairpersons. In addition, there are details of an annual three-day retreat, which has been held for five years, that includes applicant interviews, lectures, and interactive grant writing sessions. Finally, there is a description of the advisory board meeting for review of applications. This program has been extremely successful. Five years ago, when this NRCDP was first awarded, a search of NINDS data revealed only 4 neurosurgeons with active K08 awards. Currently, there are a total of 12 neurosurgeons with active K08 awards. Of the currently active K08 awards, four are scholars of this NRCDP program and two were applicants. In other words, over the five-year span of the NRCDP, there has been roughly a 300% increase in neurosurgeon K08 awards. Moreover, 50% of all current NINDS K08?s, awarded to neurosurgeons, are affiliated with the NRCDP either through direct support or through participation in the program. Another three alumni obtained independent federal funding through other mechanisms. If the scholars funded in 2016 are excluded from the analysis, which is reasonable given that they have only been funded for a few months, then 7 out of 10, or 70%, of scholars have successfully obtained independent federal funding in a relatively short period of time. In our opinion, this is an excellent success rate. Perhaps as importantly, the NRCDP has fostered a healthy and burgeoning community of junior neurosurgeon-scientists that will form the basis for reinvigoration the long tradition of scientific research in neurosurgery.

Recent investigations have identified a new signal transduction pathway, termed the sphingomyelin pathway, that may mediate the action of tumor necrosis factor (TNF)-alpha and interleukin 1 (IL-1) Beta. This pathway is initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the action of a neutral sphingomyelinase. Ceramide functions as a second messenger stimulating a Ser/Thr proteins kinase termed ceramide- activated protein kinase to transduce the signal. Three lines of evidence support this notion. Firstly, the sphingomyelin pathway is activated within seconds to minutes by TNF in HL-60 cells and by IL-1 in human dermal fibroblasts and mouse EL4 T helper cells. Secondly, cell- permeable ceramide analogs bypass receptor activation and directly mimic cytokine action. Thirdly, the effects of TNF and IL-1 on this cascade have been reconstituted in cell-free extracts indicating tight coupling of this pathway to the respective receptors. This grant proposal focusses on the elucidation of the role of ceramide-activated protein kinase in this signaling cascade. Until now, this activity has only been defined as a Mg2+ dependent, membrane-bound activity capable of phosphorylating a peptide derived from this amino acid sequenced surrounding Thr669 of the epidermal growth factor receptor. This is the same site recognized by mitogen-activated protein (MAP) kinases (also known as extracellular signal-regulated protein kinases). Hence, ceramide-activated protein kinase may belong to an emerging family of proline-directed Ser/Thr protein kinases, which includes MAP and cdc2 kinases. These kinases recognize substrates containing the minimal motif, X-Thr/Ser-Pro-X, where the phosphoacceptor site is followed on the carboxyl-terminus by a proline residue and X can be any amino acid. This grant application has three goals: (1) To define the structural determinants for substrates recognition by ceramide-activated protein kinase and for ceramide stimulation, and determine the intracellular localization of enzyme activity; (2) to purify ceramide-activated protein kinase; and (3) to define how known antagonist of TNF action might act upon the sphingomyelin pathway and, in particular, ceramide-activated protein kinase. It is our belief that ceramide-activated protein kinase represents a new target for pharmacologic intervention in TNF and IL-1 action. Hence, the title of this application has been changed to reflect this emphasis. Hopefully, these studies will begin the process necessary for testing this hypothesis.

Cocaine serves as a cofactor for susceptibility to HIV infection and AIDS progression. Cocaine also increases HIV-1 replication in peripheral blood mononuclear cells and enhances viral load in animal models. Furthermore, HIV positive cocaine users have lower CD4+ T cell counts and have a significant acceleration of decline of CD4+ T cells. Since CD4+ T cells are primary targets for HIV-1 infection and replication in vivo, it is imperative to understand the effects of cocaine on CD4+ T cell biology. This application proposes a potentially novel mechanism by which cocaine may increase HIV-1 replication. It has been proposed that cocaine enhances HIV-1 replication by regulating viral entry. Our preliminary data suggest modulation of HIV-1 post entry steps by cocaine. Our data also reveal that cocaine down regulates two anti-HIV cellular microRNAs (miRNAs), miR-125b and miR-328 in primary CD4+ T cells. Since these miRNAs target the 3'UTR of HIV-1 mRNA, we believe cocaine may target post-transcription steps of HIV-1 replication. Therefore, we hypothesize that enhanced HIV-1 replication and increased viral load by cocaine is mediated by down regulation of cellular anti-HIV miRNAs. Since HIV infected cocaine users have higher viral loads and increased risk of progression to AIDS, our findings will have far reaching implications in drug use and HIV biology. We will test our hypothesis by focusing on two aims. Aim 1: Determine effects of cocaine-induced down-regulation of anti-HIV cellular miRNAs on HIV-1 replication. Aim 2: Examine whether cocaine-induced down-regulation of anti-HIV cellular miRNAs activate latently infected HIV-1. PUBLIC HEALTH RELEVANCE: Drug use remains the second most common mode of exposure to HIV and illicit drugs serve as cofactors for susceptibility to HIV infection and disease progression. Although cocaine has been shown to enhance HIV infection and replication, the underlying mechanism remains unclear. Therefore, the focus of this proposal is to delineate the mechanism by which cocaine contributes to HIV/AIDS.

Project 1: Host Determinants of TB Susceptility Abstract: The outcomes of Mycobacterium tuberculosis (Mtb) infections are extremely diverse. Many infected individuals show no signs of disease. Among those that do become ill, the timing, location, and severity of pathology are remarkably variable. Genetic diversity in the host population is a major contributor to this variability, but the responsible alleles and mechanisms are poorly defined and have been difficult to address experimentally. In this project, we approach this inherently complex problem by modeling host diversity using a highly genetically diverse, but precisely genetically defined, panel of recombinant inbred mice known as the Collaborative Cross. The unique structure of this population allows the sequential evaluation of variable traits that reflect distinct aspects of pathogenesis. We took advantage of this iterative-phenotyping strategy to dissect the genetic basis of TB susceptibility at multiple levels. These prior studies implicated Kit Ligand (KitL) variation as an important determinant of TB disease in mice and humans, suggested that genetic interactions between pathogen and host are a sensitive metric for detecting new genes that alter disease progression, and highlighted the power of using ex vivo systems to dissect the interaction between Mtb and the macrophage. In the proposed project, we will continue to pursue these strategies to: Aim 1: Investigate the mechanism of KitL-linked susceptibility to TB disease in mice and humans. Aim 2: Use combinatorial mouse and bacterial genetics to dissect host-pathogen genetic interactions. Aim 3: Exploit the CC panel to dissect the Mtb-macrophage interaction. In combination, these new resources and approaches promise to provide fundamentally new insight into the role of host and bacterial genetic variation on the outcome of Mtb infection. !

PROJECT SUMMARY Thoracic aortic aneurysm (TAA) is a chronic condition that carries the risk of developing life-threatening complications such as aortic dissection (i.e., delamination of the aortic wall) and rupture (i.e., transmural tearing). The primary management objectives for TAA are to identify aortic dilation early and to surgically replace the aorta before it reaches a size that predicts future complications. It is estimated that 3 million patients in the US alone would meet criteria for aortic imaging surveillance based on current guidelines. Improved imaging surveillance strategies are needed to address the health and economic impacts of TAA. However, the reliability of standard aortic diameter measurements from CT images remains low. In this proposal, we will utilize a recently developed image analysis technique, Vascular Deformation Mapping (VDM), to perform semi-automated, three-dimensional assessment of aortic growth in patients with TAA undergoing imaging surveillance. This novel approach utilizes advanced computerized analysis techniques that make full use of the rich, high-resolution, volumetric data generated by modern CT scanners, to generate a more comprehensive and reliable assessment of aortic growth. VDM has the ability to significantly improve the speed and reliability of TAA growth analysis, facilitate the communication of aortic growth assessment between radiologists, care-providers and patients, and generate new biomarkers of disease progression that can be used to improve patient management decisions, and ultimately patient outcomes. In this grant effort, we propose to achieve the goals of this project through our close partnership between University of Michigan (UM) and Imbio, LLC investigators. Thus the Specific Aims of this Fast-Track proposal will develop a commercial VDM software application, including the product testing and data acquisition necessary for a FDA 510(k) application.

Fabry Disease: We have investigated the protein expression profiles in cells derived from patients with Fabry disease using microarray technology. Fabry disease is secondary to deficiency of the lysosomal enzyme alpha-galactosidase A, leading to altered glycosphingolipid metabolism and accumulation that is often associated with endothelial dysfunction. Current evidence suggests that there is impairment of the vascular nitric oxide pathway, with abnormalities evident in the cerebral circulation and in the dermal vasculature of patients with Fabry disease. Some of these findings have been confirmed in a mouse model of Fabry disease and are under investigation in the Fabry patient population. Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, will open a new chapter in our understanding of the biology of lysosomal storage disorders. Additional studies are planned to investigate the role of rare lipids in the etiology of this disease.[unreadable] [unreadable] Gaucher Disease: We extended our basic investigations on the pathogenesis of the neuronopathic forms of Gaucher disease. Both glucocerebroside and glucosylsphingosine (GlcSph) are elevated in the brain of these patients. GlcSph is neurotoxic and contributes significantly to the dysfunction and destruction of brain cells. Our investigations have identified six compounds that inhibit the enzymatic synthesis of GlcSph and ranked them in the order of their effectiveness. Using compounds that reach the brain in an effective and non-toxic concentration, we propose to conduct clinical trials with these agents to improve the debilitating clinical course in patients with neuronopathic Gaucher disease. [unreadable] [unreadable] Our genomic investigations into the changes that occur in Gaucher disease have identified statistically significant increases in expression of 10-fold or more over normal in 203 genes and decreases in 78 genes. This information is critical to understanding the genesis of the hepatosplenomegaly and other pathophysiological changes that occur in patients with Gaucher disease and may lead to improved treatment strategies.[unreadable] [unreadable] Protein Transduction Domains: We are extending our strategies to extend the organ and tissue distribution of exogenous enzymes for the treatment of both Gaucher disease and Fabry disease to augment the effectiveness of enzyme replacement therapy. We have previously expressed a TAT-GC fusion protein in which recombinant glucocerebrosidase (GC) is fused to 11-amino acid peptide from the HIV-1 transactivator protein (TAT) which functions to facilitate the transport of the enzyme across the plasma membrane of a variety of cell-types in a receptor-independent manner. We have extended these studies to include additional GC constructs containing flexible spacers to increase enzyme activity in addition to a variety of TAT analogs in order to test the effect of sequence variation on the uptake of the enzyme. Further studies have also been undertaken to construct similar fusions with the enzyme alpha-galactosidase A (AGA) as a possible strategy for wider distribution of this enzyme for the treatment of Fabry disease. Efforts are underway to introduce these fusion proteins into animals to test their distribution and compare these findings with that of the native constructs. Recombinant GC-TAT and AGA-TAT were expressed in eukaryotic cells from which catalytically active, normally glycosylated enzyme fusion proteins were obtained and tested for receptor independent uptake into cultured cells. It is expected that GC-TAT will be more efficiently delivered than unmodified GC to cells in the bone marrow and lung, and perhaps additional cells that lack the mannose lectin and thereby enhance the clinical responses of patients with Gaucher to enzyme replacement therapy We anticipate that the AGA-TAT enzyme may be capable of entering heart and kidney more efficiently than the native enzyme.[unreadable] [unreadable] Enzyme Replacement Therapy: In collaboration with members of the Surgical Neurology Branch (SNB), we determined the safety of intracerebral injection of GC in non-human primates using the convection-enhanced delivery (CED) technique developed by SNB. This was followed by clinical administration of this enzyme to a patient with Type 2 Gaucher disease. Alterations in the enzyme aimed at improving the distribution and stability of the administered enzyme are currently under investigation. [unreadable] [unreadable] Gene Therapy: We are developing methods to improve gene therapy for patients with Gaucher disease and Fabry disease using adeno-associated viral vectors in conjunction with the abovementioned protein transduction domains.. The first of these constructs increased the level of glucocerebrosidase activity significantly in multiple organs and tissues when injected into experimental animals. Moreover, it very effectively transduces bone-marrow stem and progenitor cells ex vivo. We demonstrated long-term expression of GC in experimental animals following transplantation of bone-marrow-derived cells transformed by a GC lentivirus vector. It is anticipated that autologous cells transduced with this, or a related lentiviral vector, may be appropriate for gene therapy trials in patients with Gaucher disease since successful bone marrow transplantation can cure patients with type 1 (non-neuronopathic) Gaucher disease.[unreadable] [unreadable] Chaperone Therapy: We have identified a number of patients with Fabry disease in whom the reduced catalytic activity of alpha-galactosidase A is increased in the presence of the molecular chaperone 1-deoxy-galactonojirimycin (DGJ). This technique is based on the ability of certain compounds to interact with the catalytic site of mutated enzyme and escort it from the endoplasmic reticulum where it is produced through the Golgi apparatus to lysosomes. The low pH and the reduced concentration in lysosomes causes the chaperone to dissociate from the enzyme and allows it to carry out its catalytic function. We have examined the ability of DGJ to augment residual alpha-galactosidase A activity in cultured skin fibroblasts, T cells and peripheral blood mononuclear cells derived from patients with Fabry disease. A standardized protocol has been developed for the screening of patients for whom chaperone therapy with DGJ may be an effective way of increasing the enzyme levels. Further investigations are planned to evaluate variations in test conditions with DGJ as the chaperone and to test additional agents as potential chaperones for patients whose alpha-galactosidase A activity is not enhanceable using DGJ. A protocol has been initiated for a Phase 1 safety and dose-response trial with DGJ in patients with Fabry disease. When it is completed, we shall examine the clinical effectiveness of active site-specific chaperone therapy in patients with enhanceable alpha-galactosidase A activity (cf. lead investigator's report). [unreadable] [unreadable] Additional studies have demonstrated the potential for the use of chaperone therapy for the treatment of Gaucher disease. Using a similar test model as designed for chaperone therapy in Fabry disease, candidate active-site-directed molecules are under investigation. All surviving patients with Gaucher disease have residual glucocerebrosidase activity and appear to be excellent candidates for molecular chaperone therapy. Such studies underscore the possibility that small molecules may be used in place of or in conjunction with enzyme replacement therapy as it is currently practiced.

Melanoma is a malignant skin cancer with few effective treatment options for advanced stages of the disease. It is important to develop effective treatment strategies that will eliminate existing tumors and/or prevent the formation or recurrence of tumors. The work proposed in this application is to use an immunotherapy approach in the form of a gene delivery system. Electroporation (EP) will be used to introduce a plasmid DNA encoding for a cytokine into melanoma cells promoting an immune response to reduce cancer growth and prevent development. Interleukin 15 (IL-15) is a cytokine that activates natural killer (NK) cells and stimulates CD8+ T cells to exhibit long-term memory CTL activity against target cells. The research in this study focuses on delivering plasmid IL-15 (plL-15) by EP into a mouse model injected with B16.F10 murine melanoma cells. In Specific Aim 1, we will evaluate IL-15 expression levels when applying different EP parameters and different plasmid concentrations. An ELISA and real time RT PCR analysis will be used to detect protein levels and mRNA levels produced by the tumor cells, respectively. In Specific Aim2, the high and low expression levels of IL-15 obtained from Specific Aim 1 will be used to correlate expression levels of IL-15 with tumor regression. We will analyze the local and systemic cellular infiltrate stimulated by plL-15/EP using FACS, immunohistochemistry, Luminex bead array, TUNEL and ELISPOT to understand the reduction of tumor growth. A depletion study using monclonal antibodies will be used to demonstrate the roles that innate NK cells and adative T cells play when they are separately depleted. The reaction and duration of the response by these cells in the absence of one another will be evaluated when stimulated by the production of IL-15 from tumor cells. ELISPOT and FACS analysis will be used to demonstrate the results from the depletion studies. In Specific Aim 3, we will also determine the extent of IL-15 expression levels that provide viable treatments for metastatic disease using a two-tumor model. The cellular systemic responses to intratumoral treatments will be analyzed using immunohistochemistry, ELISPOT and ELISA. The relavance of this research is that the administration of plL-15/EP will provide promising stimulation of the immune response against tumor cells without causing toxicity or deleterious effects to the surrounding tissues. These experiments can promote the development of a potential anti-cancer therapy to block cancer metastasis as well as the recurrences of new diseases.

A traditional view of metastasis is that it results from a process similar to Darwinian evolution involving the natural selection of tumor cells that are capable of migration and survival during treatment and at distant sites. In this model the selection of tumor cells exhibiting stable genetic changes occurs, the selected cells are very rare, local and cause metastasis late in tumor progression. The recent development of new technologies, including high-density microarray based expression profiling, multiphoton intravital imaging and the collection and characterization of migratory tumor cells from live tumors and bone marrow disseminated tumor cells (DTCs) from patients, have challenged this traditional model of metastasis. The new technologies indicate that metastatic ability is acquired at much earlier stages of tumor progression than predicted by the Darwinian model, is encoded throughout the bulk of the primary tumor, it is highly plastic and involves transient changes in gene expression. These results have led to the micro-environment model of metastasis. The micro-environment and Darwinian models can be reconciled if tumor progression resulting from the selection of stable genetic changes in the primary tumor during progression, contributes the micro-environments necessary to induce the transient changes in gene expression that support the invasive and metastatic phenotype. That is, the tumor micro-environment initiates the transient epigenetic expression of genes that induce tumor cell migration, survival and metastasis. Examples of such micro-environments in breast tumors are extracellular matrix density, inflammation, and hypoxia. To study these micro-environments and their effects on metastatic phenotype, we have assembled a multidisciplinary team who will collaborate using their special expertise to: 1. fate map tumor cells to determine if tumor cells migrating from different spontaneous, and nano-device generated soluble factor-derived micro-environments, have different migration, dissemination, dormancy and growth patterns in target organs. 2. Determine the spatial and temporal extent and functional consequences of these micro-environments at single cell resolution in vivo in primary tumors and in DTCs 3. Isolate and characterize the metabolomics, genomics and epigenomics of special populations of tumor cells such as the migratory and dormant tumor cells in disseminated locations. 4. Investigate ECM-dependent migratory/invasive, dormant and proliferative tumor cell phenotypes. 5. Extend key observations to human breast and head and neck squamous tumors.

Support is requested for 9 predoctoral and 6 postdoctoral positions to establish a training program in the interdisciplinary area of Pharmacoinformatics. This program is proposed because current approaches to the discovery of new drugs have not fully capitalized on advances in bioinformatics, genomics, computing and other fields, and consequently, the rate of drug discovery has lagged behind breakthroughs in these areas. The pipeline of new drugs under development has not increased in recent years, and a very small number of new drags are thus responsible for bearing the enormous costs of an increasingly expensive process that evolved prior to development of massive amounts of new information and the ability to integrate it into usable and accessible systems. We propose a comprehensive, integrated program to train the next generation of scientists to: (1) use informatics approaches to identify biomolecules that are candidates for drug targeting, (2) use structural genomics, chemoinformatics, and structure-based design to develop families of chemicals to target selected biomolecules and structures, and (3) use ADMET (absorption, distribution, metabolism, elimination, and toxicity) profiling and computational predictions of efficacy to select viable drag candidates prior to biological testing. Training will emphasize in silico approaches to achieve these goals. Inter-institutional training will be conducted by sixty-three training faculty from the six institutions of the Gulf Coast Consortia (Baylor College of Medicine, M. D. Anderson Cancer Center, Rice University, the University of Houston, The University of Texas Health Science Center at Houston, and The University of Texas Medical Branch at Galveston) in the Houston -Galveston area and will utilize research training with co-mentors, didactic courses, seminars, retreats, and career development activities. Predoctoral trainees will matriculate and receive first year support at their home institutions, and stipends will be awarded on a competitive basis for years 2-4 of graduate training. The home institution will award the Ph.D. Postdoctoral trainees will receive support for two years maximum. The proposed program is designed to train future leaders in drag discovery research.

PURPOSE OF RESEARCH: The goal of this project is the development and field-testing of a screening procedure to assess social and emotional dysfunction in populations of four- and five-year-old children, in order to allow for prompt further evaluation and early intervention. METHOD: A screening battery was developed which includes assessments of health, developmental history, and cognitive and sensory functioning. A Behavior Checklist was designed to tap behaviors considered by clinicians as indicators of social and emotional dysfunction for this age group. The battery was piloted with over 800 children in 1976. After revision it was administered to 750 subjects in 1977. A subsample of 85 children was seen for clinical interviews, and the results were compared to the screening procedure. Follow-up outcome data has been collected for the 1977 sample which will serve to evaluate the screening procedure. RESULTS AND FUTURE PLANS: A criterion validation study indicates that disturbed and normal children are significantly differentiated (p less than 04) on the Behavior Checklist. Further analysis will establish the predictive accuracy of the screening procedure as it relates to measures of the child's functioning at follow-up. Recommendations for screening policy, as well as for providing identified service needs, will be made after the data is analyzed.

The Howard University Graduate School proposes to continue its focus on the development of a new cadre of biomedical scientists at the institution. To this end, the MBRS SCORE program is requesting support for 18 new projects from faculty who have not previously participated in the MBRS Program. Seventeen of these new projects are from junior faculty (non-tenured), several of whom have just joined the faculty this academic year. These investigators are from five basic science an| two clinical departments in the College of Medicine, as well as from two undergraduate departments with strong graduate training programs. This application contains both regular (18) and pilot (4) projects. The mantra for the proposed program is "ROAD MAP TO R01 SUPPORT". Building on some of the success of our current SCORE participants (only 3 are returning in this application, as some have moved on to other individual support -one R01);the program's expectations are being raised leading to the following goals of opportunity: GOAL (1);To improve the scientific productivity of SCORE faculty. The measurable objective (A): That the publication rate of the participating faculty be increased overall and throughout the proposed project peroid. Measurable objective (B) that all of the continuing program investigators submit an R01 application by the end of the 02 year of funding and (C) that 50% of the new participating investigators will have submitted R01 applications, or submitted to an equivalent agency by the end of the proposed period. GOAL (2): To enhance the professional progression of SCORE faculty through the provision of essential research support. Measurable Objectivie (D): during the proposed period, nontenured junior faculty who are Pis on regular projects will make significant progress on each of the meaures of productivity( publications and grant submissions) which will enhance their probability of earning tenure.

The proposed Charity Hospital-Tulane-Louisiana State University (LSU) GCRC will serve as a facility for patient-based applied and basic research. The specific aims of the GCRC are: (1) to provide a well staffed facility that will allow in-hospital and outpatient care for patients who are subjects in research projects; (2) to allow these projects to be performed under controlled conditions with scientific accuracy; (3) to support a Core Laboratory which will provide assays for many of the researchers who use the facilities as a measure to increase productivity and reduce costs; (4) to improve the quality of patient care in the overall Charity Hospital- Tulane-LSU Medical Complex by applying the advances made in these clinical research endeavors to disease diagnosis and therapy; (5) to provide training in the methodology and design of good clinical research projects as a form of teaching for post-graduate research residents, interns, medical students, and fellows, nurses and other paramedical personnel in the facility. The program stresses: (1) a strong interdisciplinary effort by both medical schools and by several departments within each school; (2) a strong letter of commitment from Charity Hospital and the participating medical schools with regard to housing of the unit (Charity Hospital) plus renovation costs; and (3) quality (predominantly NIH funded or pending funded projects) by most of the leading investigators in both medical schools and at Charity Hospital. The Charity/Tulane/LSU GCRC will be predominantly an adult unit but with a small pediatrics and "contagious" areas within the same facilities.

The goal of this proposal is to study the effect of sympathetic innervation on the formation of 21 and 22 adrenoceptor (AR) signaling complexes in the heart. We will investigate how these complexes either facilitate or restrict receptor signaling; we will identify the structural domains of the receptors that are needed for interaction with the other components of the signaling complex; and we will identify cellular proteins that define these subtype-specific signaling complexes. 21ARs and 22ARs are prototypical G protein coupled receptors (GPCRs), the largest family of hormone and neurotransmitter receptors in the human genome. These receptors are essential for the physiologic regulation of cardiac function in response to catecholamines (adrenaline and noradrenaline) released from sympathetic nerves. Recent studies suggest that 21ARs and 22ARs play distinct roles in the pathogenesis of heart failure, a growing health problem in the United States. We have developed an experimental system to study the important interface between sympathetic nerves and the heart using co-cultures of neonatal cardiac myocytes and sympathetic neurons. Our preliminary studies show that 21ARs and 22ARs have differential subcellular targeting relative to these synapses, and that signaling complexes form at the sites of synapse formation. The following aims are designed to characterize these signaling complexes. Aim 1. Determine the structural basis for subtype specific targeting and trafficking of 21ARs and 22ARs in cardiac myocytes. Aim 2. Determine the functional significance of subtype-specific localization of 21ARs and 22ARs relative to sympathetic synapses. Aim 3. Characterize protein components of the 21AR and 22AR signaling complexes in cardiac myocytes innervated by sympathetic neurons. Aim 4. Determine the functional significance of interacting proteins identified in Aim 3 on 21AR and 22AR signaling and trafficking in cardiac myocytes, and verify their existence in signaling complexes in the adult heart. The proposed studies will provide new information about how the brain regulates heart function. We will characterize the mechanism by which noradrenaline and adrenaline released from sympathetic nerves alters heart function by activation of two specific adrenergic receptors. This research will further our understanding of the development of diseases such as heart failure and sudden death.

The Analytical Core (B) will be located in the Biomedical Biological Sciences Building, University of Kentucky. The Analytical Core will consist of the Director and research personnel for performing services of resolution and profiling of analytes using mass spectroscopy and for quantifying analytes using luminex platforms. An important part of Core activities will include methods development to analyze specific molecules of interest, and for profiling lipids within animal and human tissue.

Significance Recombinant SHIVenv clones are a means to analyze the role of HIV-1 env genes in vivo for viral transmission across mucosal membranes and for SAIDS pathogenesis. Additionally, vaccines based on HIV-1 immunogens can be tested for efficacy by challenge with such recombinant viral clones. Objectives An animal model to study both HIV-1 infection and AIDS pathogenesis is not available. To analyze function of specific HIV-1 genes in vivo, SIV/HIV-1 recombinant viruses (designated SHIV) have been constructed by replacing genes in the pathogenic clone SIVmac239 with counterpart HIV-1 genes. We have made SHIV clones containing the envelope (env) gene of various HIV-1 subtype-B isolates, and have analyzed these recombinant viruses in vivo in juvenile and newborn macaques. Results A pathogenic SHIV (designated SHIV-33A), containing the env gene of HIV-1-SF33, was obtained by one passage in a juvenile rhesus macaque. SHIV-33A produced simian AIDS in juvenile macaques when given by either the intravenous or mucosal membrane (oral and vaginal mucosa) routes. In addition, newborn macaques (2 days of age) also developed simian AIDS after intravenous inoculation. Sequence analysis of the env gene of SHIV-33A revealed about 15 amino acid changes, relative to the input SHIV-33 clone. All animals showing immunodeficiency disease also exhibited a rapid and sustained decline in CD4 T-cells in both peripheral blood and lymph nodes. In vitro analysis of SHIV-33A revealed increased cytopathicity and increased replication in tissue culture cells. Future Directions Intragenic SHIV-33 recombinants and point mutants, involving the HIV-1SF33 env gene, are being constructed to determine which sequence change(s) accounts for viral adaptation and pathogenesis. Studies will also be performed to determine the mechanism of rapid depletion of CD4 T-cells in the acute stage of infection with the pathogenic SHIV-33A strain. Functional domains of SHIV-33A will be analyzed by constructing env genes with point and deletion mutations; these mutant viral clones will be examined in tissue culture cells and in rhesus macaques. KEY WORDS HIV-1 genes, AIDS pathogenesis, SHIV clones FUNDING NIH Grant AI41907

Prevention strategies have been developed to take advantage of the metabolic and behavioral risk factors for type 2 (non-insulin- dependent) diabetes which have been identified in research with the Pima Indians and elsewhere. Obesity, impaired glucose tolerance, hyperinsulinemia, physical inactivity, and high fat diet have been implicated as risk factors for type 2 diabetes. Since these factors are potentially reversible with behavioral and pharmacologic therapy, strategies for prevention have been developed and will be tested. This project is a multicenter randomized clinical trial of prevention of non-insulin-dependent diabetes mellitus in overweight persons with impaired glucose tolerance known as the Diabetes Prevention Program. The coordinating center is at the George Washington University. During the project year recruitment was completed and treatment of volunteers has continued in four American Indian communities in collaboration with the Indian Health Service. - Diabetes mellitus, prevention, clinical trial - Human Subjects

Treatment of Central Vein Thrombosis remains unsatisfactory. However, there is strong evidence from a recent study that quick, aggressive therapy with Urokinase for occlusions of recent onset, can produce marked benefits. Prognosis of these particular cases has been considered poor - therefore, all work on this subject is of considerable importance. We propose a blinded randomized study whereby our objective will be to enhance knowledge of effectiveness of fibrinolytic therapy in Central Retinal Vein Thrombosis - utilizing Urokinase - a plasminogen activator and fibrinolytic enzyme. The Urokinase is supplied by the National Heart and Lung Institute, and is derived from the stockpile of the Urokinase subcommittee which approved of this protocol and hence released the amount of urokinase sufficient for this project. We aim to compare anticoagulant therapy and the use of Urokinase therapy. All patients of less than 14 days onset of symptoms diagnosed as Central Retinal Vein Thrombosis will be considered for this study. Excluding criteria will be pregnant females and those in the child bearing age unless they have a negative pregnancy test and are on adequate contraception; all uncontrolled hypertensive patients with blood pressures exceeding 150/100; those patients with a history of bleeding diathesis, or recently active peptic ulcer; recent surgery, or history of significant complicating disease such as carcinoma or cerebral vascular accident. Intravenous Urokinase or Heparin will be given via infusion pump, with close monitoring by coagulation laboratory of fibrinogen level; prothrombin, thrombin, and partial thromboplastin times; euglobulin lysis time and level of fibrinogen degradation products (FDP). Any untoward bleeding will be treated with epsilon amino caproic acid for the Urokinase group and protamine for the heparin group.

Our research is directed at epithelial differentiation in normal and pathologic states. This grant focuses on the roles of vitamins and hormones, as exemplified by retinoids and thyroid hormones, in the control of keratin synthesis and in the process of keratinization. For decades, clinicians have recognized that thyroid hormones and vitamin A had profound effects upon normal and abnormal skin and within recent years the interest in the use of vitamin A and its derivatives on diseases of keratinization has become widespread. At the same time studies of other systems have defined on cellular and molecular levels the mechanism by which these substances effect regulation of gene expression; they bind to specific receptors which in turn react with recognition elements in the non-coding regions of DNA to profoundly affect transcription. The experiments will focus upon several genes for epidermal keratins whose expression we have shown to be negatively regulated by both retinoic acid and its receptor as well as thyroid hormone and its receptor. The assays used will be based upon transfection involving regulatory regions of keratin genes linked to a reporter gene, either chloramphenicol acetyl transferase for quantitative studies or luciferase for localization studies. The recognition elements in the keratin gene for both receptor complexes will be identified through deletion studies. Specific point mutations will define their exact positions. Throughout these studies, the polymerase chain reaction (PCR) will be used to amplify appropriate areas of DNA. Parallel studies will be undertaken in cell lines and primary cultures of epithelial cells. Finally, the effects of retinoids and thyroid hormones on differentiating epidermis in tissue culture will be determined. Thus, our aims in the 5 years for which we are requesting support will be: 1. to identify and characterize the recognition elements (REs) for the nuclear receptors of retinoic acid and thyroid hormone in human keratin genes - basal cell-specific K#5 and K#14, stratum corneum-specific K#10, hyperproliferation-associated K#6 and K#16, and the simple-epithelia- specific K#8, K#17, K#18 and K#19. 2. to define the molecular mechanisms by which retinoids and thyroid hormones regulate keratin gene expression and to delineate the interactions among the two classes of mediators and their receptors; 3. to analyze the effects of retinoids and thyroid hormones on epidermal differentiation in vitro. Our proposal, which will define the mechanisms of action of retinoids and thyroid hormones in epidermis, has important implications for health care for it will ultimately lead to understanding of the normal and pathologic processes in skin and should indicate the potential of new therapeutic agents.

The proposed research is designed to study the mechanism of DNA replication using T even bacteriophage infected E. coli as a model system for the investigation with emphasis on the studies of the initiation process. T4 and T6 DNA-delay proteins, which have been defined genetically and biochemically as needed for the specific initiation process of the respective phage have been purified. They form a complex which has ATP-dependent topoisomerase activity of untwisting superhelical DNA. Because of the critical role DNA topoisomerase plays in DNA replication, the mechanism of action, the subunit strucutre and the DNA sequence of this novel enzyme, which we have discovered, will be investigated in detail. On the other hand, the origin(s) of T4 DNA replication which we proposed to be the intended target of the initiation proteins will also be characterized with respect to its structure and function. We plan to carry out experiments designed to test the hypothesis that the initiation process is mediated through direct interaction of the initiation proteins with the origin of DNA replication since both components have now been purified and isolated in our laboratory. This will include the use of T2, T4 and T6 initiation proteins of the DNA-delay gene products in a comparative study to demonstrate the specificity of the initiation process.

Protein kinases mediate many cell signaling events, and their tight control is essential for regulating essential processes ranging from cell division to energy metabolism. Thus, it is not surprising that protein kinases are directly or indirectly involved in many diseases and that kinases are key drug targets. For example, Src kinase was the first identified proto-oncogene and the formation of a de-regulated Abl fusion protein (BCR-Abl) is the cause of disease in 95% of patients with chronic myeloid leukemia. X-ray crystal structures have shown that the same kinases can obtain an active and various inactive conformations, implying that kinases are inherently flexible. How the active and inactive states are stabilized and how the states interconvert are key questions in understanding kinase regulation. Because X-ray crystal structures provide only static snapshots, we will use nuclear magnetic resonance (NMR) experiments to study the time scales and amplitudes of structural interconversions in Abl and Src kinase domains. [unreadable] [unreadable] BCR-Abl is the target of the clinically highly successful drug imatinib (Gleevec(r), Novartis) in the treatment of chronic myelogenous leukemia (CML). Why does imatinib bind and inhibit c-Abl but not the structurally closely related c-Src kinase? The crystal structure of Src in complex with imatinib shows protein-drug interactions similar to that of Abl, even though the affinity of imatinib for Src is orders of magnitude lower than for Abl. Because imatinib binds only to the inactive conformation of the kinase, drug binding is intimately related to the interconversion between active and inactive states. The goal of this study is to examine whether differences in this interconversion underlie the differential sensitivities for imatinib. Therefore, we will compare the time scales and amplitudes of backbone motions between Src and Abl kinases in the presence of imatinib by NMR experiments. In preparation for these dynamics experiments, we have established expression systems and NMR conditions and will next pursue the assignment of the Src and Abl NMR spectra. [unreadable] [unreadable] Kinase inhibitory drugs such as imatinib have a great therapeutic potential because of the many signaling events that protein kinases mediate. However, these drugs have to be exceptionally specific for their target kinase and resistance mutations can render these drugs ineffective as seen in leukemia patients under imatinib treatment. The proposed experiments will clarify how inhibitors such as imatinib exploit the characteristic movements of kinase proteins, rather than just their structures, to achieve specificity. Furthermore, the results will have broader impact on the understanding of the fundamental mechanisms of kinase regulation and of drug resistance mutations that are known to arise in cancer patients undergoing kinase inhibitory treatment. [unreadable] [unreadable] [unreadable]

Major depressive disorder (MDD) is characterized by reductions in the density and size of neuronal and glial[unreadable] cells in prefrontal cortex. We find these cellular changes to be age-dependent as younger depressed show[unreadable] prominent reductions in the density of glial cells (astrocytes), whereas older depressed have marked[unreadable] reductions in the density of pyramidal (presumably glutamate) neurons and calbindin-immunoreactive[unreadable] interneurons (mostly co-localizing GABA). Since, astrocytes regulate concentration of glutamate, an excess[unreadable] of which is neurotoxic, we propose that an early deficit in astrocytes in MDD could lead to an increase in the[unreadable] extracellular concentration of glutamate and to a reduction in pyramidal and GABA neurons later in life.[unreadable] Hence, there may be imbalances in GABA/glutamate homeostasis that are consistent with neuroimaging[unreadable] studies revealing changes in levels of GABA and glutamate in MDD which are reversible with antidepressant[unreadable] (SSRI) treatment. Cortical neurons are regulated in complex ways by serotonin acting (at least) at serotonin-[unreadable] 1A and -2A receptors located on these neurons and astrocytes. Pathology in ascending serotoninergic[unreadable] axons and postsynaptic receptors may be related to the activity, number and size of glutamate and/or GABA[unreadable] neurons and astrocytes in MDD. To date, there have been no studies on the expression or localization of[unreadable] serotonin receptors on specific cortical cell types in depression.[unreadable] The overall hypothesis is that in depression there will be age-dependent reductions in the density of[unreadable] astrocytes, glutamate pyramidal neurons and GABA interneurons, and that the expression of regulatory[unreadable] serotonin-1A and -2A receptors on these cells will be altered. These cell reductions will also be correlated[unreadable] with an age-related loss of serotonin innervation in prefrontal layers. To test these hypotheses, we will[unreadable] directly identify and quantify the packing density of astrocytes and glutamate and GABA neurons expressing[unreadable] mRNA for specific proteins (Aim 1). Moreover, we will assess the integrity of the serotonin system regulators[unreadable] (postsynaptic receptors and presynaptic axons) of prefrontal cells by estimating the proportion of cell types[unreadable] expressing mRNA for serotonin-1 A and -2A receptors (Aim 2), and the density of serotonin axons[unreadable] expressing the serotonin transporter (Aim 3). Double in situ hybridization, immunohistochemistry and 3-D[unreadable] cell counting techniques will be used in the same postmortem tissue sampled from the prefrontal cortex of[unreadable] younger and older subjects with MDD and non-depressed controls as used in our cell counting studies.[unreadable] This project will identify the cellular substrates of glutamate, GABA and serotonin interactions in the[unreadable] cortex and their potential role in the etiology, pathophysiology and age-related progression of depression. It[unreadable] may also reveal novel targets for preventing depressive illness and better antidepressant drug treatment.[unreadable]

Mathematical performance in the United States is significantly falling behind that of many other countries and failing the expectations of our nation's education leaders. This project, Number Concepts and Automaticity, is designed to target predictable factors in mathematical failure and provide the right guidance and practice to overcome deficiencies using a computer software tool. It is based on leading research in math education for elementary students. The project will focus on developing mathematical models for children, bringing hose models into working memory, and building automaticity with addition and subtraction problems. The software tool created in the project comprises a Number Concept Builder that will tie virtual manipulatives to their numeric values, an Automaticity Builder that will help students increase their ability to recall concepts from retrievable memory, and an Assessment Module that will measure student progress. The Phase I feasibility study will involve 2nd grade students. A small formative field test will guide the software tool prototype development in its earliest stages. The completed prototype will be introduced into school classrooms as part of the standard number sense curriculum. Students will participate in a controlled experiment woven into their standard classroom activities. Students with physical disabilities will engage in a single subject study to guide product accessibility. This study will seek to prove that the software can accurately measure automaticity in individual students' performance and will verify that the software can be used to increase automaticity. The study will also attempt to reveal automaticity for students with physical disabilities who have slow response times due to their impairments. Future work in Phase II and beyond will include the needs of students in throughout grades K-5 and across more areas of the math curriculum. [unreadable] [unreadable]

In rhesus macaques, a SIV/HIV-1 chimeric virus (SHIV) containing the env gene from HIV-1 89.6 is capable of initiating a systemic infection following mucosal or systemic inoculation. Previous Infection with SHIV89.6 protects 60% of rhesus monkeys from uncontrolled replication of SIVmac239 after intravaginal inoculation. Understanding the nature and anatomic location of the adaptive immune that confers protection to SHIV "immunized" monkeys would be significant contribution to AIDS vaccine development. The overall goal of the Primate Core is to facilitate the design and execution of the in-vivo primate studies that will be the basis of all 3 projects in the program. All the in-vivo studies will be carried out at the California National Primate Research Center (CNPRC).This Core will obtain mature, multi-parous, female rhesus monkeys, immunize the monkeys intravenously with virulence-attenuated SHIV89.6, administer immunomodulatory agents to monkeys and challenge the monkeys intravaginally with defined stocks of SIVmac239; determine vRNA levels and viral population complexity in plasma and tissues of the monkeys after SIVmac239 challenge; characterize CD4+ T cell levels in plasma and tissues of the monkeys after SIVmac239 challenge and characterize body weight levels of the monkeys before and after SIVmac239 challenge. This Core is essential for the completion of the proposed studies.

Obesity is a world-wide health problem that is associated with metabolic syndrome, including insulin resistance and the development of non-alcoholic fatty liver disease (NAFLD). NAFLD is characterized by hepatic steatosis and varying degrees of inflammation and fibrosis. Increasing evidence suggests that saturated free fatty acids (FFA), by promoting insulin resistance and mediating the progression from simple steatosis to steatohepatitis, are a causative link between obesity and NAFLD. However, the molecular mechanisms responsible for the effects of saturated FFA are incompletely understood. Our long-term goal is to understand the molecular mechanisms by which saturated FFA contribute to the development of NAFLD. Saturated FFA are a major source of metabolic stress that induce activation of the c-Jun NH2-terminal kinase (JNK) pathway, and gene deletion and pharmacological inhibition have established an important role for the JNK signaling pathway in obesity-induced insulin resistance and the pathogenesis of NAFLD. Our preliminary studies identified the MAP3K mixed-lineage kinase 3 (MLK3) as an important mediator of saturated FFA- induced JNK signaling. Thus, we have demonstrated that MLK3 mediates FFA-induced JNK activation, promotes obesity-induced insulin resistance and modulates FFA-induced ER stress response in specific tissues. These data indicate that MLK3 plays a critical role in the development and progression of NAFLD, most likely through metabolic stress signaling. The central hypothesis is that MLK3 contributes to the pathogenesis of NAFLD by mediating saturated FFA-induced JNK activation, thereby inducing inflammatory gene expression, promoting insulin resistance, and modulating ER stress-induced cell death in specific tissues. This hypothesis will be tested by three complementary Aims. In Aim 1 biochemical and molecular biological methods will be used to delineate the pathway(s) required for MLK3 activation by FFA. In Aim 2 biochemical and molecular biological methods will be used to determine the role of MLK3 in the FFA-induced ER stress response. In Aim 3 reciprocal bone marrow transplantation studies will be used to examine the tissue specific role of MLK3 in the development of insulin resistance. Accomplishing the goals of this proposal will increase understanding of the role of MLK3 in the pathogenesis of NAFLD. This information will provide the foundation for the potential development of novel therapeutic strategies for the treatment of this pathology. PUBLIC HEALTH RELEVANCE: Non-alcoholic fatty liver disease is the leading cause of liver dysfunction in non-alcoholic, viral hepatitis- negative populations in Europe and the USA. Saturated free fatty acids, by promoting insulin resistance and mediating the progression from simple steatosis to steatohepatitis, are a causative link between obesity and NAFLD. The proposed study will increase understanding of the molecular mechanisms by which saturated FFA contribute to the development of NAFLD. This information may suggest novel therapeutic strategies for the prevention of NAFLD.

The ability of steroid hormones to influence profoundly the excitability of the CNS is well documented. The broad objective of this proposal is to explore pharmacologically the molecular mechanisms whereby steroids modulate CNS excitability. Abnormal activation of amino acid receptors has been proposed to play a role in the etiology of psychiatric disorders such as anxiety, depression and schizophrenia. Understanding the mechanisms of steroid actions on the CNS may lead to new strategies for the treatment of psychiatric disorders. During the course of our studies we came upon the unexpected finding that pregnenolone sulfate (PS), an abundant neurosteroid, acts as a positive allosteric modulator at the NMDA receptor while inhibiting the kainate, AMPA, glycine, and GABA responses. A major focus of the research plan will be to test our working hypothesis that steroids such as PS regulate the balance between excitation and inhibition on neurons derived from the vertebrate CNS by acting on excitatory and inhibitory amino acid receptors. Toward this end, whole-cell voltage-clamp and current-clamp techniques will be utilized to character electrophysiologically the effects of PS and related steroids on excitatory and inhibitory amino acid receptor-mediated responses of embryonic chick spinal cord neurons maintained in primary monolayer cell culture. The proposed study has three major parts: First, we will screen a series of steroids for activity on amino acid receptor-mediated currents. Secondly, we will determine the potency, efficacy, and mode of action for PS and each steroid identified, with the goal of elucidating the mechanism(s) of steroid action(s) through a study of their structure-activity relationships. In particular, we will focus our studies on PS and on other steroids that are active at NMDA and non-NMDA glutamate receptors. Finally, the effects of steroids on excitatory and inhibitory synaptic transmission at single synapses will be investigated. In the long run, we will determine whether chronic treatment with active steroids such as PS, progesterone, and reduced metabolites of progesterone induces functional changes of excitatory or inhibitory amino acid receptors and of synaptic transmission at identified synapses. These studies will provide a strong basis for evaluating the role of steroids as modulators of neuronal function, and a foundation for development of novel steroid-based drugs for the treatment of psychiatric disorders.

The goal of the work described in these studies is to elucidate how critical extracellular and intracellular domains control signaling by notch receptors. The extracellular domains of all notches contain three iterated LIN12 repeats, which maintain notch receptors in their resting state prior to ligand binding. Ligand-induced proteolysis releases the intracellular portion of notch (ICN) and allow it to translocate to the nucleus, where its RAM and ankyrin-repeat domains interact with two additional factors, RBP-JK and mastermind (mami 1) to form a transcriptionally active ternary complex. The specific aims of this research plan, which will provide important new insights into the structure and function of the LIN12 and ankyrin repeat domains of notch, are to: [unreadable] [unreadable] 1. Determine how LIN12 modules prevent ligand-independent activation of notch receptors. (a) Determine high-resolution solution structures of key modules and module pairs from a Notch LIN12 domain, analyzing the structural interactions of these modules with each other and with neighboring domains. (b) Identify contacts between the LIN12 domain and the NTM extracellular stub that maintain the integrity of the Notch heterodimer, and (c) Test the functional importance of specific intermodular and LIN12-NTM contacts. [unreadable] [unreadable] 2. Determine the essential domains and residues required for assembly and function of thenotch/RBP-Jkappa/maml1 signaling complex. (a) Determine the minimal protein domains required for reconstitution of an ICN-mam11-RBP-Jkappa--DNA complex. (b) Identify contacts that contribute to energetic stabilization of ICN complexes and determine how RBP and RAMANK cooperate to recruit mamli to form a ternary complex on DNA. (c) Test the function of specific contacts responsible for stabilizing the ternary DNA-binding complex.

Project Summary/Abstract The goal of this consortium is to establish tools that can be used as biomarkers and/or sensitive and reliable objective assays of social impairment in autism spectrum disorder (ASD) clinical trials. Specifically, we aim to accelerate the development of effective treatments for social impairment in ASD by validating outcome measures that will be sensitive and reliable assessments of response to treatment and EEG and eye-tracking (ET) biomarkers that can be used to reduce heterogeneity of samples via stratification, indicate early efficacy, and/or demonstrate target engagement. The consortium will conduct a naturalistic, longitudinal study of preschool (3-5 years) and school aged (6-11 years) children with ASD and typical development (TD) with IQ ranging from 50- 115. Children will be assessed across three time points (T1: Baseline, T2: 6 weeks, T3: 24 weeks) using clinician, caregiver and lab-based (LB) measures of social impairment, along with a battery of conceptually related EEG and ET tasks and independent ratings of clinical status. This battery measures key facets of social- communication in ASD using well-validated paradigms appropriate for this developmental and cognitive range. Five Collaborating Implementation Sites (?Sites?), all highly experienced in multi-site collaborative clinical research using the methodologies proposed here in both typical and atypical development, will contribute equally to recruitment, screening, diagnosis, testing, and longitudinal assessment. The Data Acquisition and Analysis Core (DAAC) will oversee consistent application of scientific standards and methodological rigor for standardized data collection, processing, and analytics. The Administrative Core will oversee the operations of the Sites, the Data Coordinating Core (DCC), and the DAAC and coordinate with federal and private partners to achieve the aims of this cooperative agreement. The will utilize informatics and technology to develop, maintain, and monitor a robust, secure, HIPAA-compliant data collection, coordination, and storage system to streamline communication and data flow throughout the consortium and ensure organized, secure data management, quality control, and reliable upload to the National Database for Autism Research and NIH/NIMH Data Repositories. The DCC benefits from the Yale Center for Clinical Investigation's quality assurance experience and the existing multi-site ASD research data systems developed by Prometheus Research. The DCC will: 1) Anticipate, respond to, and fulfill the informatics needs of the consortium in the design, harmonization, and implementation of data coordination and innovative data collection methods and tools across the Sites, DAAC, and Admin Core; 2) Provide state-of-the-art methods for: a) consortium communication; b) data management, site monitoring, privacy, and security within the consortium and across Sites; and c) dissemination of information to its stakeholders.

During the last fiscal year, the following advances were made: 1) Biological activity of stem cells The Section continues to try to bring some clarity to the mesenchymal stem cell field. The concept of a post-natal mesenchymal stem cell (MSC) originated from studies focused on bone marrow stromal cells (BMSCs), which are non-hematopoietic adherent cells, a subset of which are skeletal stem cells (SSCs), able to form cartilage, bone, hematopoiesis-supportive stroma, and marrow adipocytes based on rigorous clonal and differentiation assays. Subsequently, it was speculated that BMSCs could form other mesodermal derivatives and even cell types from other germ layers. Based on BMSC surface markers, representative of fibroblastic cells, and imprecise differentiation assays, it was further imagined that MSCs are ubiquitous and equipotent. However, MSCs do not have a common embryonic origin and are not a lineage, but recent studies indicate that they are tissue-specific stem/progenitor cells. These cells share cell surface features owing to their fibroblastic nature, but they are not identical. They display different differentiation capacities based on their tissue origin, but do not trans-differentiate outside of their lineage, based on rigorous assays. For these reasons, the MSC term should be abandoned. Tissue-specific stem/progenitor cells provide the opportunity to devise methods for tissue regeneration by the cells themselves (tissue engineering). Their use in other forms of regenerative medicine based on paracrine, immunosuppressive, and immunomodulatory effects is far less clear (Robey, Faculty 1000, 2017). Human umbilical cord blood (CB) has attracted much attention as a reservoir for functional hematopoietic stem and progenitor cells, and recently, as a source of blood-borne fibroblasts (CB-BFs, also called cord blood-derived MSC). In a previous study, it was demonstrated that bone marrow stromal cell (BMSC) and CB-BF pellet cultures make cartilage in vitro. In addition, BMSC and CB-BF pellets remodeled into miniature bone/marrow organoids in vivo, suggesting that CB-BFs can support the Hematopoietic Stem Cell (HSC) niche. Compared with BM-ossicles, CB-ossicles showed a higher proportion of red marrow vs. yellow marrow. Marrow cavities from CB- and BM-ossicles included donor-derived CD146-expressing osteoprogenitors and host-derived mature hematopoietic cells, clonogenic lineage-committed progenitors, and HSCs. Furthermore, human CD34+ cells transplanted into ossicle-bearing mice engrafted and maintained human HSCs in the niche. These data indicate that CB-BFs are able to recapitulate the conditions by which the bone marrow microenvironment is formed and establish complete HSC niches, functionally supportive of hematopoietic tissue (Pievani et al, Dev, 2017). 2) Role in disease With regards to fibrous dysplasia of bone (FD), often in conjuction with the McCune-Albright Syndrome (over-active secretion of some hormones), a rare opportunity presented itself to study radiographs and tissue samples (removed during corrective orthopaedic surgery) from a single patient with FD/MAS. These radiographs and samples were collected at different times following intermittent treatment with intravenously administered pamidronate. In children, such treatment results in the formation of sclerotic bands (zebra lines) due to binding of the bisphosphonate to a mineralized surface and subsequent temporary decrease in bone resorption at the time of treatment. By way of contact microradiography, back-scattered electron microscopy and histological analysis, it was found that zebra lines formed only where the bone was normal, abruptly stopped at the interface between normal and FD bone, and were completely absent in FD bone, due to the inability of bisphosphonate to bind to unmineralized osteoid, a prominent feature of FD lesions. These results suggest that lack of zebra lines is indicative of the presence of FD bone, which may be useful in evaluating the initiation or expansion of FD lesions, and support the concept that bisphosphonates may be ineffective in FD due to their lack of binding to FD osteoid (Corsi et al, Skel Radiol, 2017). 3) Stem cells in tissue engineering and regenerative medicine Previously, a process was established for the ex vivo expansion of BMSCs under conditions that would maintain their biological properties. Human BMSCs are being manufactured around the world using many different methods (and for many different clinical applications), but little is known about the spectrum of manufacturing methods used, and their effects on BMSC characteristics and function. Eight centers (including one at the NIH CC) using or developing Good Manufacturing Practices (GMP) were surveyed as to their production methods. Among the 8 centers, all used bone marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression analysis (Agilent Chip Whole Human Genome). Among the 24 BMSC lots from the 8 centers, intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from all centers into five distinct clusters. Sufficient numbers of BMSCs from 6 of the 8 centers were available for further testing of their ability to form bone and support hematopoiesis (defining features of BMSCs) by in vivo transplantation. Cells from all 6 centers tested formed bone, but the quantity formed was highly variable, and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics, including their ability to form bone and support hematopoiesis. Although the number of centers and samples evaluated is small, the data point to a need for establishing rigorous criteria by which to establish SSCs/BMSCs in scaled-up culture conditions in order to maintain their biological properties. (Liu* and de Castro* et al, Scientific Rep, 2017, *co-first authors),

Opsin synthesis was measured in miniature poodles affected with progressive rod-cone degeneration. Photoreceptors develop normally but begin to degenerate after the dog is fully grown. At all ages studied the rate of rod outer segment renewal was about half the normal value. Opsin synthesis, however, occurred at the normal rate until advanced stages of the disease when photoreceptor cell death was apparent. Thus, the defect may involve photoreceptor membrane assembly rather than synthesis. Peritoneal macrophages from RCS rats exhibited normal phagocytic capabilities toward bovine or rat rod outer segments as well as toward RCS retinal debris. Thus, the genetic defect is expressed in the RCS pigment epithelium but not in all phagocytic cells.

Program Director/Principal Investigator (Last, First, Middle): Abstract The Instrumentation Module provides design, fabrication and modification capabilities for project-specific, customized and software-flexible vision research apparatuses that are not commercially available. During the past five years, the Module fabricated many in-house, tailor-fitted and specially-designed devices that have played crucial roles in generating many high-impact and innovative vision research results. In the next grant period, this Module will continue its indispensable role in supporting vision research projects by providing new mechanical, electronic, optic and computer-based design and fabrication services and to develop highly innovative, high-impact research tools, such as the chronically implanted tetrode array recording chambers, the virtual reality visual-vestibular stimulating system, and the forced-choice, Bayesian adaptive protocol-driven optokinetic reflex machine. All these devices will be constructed and repeatedly modified, fitted and adjusted before and during experiments by the in-house machine shop and electronic/optics/computer shop. Therefore the Instrumentation Module is not only a necessity of many Vision Core labs, but also an integrated part of the research process and scientific progress. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

DESCRIPTION: (Applicant's Description) This application deals with the structural biology of DNA 0-alkylation damage. As part of the applicant's long-term objectives of understanding the molecular recognition of damaged DNA, he proposes to determine the solution structure of DNA duplexes containing alkyl lesions and base analogs currently used in chemotherapy. He will establish the three-dimensional structure of the repair protein: 06-methylguanine methyltransferase (Ogt), free and after reaction with enzyme inhibitors, and of the damaged DNA/protein complex. Alkyl transferase activity has been correlated with increased sensitivity to mutations induced by methylating agents. Tumor cell lines expressing high levels of alkyl transferase activity are resistant to the action of therapeutic methylating and chloroethylating agents. In addition, alkyl transferase inhibitors used in combination with methylating chemotherapeutic agents overcome the resistance of some tumor cell lines to the drugs. The information resulting from this project will increase our knowledge of chemical mutagenesis and DNA repair and facilitate the rational design of new alkyl transferase inhibitors, which can be used in cancer therapy. Using phosphoramidite chemistry, he will synthesize large quantities of oligodeoxynucleotide duplexes containing the base analog S6-thio-2'-deoxyguanosine and S6-methyl-6-thio-2'-deoxyguanosine paired opposite to deoxycytidine and thymidine. Their three-dimensional structure will be established by NMR spectroscopy and computational methods. He will use recombinant DNA technology to obtain large quantities of highly pure Ogt protein in natural abundance as well as enriched with 15N and 13C. His solution structure will be determined by using multinuclear -- multidimensional NMR spectroscopy and computational methods. Similar methods will be used to determine the structure of protein after reaction with the enzyme inhibitors 06-methylguanine and 06-benzylguanine.

A common side effect of an illness such as an infection is appetite suppression, which can quickly lead to malnutrition. Because malnutrition extends hospital stay, increases costs and exacerbates mortality, nutritional support (NS) is instituted. Unfortunately hyperglycemia is common in infected individuals and hyperglycemia negatively impacts on morbidity and mortality. We discovered that in healthy subjects the liver and muscle adapt to sustained NS by increasing their capacity to take up and oxidize glucose. As glucose is a major caloric source in NS, this adaptation is essential to minimize hyperglycemia. Infection impairs this adaptive response to NS and the current therapy administration of exogenous insulin cannot reverse this impairment. We also discovered in response to NS plasma glucagon decreases and liver derived and plasma FGF-21 increase. In addition if glucagon is increased as occurs with an infection, hepatic FGF-21 and plasma FGF-21 decrease and both hepatic and muscle carbohydrate oxidation are decreased. As FGF-21 is known to augment tissue glucose uptake our hypothesis is that, during NS, infection induced hyperglucagonemia limits FGF-21 availability and that together these changes attenuate tissue glucose uptake. We will use state of the art tracer and controlled metabolic studies to assess glucose and lipid flux in healthy and infected animals. In Aim 1 we will determine if glucagon mediated suppression of hepatic FGF-21 secretion is responsible for the decrease in muscle glucose uptake and determine whether Gs and/or Gq dependent signaling (using designer receptor technology) in the liver can explain the glucagon-mediated decrease in muscle glucose uptake. In Aim 2 we will determine if antagonizing glucagon can reverse the infection-induced impairment in tissue glucose uptake during NS. We will also determine if liver derived FGF-21 serves a protective role during infection and whether replacement of FGF-21 can sustain tissue glucose uptake during infection.

All eukaryotic organisms have conserved a specific set of mechanisms for receiving and responding to extracellular signals. In most cases, these mechanisms depend on the presence of cell membrane receptors that act as signal discriminators. Such receptor systems mediate sensory transduction in the adult organism and are also employed to relay signals of developmental significance. The most ancient and diverse signalling system involves the use of a group of structurally related extracellular receptors that couple to intracellular second messenger generating systems by means of G proteins. Studies in simple organisms such as yeast point out the significance of this form of sensory transduction in mediating cell determination events during development. The aim of our studies is to investigate to role of metazoan G protein coupled receptor systems in relaying signals of developmental consequence. For these studies, we will employ the fruit fly Drosophila as an experimental system because of the wealth of genetic and molecular tools it offers as well as the easy accessibility of well--described developmental stages. We will focus specifically on the role of G protein-coupled receptors that are expressed at the earliest stages of development. A Drosophila homolog of vertebrate tachykinin receptors and a novel receptor, both expressed early in development, have been identified. These receptors will allow us to test hypotheses concerning the participation of individual receptor systems in early and cell-specific developmental responses. In addition, we will continue to identify CDNAS encoding a limited number of receptors which we predict are expressed early in embryogenesis and may therefore participate in initial determination events. The expression of each receptor within the context of well characterized developmental pathways will be analyzed using tools such as specific antibodies. These studies will allow us to predict specific roles for each receptor in mediating early developmental events or the development of specific populations of cells. Using this framework, specific predictions will be tested by the manipulation of the expression of each receptor by genetic techniques. Using this system, we will be able, for the first time, assess the potential of individual G protein coupled receptor systems to mediate the transduction of developmentally important signals.

PROJECT SUMMARY Retinal ischemia is a major cause of vision loss in common retinal disease conditions including diabetic retinopathy, glaucoma, retinopathy of prematurity, and vein occlusion. This project aims to define the mechanisms of retinal ischemic injury and identify new therapeutic targets. My long-term career goal is to pursue a distinguished career in vision research and academia. I will achieve this through establishing a strong independent research program in an academic institution that promotes interdisciplinary biomedical science and translational research. My short-term goal is to attain intensive training and supervised career development skills that are required for my career transition to become an independent investigator. Securing this award will provide me with the necessary training to achieve my short- term goals and will be the first step towards independence and achieving my long-term goals. My mentor's lab has demonstrated the involvement of the arginase enzyme in retinal neurovascular diseases. Arginase has two isoforms. Building upon the lab's finding that the mitochondrial isoform, arginase 2 (A2), has a deleterious role in retinal ischemia-reperfusion (IR) injury, I developed a project focusing on the neurovascular protective role of the cytosolic isoform arginase 1 (A1). My recently published paper shows a neuroprotective role of A1 expression in myeloid cells. Arginase competes with nitric oxide synthase (NOS) for their common substrate L-arginine. Nitric oxide (NO) produced by inducible NOS (iNOS) causes neurovascular degeneration. I predict that A1 upregulation in myeloid cells limits iNOS-derived nitrative and oxidative stress and reduces inflammation through its downstream metabolites ornithine and putrescine. Putrescine is the precursor of polyamines and it is formed from ornithine by ornithine decarboxylase (ODC, the rate-limiting enzyme in polyamine biosynthesis). These metabolites have been shown to promote reparative myeloid cells through chromatin modification. In line with this, my preliminary data show that histone deacetylase (HDAC) 3 is increased in the absence of A1 in both IR-injured retinas and stimulated macrophages. HDAC3 is essential for macrophage inflammatory gene transcription and it has been shown to suppress A1 expression. Herein, I propose a novel suppressive effect of A1 on HDAC3. My central hypothesis predicts that myeloid A1 protects against retinal IR injury through ODC-mediated suppression of HDAC3. I will be using mice with myeloid-specific deletion of A1, ODC and HDAC3, as well as the investigational drug, BCT-100 (a PEGylated form of arginase 1), together with primary macrophages isolation and treatment with inhibitors for HDAC3 or arginase downstream enzyme, ODC. My goal is to achieve the following objectives: A) Determine the effect of manipulating the arginase pathway on myeloid cells infiltration / activation in retinal IR injury and the therapeutic potential of BCT- 100. B) Describe the cross talk between the arginase pathway and HDAC3 and determine whether A1 in myeloid cells mediates its protective effect through suppression of HDAC3.

The purpose of this project is two-fold. The first aim is to develop a coordinated series of instruments, primarily checklists, to elicit descriptions of the child from the several perspectives of parents, teachers and clinicians. The items on the checklists are to be weighted in terms of the relative severity of psychopathology represented as judged by a national sample of mental health professionals. Application of these weights to subscores derived from checklist content clusters will produce clinically meaningful diagnostic profiles of individual children. Treatment planning and outcome evaluation forms also are being developed. The second aim in terms of timing and the major substantive goal is to evaluate the course of psychopathology in children aged 6 to 18 years who have experienced selected treatment and no-treatment regimens. An initial, six-month and 15-month assessment of each child will be made. Separate data analyses are to be performed for children categorized in terms of a few major types of disorder and for normal school children. The independent variables will be age of child, type of treatment (or no treatment), and severity of psychopathology. The dependent variables will be checklist scores, some of which will reflect changes in total symptomatology while others will reflect changes in target complaints as identified from the diverse vantage points of parents, teachers and therapists.

This research application is in response to #RFA-DA-06-004. At the heart of drug and alcohol abuse is bad decision-making, especially in response to social influences. This decision-making defect is highly reminiscent of the social decision-making impairments that characterize neurological patients with damage to ventromedial prefrontal cortex (VMPC). The VMPC patients provide a neurological model of social decision- making gone awry. There is evidence at both preclinical and clinical levels that the VMPC is a key brain region in drug abuse, and we propose that VMPC dysfunction and defective social decision-making may characterize individuals who abuse and become addicted to drugs and alcohol. In this research application, we plan to conduct studies in neurological patients in order to flesh out the neurobiological underpinnings of social decision-making, taking advantage of the unique Iowa Patient Registry that contains neuropsychological and neuroanatomical data for thousands of patients. A particular focus is on gender differences. We have intriguing preliminary findings suggesting that there might be an important sex-related difference in the relationship between the VMPC and social decision-making: in men, the right VMPC might be critical, whereas in women, the left VMPC might be critical. The experiments will test three specific aims: (1) To determine whether there is sex-related functional asymmetry of the VMPC in regard to social decision- making, using tasks such as the Ultimatum Game, Trust Game, Iowa Gambling Task, and Ellsberg Tasks; (2) To extend the investigation of sex-related functional asymmetry to other brain structures known to be critical for social and affective processes related to decision-making, namely, the amygdala and the insular cortex; (3) To investigate developmental influences on the relationship between the VMPC and social- decision making and emotional processing, by studying patients who incurred VMPC damage early in life. The experiments will furnish important new information about the neurobiological underpinnings of social decision-making and emotional processing. The link with substance abuse is direct: the decision-making deficits in VMPC patients have striking similarities with those evident in drug and alcohol abusers and addicts ("myopia for the future"). Thus, our research could help pinpoint sources of neural dysfunction that contribute to bad decision-making of the type that characterizes drug and alcohol abuse. Public health relevance: This research will help us understand how various parts of the brain are important for social decision-making and emotional processing, whether there are gender differences in these brain- behavior relationships, and how these relationships develop. The research could help inform treatment and prevention of drug and alcohol abuse disorders, as well as social conduct disorders, personality disorders, and anxiety and mood disorders, all of which have high comorbidity with drug and alcohol abuse. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Adapted from the application) The main goal of this core is to support and optimize the activities of the investigators performing studies with animals. This core will provide services in a. breeding and maintaining normal, congeneic, and gene-deficient mice with reproducible enteric microbiota and clinical disease in both conventional and specific pathogen-free housing, and b. generating germ-free and mono-associated infected mice. This core will provide support to Projects 2, 3 and 4.

The proposed International Collaborations in Infectious Disease Research renewal program comprises a multidisciplinary and coordinated set of epidemiological, clinical and laboratory research activities in enteric disease, and will specifically focus on Giardia lamblia, Cryptosporidium, and E. coli infection in children. A major integrating theme will be to determine relationships between manifestations of infection in the host, and characteristics of the host and of the enteric pathogen. We will explore how infectively of each agent, and clinical expression of infection relate to the environmental, nutritional, and immunological status of the child, and to genetic and/or antigenic variability of Giardia lamblia, Cryptosporidium, and E. coli. The proposed ICIDR will be organized as five research projects and six core components and will be a collaborative thematically cohesive program among investigators from US institutions (Columbia University, Cornell University, University of Maryland, Centers for Disease Control, Univacs, ImmuCell, Merck) and institutions in Israel (Ben-Gurion University, Hebrew University, Army Health Branch Research Unit). Project 1 will prospectively follow 240 Bedouin infants from birth to age 2 years, to examine the environmental, host, and organism characteristics and the interactions between them, leading to the range of clinical manifestations associated with the three organisms. Using novel methods of pathogen identification and characterization (ELISA assays, DNA probes), genomic karyotyping, hygienic intervention protocols, and in depth analysis of nutritional/immune/environmental status, we will establish the time course and causality of these relationships, and also how asymptomatic Giardia lamblia carriage diminishes infections with other pathogens. Since Project 1 will not provide a sufficient number of cases to explore these relationships in children with severe clinical manifestations, Project 2 will identify environmental/host/organism factors associated with diarrhea in sick hospitalized children. Project 2 will also specifically assess two therapeutic interventions (azithromycin and hyperimmune anti-Cryptosporidum colostrum immunoglobulin) on the course of cryptosporidial diarrhea in a hospitalized population. Project 3 will probe the distribution of strain diversity of Giardia lamblia and Cryptosporidium using karyotype analysis and other strain markers; utilize PCR approaches for identifying and characterizing Giardia lamblia, and study in vitro cytopathogenic effects of Giardia lamblia on human intestinal epithelial cell cultures and lymphocytes. Project 4 will focus on the cellular immune response in infants in infants and young children, in relation to symptomatic and asymptomatic infections with the three groups of organisms with particular emphasis on development of different lymphocyte subpopulations e.g. 'memory cells'. Project 5 will provide an in depth understanding of the basic molecular genetic pathways underlying the high frequency of mutation and chromosome rearrangements in Giardia lamblia, and its variable gene expression. The Core components include: (a) ICIDR administration, (b) a Visiting Investigator Program, (c) Microbiology, (d) Giardia lamblia Serology, (e) Cryptosporidium/E. coli Serology, and (f) a Biostatistics center. The proposed ICIDR Program provides a unique opportunity to examine, in a Middle Eastern population with diarrheal disease patterns of developing nations, the role of Giardia lamblia, Cryptosporidium and different categories of E.coli in diarrheal diseases, and contribute knowledge essential for development of new intervention strategies.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents, killing more pediatric patients every year than any other malignancy. When children with ALL relapse the survival rate is less than 50%, and this has not improved for more than 30 years. Although ALL is less common in adults, it is more deadly, reaching 5-year survival rates of less than 12% in people over 65, an age when tolerance for toxic therapies is poor. The majority of ALL cases in children and adults are the B-cell type (B-ALL) and those with the poorest outcome are characterized by overexpression of the CRLF2 gene (CRLF2 B-ALL). This type of leukemia is particularly devastating in Hispanic children where it occurs 5 times more often than in other children. In adults, CRLF2 B-ALL makes up 1/3 of all B-ALL cases. A safe and effective treatment capable of targeting CRLF2 B-ALL would have a significant health impact in the United States and in Central and South America where rates of CRLF2 B-ALL are even higher. We have discovered a novel biologic that selectively targets the signaling pathway that produces CRLF2 B-ALL while sparing normal immune cells. The proposed studies will establish the therapeutic efficacy of this novel biologic in patient-derived xenograft (PDX) models of high-risk ALL. Scientific premise: Our preliminary studies show that high physiological levels of the biologic exert an anti-leukemia effect against CRLF2 B-ALL and establish a likely mechanism for these effects. To advance the use of the biologic in clinical trial for the treatment of CRLF2 B-ALL, it is essential to establish the dose and efficacy of the biologic as a single agent and as a part of combination therapy in preclinical studies. The overall objective of the current proposal is to establish proof-of-principle for the use of the biologic and its efficacy in the treatment of high-risk CRLF2 B-ALL. The achieve this objective we will: Aim 1. Establish the dose of biologic required to achieve normal and therapeutic blood plasma levels in PDX mice and Aim 2. Evaluate the in vivo therapeutic efficacy of the biologic as a single agent and in combination therapy using PDX models of high-risk B-ALL. At the end of the proposed work we will have: 1) Determined the dosing required to generate PDX with normal and therapeutic levels of recombinant human biologic, 2) Assessed in vivo functional effects and toxicity of the biologic, 3) Evaluated the efficacy of the biologic as a single agent in the treatment of CRLF2 B-ALL using PDX models; 4) Evaluated the efficacy of the biologic to prevent relapse and restore normal B cell precursors in combination with standard of care therapy in PDX models of CRLF2 B-ALL. This work has the potential to produce a more effective and less toxic treatment strategy to improve survival in patients with high-risk CRLF2 B-ALL.

ABSTRACT Mossy cells (MCs) of the dentate gyrus (DG) are glutamatergic neurons that are considered to be important to normal function and their injury has been suggested to contribute to neurological and psychiatric disorders, as well as deficits after traumatic brain injury. Anatomical and slice electrophysiology studies have described MCs in detail, but there is a gap between these studies and understanding how MCs contribute to DG-dependent behavior in vivo. To address this issue, we began with a simple approach: mice were engaged in behaviors related to DG function, and MCs were examined afterwards using the neural activity marker c-fos. We quickly found that simply exploring novel objects led to a large increase in MC c-fos immunoreactivity (ir). Interestingly, most c- fos-ir did not increase in most of the other DG neurons, suggesting preferential activation of MCs by novelty. However, there was one area of the DG where c-fos-ir was consistent: a subset of GCs in dorsal DG. In contrast, the majority of MCs with c-fos-ir were ventral. Because the main projection of ventral MCs is to dorsal GCs, these data suggest that ventral MCs excite dorsal GCs. This circuitry helps explain how normally quiescent GCs become activated in dorsal DG, which is considered essential for cognitive functions of the DG. In Aim 1 we will use optogenetics to test this hypothesis, taking advantage of new mouse lines that have targeted Cre recombinase to MCs. We will also ask if dorsal MCs have effects analogous to ventral MCs, i.e., dorsal MCs contribute to ventral DG functions. In Aim 2 the underlying circuitry will be addressed. We suggest that optogenetic excitation of MCs in a normal adult mouse will recapitulate the results with c-fos: MCs excite proximal GCs weakly but distal GCs in a more robust manner. This idea has been supported by data from slices that were cut at an angle to preserve MC axons, and will be tested further in Aim 2 using voltage imaging and microelectrodes. In Aim 3 we will address the hypothesis that a large number of the distal GCs that are activated by MCs are immature. That hypothesis supports a previously published study showing that MCs are a primary source of afferent input to young GCs that are born in adulthood. This is potentially important because immature GCs are considered central to DG functions. Therefore, we will address the additional hypothesis that MCs activate adult-born GCs primarily in distal locations, leading to stronger excitation of distal GCs than proximal GCs. By providing afferent input to immature GCs, MCs could play a critical role in behaviors associated with adult DG neurogenesis. Together these experiments will significantly advance our understanding of DG circuitry and its contribution to behavior. Because MC injury is associated with several disorders, these experiments will also shed light on impairments in DG functions in those pathological conditions.

Pain is a common co-morbidity for HIV-infected patients. Prevalence studies suggest that, on average, half of all HIV-infected persons suffer pain. Chronic pain can lead to heavy alcohol use among HIV-infected persons, which may in turn be a barrier to treatment/control of HIV and contribute to spread of HIV. Thus there is an urgent need to address pain among persons with HIV. Opioid receptor antagonists such as naltrexone and nalmefene, which are licensed for treatment of alcohol use disorders, show promise as being effective and safe treatments for chronic pain among persons with HIV. This study will pilot test novel pharmacotherapies (opioid receptor antagonists) to improve chronic pain among HIV-infected heavy drinkers, and will explore the hypothesis that the mechanism of action for improving pain is through decreased inflammation. The specific aims of the research are: UH2/Aim 1: To assess the feasibility, tolerability and safety of using opioid receptor antagonists (low-dose naltrexone and nalmefene) to treat pain among HIV-infected persons with heavy alcohol use and chronic pain; UH3/Aim 2: to perform a 3-arm pilot randomized, double-blinded, placebo-controlled study of low-dose naltrexone and nalmefene vs. placebo among HIV-infected persons with heavy alcohol use and chronic pain to provide estimates of their effects on: 1) pain (both self-reported and experimental/cold pressor test; 2) inflammation (i.e., levels of inflammatory cytokines IL-6 and TNF-?); and 3) measures of HIV control (CD4 count and viral load). The results of this study will provide preliminary information (tolerability, effect size, etc.) to design a larger RCT of low-dose naltrexone and/or nalmefene for chronic pain among persons with heavy alcohol use. We choose to conduct this research in St. Petersburg, Russia, given that: 1) nalmefene is licensed in Russia, but not currently in the US; 2) patients are seldom on chronic opioids (which are contraindicated to use with opioid receptor antagonists) due to the unavailability of opioid agonist therapy for addiction and restricted use of opioids for pain; and 3) a high prevalence of heavy drinking and HIV exists in Russia. Addressing chronic pain is a high priority for patients with HIV, and therefore this application is highly ?patient-centered? as well as innovative. Given the US epidemic of opioid use disorders, new pharmacotherapies without addictive potential are desperately needed for HIV-infected persons with chronic pain and alcohol problems.

Principal Investigator/Program Director (Last, first, middle): Zeng, Fan-Gang Tinnitus is the perception of sound in the absence of external sound. Tinnitus is a significant public health problem that affects 50 million Americans. Severe tinnitus disrupts daily functions from sleep to work, often leading to anxiety, depression and lowered quality of life. Despite significant advances in research and development, presently there is no cure for tinnitus. The present application uses noninvasive electric stimulation in the ear and minimally-invasive electric stimulation to the round window or promontory for safe and effective treatment of tinnitus. One innovation is to evaluate stimulation sites and patterns that evoke auditory sensations while minimizing non-auditory sensations. Another innovation is to provide two novel tinnitus treatment options, especially for those who still have significant acoustic hearing and cite tinnitus, and not hearing loss, as the main indication. In the preliminary study, 10 minutes of round window stimulation completely silenced the tinnitus not only during stimulation but also for 5 hours after the stimulation in a subject who had suffered from tinnitus for 15 years. Successful completion of the present work can lead to safe and effective medical devices for tinnitus treatment. Project Summary

Musculoskeletal symptoms, such as arthritis, tendonitis and bursitis, are very common in Systemic Lupus Erythematosus (SLE). Pain and disability from these symptoms can be significant and current treatment options for musculoskeletal pain in SLE include non-steroidal anti-inflammatory medications (NSAIDS), narcotic analgesia and corticosteroids. Each of these is limited by significant potential toxicitie including gastrointestinal bleeding and kidney toxicity in the case of NSAIDS, somnolence, constipation and nausea for narcotics and osteoporosis, osteonecrosis, diabetes, cataracts and atherosclerosis for corticosteroids. Ajulemic acid (AjA) is derived from a class of cannabinoids that retain analgesic and anti-inflammatory properties without the psychotropic effects characteristic of tetrahydrocannabinol (THC; marijuana). AjA has been shown to have potent pain- relieving effects, without psychotropic effects, in animal studies and a clinical trial in human subjects with chronic neuropathic pain. Anti-inflammatory effects of AjA have also been demonstrated in animal models as well as in human subjects and in vitro studies of human inflammatory cells. Thus far, a total of 123 subjects (healthy individuals and patients with neuropathic pain) have received AjA with no serious side effects. The overall purpose of this Phase IIa clinical trial is to evaluate the safety of AjA in SLE patients with mild to moderate musculoskeletal pain and to determine an optimum dose of AjA that will provide maximum benefit and minimal toxicity. Clinical improvement will be determined by assessment of SLE disease activity and changes in pain scales. Studies are also planned to evaluate the mechanisms of action of AjA on circulating inflammatory cells. Based on pre- clinical studies already completed in our laboratory, we expect that AjA will decrease the expression of certain pro-inflammatory proteins in peripheral blood cells and increase the expression of other proteins that enhance the resolution of inflammation.

We have demonstrated that Raman spectroscopy, a noninvasive technique yields significant information on both structural and chemical properties of the intact transparent crystalline lens. It is our objective to ascertain the feasibility of applying this technique to experimentally and naturally occurring cataracts. Our preliminary studies of cold-cataracts have demonstrated that Raman spectroscopy can be applied to provide information about cataractogenesis. Upon determining the Raman spectra signatures of cataracts, we will develop an in vivo method of studying the lens. Such an in vivo measurement of the Raman spectrum may suggest biochemical changes that occur in the lens before cataracts actually develop. An understanding of these biochemical alterations should produce significant information which is crucial to the development of methods for the prevention and reversal of cataract.

Attachment of bacteria to the host tissue represents the first critical step in a process that may lead to clinically manifested infections. Extracellular pathogens attach via specific surface located MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) to components of the extracellular matrix. We have previously demonstrated that many Gram-positive bacteria can adhere to collagen and we identified collagen binding MSCRAMMs on S. aureus and E. faecalis called CNA and ACE, respectively. We found that these MSCRAMMs can bind to multiple sites in collagen. The structure of the collagen binding domain of CNA was determined using X-ray crystallographic methods and found to contain a collagen binding extended trench on its surface. Furthermore, we found that vaccination of mice with recombinant CNA protected the animals against S. aureus induced septic death. These results form the basis for the now proposed studies where we want to identify additional collage binding MSCRAMMs and determine the structures of CNA and ACE. We hypothesize that these MSCRAMMs and other collagen adhesion receptors such as the integrins, contain trenches on their surface that represent the collagen binding sites. This hypothesis, as well as its mechanistic implication for collagen binding, will now be examined. We will identify binding sites in collagens for different adhesion receptors and characterize the interaction of synthetic triple helix collagen peptides containing these binding sites with the receptors. Finally, we will analyze in detail the established role of CNA as a virulence factor in septic arthritis and locate the protective epitopes in this MSCRAMM.

Apoptosis occurs under a variety of physiological and pathological conditions. It is essential for maintaining the homeostasis of the skin, and inappropriate apoptosis has been implicated in the pathogenesis of a multitude of skin disorders. Apoptosis mediates the decrease in cellularity during the transition between granulation to scar tissue, and aberrant apoptosis during wound healing has been attributed to the development of hypertrophic scars and keloids. Apoptosis may protect against the development of UV-induced skin cancer by selectively destroying sun-damaged keratinocytes. Mutations in the components of the surveillance machinery, such as p53 and bd-2, provide cells with an opportunity to escape from apoptosis and develop into nonmelanoma skin cancers. Early onset of apoptosis has been observed in diabetic wounds, contributing to blunted wound healing. Thus, manipulations which alter the onset or extent of apoptosis can potentially provide therapeutic interventions for the treatment of a variety of skin diseases. It is therefore important to delineate the molecular pathways and regulation of this multistep process. In this grant, I propose to use a variety of ways to manipulate the actin cytoskeleton and determine how actin remodeling contributes to UV- induced apoptosis in keratinocytes. I will examine the role of gelsolin, a Ca2+-activated actin filament severing protein which is recently identified as a direct and predominant substrate of caspase-3, in UVB-mediated apoptosis. This project is a new direction for my laboratory, but is very appropriate and timely because very little is known about the involvement of the actin cytoskeleton in apoptosis, even though the cytoskeleton is an important structural and regulatory machine. Its derangement is likely to contribute to the pathophysiological of many skin diseases. We have the tools and expertise to investigate this very important basic biological problem, and anticipate that our findings will suggest novel approaches for the treatment/or prevention of many skin diseases.

In the present project we are attempting to identify the parameters of intermediary metabolism that may be of importance for cellular radiation sensitivity, and to utilize the knowledge gained to devise improved schemes for enhancement of radiation response. Initially, we will attempt to poise cells in vitro at high and low levels of potential intracellular radical scavengers, reduced pyridine nucleotides, non- protein sulfhydryls and endogenous substrates, in the presence and absence of oxygen, and determine the radiation sensitivity of the system in terms of cell survival. Of particular value in this study are chemicals that sensitize under anoxic conditions. The use of these chemicals will enable us to control cellular electron transfer as well as the level intracellular scavengers in order to determine the importance of reductive metabolism in repair of radiation damage. Sensitizers will also be used in combination with insulin, glucose and phosphate, agents known to alter cellular radiosensitivity as well as cellular redox potential. We plan to assay radiation response with both tissue cultured cells and animal ascites tumors.

The proposal describes the design of an adaptable computer language called AID, to be used for Assistance In Decision making for acute care of trauma and burn victims. The system should overcome the underutilization of complex data now collected by ICU information systems by providing up-to-date medical consultation to users unfamiliar with their interpretation and clinical significance. The language has been designed to provide powerful capabilities for retrieval from computer medical records so that consultation may occur without time-consuming data entry by physicians, and to provide interpretations of varying degrees of certainty when data are missing. The system will permit a useful structuring of the computer record so that significant findings are presented opportunely and concisely. The initial clinical applications are for ventilatory management, head trauma, and fluid/electrolyte balance. Development of decision algorithms in these areas should lead to a more basic understanding of the pathophysiology and treatment of trauma.

The association between human leukocyte antigen (HLA) B27 and ankylosing spondylitis (AS) is among the strongest associations known between a disease and a genetic marker. Genes, both within and outside the major histocompatibility complex, are believed to account for more than 90% of the population variance in AS. While these associations clearly indicates strong genetic contributions to the susceptibility to AS, whether genes influence the severity, of AS is unknown. The severity of AS varies widely among patients, with some having severe persistent joint inflammation, functional limitation, premature work disability, and early spinal fusion. We hypothesize that the severity of -AS is genetically determined, and that genes that influence susceptibility to AS, genes that influence the severity of inflammatory responses, and genes regulating transforming growth factor-beta are among the genes that also influence its severity. We propose a model of AS severity in which genetic differences among patients influence the immune response and consequently the persistence and severity of joint inflammation. Persistent severe joint inflammation thereafter leads to spinal ankylosis and poorer long-term health outcomes. We will test this model in two stages. First, we will identify genetic markers associated with worse radiological outcomes, greater functional disability, work disability and need for total hip arthroplasty in a retrospective cohort study of 400 patients with AS of 20 years or longer. Second, we will test these genetic markers for associations with persistent active inflammation in a prospective cohort study of 200 patients with early AS who will be repeatedly assessed over 2 years. The model will be supported if the genetic markers that are associated with persistent active inflammation are also associated with poorer long-term health outcomes. In addition, the prospective cohort will enable the development and assessment of new clinical and radiographic measures of disease activity in AS for use in clinical trials. Understanding the contribution of genetic factors to differences in the severity of AS and in the health outcomes of patients would further our understanding of the pathogenesis of AS, provide important prognostic information, and could identify subgroups at high risk for poor outcomes who could then be targeted for more aggressive interventions.

Although there is very high interest in the demography and welfare of families headed by lesbian and gay (LG) parents, there is currently very limited longitudinal research on LG families or the transition to LG parenthood. Most of the extant literature is descriptive, comparative, or limited to distinct subgroups of LG parents. Of this research, demography studies that examined trends in LG family structures have rarely considered the family and individual processes that may account for changing trends in LG family formation; meanwhile developmental studies of LG parents have rarely considered the ecological and cultural influences on family and individual functioning, nor systematically considered how family structure may impact the family dynamic. Lastly, no studies that I am aware of have integrated family demography and biosocial approaches to understanding how stress is related to ecological and individual or family decisions and behaviors in LG families. The current research will use both secondary data analyses of longitudinal national datasets such as the U.S. Census and American Community Survey to examine within-state changes in rates of LG family structures over the past 10 years and how these changes correlate with state-level sociopolitical climates and public policies pertinent to LG family formation. This will be followed by a pilot study that will explicitly examine how sociopolitical and ecological factors ar associated with decisions regarding family formation and LG family functioning as mediated by individual mental health, relationship stability, and stress load. I have outlined 3 training goals that are critical for the success of this timely and innovative research study. The first goal is t develop conceptual and methodological expertise in the study of family demography, with an emphasis on contextual influences on family formation for lesbian and gay parents. The second goal is to increase my knowledge and expertise in using established national datasets such as the U.S. Census and the American Community survey to conduct secondary data analyses pertinent to the study of LG families. The third goal is to conduct a multi-state pilot using a biosocial model of individual and family formation and functioning to understand how contextual factors are related to LG individuals' well-being, decisions regarding the timing, method, and location for family formation, and parental functioning in preparation of submitting an R01 proposal on this topic in the later years of this K01 funding period. I have established an extremely strong and accomplished mentorship team that will guide my training and collaborate on each phase of the proposed research. In consultation with my mentors and consultants I have established a training plan that includes (1) mentor-based supervised training and directed activities, (2) formal graduate level coursework and short courses, and (3) participation in relevant research seminars, colloquia and scientific meetings. This training and pilot research will provide me with the skills, experience, and preliminary data necessary to establish an independent research program on this highly significant and innovative topic.

The efficacy of medical therapy is usually best demonstrated by randomized clinical trials but results of full-scale trials may be unavailable or may not be applicable to single patients. Single patient trials have been proposed to solve therapeutic dilemmas in dividual patients but are difficult for the clinician to perform. A single-patient clinical trial service will be established at a university hospital. The participants will publicize the role of single-patient clinical trials and teach clinicians about their applicability to certain therapeutic problems. They will assist clinicians in designing, executing, and interpreting randomized, crossover, controlled trials in appropriate patients. The value of the trials in therepeutic decision making for patient well being will be assessed on a case-by-case basis. The feasibility of the service will be assessed based on the value of single-patient clinical trials to patient care and the resources expended in providing the service. If successful, the service could become an ongoing, self-sustaining activity.

This proposal focuses on those drugs that are commonly utilized for the treatment of adult solid tumors, including both modern "targeted" oncology drugs, as well as cytotoxic agents. This includes studies of new candidate genes identified through recently completed genome-wide studies, both clinical and in the HapMap lymphoblastoid cell lines (LCLs). A central strategy of the proposal is the development of endophenotypes that are intermediate between clinical phenotypes, such as adverse events and measures of efficacy, and the genetic variants that affect their risk. All research projects are classified as primarily phenotype-centric, drug-centric, or gene-centric. The phenotype-centric studies will focus on markers of gene activity, such as the transcriptome, or potentially serum proteomic markers. The drug-centric studies will focus on drugs of importance for adult solid tumors. The gene-centric studies generally focus in a detailed way on candidate genes identified through these other studies. The overall goal is to identify functional relationships at the intersection of drug, gene, and phenotype, particularly those phenotypes that are affected by one or more functional polymorphisms when patients are treated with a specific anticancer drug (at a clinically relevant dose). Mathematically, this can be described by a multidimensional matrix (to be made publicly available through a PGScore database) of variation in a specific gene (or polymorphism) on the effects of a drug as measured by a specific phenotype. A consistent framework is utilized to address questions relevant to six interrelated Themes (Cytotoxicity, Transcriptome, Irinotecan, Angiogenesis, UGT, and EGFR), supported by two Platforms (Clinical Studies and Functional Studies) and four Cores (Management, Genetics-Informatics-Statistics, Liver, and LCL). Extensive collaborative clinical studies are proposed with CALGB, as well as other current PGRN Groups. Other ongoing collaborations will be continued, including studies of glucuronidation and LCLs. In addition, a new Network Resource is proposed. Cell Lines as a Resource for Pharmacogenomic Studies. PUBLIC HEALTH RELEVANCE: There is marked variability in pharmacological response to anticancer agents, which historically have been characterized by severe toxicity and inconsistent efficacy. This proposal aims to characterize the genomic basis for this variability through a series of interrelated studies addressing a breadth of modern and classical anticancer agents. This will include both laboratory and translational clinical studies.

There are two main objectives of this research: (1) to develop the methodology - utilizing both instrumental and radiochemical neutron activation analysis - to determine selected element concentrations in cow's milk, infant formula, and human milk; and (2) to compare these element concentrations in pooled samples of cow's milk and infant formula to individual samples from human volunteers (20-30) sampled over that period of time prior to use of supplemental foods (3-6 months). The elements expected to be determined are Fe, Cu, Mn, Zn, Co, Mo, Se, Cr, As, Sb, Cs, Rb, Eu, K, Ca, Br, Na.

The current proposal, for the first time, addresses the role of neuro-immune activation within the central nervous system as a plausible mechanism underlying the visceral pain component of many common functional gastrointestinal disorders (FGID) including irritable bowel syndrome (IBS). Although increasing evidence has emerged for the role of spinal glia in the mechanisms mediating persistent pain, its implication in visceral pain has not been evaluated. We propose the general hypothesis that chronic stress, known as a key factor in the first onset or exacerbation of IBS symptoms, triggers the activation of a spinal network comprising neurons and immune cells (glia), playing a central role in the modulation of visceral nociception. Using a rat model of chronic water avoidance stress, we propose to characterize the role of stress-induced spinal glia activation in the modulation of visceral sensitivity and determine the molecular pathways engaged in the initiation and maintenance of visceral hyperalgesia. Our first specific aim is to assess the temporal profile of spinal glia activation during and after chronic stress and to test the ability of inhibitors specific of glia to reduce stress- induced visceral hyperalgesia. The second specific aim relates to the characterization of molecular mechanisms linking chronic stress and spinal glial activation. The experimental design proposed in this application includes the behavioral assessment of visceral sensitivity in response to different pharmacological treatments (antagonists, agonists, oligonucleotide antisenses), combined with in vitro analysis using Western blotting, multiplex ELISA, immunohistochemistry and quantitative RT-PCR. The long-term goal of the proposed studies is the characterization of signaling pathways underlying the observed endocrine-neural- immune interactions using selective knockout animals and glia/neurons co-cultures. Targeting specific mediators of the glial-neuronal crosstalk may provide an innovative approach for the development of novel therapeutic targets for the treatment of chronic pain conditions associated with enhanced stress responsiveness. The concept of stress-induced modulation of glial-neurons signaling and its implication in long-term alteration of the sensory system may be generalized to many other stress-sensitive pain conditions, including interstitial cystitis, non-cardiac chest pain and fibromyalgia. PUBLIC HEALTH RELEVANCE: In the current proposal, we have developed the concept that chronic stress may induce activation of the immune system in the spinal cord, initiating a cascade of events that will affect the sensory system. This new approach has considerable implication for the development of novel therapeutic targets for the treatment of chronic pain conditions associated with enhanced stress responsiveness, including functional gastrointestinal disorders, for which effective treatment remains a clinical challenge.

Schizophrenia, Parkinson's disease, Tourette's syndrome, obsessive-compulsive disorder and drug addiction, are only some of the relevant clinical conditions that have been related to dysfunction of brain dopamine systems and abnormal processing of cortical input in the striatum. The robust membrane potential depolarizations ("up states") during which striatal neurons become responsive to the fine structure of cortical input, are synaptically-driven events invoked by the cortex itself and probably shaped by dopamine-regulated membrane currents. The suggestion that the striatum behaves as an "action selection" network, raised interest in understanding how dynamic patterns of cortical activity are represented in the striatum and how this representation is modified by changes in dopamine neurotransmission. By simultaneously recording the population activity of cortical neurons and the membrane potential of striatal neurons, we have recently clarified some aspects of the temporal dynamics of up states. In this proposal, we plan to analyze the representation of spatial variations of cortical activity on striatal neurons by recording field potentials and multiunit activity from multiple cortical sites together with the membrane potential of striatal neurons in anesthetized rats. Our main goal is to understand how spatially-distributed and dynamically-changing patterns of cortical activity are reflected in the membrane potential (as an index of synaptic input) and firing pattern (as an index of neuronal output) of striatal neurons. Furthermore, we will investigate the impact of cortical activity on striatal neurons in genetically-modified mice lacking functional D1 or D2 dopamine receptors. This research will be done primarily in Argentina, at the Buenos Aires University School of Medicine, in collaboration with M. Gustavo Murer, as an extension of NIH grant # R01MH060131.

This project on human immunogenetics of the major histocompatibility complex is an integrated investigation of immunological, genetic, and biochemical aspects of the HLA complex and closely linked genes. The overall objectives for this project are to obtain a better understanding of the genetic factors responsible for allograft reactions, to identify the immunological mechanisms involved in such reactions, and to understand the relationship between the HLA complex and genetic factors predisposing to diseases. The studies are divided into four projects. (1)\Cell surface phenotype and function of human alloactivated lymphocytes. This project utilizes the techniques of T-lymphocyte cloning for the analysis of lymphocyte diversity. (2)\In vitro production by human B lymphocytes of human monoclonal antibodies detecting cell surface antibodies is being investigated. (3)\Serological, biochemical, and genetic analysis of the human equivalent of the thymus-leukemia (TL) antigen will be undertaken and compared with the murine counterparts. (4)\The relationship between a disease-causing gene and HLA will be investigated using congenital adrenal hyperplasia due to 21-hydroxylase deficiency as a model. This project utilizes molecular genetic tools for the analysis of the genes in the HLA region. The major emphasis in the studies of alloreactivity during the last year has been the interactions between cell surface determinants on allocytotoxic-T cells and specific epitopes on cell surface antigens on the target cells. These studies have involved allocytotoxic, human T-cell clones. The conclusions are based upon the combined analysis of biochemical studies of MHC antigens using murine anti-HLA monoclonal antibodies, analysis of T-lymphocyte differentiation antigens with monoclonal antibodies and blocking studies of allocytotoxicity with these two sets of antibodies. The investigations have revealed a distinct but complex pattern. It has been demonstrated that multiple HLA-D-related class II antigens all carry allotypic determinants which can be recognized by cytotoxic T cells. The contribution of different T-cell differentiation antigens in the cytotoxic reaction against class I and class II antigens is also complex. While the T4 positive T cells primarily recognize class II antigens and T8 positive T cells primarily recognize class I antigens, this correlation is far from complete. The second major progress within this project on human immunogenetics has been achieved in the molecular genetic analysis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OH-Def.). We have during the last year cloned and characterized a cDNA for adrenal 21-hydroxylase. This cDNA has been used to map two 21-OH-genes within the murine H-2 complex. These genes are located within the S-Region of H-2 and very close to H-2D. The same cDNA probe has been used to characterize the 21-OH genes within the HLA complex. At least one of these two genes are deleted in the HLA-Bw47 positive HLA haplotypes found in some patients with the severe, salt-wasting form of 21-OH-Def. These studies indicate that CAH due to 21-OH-Def. is a defect in the structural gene for adrenal cytochrome P450 21-hydroxylase. (CS)

Continuing biological evaluation of potential anticancer and antisepsis chemotherapeutic analogues of members of the hydrolyzable tannin family of secondary plant metabolites is proposed. Characterization of the immunomodulatory properties of these species will proceed. Mechanism-of-action investigations at the molecular, cellular, and whole animal levels, coupled with structure/activity studies, are designed to reveal the basis for both up-regulation and down-regulation of cytokine (IL-lb and TNFa) secretion by certain tannins. A novel tumor targeting strategy for tannin delivery will be explored. Appreciation of the molecular-level details responsible for either triggering or suppressing cytokine release may serve as the basis for designing tannin-inspired chemotherapeutic agents for diseases as diverse as cancer and septic shock. Ongoing synthesis studies directed toward the potent marine anticancer principle diazonamide A will be brought to completion. In addition to providing a supply of this scarce material for further biological evaluation, successful execution of the plausibly biomimetic synthesis route will provide insight into the otherwise obscure biosynthesis of this complex peptide-based secondary metabolite. Finally, new studies on the synthesis of the architecturally complex 20S proteosome inhibitor TMC-95A and rationally designed analogues will be pursued. A possibly biomimetic (modified) tryptophan oxidative cyclization forms the centerpiece of the approach, and acquisition of the target and the aforementioned analogues will enable mechanism-of-action studies to move forward. Inhibition of the 20S proteosome can form the basis for chemotherapeutic intervention in a variety of disease states, including cancer, cachexia, and sepsis. The search for selective 20S proteosome inhibitors among the Ntn-type proteases will be advanced by these investigations.

The present study is a continuation and further development of a novel program of research designed to reduce the onset and extent of drinking by students during their first year of college, through provision of efficacious individually oriented interventions prior to college entrance. We aim to influence the drinking behavior of college-bound high school athletes during the spring of their senior year of high school, by enhancing the influence of their parents and/or by introducing them to college student athletes who will serve as peer counselors to provide a brief motivational feedback intervention. Athletes represent a high-risk and understudied group, and existing research indicates high school athletes drink as much as or more than other students. Research also indicates that for many students, excessive drinking in college is a continuation or exacerbation of high-school drinking tendencies. However, almost all current approaches in the college-drinking domain are based on implementing interventions while the students are at college. Further, there have been no theory-driven, systematic interventions targeting high school athletes so as to prevent alcohol misuse as they transition to college. We intend to implement two early interventions for high school student athletes: (1) a Parent-Based Intervention (PBI) based on the work of Turrisi and colleagues and (2) a Brief Peer-delivered Motivational Intervention (BPMI) based on the work of Larimer and colleagues. There is sufficient empirical evidence demonstrating the efficacy of these interventions in college and older adolescent samples to warrant an examination of their unique and combined additive effects for a high-risk high school athlete sample. The integration of these two research programs will provide a unique theoretical framework and a rich database from which to assess the benefits of parent and peer-delivered intervention approaches at this late stage of adolescent development. Thus, the research will investigate, through the use of a factorial design, the unique and combined utility of the PBI and the BPMI to reduce alcohol onset, usage and negative consequences among high school athletes so as to ultimately reduce college student drinking; identify demographic and psychological characteristics of high school athletes for whom the interventions are more effective versus those for whom the interventions are relatively ineffective; and examine processes by which the interventions influence use and negative consequences for students.

Although percutaneous transluminal coronary angioplasty (PTCA) appears to be a relatively safe and effective procedure, reported complications include coronary occlusion and dissection with or without acute myocardial infarction. We reported this complication of PTCA. A 77-year-old man with unstable angina pectoris by coronary angiography had focal severe stenosis of a dominant right coronary artery, a normal left coronary system, and normal left ventricular function. PTCA of the right coronary artery was performed. The tight stenosis prevented appropriate placement of a 2-mm. Gruntzig catheter; therefore, the narrowing was dilated initially by multiple inflations of a Simpson catheter advanced across a guide wire, and subsequently by a 3-mm. Gruntzig catheter at 4 atm. Post-PTCA angiograms demonstrated mild residual stenosis. Coronary angiography 3 months later disclosed significant stenosis again at the site of the previous dilatation procedure. Repeat PTCA was performed. Multiple inflations of a 3-mm. Gruntzig catheter at a maximal pressure of 9 atm reduced the stenosis to about 60% diameter reduction. A 3.7-mm. Gruntzig catheter was then positioned and inflated 5 times at a maximal pressure of 10 atm. On the fifth inflation, the proximal end of the balloon ruptured. Angiography then demonstrated extravasation of contrast material into the epicardial tissue plus run-off into the distal right coronary artery. The heart rate and arterial blood pressure quickly decreased; blood was drained from the pericardial sac, but fatal cardiac arrest ensued. At necropsy, the heart weighted 265 g. The epicardial adipose tissue surrounding the right coronary artery was hemorrhagic and an elliptical rupture site measuring 0.6 cm. in the long axis was present in the right coronary artery 3.5 cm. from the ostium. Radiographs of the major epicardial coronary arteries disclosed focal calcific deposits.

Eukaryotic cells show a high degree of macromolecular organization in which specific components are segregated into discrete structures with specialized functions. Appropriate model systems have been chosen to study the mechanisms by which proteins reach their final destination within organelles or membranes. 1. We have developed an in vitro model system using cultured epithelial cells of renal origin in which we will investigate, using biochemical, biophysical and ultrastructural methods, how tight junctions are formed and plasma membrane polarization is established during monolayer formation and after junctions are opened by experimental procedures. 2. In the same cell system production of enveloped viruses occurs in a polarized fashion. We will therefore be able to investigate the mechanism by which viral envelope glycoproteins are segregated into specific areas of the plasma membrane. 3. In vitro studies on the biosynthesis of selected membrane and organelle proteins will be carried out to identify features of the primary translation products which may be involved in directing these proteins to their functional location. The role of post-translational modifications carried out by intracellular membranes will be examined. It is hoped that these studies will contribute to a better understanding of membrane changes which may accompany cellular and organismic senescence.

Project Summary The proposed work focuses on identifying novel miRNA drivers in cancer and utilizes this and other knowledge to develop miRNA-based therapeutics. This Pathway to Independence award application includes a mentored career development plan for the transition of the candidate, Dr. Andrea Kasinski, into an independent investigator, as well an accompanying research plan describing the proposed experiments on discovering miRNAs that can potentiate KRAS-driven lung adenocarcinoma and exploring novel therapeutic strategies for re-expressing tumor-suppressive miRNAs, which includes a high-throughput screen to identify a small molecule that can restore miRNA processing and combinatorial miRNA therapies for sensitizing tumors to conventional chemotherapies. The candidate, Dr. Kasinski, is a postdoctoral fellow at Yale, in the lab of Dr. Frank Slack in the Department of Molecular, Cellular, and Developmental Biology. The work leading to her graduate degree in Genetics and Molecular Biology at Emory University was conducted in the lab of Dr. Haian Fu in the Department of Pharmacology and focused on targeting cell survival signaling for therapeutic development. In the Fu lab, Dr. Kasinski performed two very distinct projects that were interrelated based on the ultimate goal of developing targeted therapeutics: in one project Dr. Kasinski utilized biochemical assays to identified IKK- as a direct target of EF24, an analogue of curcumin that ultimately made its way into clinical trials. For the second project a series of genetic and molecular techniques were used to evaluate the transcriptional regulation of an oncogenic 14-3-3 family member, with the hope that this knowledge might spearhead subsequent studies to block 14-3-3 expression. The mentoring and career development plan will supplement her background, which is evenly split between genetic, molecular biology, whole animal studies and small molecule screening, with training and instruction in each, and in the particular areas that this project involves: murine biology, cancer biology, cell culture technique and high-throughput drug screening. Dr. Kasinski's goal is to become a faculty member in an interdisciplinary biosciences, cancer biology, or similar department at an academic, private, or government facility, in which she can research the biology of miRNAs in cancer and work to advance miRNA-based therapies. This research on miRNAs involvement in cancer requires that an innovative and selective screen be performed which Dr. Kasinski is actively pursuing first in cell culture and will advance into animal models. This assay in soft agar is selecting for miRNAs that can specifically cause normal human lung bronchial epithelial cells to become transformed. MiRNAs identified from this assay will be evaluated further in cell culture and ultimately in vivo. The proposed study involving miRNA therapeutics is two fold. The first takes into account the interaction of let-7 and LIN-28. Dr. Kasinski is in an active collaboration with Dr. Haian Fu, Co-Director of the Chemical Biology and Drug Screening Center at Emory University, to perform a high throughput screen (HTS) to identify inhibitors of this protein-RNA interaction. Hits from this HTS will be evaluated for cell permeability, kinetics and in vivo therapeutic potential, by several independent avenues of investigation. Finally building on Dr. Kasinski's current findings that miR-34 and let-7 represent valid therapeutic options for non-small cell lung cancer, she will evaluate these miRNA therapies in combination with currently used chemotherapies and targeted-therapeutics in cell culture, xenografts and in the Kras;p53 double mutant. This work is novel, timely and has clear and significant implications for human health and survival. The identification of miRNAs and targeted-therapies to perturb miRNA imbalance that occurs in cancer is invaluable to the fields of cancer and miRNA biology, and has direct clinical application.

The malaria parasite Plasmodium falciparum has a complex life cycle involving two hosts. P. falciparum gametocytes are the form of the parasite which undertakes the transition from the human to the mosquito host. The developmental changes required for this transition are poorly understood. There is, however, some evidence for a shift in metabolic energy generation from glycolysis in the human host to respiration in the mosquito host. The primary goal of the proposed study is to test the hypothesis that P. falciparum mitochondria undergo developmental changes during gametocytogenesis that allow them to become functionally competent for respiration. A secondary goal is to determine if the timing of biochemical changes in developing gametocyte mitochondria correlate with changes in P. falciparum sensitivity to the antimalarial drug primaquine. The long-term goal of this study is to elucidate the mechanisms by which gametocytes regulate changes in metabolism in preparation for the shift in hosts. There are three specific aims for the proposed study. The first specific aim is to develop assays to measure the expression of marker proteins representing the three major mitochondrial activities. The marker proteins selected are cytochrome b, which is required for electron transport, mitochondrial malate dehydrogenase, which is required for the tricarboxylic acid (TCA) cycle and for the malate shuttle, and cytoplasmic malate dehydrogenase, which is required for the malate shuttle. These assays will require preparation of nucleic acid and antibody probes for each marker protein. The second specific aim is to characterize the biochemical changes in P. falciparum mitochondrial during the transition from the human to the mosquito host. To accomplish this, the expression of the marker proteins will be measured in asexual erythrocytic parasites, in parasites in early stages of gametocyte development, in parasites in late stages of gametocyte development and in zygotes. The third specific aim is to determine if there is a correlation between the biochemical changes in developing gametocyte mitochondria and the development of primaquine sensitivity. To accomplish this, a vital dye will be used to first determine how mitochondrial structure and transmembrane potential change during asexual and sexual developmental stages. Then the vital dye will be used to assay these same stages for the effects of primaquine on mitochondrial potential and cell viability. These studies will increase our understanding of the role of P. falciparum mitochondrial biogenesis in the transition from a human to a mosquito host.

Cardiovascular disease remains the leading cause of death in the United States and vascular calcification and diabetes are both independent risk factors for incident cardiovascular disease. Fetuin-A is a hepatic secretory protein found in high concentrations in human serum and our preliminary studies suggest that feutin- A may simultaneously inhibit vascular calcification and promote insulin resistance and diabetes. We propose to explore the relationship of fetuin-A with the longitudinal development and progression of vascular calcification and diabetes in the Multi-Ethnic Study of Atherosclerosis (MESA) cohort. The research evaluates biological mechanisms interlinked by a single protein and will provide new insights to cardiovascular biology and diabetology simultaneously. Conducting this research within MESA has a number of advantages. MESA stored sera from the baseline visit, allowing fetuin-A measurement. MESA extensively measured subclinical cardiovascular disease;therefore, all participants already have measurements of vascular calcification, vascular function, and metabolic parameters. Additionally, MESA participants were followed longitudinally, allowing for evaluation of the associations of baseline fetuin-A concentrations with incident vascular calcification and diabetes. We propose to take advantage of this rich resource to explore the following primary aims: (1) To determine the cross-sectional and longitudinal associations of fetuin-A with vascular calcification and vascular function;and (2) To determine the associations of serum fetuin-A concentrations with adiposity, impaired fasting glucose, and diabetes mellitus. This research will take advantage of the hypothesized countervailing effects of fetuin-A on vascular calcification and diabetes to provide new insights to cardiovascular disease pathogenesis. PUBLIC HEALTH RELEVANCE: While cardiovascular disease and diabetes represent common and morbid diseases, our understanding of their pathogenesis remains incomplete. The results of this epidemiologic study will provide novel insights into their pathogenesis and may ultimately identify novel therapeutic targets for their prevention or treatment.

PROJECT SUMMARY Stroke is the fifth leading cause of death and the leading cause of chronic disability in the United States. Each year approximately 780,000 people suffer an acute ischemic stroke (AIS) in the United States at a cost of over $60 billion annually to the US health care system. Up to half of these strokes are due to thromboembolic occlusion of a large cerebral artery potentially accessible by catheter for endovascular treatment. Emergent large vessel occlusion (ELVO) AIS is particularly devastating and, due to the amount of thrombus burden in the arteries at the base of the brain in this disease, intravenous tPA is often insufficient to recanalize the cerebral arteries, reperfuse the brain tissue, and prevent irreversible infarction. In 2015 five randomized controlled clinical trials demonstrated the clinical superiority of catheter-based endovascular embolectomy to medical therapy alone in the first six hours after stroke onset. This has transformed the stroke community, with efforts to organize comprehensive stroke centers to treat ELVO patients rapidly along the lines of heart attack or major trauma treatment in the cardiology and trauma communities, respectively. Fewer than 10% of ischemic stroke patients receive treatment largely due to the narrow recommended time window for therapy within 3-6 hours of stroke onset. The basis for this recommendation is that the longer brain is deprived of blood flow, the more likely it is to infarct, and the more infracted brain there is, the more likely it is to hemorrhage after opening the occluded artery. Advances in neuroimaging are shifting the paradigm from ?time is brain? to ?physiology is brain?. Perfusion imaging (CT and MRI) and diffusion weighted MR imaging (DWI) now allow detection not only of infarcted brain but also of brain that is potentially salvageable if blood flow can be restored. MR DWI is the gold standard in medical imaging for identifying irreversibly infarcted brain (core infarct). Brain can progress from reversible but severe ischemia to irreversible infarction over the matter of minutes. Whereas current acute stroke diagnosis occurs by CT or MRI, treatment requires moving the patient to an X-ray angiography suite for endovascular therapy, leading to delays in care and variability in clinical outcomes. Current X-ray guided endovascular stroke treatment procedures have limitations that could be overcome using MRI guidance, including real-time tissue viability assessment and, in some cases, enhanced catheter navigation. We propose to develop the endovascular tools needed to treat patients safely and effectively whether in the strong magnetic field environment of MRI or the high ionizing radiation environment of X-ray angiography. Devices that are safe to use in the 3T MRI environment would allow patients to be treated in any standard clinical MRI scanner, thus allowing real- time assessment of brain viability during endovascular intervention. The strong magnetic field of the MRI scanner provides a unique opportunity to develop remote-controlled catheters that can navigate through the vasculature by creating magnetic forces on their tips. We have previously developed such magnetically assisted remote controlled (MARC) catheters for use in larger vessels of the body and neck; we now propose to develop smaller, more flexible versions for use in the vessels of the brain. Success in this project could transform the treatment of diseases like AIS that benefit from real-time physiologic tissue monitoring during therapy, thereby maximizing benefits and minimizing risks of treatment.

A population of diabetics, both juvenile-onset and adult-onset, either on diet, insulin or oral hypoglycemic agents, will be screened using the photon absorption technique to detect the incidence of low bone mass. The presence of abnormally low mass will be correlated with indices of diabetic control. From the population of diabetics screened, a group of patients with low bone mass and a group with normal bone mass will be chosen for more extensive studies of calcium absorption, parathyroid hormone and vitamin D metabolism, in an effort to uncover the etiology of this abnormality. In a small population of patients with the most severe loss of bone, iliac crest biopsies will be performed for quantitative histomorphometry. One-hundred patients with diabetes who present for the first time to the clinic will be entered into a prospective study in which changes in bone mass, measured by both the photon absorption technique and radiogrammetric and radiodensitometric techniques, will be followed. Changes in bone mass will be correlated with level of diabetic control, vitamin D metabolities and parathyroid hormone levels.

The objective of this proposal is to research the technical and commercial feasibility of a novel wrist brace that reduces the risk for wrist fracture by resisting wrist terminal extension while otherwise allowing unconstrained movement throughout the normal range of motion. The novel wrist brace will be designed, fabricated, and validated in our laboratory under simulated impact conditions. The long-term goal of the product is to determine the efficacy of the brace on snowboarding and in-line skating populations using a prospective, randomized, longitudinal study. Wrist braces offer limited protection against distal radius fractures, yet they have achieved limited acceptance, particularly among youths, because they are bulky and limit normal range of motion. Research has also demonstrated that they do not prevent fractures under high impact energy loading conditions. We postulate that wrist guard usage will increase significantly with a device that is less constraining and which offers increased protection against distal radius fractures

A study of the role of blood proteins and receptor-mediated endocytosis in the metamorphosis and egg formation of insects is proposed. Vitellogenin, lipophorin, and three hexameric storage proteins, which comprise most of the blood proteins of Hyalophora pupae, are to be isolated and labelled for use as physiological probes. A series of tests is designed to assess whether the probes are handled by the tissues of the insect in the same manner as before isolation and labelling. The fates of the probes when presented to developing tissues either in situ or on isolation in a culture system will be examined. It is anticipated that each protein will prove to be endocytosed by a particular tissue and at particular stages of development, and that it will either be incorporated into cellular structures intact, as in the case of vitellogenin, or hydrolyzed to release amino acids for use in synthesizing other tissue proteins. The binding selectivity of each target cell for the five probes will be determined, and since the first two to be isolated have proven to be mannose-rich glycoproteins, the role of sugars in ligand binding and endocytosis will be studied.

The objective of this proposal is to bring to bear state of the art theoretical methods to the study of the mechanisms of ribozyme catalysis and the factors that regulate reactivity. An overarching theme in the proposal is to facilitate active collaborations with a network of experimental groups in order to progress toward a consensus view of mechanism that may, ultimately, contribute to a deeper understanding of more complex cellular catalytic RNA systems such as the ribosome and spliceosome. The proposal focuses on application to a series of archetype ribozymes that catalyze site-specific phosphodiester bond cleavage (and ligation) but that have different active site architectures and catalytic requirements. As a baseline, the same reaction will be studied in a non-enzymatic dinucleotide model in solution, and catalyzed by the protein enzyme analog, RNase A. The tandem study of alternate mechanistic strategies and the factors that govern reactivity will provide penetrating insight into the rational design of new biomedical technology. However, these applications demand new methodological advances. Of key importance are accurate and efficient quantum mechanical/molecular mechanical (QM/MM) methods for catalysis, reliable molecular simulation force fields for RNA and metal ions, and efficient methods for sampling free energy surfaces and conformational transitions. Toward this end, we propose to: 1) improve the QM, MM and QM/MM models for ribozyme catalysis, 2) develop a new QM/MM method for prediction of pKa shifts in ribozymes;3) develop a novel free energy expansion approach to extend our QM/MM methods to the ab initio level;4) develop efficient path methods to study chemical mechanisms and conformational transitions in ribozymes. In this way we hope to greatly extend the predictive capability and range of application of state of the art theoretical methods to RNA catalysis. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to use quantum mechanical and molecular simulations methods to study the mechanisms whereby molecules of RNA catalyze important chemical reactions. The insight gained by these studies will enhance our understanding of the fundamental role RNA plays in cells and facilitate the design of new RNA-based biomedical technology.

Discover new drugs with anti-Candida activity from four unique sources: antimicrobial-producing microorganisms, plant tissue cultures, "immunoliposome"-encapsulated anti-Candida drugs, and novel cell wall inhibitory analogs from syntheticchemical efforts of our laboratories. In vitro models for initial rapid evaluation of potentially active drugs from thesources mentioned above will be used. These in vitro models will allow us to ascertain MIC values by established methods. Since the fungal cell wall contains enzymes and components not found in human tissue, the evaluation screens will focus on compounds that inhibit fungal cell wall synthesis to develop a drug that is pathogen-specific. Animal model evaluation of potential drugs based on in vitro tests will utilize an established mouse animal of candidiasis. Parameters of the animal model systems will be varied to determine the method and route of administration, timing and dosage necessary for anti-Candida activity, and toxicity.

To elucidate the mechanisms involved in the localization of neurotransmitter receptors under nerve terminals, we will analyze the interactions between acetylcholine receptors (AChRs) and cytoskeletal components in the nicotinic post-synaptic membranes isolated from Torpedo electric organ. In those isolated membranes, as at the vertebrate neuromuscular junction, AChRs are constrained and unable to diffuse freely, but upon removal of peripheral proteins from that membrane, constraints on AChR mobility are effectively removed. It is our hypothesis that interactions between AChRs and peripheral membrane proteins serve to restrict receptor mobility, and we will characterize the structures of those peripheral proteins and identify the protein interactions that account for AChR immobilization. By immunological techniques four peripheral proteins (43K, 58K, 87K, and 270K) have been identified on the cytoplasmic face of the Torpedo post-synaptic membrane and also at the vertebrate neuromuscular junction. Experiments will focus on the 43K protein, which is present in stoichiometric amount with AChRs, and the 270K protein that has been shown by immunological criteria to be related to dystrophin, the protein product of the Duchenne/Becker muscular dystrophy gene locus. Immunoelectron microscopy will be used to identify the filamentous cytoskeletal elements associated with the innervated surface of the Torpedo electrocyte and to determine the disposition of 27OK/dystrophin as well as 58K and 87K proteins relative to AChR/43K, the lipid bilayer and the cytoskeleton. Biochemical and immunochemical techniques will be used to define the mechanism of association between 43K protein and AChRs and also with lipid. The 270K protein will be isolated and characterized by protein microsequencing to determine whether that protein contains all the structural domains of muscle dystrophin. Biochemical and immunochemical techniques will be used to determine whether 27OK/dystrophin interacts directly with AChRs or other integral membrane proteins of the post-synaptic membrane and to determine whether it binds to the actin cytoskeleton. To determine whether 58K and 87K proteins are related to previously described muscle proteins, they will be isolated and characterized by protein microsequencing.

Studies have shown that the loss of fragile X mental retardation protein (FMRP) causes a mental retardation condition known as fragile X syndrome. The protein contains two RNA binding domains known as K homology domains (KH domains). The specific function and target of these RNA binding domains are not known nor is it known why the loss of FMRP leads to mental retardation. Therefore to understand the molecular function of the FMRP, one needs to first study these domains. It is the aim of this proposal to identify RNA targets and RNA binding characteristics of the first KH domain in FMRP. The SELEX procedure (Systematic Evolution of Ligands by Exponential Enrichment) will be used to identify RNA targets. SELEX is an in vitro selection procedure that identifies high affinity nucleic acid ligands for a given protein. The sequences of the ligands obtained from SELEX are not necessary that of the in vivo target but they can be used as templates in searching genome sequences for possible targets. Once a high affinity target is identified, phage display will be used to investigate the binding of the KH domain to the target. By displaying mutant proteins on the coat of the phage, phage display enables efficient screening and selection of a large pool of mutants. The purpose of the mutation analysis is to identify the residues that participate in RNA binding. Identifying potential RNA targets and regions of the KH domain responsible for RNA binding may help to elucidate the physiological function of the FMR protein.

Biological therapy of cancer is defined as therapy directed at biological functions of the host or the tumor with change host tumor interactions in favor of host destruction of tumor cells or regulation of tumor cell proliferation. Biological response modifiers include immunotherapeutic agents, interferons, other cytokines, maturation and differentiation factors and vitamins and other nutritional factors. Augmenting agents include natural or synthetic substances which stimulate various host defense mechanisms such as macrophage tumoricidal activity, NK cells, interferon production, etc. In this project, we will investigate, in control Phase I and II studies, therapy with "second generation" augmenting agents of defined chemical structure. Optimal doses and schedules will be sought using a unique statistical approach and various assays of host defense activation including interferon induction, macrophage, NK cell and RES activation. Once optimal "augmenting" doses are defined, the agents will be introduced into Phase II trials (alone or in combination with conventional therapy) to determine 1) if the agent will increase the remission rate, duration, and survival, 2) if the degree of "augmentation" correlates with the therapeutic effects. Initially, MVE-2 (the 15,500 MW fraction of pyran copolymer) and ABPP (2-amino, 5-bromo, 6-phenyl pyrimidinole--an orally active interferon inducer) will be studied. These serve as models for the study of macrophage activators and interferon inducers. Each year of the project, two new augmenting agents will be introduce into Phase I trials and two will be transferred to Phase II trials. These studies should establish the therapeutic activity of a variety of augmenting agents for cancer management.

Studies were performed to investigate the effects of chronic, low level pre-and postnatal lead exposure on immune functions in rats. Weanling female rats were exposed to lead (as lead acetate) in their drinking water at 0, 25, and 50 ppm for 7 weeks. At the end of 7 weeks they were mated with untreated males and continued on the same dosage throughout gestation and lactation. The offspring of these females were weaned at 21 days of age and continued on the same lead exposure regimen as their mothers. These offspring were used in immune surveillance procedures between 35 and 45 days of age. Lead exposure at the levels employed had no statistically significant effect on growth and did not result in overt signs of toxicity. Thymic weights were significantly decreased in both males and females of the two lead dosage groups. Furthermore, lead exposure resulted in suppression of responsiveness of lymphocytes to mitogen stimulation and in reduced delayed hypersensitivity responsiveness. A marked depression in the antibody response to sheep red blood cells (SRBC) as well as decreased serum IgG levels were observed. Serum IgM and IgA levels were normal.

The goal of these studies is an in-depth investigation of the thymus derived lymphocyte and its products. Lymphotoxin (LT), a product of antigen or mitogen activated T cells, is being studied as a model lymphokine. The long term goals are to study LT's cell of origin, biochemical nature, mechanism of action, relationship to other lymphokines, regulation and role in vivo. Murine lymphotoxin cDNA has been cloned with mRNA from an interleukin-2 maintained T cell clone. Its gene has been isolated and mapped to chromosome 17. The specific aims of the next 5 years are to analyze the structure of the LT gene, to precisely localize it on chromosome 17, to determine the linkage relationship of the genes for LT and tumor necrosis factor (TNF), to obtain maximal expression of LT cDNA, to analyze regulation of LT and TNF, to study expression of the transfected LT gene and to analyze the 5' regulatory sequence of the LT gene. Goals will be accomplished by sequencing the gene, by identifying LT promoter activity, and by expressing LT DNA in prokaryotic and eurkaryotic cells. Interleukin-2 maintained T cell clones will be exposed to several inducing agents and their LT and TNF mRNA analyzed by Northern blots. The murine LT gene will be ligated to strong promoters and its expression studied in human T cell tumors. Similar vectors will be used to study the effect of LT gene overproduction in transgenic mice. Such mice will be analyzed for autoimmune pathology. If the LT gene is overexpressed in the T cells of these mice, and those cells killed, a murine model for AIDS will be available. LT 5' regulatory sequences will be ligated to a neutral gene, chloramphenical acetyle transferase, in order to study regulation of the LT gene in the absence of effects attributable to the product itself. Studies with transfected T cells, B cells, macrophages and transgenic mice will provide insigt into the nature of T cell specificity of LT expression and eventually lead to an understanding of the molecular basis of LT gene activation by antigen.

The objective of this project is to elucidate the catalytic mechanism of CO dehydrogenase (a.k.a. acetyl-CoA synthase - ACS), emphasizing the structure and function of the unique Ni-Fe-S clusters contained therein. The enzyme is an alpha-2-beta-2 tetramer that catalyzes two reactions; the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA. The former occurs at one Ni-Fe-S cluster in the beta subunit, while the latter reaction occurs at another Ni-Fe-S site in alpha. A tunnel through which CO migrates connects the two sites. The reaction mechanism is "organometallic" in nature; how does Ni promote this within an aqueous protein matrix? Mechanistic steps occurring at the two sites are synchronized, making it an attractive system to investigate details of metabolic channeling and active-site coupling. ACS is useful biotechnologically; organisms containing it reduce atmospheric levels of CO and degrade TNT. ACS is found in Clostridium difficile, a pathogenic organism responsible for the deaths of about 2000 people annually. Given the complexity of this enzyme, the general strategy used in this project will be to study each activity separately, and then compare observed properties with those of the bifunctional enzyme; such an approach may reveal some of the complexities of channeling and catalytic coupling. This approach is possible because isolated recombinant alpha subunits able to catalyze the synthesis of acetyl-CoA can be prepared, as can a homolog of the beta subunit that is able to catalyze CO/CO2 redox. Many of these developments (recombinant biosynthesis of active alpha, discovery of the tunnel and of active site coupling) arose from efforts of the previous granting period which resulted in 11 publications. The methodologies used for the project include enzyme kinetics (stopped-flow, rapid freeze-quench, and steady-state), spectroscopy (EPR, Mossbauer, and XAS), and X-ray diffraction. A rapid-freeze-quench instrument that allows samples to be prepared under reliably anaerobic conditions has been constructed, and should be an improvement over existing technology. Kinetic data will be simulated, ultimately leading to a comprehensive mechanistic model describing the general kinetic behavior of the enzyme. Crystals of alpha have been obtained, suggesting that an X-ray diffraction structure may be achievable. These studies require repetitive purification of at least 5 different proteins; funding for a preparative FPLC/HPLC is requested.

MeCP2-duplication/triplication syndrome is a devastating neurodevelopmental disorder mostly affecting boys and is caused by duplication (or triplication) of the region in the X-chromosome containing the gene encoding MeCP2 (methyl-CpG-binding protein 2). MeCP2 binds to methylated CpG sites on DNA and recruits transcription complexes to modulate gene expression. Because MeCP2 modulates a multitude of genes, it is not surprising that pinpointing specific functions has proven difficult. In additio to MeCP2-duplication syndrome, loss-of-function mutations in MeCP2 account for about 96% of Rett Syndrome cases, another devastating neurodevelopmental disorder that is typically seen in girls. Because of the strong neurologic component seen in both Rett and MeCP2-duplication syndromes, MeCP2 was long assumed to be primarily important for neurons. However, recent studies have shown that other cells, such as astrocytes and microglia in Rett syndrome, and T cells in MeCP2-duplication syndrome, also play a significant role. This understanding opens up the possibility for treatment modalities beyond direct neuronal intervention, which has been a problematic approach due to the complexity and risk involved in any manipulation of neurons themselves. Here, we propose to test the hypothesis that MeCP2 overexpression causes defects of adaptive immunity that lead to excessive, pathologic innate inflammation, ultimately contributing to morbidity and mortality. Preliminary data in MeCP2Tg3 mice, which overexpress MeCP2 at 3-5 fold normal levels, reveals that as disease progresses, peripheral organs undergo significant weight loss associated with gross pathologic appearance and immune infiltrates. However, immune-privileged organs (brain and testes) are protected from these changes. This suggests that a peripheral destructive immune process is contributing to pathology in MeCP2Tg3 mice. In addition, MeCP2Tg3 mice have significantly increased mortality from influenza infection. In an influenza model, MeCP2Tg3 mice exhibit a deficient CD8+ T cell response and excessive innate inflammation without an apparent defect in viral clearance; we intend to test the hypothesis that CD8+ T cell defects result in a compensatory increased innate response, which causes pathology leading to increased death. The first aim is to test the hypothesis that dendritic cell malfunction contributes to defects of the adaptive immune response using both in vitro and in vivo assessment of antigen presentation and T cell activation. The second aim in this proposal will test the hypothesis that a deficient CD8+ T cell response contributes to increased mortality in MeCP2Tg3 mice via an excessive innate immune response. This will be tested by in vitro assessment of CD8+ T cell function and in vivo assessment of CD8+ T cell response to influenza. The third aim is to ameliorate death from influenza in MeCP2Tg3 mice via either bone marrow transplant or suppression of the excessive innate inflammation. Ultimately, results obtained from this work may lead to immune-based treatment strategies for patients with MeCP2-duplication syndrome.

This project is part of a long-standing collaboration with the laboratory of Dr. Ettore Appella (LCB/NCI), in which the Wip1 protein was discovered. Initial characterization resulted in determining two classes of phosphorylated substrates, involving many proteins involved in cell growth regulation. The first has a diphosphorylated sequence motif (pT-X-pY), such as in p38 MAP Kinase, while the second has a mono-phosphorylated sequence motif (p(S/T)Q), such as in the p53, Chk1/2 and ATM proteins. By development of an atomic-scale computer model of the extended active site of Wip1 and a series of mutagenesis experiments, we were able to reveal the structural basis for the range of substrate specificity. This lead to the development of a cyclic peptide molecule that competitively inhibits Wip1, the first inhibitor of any kind for this family of enzymes. We then pursued development of a more drug-like, small molecule inhibitor in collaboration with Dr. Daniel Appella (LBC/NIDDK), who specializes in synthetic chemistry. The resultant small molecule is based on a pyrrole ring scaffold, with 5 different emanating sidechains to mimic the amino acids of the cyclic peptide. While successful, the final inhibition constant was still only in the low micromolar range. To further this effort, we returned to optimizing the cyclic peptide inhibitor. By multiple iterations of design and testing, we were able to drastically increase the binding affinity, resulting in an inhibition constant of 110 nM. The structural modeling involved in this process revealed both important new interactions in the extended active site, and the role of the proximal B-loop in binding substrate and regulating activity. Since the B-loop is unique to the Wip1 member of the PP2C family, its role was previously unknown. We are now applying these lessons to designing a new generation of pyrrole-based inhibitors. We are also pursuing generating sufficient Wip1 protein to determine the structure by X-ray crystallography, which will greatly aid in inhibitor optimization. Recently, we have verified the requirement of binding a 3rd magnesium ion for activity of Wip1 and the related PP2Ca homologue, and used deuterium exchange mass spectroscopy to study the functional conformational changes. We are currently determining the crystal and NMR structures of PP2Ca/cyclic peptide inhibitor complexes. Recently we used Deuterium Exchange Mass Spectroscopy to study the functional structural changes of Wip1 and the PP2Ca homologue. We have also determine the crystal structure of PP2Ca with a bound substrate.

Uncontrolled hyperbilirubinemia (jaundice) in neonates has long been known to lead to neurological dysfunction including irreversible athetoid cerebral palsy with speech, ocular and hearing impairments, and even death. Contemporary management is based upon monitoring the total serum bilirubin (TBS), taking into account other clinical parameters such as birth weight and gestational age, and administering effective treatment (phototherapy and/or the rare exchange transfusion), if dictated. Unfortunately, the trend towards discharge of apparently healthy neonates from the hospital very soon after birth has made the management of subsequent jaundice more difficult in that population. A long suggested, but not used, better predictor of the neonate's risk for neurological sequelae due to elevated bilirubin is a measure of the capacity to sequester bilirubin in the blood compartment by its binding to serum albumin. The concentration of unbound bilirubin, the driver for bilirubin escaping from the vasculature, can be calculated from the TBS and binding capacitl. Presently existing methods for assaying binding capacity and unbound bilirubin are not facile. However, all these parameters can be directly measured simply in a very small volume of whole blood with a special purpose fluorometer, the hematofluorometer, first described years ago, by making use of the natural fluorescence of bilirubin bound to albumin. This technology is amenable to point-of-care use. The aims of this project are to transform the modernized and miniaturized hematofluorometer developed in Phase I into a product suitable for operation in various point-of-care environments, including the intensive care and healthy baby nurseries, the neonatal inpatient clinic, and the pediatrician's office. The first aim is to optimize the basic optical and electronic design: redesin the electronics to support the hospital information management requirements, such as a bar code reader, printer, and interface with a computer, either directly or via the local internet. The second aim is to develop a reagent kit that is easy to use and inexpensive. Phase I work demonstrated that significantly more work is needed to design a kit meeting these requirements, and then scaling this design up into a product that can be mass produced. The third aim is to test the instrument with neonate blood samples in a clinical environment to demonstrate that it well suited to meet the needs. With this goal in mind, Stanford University's Medical School and Children's Hospital has agreed to participate as a subcontractor to evaluate the new technology. With these proposed improvements, the instrument will be ready for the next stages: releasing it to the market for immediate R&D uses and clinical studies, and eventual approval by the FDA for general use.

The long-term objective of the Animal Health Resources Program is to facilitate biomedical research at the Fred Hutchinson Cancer Research Center by providing quality animal care, by offering a broad range of technical (e.g., surgical) support services, and by eliminating complications of animal research due to intercurrent disease. The specific aims of this proposal are threefold. First, we plan to upgrade the general level of animal care throughout our facility and to ensure future accreditation by replacing worn and unreliable equipment (an autoclave and ten dog runs). Second, we propose the renovation of existing space for a much needed surgical facility for dog and rabbit studies, currently conducted off-site or not at all. Third, we will expand our current, disease-free microisolator rodent housing system through the purchase of 100 additional cages for rats. Support as requested will enable the Animal Health Resources to upgrade general operations, maintain accreditation standards, and meet its service goals for the coming years.

The Kansas City Clinical Oncology Program (KCCOP) expects to transition to NCORP-KC. It is a very successful non-profit organization conducting clinical trials in cancer therapy, cancer symptom prevention. Quality of Life, and cancer control since 1983 with uninterrupted funding by the National Cancer Institute. The program is located in the Kansas City metropolitan area and serves the needs of both Kansas and Missouri residents spread over 16 counties and 2.5 million citizens. The program is comprised of seven component area hospitals and 52 investigators representing specialties well beyond medical, surgical, gynecologic, and radiation oncology. The other areas of expertise include outcomes research, internal medicine, healthcare administration, healthcare informatics, behavioral science, rehabilitation and survivorship, palliative care, neurology, gastroenterology, genetic counseling, pathology, cellular therapy, laboratory science, and radiology. The program has provided participation opportunities to over 4,200 patients and volunteers in cancer-related trials since its inception. Over the last five years, an average of 235 new individuals have participated per year. The program has maintained excellent quality of research as confirmed by patient satisfaction and reports from external audits. The program derives its cancer trials from SWOG, NRG, ALLIANCE, CTSU, MD Anderson, URCC, and SunCoast research bases of the National Cancer Institute. NCORP-KC wishes to thank its participants and extend its service by entering a new era of Cancer Care Delivery Research while continuing to conduct high quality trials in cancer therapy, cancer control, screening, imaging, and post-therapy surveillance. The program monitors trends and implements measures for continued improvement. It balances protecting patients, their safety, and health with providing the broadest possible access to trial activation and minority outreach participation with the help of a highly responsive and responsible support staff.

Positron emission tomography (PET) studies of neuroreceptors in vivo in man hold great promise in helping to identify the role of receptor defects in many diseases. This research is aimed at developing radiopharmaceuticals which localize in discrete regions of the brain based upon their selective affinity for the postsynaptic dopamine D2 receptor. 18F- and 11C-labeled spiperone and its analogs continue to be used for PET studies of the D2 receptor despite problems such as non-selectivity, high non-specific binding, and the difficult radiochemical synthesis of ring fluorinated spiperone. The absence of a substantially better D2 ligand is a primary reason for the use of spiperone. The work proposed here is aimed at the synthesis and evaluation of agents possessing substantially improved properties over spiperone. The goal of this work is to demonstrate the applicability of these agents for future in vivo studies in human subjects. It is anticipated that the development of these radiopharmaceuticals, their in vivo characterization, and their use will eventually aid in the study of Parkinson's disease and schizophrenia in which abnormalities in dopaminergic transmission are believed to play a role. In addition, these agents may well prove useful in studying various changes of the D2 system in aging. Non-radioactive phenyl ring fluoroalkylated benzamide compounds will be synthesized and the characterized in vitro using competitive displacement binding techniques. Those agents possessing suitable in vitro binding properties will be further assessed in vivo to determine their potential as radiopharmaceuticals for quantitative PET studies of the dopamine D2 receptor. The desired in vivo characteristics of these ligands include: good brain penetration to provide adequate counting statistics for PET studies; regional localization in D2 receptor rich areas; retention of activity in those specific areas to allow for adequate PET data acquisition; rapid clearance of non-specific uptake in non-target areas such as the cerebellum; pharmacological specificity for the dopamine D2 receptor; uptake site saturability; absence of lipophilic metabolites in the blood; and few metabolites in the brain. Those compounds which demonstrate a selective, potent binding site affinity to the D2 receptor will be radiolabeled with tritium. The tritium-labeled compound will be used in further studies to assess in vitro binding parameters of the agent. Those compounds displaying promising characteristics in vitro and in vivo will be labeled with high specific activity 18F (a short-lived positron emitter with a 110 min half-life) and utilized in PET regional localization investigations in monkeys. The dopamine D2 site densities and dissociation binding constant for the agent will be determined. PET studies in animals will determine the efficacy of the compounds as potential dopamine D2 receptor ligands for investigations in human subjects. A series of pilot experiments will be conducted in normal subjects to assess the properties of the 18F-labeled ligands in vivo to determine dopamine D2 binding parameters.

Core Component D serves three important purposes. The first purpose of this Administrative Core is to provide all Projects and Cores with appropriate administrative and secretarial support to efficiently carry out the scientific goals of the Program Project. These essential services are not provided by the Massachusetts General Hospital. The key responsibilities of this core include: (i) general administration;(ii) fiscal oversight (budgeting, purchasing);(iii) communication and coordination of data flow;(iv) seminar series coordination and travel arrangements;and (v)preparation of manuscripts and reports. The second purpose of this core component is to closely monitor the activities of the Cox-7 Animal Colony, which is the chief facility of Core C. The Surgical and Animal Core (Core C) requires additional administrative effort for the following reasons: (i) it must adhere closely to additional regulations for breeding and maintaining animals;and (ii) it is responsible for the coordination and submission of all animal protocols. The third purpose of Core D is to provide the overall scientific direction for the Program Project. This is achieved by close interaction with: (i) the Scientific Steering Committee;(ii) External and Internal Advisory Committees;and (iii) Consultants for individual projects and cores. These groups regularly evaluate the progress and agenda of the PPG. The input of these advisory groups is discussed at the monthly Scientific Steering Committee Meetings and is incorporated in specific projects and cores.

Dramatic expansion of biomedical data in the post-genomic era on various organisms, including humans, has disclosed the enormous complexity of biological systems and increased our knowledge of many disease processes. This is particularly evident in reference to neoplastic disorders where molecular technologies on a genome, epigenome, transcriptome and proteomic scale have disclosed unique molecular pathways and their networks that provide significant information concerning therapeutic strategies and clinical outcomes. This in turn has affected the standard of practice of many medical disciplines including pathology. The newly developed concept of personalized and targeted therapies in cancer medicine increased and changed the traditional roles of pathology as a clinical discipline. The expanding molecular testing of tumors, with complex technologic approaches, requires a redesign of the conventional microscopic pathology services. This T32 proposal is intended to provide resources to train a new generation of pathologists via laboratory-based research training that will complement the current Accreditation Council for Graduate Medical Education-accredited clinical fellowship programs of the Department of Pathology at The University of Texas MD Anderson Cancer Center. The goal of this program is to train the next generation of physician/scientist pathologists capable of combining the conventional role of diagnostic pathology with investigative molecular approaches. To accomplish this goal, a focused 2-year research program will pair trainees with experienced educators who are also accomplished translational researchers. The trainees will work in their mentors' laboratories training in laboratory methods, processes, and team orientation needed in today's translational research. The graduates of this program will complete research projects aimed at improving patient care with patient-directed personalized treatment, concluding with the submission of a manuscript to a peer-reviewed journal. Coursework and a wide range of educational opportunities at The University of Texas MD Anderson Cancer Center will complement the laboratory research training. Combined with subspecialty clinical training, this program will produce extremely well-qualified academic, research-oriented pathologists who can, by themselves, be independent researchers and future academic mentors.

Previously, Dr. Elder and his colleagues used comparative modeling of FIV and HIV aspartyl proteinases (PRs) to identify specific amino acid residues that interact with the substrate binding pocket in a unique fashion in each protease. They have discovered that HIV protease and FIV protease are very similar structurally but that each has certain unique characteristics which result in strongly preferential homologous gag substrate cleavage. The hypothesis behind the proposed work is that an in-depth understanding of both the similarities and the differences between two distantly related lentiviral proteases will promote development of protease inhibitors that may be efficacious against both FIV and HIV proteases but at that same time, relatively homologous resistance-proof. To explore this hypothesis, Dr. Elder and his colleagues will focus on substrate binding pocket interactions. They will use site-directed mutagenesis to replace FIV-specific residues with HIV-specific residues, both alone and in combinations predicted to be interactive in the three-dimensional structure. Mutant proteases will be expressed in bacteria, purified, refolded. These mutant proteases will be biochemically defined as to their substrate specificities using both natural and synthetic peptide substrates and as to the kinetics of their inhibition by a panel of previously defined HIV protease inhibitors. Interesting mutants will be placed in the context of infectious virus, and examined in cell culture for their potential to be inhibited by, or develop resistance to, known protease inhibitors during virus passage. Where appropriate, the three-dimensional structure of interesting mutant proteases can be examined by Dr. Alex Wlodawer.

Ivermectin has been shown in an open, in-hospital trial in South India to be effective in clearing microfilaremia in patients with bancroftian filariasis at dosages as low as 25-mcg/kg given once orally. A second, blinded, placebo-controlled trial of ivermectin and the currently used drug, diethylcarbamazine (DEC), has indicated that the efficacy of ivermectin and DEC were equivalent for 3 months, but by 6 months the ~30% microfilarial recurrence rate of the ivermectin-treated patients was significantly greater than the ~15% rate of the DEC group. Side effects were qualitatively and quantitatively identical for both groups. Thus, though ivermectin appeared somewhat less effective than DEC at 6 months, its single-oral-dose mode of administration with toxicity no greater than that of DEC means that this drug should engender greater patient compliance and, therefore, be much more effective in mass-treatment filariasis control programs than DEC. Observations on 200 Peace Corps volunteers going to Loa loa endemic areas of Africa have concluded after three years of a placebo- controlled chemoprophylaxis trial using weekly doses of DEC. In Gabon (where exposure to the parasite was heaviest) 30% of individuals in the placebo group developed overt clinical disease compared to none in the DEC-treated group (p<.02) and 50% became seropositive in the placebo group vs 13% in the DEC group (p<.02). No significant side effects were seen. These findings indicate that DEC given orally once-weekly can be an effective, acceptable chemoprophylactic for preventing loiasis.

Ion channels are important drug targets and unintended drug interactions with ion channels are also of critical importance, requiring the screening of all drug candidates. Conventional high throughput screening processes for soluble proteins are problematic to apply to ion channels because the channels must be incorporated into a lipid bilayer membrane and transport an ionic current to display their functionality. There are currently no high quality, high throughput assays for ion channel drug screening. Recent efforts to increase throughput have resulted in automated patch clamp systems but these are still over two orders of magnitude lower throughput than conventional drug screening technologies, requiring costly instrumentation, cells, and consumables. For existing methods of ion channel screening, there is a gap in information quality, throughput, and cost. An alternative method of ion channel measurement involves reconstituting them in artificial lipid bilayer membranes. In recent research at UCLA led by the PI, a new high-freezing point lipid membrane composition was developed enabling it to be frozen. When frozen, we showed that it was sufficiently robust to withstand shipping, a major breakthrough. These membranes, when packaged in inexpensive chips, have the potential to significantly change the way ion channel screening is done. Our company, Librede Inc., was formed by the UCLA team to further develop and explore the commercial potential of this technology, the first steps of which are proposed here in this Phase I SBIR proposal. Our ultimate goal is to create an inexpensive, disposable chip containing arrays of lipid membranes to enable low cost high throughput screening of ion channels. In the preliminary work at UCLA, the technology was demonstrated with the shipping of small numbers of membranes, with a net yield of 30%. In the proposed work, we will design and fabricate large array chips containing 48 membranes compatible with industry standard 96 well fluid handling robotics and pipetters. With these chips, we aim to demonstrate the viability of the membrane technology over a much larger scale producing over 3000 membranes. To increase their commercial viability, we will also seek to increase the yield by systematically changing the lipid concentration, solvent composition, solution volume, and thawing temperature. Although this space is potentially very large, the membrane arrays will electrically probed in an automated fashion using a custom-built electrical interface and multiplexer. We will use the materials and designs from Phase I to contract with a plastics manufacturer in Phase II to create an inexpensive, injection molded prototype. At that point, we will have demonstrated a chip able to perform industry standard ion channel screens for a significantly lower cost. These membrane array chips have the potential to increase throughput by several orders of magnitude and similarly decrease cost by several orders of magnitude-as a result, transforming the process of ion channel measurement and screening. PUBLIC HEALTH RELEVANCE: Measurement of ion channel interactions with drugs is a key process in drug discovery and drug safety screening. Due to the difficulty in working with ion channels, the existing processes used are slow, laborious, and expensive. Our team has recently developed a platform for ion channel measurement which is much less expensive and much easier to use, based on lipid membranes that can be shipped, a world first. We propose here to develop inexpensive membrane array chips and use them for large scale production, demonstrating the feasibility of this technology for ion channel screening.

Summary: One of the main reasons that promising drugs fail to live up to their potential for ameliorating disease is unforeseen toxicity. The ability to monitor, and diagnostically predict which compounds may engender toxicities from a large lead compound portfolio, will have a dramatic impact on pharmaceutical development and the FDA's ability to monitor and ensure the safety of new drugs or new combination therapies. In the past, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been the major technology utilized by experimentalists to separate and analyze proteomes. This technology, although a powerful method for characterizing thousands of proteins from any given sample, is low-throughput, insensitive, and has limitations with separating low molecular weight and highly basic proteins. Our laboratory accepted the challenge of developing novel methods and tools to utilize proteomic information in the discovery of protein biomarkers of toxicity. Of critical importance, our laboratory invented (patent-pending) and developed, through a collaboration with Correlogic Systems, Inc., a heuristic pattern recognition algorithm which can import the complex data streams produced by SELDI and rapidly identify (in up to 20th-dimensional space) patterns of proteins that are diagnostic for human cancer in serum or cell lysates.. A component of our research efforts focuses on the applicability of proteomic technology to identification of changes in protein patterns that could predict drug toxicity. Through a collaboration with Dr. Frank Sistare's group at the center for Drug Evaluation and Research (CDER), Dr. Petricoin obtained sera from animals treated with different drugs, with known toxicities, as well as sera from control animals. These sera were analyzed by SELDI using 1 ul of serum and each sample was analyzed in quintuplicates. Protein profiles were generated in less than 2 weeks. The SELDI profiles were then normalized and analyzed by the novel heuristic cluster algorithm, developed by the Petricoin laboratory, to identify protein patterns that were diagnostic for toxicity. The results of the analysis were remarkably clear and intriguing. Importantly, this tool was able to identify drug-induced toxicity over the vehicle alone background 100% of the time in the blinded test set. Critically, the proteomic profile proved to be more sensitive than histopathology or ELISA analysis in detecting toxicity at lower doses and at consistently earlier time points. Each of these profiles appears to be drug-specific, although the data are currently being tested to assess whether a global toxicity pattern can be uncovered.

The Center for Neural Science at New York University is a premier site for research on functional aspects of vision. Eighteen investigators from the Center request support for their vision research through continuation of a Core Grant. They are bound together by their research Interests In the neural and behavioral bases of vision and visually guided behavior. The proposed Core will comprise of four modules, each of which will benefit research in the research areas represented by program faculty: 1. A Visual Displays Module which will provide support for the development of state-of-the-art software for the generation of a wide variety of visual stimuli for psychophysical, neurophysiological, and brain experiments. 2. A Design and Fabrication Module which will provide expertise to design and build novel electronic and mechanical devices such as theose required for multielectrode recording in the visual cortex, noninvasive methods of eye movement recording, and Interface devices for behavioral and blloglcal experiments. 3. A Neuroanatomy and Molecular Biology Module which will provide expert support for the processing of neuroanatomlcal material at both the light and EM level, for the analysis and localization of electrophysiological recording sites In histological material, and for the creation of transgene-containing viruses to modify neuronal funtlon experimentally. 4. A Functional Imaging Module which will provide support for hardware and software services for users of the Siemens MRI scanner and associated devices In the Center for Brain Imaging, giving access for Core researchers to functional and structural Imaging capabilities for human and animal research.

The proposed studies represent an extension of an ongoing program of research. The major objectives of this research are to describe the extant communication abilities of children and adults with severe to profound mental retardation; and to identify the factors that are associated with the attainment (or nonattainment) of specific communication and language abilities by such individuals. The proposed studies will address five specific aims related to these overall objectives that have been identified on the basis of the applicant's current and previous research. All of these aims relate to a proposed model of communication development in this population that identifies a number of critical "cusps" that have apparently been traversed by some individuals in the course of their communication development; but that represent the end point of that development for other individuals. The first three specific aims are: 1) To describe the extant communication abilities of a sample of young prelinguistic children with severe to profound mental retardation; 2) To describe the rate and sequence of communication skill development by these same subjects over a five year observational period; and 3) To determine whether specific child and/or environmental variables are significantly related to differential communication skill attainments by these subjects at the end of this observational period. These three aims will be addressed through a five year, repeated measures design involving 25 prelinguistic young children (ages 4 to 5 years at onset of observations) with severe to profound mental retardation. A battery of measures, including standardized assessments and direct observations of parent-child interaction, will be administered to each subject at least every six months, and more often with subjects who are arriving at the identified cusps at a faster pace. Specific aims 4 and 5 are to determine whether specific intervention procedures are effective in helping individuals with severe mental retardation make the transition from presymbolic, distal gesture communication to referential symbolic communication (Aim 4); and across one or more other critical cusps, including that between contact gesture and distal gesture communication, and/or that between single symbol and linguistic (syntactic) communication (Aim 5). Subjects in these studies will include both children and young adults; and single subject research designs will be used to determine the effectiveness of specific treatment approaches for each subject.

We conduct Monte Carlo experiments under the model of random censorship to investigate the small sample behavior of different standard deviation formulas for the Kaplan-Meier estimator. The simulation studies include the Greenwood formula, Peto's formula and an alternative modified Greenwood formula. The results of the simulation showed that the Greenwood formula underestimated the true variance more than 90% of the times. Peto's formula overestimated the true variance when the survival rates were less than or equal to 0.5. The accuracy of the three different variance estimators is investigated using the relative bias, mean square error and confidence coefficient criteria. the absolute value of the relative bias for the modified Greenwood formula was the smallest among all three different variance estimators. On the basis of the mean square error criteria, the differences were negligible between the three different variance estimators. The Greenwood formula did not approach the confidence coefficient 0.95 in any censored observation. The confidence coefficient of Peto's formula was closer to 0.95 than the other two variance estimators. The sample sizes, censorship levels and distributions of the random variable all influenced the accurancy of the variance estimators.

The overall objective of this research is to elucidate the molecular mechanism(s) by which estrogen mediates vascular lesion formation. Occlusive vascular lesions are characterized pathologically by endothelial cell hyperplasia and aberrant smooth muscle cell growth. Estrogen therapy is associated with a worsening of atherosclerosis. Until now adverse estrogenic effects have been explained by the dysregulation of the estrogen receptor (ER). Although the ER is required for the growth of cells, we consider ER actions as a late event when compared to the rapid formation of estrogen-induced oxidants that initiates the early signal for cell growth. We have shown that estrogen-induced oxidants signal endothelial cell proliferation and blood vessel formation. Since pathogenic blood vasculature formation contributes to the etiology of vascular disease, we postulate that exposure to estrogen therapy is harmful to the human cardiovascular system. This revised proposal will examine the role by which 172-estradiol (E2) activates a redox sensitive Pyk2 signaling pathway involved in the pathology of vascular lesions. Therefore, the objective of this proposal is to elucidate the molecular mechanism by which estrogen induces vascular lesion formation. The hypothesis of this proposal is that a Pyk2/GPR30/Vav1 complex mediates pathogenic blood vasculature formation in response to exposure to estrogen. We have formulated the following inter-related, focused specific aims to test our hypothesis: Aim 1: To determine whether estrogen exposure initiates Pyk2 protein-protein interactions resulting in a Pyk2/GPR30/Vav1 complex. Aim 2: To determine the mechanism by which estrogen-induced Pyk2 protein- protein interactions signal Id3 to block p21Cip1 expression. The long-term goal of this research project is to determine whether in vivo estrogen-induced Pyk2 signaling controls pathogenic blood vasculature formation. However, this long-term goal is not the purpose of this SC3 grant proposal; rather we intend to generate preliminary data from this SC3 Award that will be used to apply for successful NIH R01 funding in which we will include the determination of in vivo whether estrogen-induced Pyk2 signaling controls the formation of human vasculature in a mouse model. The PI's career goal is to become an independent investigator in the field of cardiovascular toxicology. The PI plans to achieve this objective by investigating the molecular mechanism by which estrogen induces vascular lesion formation. Florida International University offers a rich academic environment and well-respected research infrastructure that will provide a supportive setting to foster the PI's career development. The findings from the proposed research will contribute to a more profound understanding of how estrogen therapy mediates the formation of occlusive vascular lesions. This, in turn, will give new insights into the mechanisms by which estrogen increases the risk of occlusive vascular lesions and will identify novel targets for the treatment of vascular lesions in individuals exposed to these compounds.

Songbirds represent one of the few animal model systems for the study of the acquisition of human speech. Birds learn their song during development, and have a well-delineated forebrain circuitry devoted to learning and producing song. Area X is a part of the neural circuitry involved in song acquisition and modulation. Area X is also a part of the avian basal ganglia and receives a strong input from midbrain dopaminergic nuclei. Cells in Area X respond to changes in social context: their activity changes depending on whether males are singing in isolation or singing to a female. The proposed studies will investigate the role of dopamine in modulating neural activity in Area X under different social contexts, and how changes in activity in Area X may affect the responses of other nuclei in the learning pathway. Given both the homology and similarity between Area X and mammalian striatum, as well as the ecological and behavioral relevance of the bird song system, the merging of the two fields of study would provide considerable information toward understanding plasticity and song production in birds, as well as insight into striatal function in a behaviorally relevant paradigm. [unreadable] [unreadable]

The inflammasome has recently been described as an important protein complex in which a non-NFkB- mediated signaling pathway leads to up-regulation of key pro-inflammatory cytokines, including interleukin (IL)- 1b and IL-18. Members of our group recently reported that the inflammasome plays a critical role in pro- inflammatory response in murine sepsis, and this response is dependent upon mitochondrial integrity and an intact autophagy response to injury. We now present data supporting an important role for the inflammasome in predicting severity and mortality during human infection-related ALI/ARDS. Moreover, our preliminary animal studies demonstrate that statins exacerbate lung injury and inflammation via activation of the inflammasome. We therefore hypothesize that activation of the inflammasome plays a critical role in the development of infection-related ALI/ARDS and that statin administration may increase inflammasome-related downstream cytokines during lung injury. In particular, our collaboration with the ARDSnet SAILS trial investigators (a randomized trial of statins vs. placebo in infection-related ALI/ARDS) in this Ancillary Studies in Clinical Trials RFA (HL-12-012) provides a unique opportunity to obtain additional collection of key human samples to address these important processes. We therefore propose: Specific Aim 1: To determine gene expression and protein levels of the inflammasome during infection-related ALI/ARDS using prospectively collected blood (n=100) and banked plasma samples (n=600) from placebo- and statin-treated SAILS subjects. Gene expression and protein levels of the inflammasome will be correlated with 60-day mortality and additional SAILS trial secondary outcomes. We hypothesize that circulating inflammasome levels will serve as a biomarker of severity and mortality of infection-related ALI/ARDS and that inflammasome levels in statin-treated subjects will correlate with clinical outcomes. Specific Aim 2: To determine the cellular localization of expression of the inflammasome complex and role of inflammasome activation on cellular responses and function, using primary neutrophils and monocytes isolated from prospectively enrolled placebo- and statin-treated SAILS subjects (n=100), as well as primary cells isolated from control ICU subjects (n=100). We hypothesize that determining which circulating cells are the predominant source of inflammasome activation will increase sensitivity of the correlation of inflammasome levels with clinical outcomes and that localized activation of the inflammasome will result in mitochondrial dysfunction that will be enhanced in the presence of statin administration.

Despite aggressive surgical resections, high-dose radiation therapy, and chemotherapy delivered at toxic doses, the vast majority of patients with malignant brain tumors survive less than one year making conventional therapy for malignant brain tumors the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. Moreover, the failure of these treatment modalities to be tumor-specific at the molecular level, results in inevitable damage to surrounding normal brain that incapacitates patients treated with these traditional modalities. The inherent specificity of immunologic recognition offers the prospect of targeting malignant cells more precisely. Within our program, direct injection of 131-I-labeled, operationally-specific, monoclonal antibodies (MAbs) into brain tumor resection cavities delivers extremely high radiation doses to tumor cells around the resection cavity and has produced promising results in Phase II clinical trials. These MAbs diffuse only short distances beyond the cavity, however. Therefore, most of the radiation extending beyond the cavity is not specifically targeted to tumor cells and the radiation dose delivered beyond the cavity declines exponentially from the cavity interface. As a result tumor cells that are known to infiltrate the brain for significant distances beyond the cavity are sub-optimally treated and lethal tumors always recur within 2 cm of the radiated re section cavity. Continuous microinfusion is a promising technique that allows homogeneous delivery of even large molecular weight molecules at high concentrations throughout large areas of the brain. Although this technique may enhance the delivery of 131-I-labeled MAbs and other therapeutic agents to diffusely infiltrating malignant brain tumors and reduce recurrence rates, the parameters that govern this technique and its limitations have not been defined. One of the major goals of this proposal is to define these parameters. In addition, this proposal is designed to investigate whether targeted radiotherapy might be improved through the use of human chimeric MAbs with increased biostability and the use of high linear energy transfer radioisotopes, such as 211-At, with greater relative biological effectiveness.The hypothesis to be tested in this proposal is that continuous microinfusion will widely deliver operationally tumor-specific monoclonal antibodies conjugated to 131-I or the alpha-emitter 211-At such that they will be specific and potent therapeutic agents against malignant brain tumors with major reductions in toxicity to normal brain.

The neuronal ceroid lipofuscinoses (NCL) comprise a group of poorly understood inherited neurodegenerative diseases of children and adults. Although canine and ovine models of this disease are available, they are not economical to maintain, difficult to study genetically, and not easily manipulated experimentally. Two mouse models of NCL have been discovered. Both develop severe neurological disease; both have numerous neuronal cytoplasmic autoflourescent inclusions. Ultrastructurally these inclusions consist of stacked membranes identical to those found in the canine model of NCL and similar to inclusions considered diagnostic of human forms of the disease. One of these mouse mutants, juvenile bare (jb), has been mapped to the proximal part of Chromosome 7; the genetic linkage of the second mutant, motor neuron degeneration (Mnd), will be established, and candidate genes for both mouse diseases will be sought. These mutants will be studied to characterize the progression of lesions in affected organs, particularly the brain, during the life span. Early diagnostic lesions will be sought. The basis of the biochemical defect will be analyzed by isolating and sequencing the stored lysosomal protein(s) and searching for abnormal lysosomal enzymes. A search for other models of NCL will be conducted among the dozens of known mouse mutants with blindness, seizures, diffuse neuronal degeneration and known lysosomal disorders. Finally, the mutant models will be made available to other investigators. It is hoped that studies of these models will provide important insights into the devastating pathogenesis of Batten disease.

The Xenotransplantation Core provides specialized, on-demand surgical services for the transplantation and evaluation of human fetal tissues in immunodeficient animals in support of the research projects carried out by the Formative Center. The core is responsible for the coordination of immunodeficient animal procurement (nude rats) with anticipated availability of fetal tissues, and sterile renal subcapsular or subcutaneous transplantation of appropriate fetal source tissue upon availability. Our group has demonstrated that fetal testis, handled and processed in the usual manner by hospital staff, is histopathologically responsive to selective toxicant exposure when implanted under the kidney capsule of nude rats. The Xenotransplantation Core facilitates the expansion of this past work into other fetal tissue types and environmental impacts by centralizing development of the xenografted animal model in support of the three primary research areas of the Formative Center: Metabolic Syndrome. Prostate Imprinting, and Lung Remodeling. The Xenotransplantation Core will function as the site of the initiation of the experiments carried out in the Formative Center's Research Projects by providing an on-demand service that coordinates with the Tissue Procurement Core to transplant fetal tissue as it becomes available into xenogeneic hosts. As such, the Core will maintain host animals for instant availability, and its personnel will have the shared responsibility of performing surgical requests within a time frame that allows confidence in graft viability upon implantation. The Xenotransplantation Core will pursue these Specific Aims: Specific Aim 1 - Provide animal surgery and model development services Specific Aim 2 - Interact with research investigators to initiate animal experiments in support of the Formative Center's research projects Specific Aim 3 - Implement current and emerging surgical techniques in xenotransplant model development and training of technical staff

Lung cancer is diagnosed late, has a 5 year survival of only 15%, and kills more people than colorectal, breast and prostate cancer combined. Less than 30% of lung cancers are resected and the majority of patients are treated with cisplatin and ionizing radiation (IR). We show that inhibition of DNA damage signaling by ATR kinase during treatment with cisplatin and IR is well-tolerated and leads to durable responses in mouse xenograft and genetic models of lung cancer. Quite unexpectedly, we show that in addition to potentiating DNA damage, two clinical ATR kinase inhibitors (ATRi?s), with unrelated structures, block expression of the immune checkpoint protein PD-L1 and increase presentation of MHC class I antigens in lung cancer cells after IR. Our finding that crosstalk exists between DNA damage signaling and immune checkpoints has not been described previously and is the focus of this proposal. Immune-inhibitory pathways, termed immune checkpoints, are coopted by tumor cells to evade cytotoxic immune cells. PD-1 is expressed on cytotoxic T cells and its ligand PD-L1 is upregulated in lung cancers. PD-L1 binding by PD-1 prevents the activation of cytotoxic T cells. Immune checkpoint blockade using anti-PD-L1 and anti-PD-1 antibodies restores anti-tumor immune responses and is emerging as an exciting lung cancer therapy. We propose that ATRi?s inhibit DNA repair and cell cycle checkpoints potentiating the DNA damage induced by cisplatin and IR while concurrently inhibiting PD-L1 expression and restoring anti-tumor immune responses. Our objective in this proposal is to define the mechanisms that connect DNA damage signaling and immune checkpoints. This objective will be accomplished by the following Specific Aims. Aim 1: To determine how ATRi?s inhibit PD-L1 expression in lung cancer cells after IR. This aim will define the contribution of ATR, ATM, IRF-1, NF-?B and p53 to PD-L1 expression after IR. Aim 2: To determine how ATRi?s increase MHC class I expression on lung cancer cells after IR. This aim will identify ATR, ATM, and p53 signaling that inhibits protein synthesis and MHC class I presentation after IR. Aim 3: To identify ATRi-induced PDL-1/PD-1 immune checkpoint blockade in lung cancer after IR. This aim will identify ATRi-induced immune checkpoint blockade in lung cancer after IR. The outcomes of these Aims will identify mechanisms that connect DNA damage signaling to immune self- tolerance. This will define a novel therapeutic opportunity to use ATRi?s to potentiate the DNA damage induced by cisplatin and IR while concurrently inducing immune checkpoint blockade.

The mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), are important regulatory neuropeptides which mediate a range of biologic responses including smooth muscle contraction, stimulation of secretion, modulation of neuronal activity, and control of cell proliferation. Previous studies from this laboratory have shown that: 1) in tumor cell lines, the prepro-GRP gene is regulated at the level of primary transcription; and 2) detectable expression is restricted to a subset of neurons in the central and peripheral nervous system, neuroendocrine cells, and derivative tumor cell lines. We plan to define the molecular mechanisms important in prepro-GRP transcriptional activation in expressing cells, and suppression in nonexpressing cells. The first step in this analysis is the identification of cis-acting prepro-GRP promoter elements important for transcription. A series of fusion genes were assembled to assay the transcriptional effects of sequence elements in the prepro-GRP promoter. In these constructs, the firefly luciferase reporter gene is placed under transcriptional control of varying lengths of the human prepro-GRP promoter region. After transfection and subsequent transient expression in various host cell lines, luciferase activity is determined for each construct containing different prepro-GRP promoter elements. Using this assay system, we have made the following observations: 1) Promoter sequences between -5000 and -1600 (the origin is defined as the initiation site for transcription) exert an approximately two-fold negative effect on transcription, while sequences between -1600 and -400 exert a weak positive effect. In this positive domain, a several hundred base long 5' upstream sequence is very highly conserved between the rat and human prepro-GRP gene (-720 to -450); and 2) The constructs behave the same in host cells that do or do not normally express the prepro-GRP gene. We conclude that promoter elements between -5000 and + 100 alone do not determine cell-type specificity of transcription regulation.

The Laboratory of Pathology (LP) supports the clinical and research missions of the NIH and the NCI by providing anatomic pathology and tissue processing services to the Clinical Center and to all of the categorical institutes. LP is CLIA-certified with a Certificate of Accreditation and is inspected every two years for compliance with regulatory standards by the College of American Pathologists. LP's clinical services provide services in surgical pathology, autopsy pathology, hematopathology, cytopathology, chromosome pathology, pediatric pathology, flow cytometry and molecular/special diagnostics. Clinical Operations is the administrative and technical core of the clinical component of the Laboratory of Pathology. It oversees the Quality Management program, the core laboratory services, human resource management for clinical and technical staff, the Laboratory Information System (LIS) and manages the clinical diagnostic tissue archives of the laboratory. As a service to investigators throughout the NIH, scientists may request tissues for research from the archive following appropriate ethical approval (from their IRB or from the OHSR). In this fiscal year through the end of July, we processed about 250 new and ongoing requests for tissue samples. This resulted in approximately 10,500 individual biospecimens (unstained slides, special stains, tissue shavings, and tissue slides processed with RNAse precautions) provided to researchers from our tissue archive. The Clinical Operations section also processed approximately 125 medical-legal requests, which involved retrieving and processing approximately 1,000 clinical slides and/or blocks that were forwarded to other healthcare or research facilities at the request of clinicians or patients. A relatively new function within LP's Clinical Operations section has been the addition of whole slide scanning of stained tissue slides. Whole slide scanning allows the indefinite storage and electronic access of patient case material, without the risk of slide loss or deterioration of stain intensity over time. Newer technologies in slide scanning also provides a platform that employs image algorithms that can accurately and reproducibly analyze immunohistochemical stains. In fiscal year 2016, LP residents and staff scanned approximately 2,800 stained whole tissue slides of: unique and interesting pathology cases; cases submitted to LP clinical pathologists which required the slides be returned to the submitting facility; clinical cases shared with staff pathologists or clinical consultants electronically rather than risking the loss of glass slides; and cases scanned for resident study sets, research or to present at conferences on and off campus. As of July, 2017, there have been more than 4,200 while slide scans performed or supported by Clinical Operations staff. In total since inception of whole slide scanning, over 20,000 slides have been scanned. The Clinical Operations section also includes oversight of the Laboratory Information System (LIS) and collection and reporting of quality management (QM) data to LP's leadership and QM Committee monthly. The LIS is part of the SoftLab system used by the Clinical Center and it interfaces with the hospital information system so that anatomic pathology results are available online. As part of the tissue request process and to support requests for pathology reports from patients and clinicians, we conduct searches of the pathology database and provide lists of cases that match the requested criteria. We currently have reports scanned from 2008 to the present and are maintaining the report archive in real-time. The LIS is also used by Clinical Operations to generate benchmark data and quality assurance statistics for managing and continuously improving the clinical diagnostic services. The quality management program monitors LP's performance with key benchmarks, such as: cytology, surgical and biopsy turnaround times; specimen adequacy, labeling and identification compliance; and quality control of special stains. Annual QM reports are provided to LP's clinical leadership to identify areas that have demonstrated improvements, but also shows areas that need continuous improvement and monitoring. Annual QM reports are provided to the NIH Clinical Center's Office of Patient Safety and Clinical Quality as one element needed to demonstrate an ongoing quality assurance and process improvement program for clinical services that is required for Joint Commission accreditation. Clinical operations also oversees pathologist-supervised tissue division for research. Patients who undergo surgery at the NIH Clinical Center frequently have tissue divided between research and diagnostic purposes, as dictated by IRB-approved protocols.The Technical Laboratory Manager maintains the lab, and Pathologists Assistants (PAs) assist in procurements, dissection, documentation of specimen accountability, and distribution of tissues to researchers and clinical sections. The TLM also serves as contract officer technical representative for LP and assists the Clinical Manager in property management, equipment procurement, and maintenance activities for the clinical and research sections of LP.