The CCR-Flow Core is an indispensable resource for NCI and contractor investigators at the Frdereick National Lab for Cancer research. Seventy investigators from 35 different laboratories have used the expertise of the flow core staff and the instrumentation available and maintained by the staff. The projects investigators have been studying include tolerization of the high-avidity T cells in the tumor microenvironment. Another study was of the critical role of tumor necrosis factor in stabilizing the CD4+Foxp3+ regulatory T cell. Regulation of the NF-B signaling pathway of pancreatic cancer stem cells was also studied. A project looking at the pivotal role of IKKalpha in spontaneous lung squamous carcinomas was also done. The influence of HLA-C expression on HIV control was also a project of study. Studies have been done on the role of DC cells in the acute infectious inflammatory process. The flow core staff has trained eighteen investigators in the first three quarters of the year to run their own samples. The instruments are used daily by the trained investigators often into the night and on the weekends. This leaves more time for the flow core staff to sort approximately 450 samples and analyze about 10419 samples in the first three quarters of this year. Without the investigators acquiring the data and analyzing their own samples, the government would have to hire full time experienced individuals to perform the same volume of work. At least 15 papers have been published, 8 papers are in press and 14 papers have been submitted in the past year using data obtained in the flow core. The future goals include keeping up with cutting-edge technology in the flow lab by expanding the pool of investigators trained to run and analyze their own samples to all of CCR in Frederick thereby expanding the use, productivity and quality of the science generated using the core's instrumentation and expertise. It is necessary to up-grade all of the instruments so that laser and fluorochrome capabilities are more interchangeable so that investigators have the flexability to use more than one instrument. This would prevent loss of data because the instrument needed was in use by another investigator or down because of needed repairs. It is critical to have this flexability in analysis of samples as well as for sorting of cells. We would also like to stay a cutting edge facility which would require at least one analyzer to be up-graded to analyze at least 28 parameters at once.


It is now recognized that T cells required 2 signals for induction of immune responses. Interruption of costimulatory signals by blocking CD28 or its two ligands B7-1 and B7-2 can induce transplantation tolerance and prevent or reverse autoimmunity in animal models. Similar data exists for blocking the CD40-ligand:CD40 interaction, a potential additional costimulatory pathway. The overall goal of this project is to understand the role of costimulation in graft rejection and determine the strategies and mechanisms by which blockade of costimulatory signals induces transplantation tolerance. Our studies of induction of transplant tolerance through blocking CD28 or CD40L have shown a complex series of relationships between their ligand, B7- 1, B7-2 and CD40 (all expressed on antigen-presenting cells), and the induction of rejection or tolerance. Specific aim #1 will focus on these questions, using selective blocking reagents and knockout animals in a murine cardiac allograft model to: examine the separate roles of B7-1 and B7-2, test the hypothesis (based on preliminary data) that B7-1 can, in select circumstances, promote tolerance interactions with CTLA4, and examine the cellular and molecular regulation B7-1 through CD40. Our data also show that delivery of additional donor antigen, in the form of splenocytes, at the time of transplantation, is required to synergize with costimulatory blockade to induce tolerance. In the absence of donor antigen, animals are transiently immunosuppressed but undergo chronic rejection. Specific aim #2 will identify the cell and MHC type which is required in the donor- transfusion to induce tolerance. We will then test the hypothesis that immunogenic allopeptides can substitute for intact cells to induce tolerance. Lastly, using the allopeptides, we will test the postulate that chronic rejection is linked to failure to tolerize to indirect allorecognition. Finally, based on data that costimulation through CD28 induces the cell survival gene bcl-x which is required for lymphocyte survival, in specific aim #3 we will use bcl-x transgenic mice to test the hypothesis that immunosuppression through CD28- blockade operates by induction of cell death and that bcl-x transgenic mice will be resistant to tolerance induction through this maneuver. These studies will have important implications for the development of implementation of therapies for transplantation and autoimmunity.


This is a Phase II randomized blind, parallel group, multicenter, therapeutic equivalence trial. The Primary Immune Deficiency (PID) patients will be randomly assigned to one of the two treatment groups: experimental IGIV (IGIV-C, 10%) or control IGIV (IGIV-S/D, 10%). A total of 152 patients will be enrolled with a confirmed diagnosis of Primary Immune Deficiency and who are on stable IVUG replacement therapy for at least three months prior to entry into the trial. Nine infusions, with one of the study drugs, will be every three to four weeks for nine months. Patients will remain on the fixed individual replacement dose and dose interval established prior to entry into the study.


PROJECT SUMMARY Emphysema is an important pathologic finding in Chronic Obstructive Lung Disease (COPD), a leading cause of death in the United States and worldwide. Emphysema is characterized by increased DNA damage and the consequences of failed DNA repair: apoptosis, cellular senescence, and inflammation. Cigarette smoke (CS) is the most important etiology for DNA damage in emphysema. Yet, emphysema does not occur universally among smokers and there is little understanding of protective mechanisms that underlie this heterogeneity. Therefore, identifying cellular responses to CS that promote DNA repair may lead to new therapies for this disease. This proposal seeks to understand the role of Macrophage Migration Inhibitory Factor (MIF) in promoting DNA repair and antagonizing the development of emphysema. MIF is a cytokine secreted in response to CS and a critical regulator of multiple signaling pathways. We previously demonstrated that circulating MIF is decreased in patients with COPD, and genetic deletion of murine MIF results in increased susceptibility to CS mediated airspace enlargement. The endothelial cells of MIF-KO mice are particularly susceptible to CS-mediated DNA damage and consequent apoptosis and cellular senescence. The source of protective MIF is unknown. Our preliminary data suggests that MIF promotes DNA repair in endothelial cells exposed to CS extract and that endothelial cells may be an importance source of protective MIF. Based on these findings, we propose the following hypotheses and specific aims: Hypotheses 1. MIF, via its receptor CD74, promotes HR and NHEJ following CS-mediated DNA damage. 2. MIF, via CD74, protects lung endothelial cells from CS-mediated cellular senescence and apoptosis by promoting BRCA1 gene expression. 3. Deletion of MIF in the endothelium is necessary and sufficient to increase murine susceptibility to DNA damage and emphysema. Aims 1. Define the DNA repair mechanism(s) promoted by MIF in lung endothelial cells. 2. Determine the role of BRCA1 in mediating MIF's protective effect. 3. Define the protective role of endothelial-derived MIF in CS-mediated emphysema. Experimental Approach To address Aim 1, we will use primary mouse and human endothelial cells and expose them to CS extract, followed by a recovery period to observe DNA repair. We will characterize DNA damage and repair using single- cell gel electrophoresis, imaging-flow cytometry (Amnis) and host cell reactivation assays. We will use silencing RNA and chemical inhibitors of MIF-CD74 binding to determine if MIF promotes DNA repair via its receptor CD74. In Aim 2, we will characterize the effects of CS on BRCA1 gene expression and protein in primary lung endothelial cells and paraffin-embedded, de-identified human lung tissue samples. We will use chromatin precipitation assays to determine the mechanism via which MIF promotes BRCA1 gene expression, and we will measure consequences of CS extract in endothelial cells, such as apoptosis and cellular senescence, to determine if MIF protects against these consequences in a BRCA1-dependent manner. We will use silencing RNA and chemical inhibitors of MIF-CD74 binding to determine if MIF promotes BRCA1 gene expression in a CD74-dependent manner. In Aim 3, we will assess the development of emphysema in mice exposed to CS with an endothelial specific deletion of MIF. We will also determine if an endothelial-specific lentiviral vector expressing MIF can rescue the susceptibility of MIF-KO mice to CS. This work will provide us with insight into the pathogenesis of emphysema in addition to the novel regulatory functions of MIF in promoting DNA repair. This grant proposes a research mentorship program at Yale University under the primary sponsorship of Dr. Richard Bucala, an expert in immunology and MIF biology, and Dr. Patty Lee, an expert in oxidant-mediated vascular injury and repair in the lungs as co-mentor. We have also enlisted the expertise of multiple Yale investigators, including two experts in DNA repair research, Dr. Joann Sweasy and Dr. Peter Glazer, Dr. Naftali Kaminski, who is an expert on translational pulmonary research, and Dr. Veronique Neumeister, who has significant expertise in immunohistology. The advisory committee members will provide scientific and career counseling, and the proposed career and research program as outlined will provide an extraordinary scientific environment wherein Dr. Sauler can launch his future independent career as a physician- scientist.


A trial to determine if the relatively new procedure called transjugular intrahepatic portosystemic shunt (TIPS) can treat fluid build up in patients who have had cirrhosis and increased pressure in the veins supplying blood to the liver. Cirrhosis effects 3.6 out of every 100 North American adults and is responsible for the loss of 10.6 million work days and 32,000 deaths annually. Fluid retention is the most common complication of cirrhosis and developes in over 60% of patients. There is a need to develop a better treatment for this problem. Fluid build up is a common complication of cirrhosis of the liver. The worsening of symptoms is often complicated by the development of resistence to diuretics (water pills), or complications limiting the use of diuretics. This fluid retention, which does not respond to treatment, is associated with discomfort, difficulty breathing, malnutrition, liver and kidney failure, and death or increased risk of death following liver transplantation. Paracentesis (repeated removal of large amounts of fluid by piercing the organ) or total removal of the fluid along with intravenous administration of albumin are the most commonly used therapies for this problem. Neither of these therapies addresses the problem causing the water retention and may cause the recurrence of retention. Neither therapy has been shown to improve the survival of patients. TIPS is the insertion of a by-pass into the vein system of the liver in order to help the flow of blood and prevent the build up of fluid in the abdomen and esophagus. TIPS avoids the risk of general anesthesia and major surgery and has been shown to resolve the fluid retention problem in most patients. Also, in some patients, the nutritional state and liver function improve, thereby making transplantation unnecessary. But, because all published studies have been done on a small number of patients, this study will directly compare TIPS with the current standard of care, the puncturing of organs. 72 patients with severe fluid retention will be seen at the General Clinical Research Center. There will be two groups with two different combinations of therapy. The patients will be seen 1,2,and 4 weeks after insertion of the shunt, and monthly for 12 months. Then they'll be seen every three months. Special diets will be assigned and lab tests will be done at every visit.


In the past year, we have continued our investigations on some of the factors that control the proliferation of cells, specifically how the microinjection of monoclonal antibodies may affect cell proliferation. In these experiments, we microinjected monoclonal antibodies into the nuclei of quiescent cells to determine their function. We have been able to show that a monoclonal antibody against a p53 protein, a transformation related protein, inhibits serum-stimulated DNA synthesis. Interestingly enough, the inhibition of serum-stimulated DNA synthesis occurs only if the monoclonal antibody against the p53 protein is microinjected in the first 2 hours after serum stimulation. Once the cells have gone for more than 2 hours after serum stimulation, they are no longer inhibited by the microinjection of monoclonal antibodies against the p53 protein. Control monoclones and other antibodies have been microinjected without any effect whatsoever on serum stimulated DNA synthesis. We have also investigated how the microinjection of monoclonal antibodies against the SV40 T antigen may affect the function of the SV40 T antigen. In these experiments, a number of deletion mutants of the SV40 genome were microinjected into quiescent cells, together with monoclonal antibodies. The deletion mutants were chosen so that different domains of the SV40 T antigen coding gene were represented. The monoclonal antibodies were selected from a battery of monoclonal antibodies which included some antibodies that recognized the amino-terminal third of the T antigen and others that, instead, recognized the carboxy-terminal third. Results clearly indicated that monoclonal antibodies against the SV40 T antigen inhibit the ability of SV40 T antigen to induce cell DNA synthesis in quiescent mammalian cells and also that the microinjection of a monoclonal antibody has no effect unless this antibody reacts with a specific antigen. In other experiments, we have looked at the effect that methylation may have on the expression of microinjected genes. As already reported, we have reconfirmed that the methylation of 5' flanking sequences may abolish the transcription of microinjected genes, whereas methylation of genes along their structural coding sequences has no effect whatsoever.


Uterine leiomyomata (UL) are diagnosed in 25-30% of reproductive-aged women, resulting in considerable gynecologic morbidity and billions in U.S. health care costs annually. African Americans have 2-3 times the UL incidence of Whites and greater disease severity. Sex steroid hormones are involved in the etiology of UL. Given that endocrine disrupting chemicals (EDCs) can alter the functioning of hormones, we propose to evaluate UL risk in relation to 3 classes of EDCs: phthalates, polychlorinated biphenyls (PCBs), and phenols, specifically bisphenol A (BPA). Our choice of chemicals is informed by their high exposure prevalence in humans and by in vitro, animal, and human data documenting their effects on reproductive hormones and processes that could influence UL risk. Existing studies of EDCs and UL are limited by small sample size, uncertain temporality due to retrospective design, and suboptimal measurement of EDCs and UL. This study will use data and biospecimens from the Study of Environment Lifestyle and Fibroids (SELF), a NIEHS prospective cohort study of 1,300 African American women aged 23-34 and free of UL at baseline (2010-2012). Questionnaire data were collected at baseline and subjects are being followed for 5 years. Every 20 months, blood and urine are collected, and ultrasounds are performed by trained sonographers to detect UL. We request support for the laboratory and statistical analysis of EDCs in relation to UL incidence. We propose a cost-effective case-cohort study design that includes 400 incident UL cases and 400 controls. We will measure urinary phthalate metabolites, serum PCBs, and urinary BPA, characterize exposures according to UL risk factors, and determine the associations of these chemicals with UL incidence and tumor characteristics. Non-persistent chemicals (phthalates, BPA) will be measured at baseline, 20 months, and 40 months. Persistent chemicals (PCBs) will be measured at baseline only. SELF provides a unique opportunity to assess the association of common EDCs with UL risk. This project has many strengths that overcome the limitations of prior studies: large sample size, prospective data collection, state-of-the-art EDC measurement, serial ultrasound screening for UL, analysis of repeated EDC measures, innovative statistical approaches for evaluating EDC mixtures, and control for multiple confounders. Pilot data show large exposure variability, providing excellent power to test the proposed hypotheses. This study of African Americans, a high-risk population for EDC exposure and UL, will provide informative data on the effects of widespread pollutants on UL, seek explanations for the racial disparity in UL incidence, and provide critical data to the public, scientific community, and policy makers.


The overall objective of this research is to understand the mechanism for the enzymatic assembly of cholesterol, the control of this process, and the relationship of this process to lipoprotein assembly in the liver. Specific objectives include the isolation and purification of Sterol Carrier Proteins and delineation of the relationship of these substances to lipoprotein assembly. Others objectives include the determination of chemical structures of intermediates in cholesterol biosynthesis, as well as, the mechanism of inhibition for hypocholesterolemic drugs.


In collaboration with Todd Golub at the Broad Institute, we have performed a luminex assay to quantitate tyrosine phosphorylation in tyrosine kinases. Active tyrosine kinases are also generally associated with increased overall tyrosine phosphorylation. Our assay captured more than 150 tyrosine kinases in a multiplex assay and detected the level of tyrosine phosphorylation in all these kinases using a pan-phosphotyrosine antibody (4G10). We used 150 human lung adenocarcinoma tumor tissue and corresponding adjacent normal lung tissue to profile tyrosine phosphorylation of these tyrosine kinases. We identified around 30 specific tyrosine kinases differentially phosphorylated in tumor tissue compared to adjacent normal lung. Examples of such potentially active tyrosine kinases are NTRK1, EPHA2, DDR. We then completed a nanostring assay in collaboration with Dr. Marc Ladanyi at MSKCC to measure transcript expression of majority of tyrosine kinases in a multiplex assay. Interestingly, as we had predicted, expression of several tyrosine kinases do not change at least at the level of trasnscription, however there is significant change in tyrosine phosphorylation, which is generally a surrogate of activation. In collaboration with Dr. Paul Meltzer in the NCI intramural program, we have sequenced around 1300 cancer-related genes, including the tyrosine kinases used in our luminex assay to identify specific mutations in a targeted manner. We are currently analyzing the sequencing data to identify somatic changes in the genes selected. The somatic mutation data is being correlated with the luminex data to identify possible somatic changes in kinases resulting in increased activity.


Osteogenesis imperfecta is a heritable disease of connective tissue that results from a wide variety of mutations of type I collagen. This grant outlines a series of protocols designed to define these abnormalities at the molecular level. It is based on the assumption that it is easier to define the mutations in collagen by studying the mRNA rather than its very complex gene. Fibroblasts derived from patients with 0I will be used as a source of RNA. Structural mutation of the alpha 1 or alpha 2 collegen mRNA will be localized using a series of end-labeled S1 probes that encompass the entire mRNA. The probes will be derived from single-stranded cDNAs cloned in M-13. The exact mutation will be defined by dideoxy sequencing of an extended cDNA initiated from a synthetic oligonucleotide primer complementary to a region 3' to the region suggested of having the abnormality as dictated by the S-1 protection experiments. Another oligo-nucleotide coding for the abnormal sequence will be constructed and used as a mutation specific hybridization probe to precisely localize the mutation to the gene. Mutations which alter the steady state level of collagen mRNA will be tested for an error of RNA processing using a novel application of the M-13 derived hybridization probe. This will involve an S-1 protection protocol to detect an accumulation of a specific unspliced mRNA, measurement of half-life of a normal and mutant cytoplasmic mRNA and solution hybridization of radiolabeled RNA from in vitro transcribed muclei. The methods that are developed in this grant will have general applicability to a wide variety of mutations involving type I collagen. The mutation-specific oligonucleotides provide the means to rapidly detect a subtle mutation in genomic DNA. It has the potential to become a very powerful adjuvant for the clinical geneticist who must counsel a family with a connective tissue disorder.


This project will address the therapy of mesothelin expressing tumors by developing and testing engineered T cells with potent antitumor cytotoxicity. Mesothelin is a tumor-associated antigen that is frequently over expressed on mesothelioma, non-small cell lung cancer, pancreatic and ovarian cancers. The strategy to be used is the "T-body" approach, which employs genetically reprogrammed, patient-derived lymphocytes transfected with a novel chimeric receptor that contains combinations of the signal transduction domains of 4-1BB (CD137), CD28, and CD3zeta as well as anti-mesothelin scFv (anti-meso-CD28-41BB-zeta). The central hypothesis to be tested is that previous trials of adoptive therapy for cancer have used insufficient numbers of cytotoxic T lymphocytes (CTL) that have shown inadequate engraftment, persistence and effector function to self antigens. Presently, we are the only laboratory in the world that is actively testing lentiviral modified T cells in the clinic, and in that trial we have demonstrated safety and prolonged lentiviral gene transfer. The following three specific aims will test the hypothesis that engineered human T cells expressing an anti-mesothelin-CD28-41BB-zeta chimeric receptor will have potent antitumor activity in vitro and in vivo by: (1) developing and optimizing the anti-meso scFv vector. The avidity and the cytosolic signaling modules will be optimized to obtain highly efficient lentiviral vectors that retarget T cells to specifically kill tumor cells that express mesothelin at low effector to target ratios in vitro;(2) carrying out in vitro experiments to optimize the effector functions of anti-mesothelin scFv CD28-41BB-zeta T bodies. Experiments will determine optimal conditions for redirected T cell serial killing, cytokine production and proliferation, and compare this to natural MHC restricted CTLs;and (3) performing in vivo experiments in immunodeficient NOD/SCID/beta2null mice xenografted with human tumors that express mesothelin. These experiments will test the hypothesis that vectors with high affinity scFv receptors and 4-1BB and CD28 signaling modules will have the most potent anti-tumor effects. Finally, the engraftment, persistence and antitumor effects of chimeric T cells given by intravenous and intraperitoneal routes will be compared using bioluminescence imaging. In summary, an outstanding team of basic and translational scientists has been assembled that will develop and test a universal T cell receptor to target some of the most common and drug resistant tumors. Lay Description. A common reason for failure of immunotherapy of epithelial tumors is that the immune system does not generate sufficient numbers of T cells to eradicate the tumor cells. It is now possible to use lentiviral vector technology to engineer T cells with potent and specific antitumor effects. This project will evaluate engineered T cells that target mesothelin that is overexpressed on both uncommon tumors such as mesothelioma and a variety of commonly lethal tumors including pancreatic, ovarian and non-small cell lung carcinoma.


Logistical support for BEE/Council Working Group on NHLBI Program Project Grant Mechanism


The isolated ventricular cell must retain the physiologic characteristics of its native tissue if it is to be useful in studying excitation-contraction coupling. A wide variability in isolated cell size, rest sarcomere length, amount and velocity of sarcomere shortening and action potential has been observed. This proposal seeks to understand this variability by correlating the function of individual cells, as examined by sarcomere motion and rest and action potentials, with the structure of the same cell. Cells will be isolated from the right and left ventricle of the rabbit and rat using enzymatic and mild mechanical dispersion. Rabbit myocardiuim will be studied because it has physiologic properties much more like those of other mammals than those of the rat. The rat will be studied because it has such strikingly different physiologic properties and has been used in many isolated cell investigations. The experiments will make use of the prominent effects induced by altering the rate and pattern of stimulation on contractility and the action potential and the characteristic changes in these effects induced by inotropic agents. Timed changes in extracellular ionic concentrations during rest and contraction will take advantage of the short diffusion distance. The proposal will provide a detailed examination of the physiological and ultrastructural properties of the same cell. This will enable us to assess whether intrinsic in situ differences in cells or the effects of the isolation procedure are the basis of cell-to-cell variability. Sarcomere shortening and action potential characteristics will be related to cell shape, size and ventricular site of origin. Species differences will allow structrue-function correlations to be made. The changes in myocardial performance produced by altering the rate and pattern of stimulation have been used in multicellular preparations to study contractility and to characterize the effects of disease. By producing a detailed quantitative description of the changes in sarcomere shortening velocity induced by altering the pattern of stimulation, this investigation will provide a basis at the cellular level for the force-interval relationship in the intact heart. By assessing the importance of extracellular calcium concentration at different times during activation and rest, this proposal will provide new data for testing models of excitation-contraction coupling. This proposal will provide a basis for future isolated cell investigations of disease-induced derangements in excitation-contraction coupling.


The University of Montana School of Pharmacy and Allied Health Sciences has a long standing history and commitment to the recruitment and training of minority students (principally Native American Indians) for degrees in pharmacy and physical therapy. The recently obtained Native American Center of Excellence (NACOE) grant builds on strength from funding obtained for the HHS-Health Careers Opportunity Program (HCOP), the NIH-Bridges to Baccalaureate Program (BRIDGES), and the NSF-EPSCoR Program to provide both the environment and the infrastructure necessary to offer degree training for under-represented minorities in pharmacy (Pharm.D.), physical therapy (M.S. and D.P.T.), and more recently in graduate education (M.S., Ph.D.). Research infrastructure has dramatically increased in the School's Department of Pharmaceutical Sciences in the past ten years to the point where the School is ranked 1lth of 85 schools/Colleges of Pharmacy in NIH funding per Ph.D. faculty. The Department has created two state-approved research centers and offers two M.S. and two Ph.D. programs. The primary goal of the Endowment Fund Program is to add at least two tenure track minority faculty through training and recruitment efforts, while perpetuating recent success in the recruitment, retention, and training of minority students in Pharmacy, Physical Therapy, and Graduate programs. Focus on minority health disparities research will continue to be expanded. These goals will be accomplished as follows: 1) creating new tenure track faculty lines for minority faculty (preferably Native American Indians), 2) Enhancing opportunities for minority students who earn the Pharm.D. or D.P.T. degree to obtain postdoctoral fellowship training in clinically relevant areas, 3) Enhancement of opportunities for minority students to pursue graduate training leading to the Ph.D. degree. 4) Perpetuation of a strong program to recruit and train undergraduate minority students, 5) Use of endowment income to leverage additional institutional/state and federal support, and 6) Progressive growth of the endowment corpus. Priorities for use of the endowment income are A) Recruitment and training of two minority PhD, students and one minority post Pharm.D. Fellow (Years 1-3) and B) Complete training and/or recruitment of two minority faculty with priority given to Native American Faculty candidates, particularly those involved in health disparities research (Years 3-5). [unreadable] [unreadable]


Chronic exposure to high concentrations of manganese (Mn) results in adverse neurological health effects commonly referred to as manganism. Manganese neurotoxicity is a significant toxicological problem resulting from the use of manganese as a gasoline additive, and in welding, metal industries, pesticide manufacturing, pharmaceutical preparations, infant food formulations, and battery production. Manganese predominantly accumulates in the basal ganglia structures and causes mitochondrial dysfunction, oxidative stress, and apoptosis. However, cellular and molecular mechanisms underlying manganese neurotoxicity are poorly understood. We have been studying mitochondrial-dependent apoptotic signaling in manganese neurotoxicity and found that protein kinase C-delta (PKCd), a member of the novel PKC isoform family, is persistently activated by caspase-3 to promote apoptosis during manganese exposure. While studying the apoptotic signaling pathway in cell culture models of manganese neurotoxicity, we also unexpectedly identified that PKCd is upregulated both in protein and mRNA levels during manganese treatment. Further analysis of the PKCd promoter revealed that two key transcription factors, NFkB and SP1, regulate PKCd gene expression. Thus, our competitive renewal proposal aims to systematically characterize this gene-environment interaction by studying novel molecular mechanisms underlying manganese-induced upregulation of the proapoptotic PKCd gene. We propose to complete the study by pursuing the following specific aims: i) To characterize upregulation of an oxidative stress-sensitive proapoptotic kinase PKCd in mouse primary neuronal cultures and animal models following manganese exposure, ii) To investigate molecular mechanisms of manganese-induced upregulation of PKCd by examining its transcriptional regulation of a PKCd promoter, and iii) To further define the functional role of NFkB and SP1-dependent PKCd upregulation in manganese-induced neuronal damage during chronic manganese exposure. Cellular, molecular, and neurochemical approaches in relevant cell cultures and animal models of manganese neurotoxicity will be used. We anticipate that the proposed study will provide comprehensive information about how environmental exposure to manganese can alter the expression of the key proapoptotic gene PKCd to augment manganese neurotoxicity, and this knowledge may advance the development of novel translational approaches for the treatment of manganese neurotoxicity. PUBLIC HEALTH RELEVANCE: Environmental exposure to excessive manganese impairs basal ganglia function, resulting in a neurological disorder relatively similar to Parkinsonism commonly known as Manganism. Manganese exposure is of serious concern due to the increased incidences of extrapyramidal neurological symptoms among miners and industrial workers, including welders. Manganese predominantly accumulates in the basal ganglia structures and causes mitochondrial dysfunction, oxidative stress, and apoptosis. However, cellular and molecular mechanisms underlying manganese neurotoxicity are poorly understood. The proposed study will elucidate the neurotoxic mechanisms underlying manganese induced upregulation of a proapoptotic kinase PKCd and its functional relevance to manganese neurotoxicity.


The Administrative Core provides the infrastructure and centralized support that will enable all four research projects to be productive and collaborative. This will be achieved by the following aims: Aim 1: Administration and fiscal management. The administrative core will provide oversight of administrative and budget matters including human resources, fiscal management, conflict resolution, institutional regulatory compliance (i.e. IRBs), and all communication with NIH. Aim 2: Communication management. The administrative core will schedule and facilitate monthly project leader and P01 staff meetings for dissemination of scientific findings, organize the annual scientific advisory panel visit and the executive committee meetings, and foster a collaborative scientific environment (advertising seminars, work-in-progress meetings, attendance at meetings to disseminate research findings within the scientific community, etc.). Aim 3: Scientific oversight. The administrative core will ensure scientific integrity such as data sharing plans and data management and assurances. The administrative core will also provide support for laboratory equipment. Aim 4: Statistical data analysis support. The Core will provide statistical support and oversight that include recommendations for experimental design to achieve desired statistical power, power calculations to determine group sizes, recommendations for data transformations to achieve forms appropriate for statistical analysis and advice regarding appropriate statistical tests to employ during the data analysis phase.


Macrophages are distributed throughout the body where they are poised to detect pathogens and to subsequently alert the immune system to the presence of infection through the production of inflammatory mediators. Inflammatory mediators are critical for pathogen control, but if produced excessively, can result in inflammatory diseases. My long-term goal is to understand the regulation of the production of inflammatory mediators by macrophages during infection. I have identified a novel type of negative regulation of the inflammatory response to pathogens through the TREM-2/DAP12 receptor complex. The specific aims of this proposal seek to define the mechanism of TREM-2 and DAP12 inhibition of inflammatory signaling in macrophages both in vivo and in vitro. The specific aims are: 1. To determine the ligand requirements for TREM-2 and DAP12 inhibition of inflammatory responses in macrophages. I will test the hypothesis that low avidity signals through DAP12 result in inhibition of inflammatory responses, whereas high avidity signals result in activation of inflammatory responses. 2. To determine the mechanism by which TREM-2 and DAP12 inhibit inflammatory signaling in macrophages. These experiments will define how DAP12 signaling results in inhibition of ERK phosphorylation and activation leading to dampening of inflammatory cytokine production in macrophages. 3. To determine why DAP12-deficient mice have enhanced innate immune responses in vivo. I will determine the mechanism for the increased innate response to Listeria monocytogenes infection in DAP12- deficient mice. I also will investigate what myeloid populations are inhibited by DAP12 signaling and whether this correlates with TREM-2 and TREM-2 ligand expression. Results from these studies should provide novel insights into how the early innate immune response to infectious pathogens is controlled. This is a topic of significance in the development of vaccines to prevent infection and immunomodulatory drugs to treat infections with pathogens relevant to biodefense, including Listeria monocytogenes, an Ml AID category B priority pathogen addressed in this proposal. [unreadable] [unreadable] [unreadable]


Despite a wide array of HIV prevention approaches and related research, there is a decade-long trend of 40,000 new HIV diagnoses per year in the U.S. (CDC, 2004; Copenhaver & Fisher, in press). Injection drug users (IDUs) remain a target population as they represent a significant vector for the transmission of new HIV infections (Avants et al., 2004; Margolin et al., 2003), which occur through preventable drug- and sex- related HIV risk behaviors. A number of evidence-based HIV risk reduction interventions are now widely available as complete intervention packages. However, very few evidence-based interventions have been designed for implementation within common drug treatment CBOs, such as methadone maintenance programs (MMPs), where many high-risk drug users seek treatment. Moreover, based on analogous efforts to disseminate evidence-based behavioral interventions for treating drug dependence (Morgenstern et al., 2001; Institute of Medicine, 1998), the few evidence-based interventions that are applicable to drug treatment CBOs are not designed to be "community-friendly" and are therefore unlikely to be implemented as intended or durable within these critical settings. Our team of investigators has developed a significantly shortened, community-friendly, version of the comprehensive evidence-based Holistic Health Recovery Program (HHRP; Avants et al., 2004; Margolin et al., 2003). The shortened version, the Community-friendly Health Recovery Program (CHRP), has demonstrated feasibility and acceptability as well as preliminary evidence of effectiveness in an uncontrolled study within a resource-limited drug treatment CBO (Copenhaver et al., in press; see Appendix). In this revised R01 application, we propose to evaluate CHRP in a randomized controlled trial (RCT). If found to be effective in the proposed trial, CHRP has the potential to be fully integrated, as designed, within many other resource-limited CBOs where large numbers of high risk drug users participate in drug treatment. [unreadable] [unreadable] [unreadable]


AMPK is a fuel sensing enzyme that is activated by hormones, cytokines, exercise, and stresses that diminish cellular energy state (e.g., glucose deprivation). In addition, metformin and thiozolidinediones, agents used to diminish insulin resistance in type 2 diabetes, have been shown to activate AMPK. Activation of AMPK increases processes that generate ATP (e.g., fatty-acid oxidation) and restrains others such as fatty acid-, glycerolipid- and protein-synthesis that consume ATP, but not acutely necessary for survival. Conversely, when cells are presented with a sustained excess of glucose, AMPK activity diminishes and these synthetic processes are enhanced. Investigations by others have demonstrated that prostate cancer cells require high rates of fatty acid and protein synthesis for their invasive growth and survival. In preliminary studies, we have observed an apparently selective inhibition of the growth of prostate cancer cells by three distinct AMPK activators, AICAR, rosiglitazone, and metformin. We have also found that these effects are associated with inhibition of fatty acid synthase (FAS), acetyl CoA carboxylase (ACC) and mTOR. Based on these findings, we will test the hypothesis that AMPK activation restricts the growth of prostate cancer cells by altering an array of cellular events. We will carry out studies with the following aims: (1) To confirm and extend our preliminary finding that AMPK activity is suppressed in prostate cancer cells and to explore to what extent this accounts for their increased growth/survival and altered cellular metabolism;(2) To determine whether agents that inhibit prostate cancer cell growth in vitro prevents the growth of prostate cancer xenografts and/or cause their regression;(3) To assess whether the uptake and oxidation of exogeneous fatty acids are imparied in prostate cancer cells, and if so to determine the mechanism(s) responsible for these abnormalities, and whether they are reversed by AMPK activation or inhibition of ACC and;(4) To explore the molecular mechanisms by which androgen and AMPK oppositely regulate the mTOR signaling pathway. Since AMPK can be activated by several anti-diabetic drugs and by certain adipocyte-derived hormones (e.g. adiponectin and leptin), success in this effort would suggest a novel, yet practical approach for treating prostate cancers in humans.


This Phase II proposal integrates emerging technologies to expand the behavioral health capabilities and technological sophistication of the Journey to Disaster RecoveryTM website. This web-based system is designed to help restore a sense of control (i.e. coping efficacy) and mastery to survivors by providing a) knowledge to assist in understanding the recovery process, b) skill training to manage trauma related distress, and c) assistance in determining the need for and access to professional assistance. This project provides three essential developments with scientific and technical impact. First, it expands the technical sophistication and capabilities of the website to make it easy to customize and more responsive to different disaster/user needs. Second, it provides a critical scientific evaluation of the site's effectiveness to enhance mental health in the acute phase of disaster recovery helping to extend the very limited current knowledge base in this area. Third, it creates important training opportunities and evaluation with disaster response groups in disaster prone areas of the United States. Our ultimate goal is to provide the most reliable, customizable, and culturally based interactive, web system for empowering disaster survivors. The specific aims for this project are: Aim 1: Generate and evaluate the effectiveness of a content management system to enable highly efficient web customization. This system will integrate with a database of stored information rendered for different users (e.g., pictures, video, and text for different ages, genders, and ethnicities). Aim 2: Extend the technological and psychological reach of the site to incorporate Smartphone technology, allowing the system to deliver real time, interactive support and skill development. This will provide extensive avenues for future scientific exploration about human computer interactions related to disaster mental health. Aim 3: Expand the scientific evaluation of Journey to Disaster RecoveryTM by conducting a large randomized controlled trial during the acute phase of disaster recovery. This will broaden the current knowledge base about Internet interventions for disasters. Aim 4: Develop a web-based training and support system that instructs disaster behavioral health counselors about how to use the site in face-to-face counseling sessions. A quasi-experimental wait-list study will test the utility of the website as a training tool with crisis counselors. Aim 5: Complete and evaluate the cultural sensitivity and relevance of the Spanish language version of the site, MI RECUPERACISN. Given the number of native Spanish speakers in disaster prone areas, a culturally sensitive Spanish version of the site is essential. PUBLIC HEALTH RELEVANCE: The research involved in this Phase II STTR application will provide critical information about the efficacy of a web-based support system for disaster survivors to reduce their psychological distress and promote empowerment. At least 50 percent of disaster survivors report experiencing moderate to severe psychological distress and face barriers to accessing care including: cost of services, lack of knowledge about where services are available, and the stigma of seeking care. Journey to Disaster RecoveryTM provides a unique resource to reach beyond these barriers by offering a highly customizable, culturally relevant, anonymous, theoretically- based system with enhanced scalability to support disaster survivors.


The overall goal of the University of Utah Molecular Medicine Translational Research Center in Thrombosis (U2M2-TRCT) is to test the hypothesis that the abnormal metabolic milieu in type 2 diabetes (T2DM) and the metabolic syndrome (MS) leads to genetic and metabolic reprogramming of platelets. This reprogramming directly contributes to the increase in platelet activation that characterizes these subjects who are at increased risk for thrombosis. The specific focus of project 2 is to elucidate the mechanisms that are responsible for the metabolic reprograming that develops in platelets of humans and mouse models of T2DM and the MS, and to determine if the associated changes in platelet metabolism will directly contribute to platelet dysfunction. Our preliminary studies reveal that diabetes alters metabolome profiles of platelets with changes consistent with increased glycolysis and TCA (tricarboxylic acid) flux and accumulation of lipid intermediates. In addition, transcriptional profiling reveals significant changes in the expression of genes that regulate mitochondrial energetics, coupling efficiency (e.g. UCP2) and mitochondrial dynamics (e.g. Mfn2). Thus we hypothesize that the metabolic milieu of T2DM and the MS increases platelet glucose utilization and mitochondrial metabolism that induces mitochondrial oxidative stress, leading to platelet activation. In aim 1 we will test the hypothesis that T2DM and the MS leads to mitochondrial dysfunction, characterized by mitochondrial uncoupling, ROS overproduction and impaired mitochondrial dynamics. In aim 2 we will determine the mechanisms by which T2DM and the MS alter mitochondrial function and will test the hypothesis that manipulation of these mechanisms (i.e., KO of platelet superoxide dismutase 2, UCP2 and Mfn2) are sufficient to modulate platelet activity. In aim 3 we will determine the contribution of increased platelet glucose utilization to platelet hyperactivation in T2DM and the MS. This hypothesis will be addressed in studies of diabetic murine models with genetic reduction or augmentation in platelet glucose transport. Collectively these studies will shed new insight into mechanisms that alter platelet metabolism and function in obesity and diabetes.


The HIV/AIDS Adolescent Risk Reduction Project (HAARP) focuses on the window of opportunity existing for some adolescents and HIV/AIDS. The proposed project is based on the hypothesis that adolescents who receive motivating information related to their vulnerability to HIV and effective practice in AIDS prevention behaviors will have lower HIV risk scores than those who do not receive the coupled interventions. The proposed project is a collaborative venture between AIDS Impact, Seattle and Western Washington University (WWU) in Bellingham, WA. The HAARP study will have three interventions and a control group: (1) a motivating trigger tape aimed at raising vulnerabilities with guided discussion; (2) a skill-building refusal skills modeling video and role play aimed at increasing self-efficacy; and (3) a combined pairing of the previous two interventions. A risk assessment will be developed and administered pre and post to all groups plus a control group. The 200 students choosing to participate will be from a pool of 650 randomly selected undergraduate students. The project will develop two video tapes and facilitator guides as intervention elements: (1) a :07 trigger tape aimed at eliciting vulnerability and providing motivation; (2) an :08 refusal skills tape providing models.


Brain abscesses represent an important medical problem despite recent advances made in detection and therapy. Because of the emergence of multi-drug resistant strains and the ubiquitous nature of bacteria, these CNS infections are likely to persist. The size of a developing abscess normally extends well beyond the original site of infection leading to damage of surrounding normal brain parenchyma. This finding suggests that the CNS antibacterial response is not down regulated in an efficient manner, resulting in chronic inflammation and large abscess lesions. They propose that a balance exists between sufficient and over-compensatory responses to S. aureus in the CNS, which dictates the outcome of brain abscess development; therefore, therapies aimed at attenuating chronic CNS inflammation subsequent to effective bacterial neutralization may result in smaller abscesses and subsequent improvements in cognitive and neurological functions. The objective of the proposed work is to examine the influences of minocycline and PPAR-gamma agonists on the pathogenesis of brain abscess development. Recently, these compounds have been found to exhibit neuroprotective effects in several models of CNS disease; however, their roles in regulating CNS infectious disease has not yet been examined. To address this objective, the following Specific Aims will be addressed: (I) to evaluate the dose- and time-dependent effects of PPAR-gamma agonists and minocycline on S. aureus-induced brain abscess development; (II) to investigate the effects of PPAR-gamma agonists and minocycline on cell migration and neuronal cell death induced by S. aureus-stimulated microglia; and (III) to examine the mechanism(s) responsible for impaired neutrophil infiltration into brain abscesses of CXCR2 KO mice and the potential effects of PPAR-gamma agonists and minocycline in the CNS compartment. In addition to its anti-inflammatory properties, the bacteriostatic activity of minocycline may augment its effects on brain abscess development. The potential multifactorial effects of these compounds suggest that they may be more efficacious compared to traditional therapies developed to counteract a single pathway in CNS diseases. These experiments should provide meaningful insights into how minocycline and PPAR-gamma agonists influence brain abscess development and will reveal whether their ability to modulate non-infectious CNS conditions extends to infectious diseases.


Enzyme-linked immunosorbent assay (ELISA) was used to quantitate interphoto-receptor retinoid-binding protein (IRBP) in retinoblastoma tumors and in human subretinal fluid (SRF) samples. In retinoblastoma tumors there was a direct correlation between the degree of differentiation of the tumors and IRBP concentration. Higher concentrations of IRBP were found only in the more recent rhegmatogenous detachments, and IRBP was absent from SRF of patients with retrolental fibroplasia. Cyanogen bromide peptides from purified bovine IRBP were purified by high performance liquid chromatography (HPLC) and several peptides were found to produce experimental autoimmune uveitis (EAU) in rats. IRBP was shown to be phosphorylated in a crude bovine interphotoreceptor matrix (IPM) wash, and the phosphorylation was of serine and/or threonine residues. Intravitreal injection of radiolabeled retinol or fatty acids into frog eyes showed both retinol and fatty acids to be bound to IRBP.


A total of over one hundred pharmacologically active alkaloids have been characterized in skin extracts of some 50 species of dendrobatid frogs. Five major classes have been eludicated and their pharmacology defined: 1) The batrachotoxins (6 compounds) are highly toxic, complex steroidal alkaloids which prevent the inactivation of voltage-dependent sodium channels in nerve and muscle; 2) The histrionicotoxins (10 compounds) are 3,7-disubstituted 1-azaspiro (5.5) undecanes, which facilitate the conversion of chemosensitive sodium-potassium channels in nerves and effector cells to the desensitized state; 3) The pumiliotoxin-C class (42 compounds) are 2,5-disubstituted decahydroquinolines, which block nicotinic receptors; 4) The pumiliotoxin-A class (24 compounds) are 6-alkylidene-8-hydroxy-8-methylindolizidines which facilitate and then block calcium-dependent excitation-secretion coupling and excitation-contraction coupling in nerve and muscles; 5) The gephyrotoxins (11 compounds) are 3,5-disubstituted indolizidines and tricyclic analogs. Certain members of this class block voltage-dependent potassium channels and muscarinic receptors. An additional 14 compounds cannot be assigned to a particular class: one of these, a pyridine derivative, represents a new class of potent analgesics: Compounds antagonizing the binding of ouabain to Na ion -K ion -ATPase were identified as bufodienolides in extracts of certain bufonid toads and frogs, while a new class of Na ion -K ion -ATPase inhibitor was present in extracts on one bufonid frog.


Despite glaring health and HIV/AIDS-disparities among American Indian and Alaska Natives (AIAN), Native Hawaiians and other Pacific Island Islanders (NHPI), there is a paucity of culturally grounded research addressing their HIV-related biomedical and behavioral health concerns. A strong network of highly trained and productive Indigenous scholars dedicated to research that is culturally grounded would contribute to ameliorating HIV-related disparities among Indigenous populations. This competitive renewal application, in response to PAR-12-273, NIMH Research Education Mentoring Programs for HIV/AIDS Researchers is designed to develop the Indigenous HIV/AIDS Research Training Program-Lauhoe (IHART2) to develop a cadre of culturally grounded Native scholars capable of serving as PIs on extramurally funded HIV/AIDS- related prevention and disparities studies with Indigenous populations. The program is based on the success of the first 5 years of the Indigenous HIV/AIDS Research Training (IHART) R25 program (under PAR-06-494), the only Native-specific HIV/AIDS research training and mentorship program in the US. IHART2 extends the reach of the original IHART program from a primary focus on indigenous North Americans (e.g., AIAN and Native Latinos), to including NHPI (e.g., Native Hawaiian, Samoans). Additionally, where IHART focused on mid-career scientists, IHART2 now targets postdoctoral and early-career scholars. Building upon IHART success, IHART2 deepens the 360 degree co-mentorship approach with flexibility for program Fellows and Mentors to co-identify HIV training, career development, and cultural needs of the Fellow. IHART2 will be housed at the university-wide interdisciplinary funded Indigenous Wellness Research Institute (IWRI; iwri.org) National Center of Excellence (NIMHD; P60 MD006909-02) at the University of Washington. IWRI is directed and staffed almost entirely by AIAN, with three of the leading federally funded AIAN HIV researchers at the helm. IWRI has the infrastructure and environment necessary to promote culturally grounded Indigenous HIV prevention and disparities science training in a manner that is not currently covered by any existing R25s. To achieve the overall objectives of IHART2, we will select 15 Fellows who will undergo a structured 24-month intensive year-round mentorship program that includes: (a) annual research institutes and writing retreats (writing retreat; Spring Research Roundtable; Summer Research Institute; grant writing workshop,); (b) other training (webinars; 4 quarterly seminars; on-site mentor shadowing; and support for attendance to HIV/AIDS training institutes; (c) technical assistance (peer review for grant submission; statistical, editorial, and technical assistance for grant applications and manuscripts for publication); (d) seed funding ($20,000 to conduct pilot studies, travel to conferences, or to buy out time); and (e) network development (via mentor networks and website). By the end of the 24 month program, Fellows will have implemented a pilot study, published HIV/AIDS articles; presented at HIV-related conferences; and developed a NIH HIV-related grant proposal.


Major depression ranks among the leading biobehavioral risk factors for cardiovascular (CV) morbidity and mortality. Research has revealed several pathways through which depression increases CV risk including health behaviors and psychosocial functioning. That depression remains a significant predictor of CV disease (CVD) after intervening to improve health behaviors and psychosocial functioning in adults with depression suggests that additional, important risk factors have yet to be identified. We have developed a conceptual model that extends previous research on depression and CV risk by considering sleep as a novel biobehavioral mediator through which depression increases CV risk. We hypothesize that sleep disturbance, including decreased sleep duration, continuity and depth and increased sleep disordered breathing contribute to the increased CV risk observed in adults with major depression. Importantly, each of these components of sleep is modifiable and may represent promising therapeutic targets for reducing the cardiovascular consequences of major depression. The proposed 5-year study will evaluate these relationships in a well-characterized cohort of 200 adults with a history of recurrent major depressive disorder (MDD) who underwent psychiatric assessments and sleep studies in our laboratory approximately 10 to 30 years ago (T1). Participants, who were medically healthy without clinical cardiovascular disease at T1, exhibited profound sleep disturbances that persisted during remission. Members of this cohort have expressed great interest in the proposed follow-up study (T2). Proposed T2 measures include follow-up psychiatric assessments and sleep studies coupled with assessment of CV risk and subclinical disease including indices of autonomic imbalance, endothelial dysfunction, preclinical atherosclerosis and the metabolic syndrome. We will also assess health behaviors, psychosocial functioning and potential confounding variables. Together, these data will provide the first test of the hypothesized paths in our conceptual model. Our primary aim is to use T1 depression and sleep data in conjunction with T2-assessed CV outcomes to evaluate whether PSG-assessed sleep disturbance attenuates the prospective relationship between depression and CV risk and subclinical disease. Our secondary aim is to evaluate both wake and sleep pathways in the same model, using data collected at T2, including objective assessment of physical activity. The use of multiple-group structural equation models will provide the opportunity to evaluate whether relationships among depression, sleep disturbance and CV risk/subclinical disease differ by age and gender (exploratory aim). In future studies, experimental approaches will be needed to establish sleep as a causal pathway, identify the biological mechanisms through which specific components of disturbed sleep in depressed individuals increase CV risk, and develop evidence-based sleep interventions to prevent or attenuate the cardiovascular consequences of major depression. PUBLIC HEALTH RELEVANCE: The proposed study will be the first to evaluate sleep as a novel pathway through which depression increases cardiovascular disease risk, morbidity and mortality. Insights into the role of sleep disturbance in the link between major depression and cardiovascular disease is essential to our understanding of the pathophysiology of this prevalent and costly disease. Because sleep represents a modifiable pathway through which depression increases cardiovascular disease risk, it holds promise for the design and implementation of effective, evidence-based interventions to prevent and/or treat the cardiovascular consequences of major depression.


We are now practised in the complex surgery of isolating and perfusing kidney from a rat. Oxygen-sensitive crystals of LiPc were placed in the cortex and outermedulla so as to measure the partial pressure of oxygen (pO2) simultaneousl y in both these regions within the same kidney, allowing us to compare these results with our in vivo model. The advantages of using a perfused system are that many parameters can be measured and tightly controlled: 1) The dependence of tissue pO2 on perfusion pressure and/or flow rate in the control kidney, the pO2 in the cortex region is high compared to that in the medulla. However, at a critical pressure, the pO2 in the medulla sharply increases to a level equal to that in the cortex. If the pressure was again increased, we found that this phenomenon was reversed. 2) The effects of nitric oxide on oxygenation- by addition of nitroprusside (a nitric oxide donor) or a blocker of nitric ox ide synthesis, we were able to control the vasodilation of vessels in the medulla, and monitor the effects on pO2. 3) Endotoxin (either by bolus dose or continuous infusion) probably induces NO synthesis in the medulla, since we observed an increase in tissue pO2 with little change in tissue oxygen consumption.


The goal of this proposal is to develop chemosensors using arrays of engineered yeast strains to mimic olfaction. The experiments I propose will lead to a better understanding of the functional organization and evolution of G protein coupled receptors (GPCRs) and to greater understanding of the molecular mechanisms of olfaction. In many organisms, olfaction appears to be based on a mechanism involving combinatorial activation of broad-specificity GPCRs. I propose to utilize the fundamental mechanisms of olfactory chemosensing to construct yeast arrays capable of serving as chemical sensors. I will take two different approaches to construct the arrays, in each case using yeast strains engineered to functionally express human GPCRs. First, I am currently utilizing directed evolution techniques to generate mutant variants of the human muscarinic and melatonin receptors. I plan to use these mutants to create receptor arrays that are capable of analyzing complex mixtures for low levels of certain analytes. I also intend to continue the efforts of our lab to functionally express olfactory receptors in yeast. I will analyze libraries of chimeras between the melatonin receptor and olfactory receptors with known ligands. Once functional receptors are isolated, I plan to develop a generalized technique for generating functional chimeric olfactory receptors.


Project Summary This project will investigate how different environmental, personal and disease related factors contribute to the experience of behavioral symptoms of patients with Alzheimer's disease (AD). Patients with AD will experience behavioral symptoms of dementia (BSD) at some point during their illness, though most patients will not experience all types of BSD or experience them in a predictable pattern. This has made development of effective treatments and symptom management approaches for AD highly challenging. Our pilot data shows a high degree of intraindividual heterogeneity in daily reports of BSD both in type of symptom and frequency of occurrence. Others report significant intraindividual heterogeneity in the rate of change in BSD over a 3-month period. It is currently unclear what mechanisms contribute to these within- and between-person variations in BSD. While BSD subsyndromes have been proposed it is unclear how the types of BSD within the subsyndromes are temporally dependent or distinct from each other. As applied to AD, the Revised Symptom Management Model, points to environmental (i.e., unmet needs, stressors), personal (i.e., genetic, microbiome, ethnicity), and disease (i.e., HSV1 infection, systemic inflammation) factors as mechanistic pathways for BSD. We hypothesize that these factors across multiple systems contribute to the mechanistic development of BSD, and thus the variability in individual BSD expression. Elucidation of these factors across multiple systems will help create personalized approaches to treatment of BSD spanning from caregiving behavioral training to pharmacological interventions. Dyads of co-residing caregivers and persons with AD or mixed-type AD (with vascular involvement) (N=162 dyads) will be recruited. Equal numbers of Caucasian and Hispanic dyads will be enrolled so that the contributing role of ethnicity can be examined. Using a micro-longitudinal design, caregivers will complete daily diaries for 30 days reporting on their observations of environmental exposures, including type, frequency, duration, and severity of BSD. At enrollment and at the start of each new week of diaries, dyads will visit the research clinic where blood and stool samples will be collected from the person with AD (for 5 samples total). BSD will be reported in the daily diary entries, with BSD type measured as presence or absence of 22 symptoms across 17 domains. We will use a multi-level model analytic framework to examine hypotheses outlines in the research strategy in order to complete the following aims: (1) Determine how environmental (i.e., unmet needs, stressors), personal (i.e., genetic, microbiome, ethnicity) and disease (i.e. HSV1 infection, systemic inflammation) factors impact probability of daily BSD, either directly or through interaction effects, and (2) Identify subsyndromes of BSD, and predictors of group membership. Impact: By competitively testing leading hypotheses for BSD, this project will elucidate criteria for distinguishing patients with BSD that are not resolvable via appropriate attention to their unmet needs. Findings from this project will inform targeted interventions to support persons with Alzheimer's disease and their family caregivers.


The capsular polysaccharides of Streptococcus pneumoniae are essential virulence determinants for this organism. They serve as the basis for serologic classification, with ninety serotypes being distinguished to date, and they are the target for protective antibodies. In both human infections and animal models, virulence is related to the serotype of the capsule expressed. The mechanisms underlying the virulence differences between serotypes are not known but may involve both the polysaccharide structures and other factors in the genetic background in which they are expressed. Molecular genetic studies have identified a common organization among the different S. pneumoniae capsule loci. This organization permits the exchange of capsular serotypes during transformation, leading to new combinations of virulence factors and potentially impacting on the efficacy of polysaccharide-based vaccines. Knowledge of the genetic components of the loci has permitted pathways for polysaccharide synthesis to be demonstrated or proposed. Often, enzymes expected to be essential for capsule production are not encoded by genes in these loci, and the necessary products are obtained from cellular pools contributing to pathways for peptidoglycan and teichoic acid synthesis. Thus, capsule expression and basic cellular functions are intimately linked. Recent studies have demonstrated a requirement for capsule in colonization but have suggested that capsule production is reduced in this environment as compared to other host environments. Expression of other factors important for adherence and sustained colonization may be elevated during colonization and reduced at other times. Coordinate regulation of these virulence factors is thus anticipated and recent studies have identified a surface component potentially involved in adherence whose expression is altered in response to changes in capsule production. In the proposed studies, we will continue the genetic analysis of virulence and capsule expression. The specific aims are to: 1) determine the effect of specific alterations in capsule type and structure on virulence, 2) characterize the requirement for capsule in pneumococcal infections and its expression in vitro and in vivo, and 3) characterize regulatory networks and cellular pathways associated with capsule synthesis.


Optimal post-seizure therapy requires anticonvulsant and neuroprotectant therapeutic strategies, as both prolonged seizures (status epilepticus) and repetitive seizures occurring over time (epilepsy) cause neuronal death in brain and cognitive decline. Our data challenge assumptions on the relevant molecular pathways for such neuronal loss, revealing that Death Receptors of the extrinsic pathway are preferentially activated following seizures, which then trigger endoplasmic reticulum (ER) dysfunction. This extrinsic pathway activation occurs before any (intrinsic) mitochondria-linked cell death pathways become activated. We show protection from extrinsic pathway activation by ER-resident anti-apoptotic Bcl-w, which functions through effects on calcium regulation, attenuation of ER based pro-apoptotic Bcl-2 family protein function and inhibition of the integral ER membrane BAP31 complex. The SPECIFIC AIMS of this project are: Aim 1. Determine the significance and mechanism of death receptor complex activation as a cause of ER dysfunction and neuronal death following seizures. Aim 2. Determine how Bcl-w protects against seizure-induced neuronal death via effects on the extrinsic pathway target: Aim 3. Show the relationship between Extrinsic and Intrinsic pathway activation by seizures. To accomplish these aims we have developed a mouse model of seizure-induced brain injury, not previously available, with continual EEC seizure monitoring, permitting electrographic seizure quantitation and distinction between injurious and non-injurious seizure types. We also use an in vitro seizure model permitting single cell, calcium imaging and culture studies. With mouse modeling we will use knockouts for down regulation of apoptosis modulatory genes under study and provide up-regulation of protective gene products with adenoassociated viruses and TAT fusion proteins. We determine the effect of pharmacological and molecular manipulation of the extrinsic cell death pathway on seizure induced cell damage. Finally, using continuous video-EEC monitoring, we will investigate how prevention of neuronal damage following seizures effects the development of an epileptic phenotype.


Two isoenzymes of amine N-methyltransferase from rabbit liver are available in homogeneous form and have been characterized. The enzymes have overlapping specificity for the transfer of a methyl group from S-adenosyl-L-methionine to any one of a very large number of amine acceptors. Acceptor molecules include those of very different carbon skeleton among which are aliphatic, aromatic and heterocyclic compounds that are primary, secondary and tertiary amines. Methylation of the tertiary amines, pyridine for example, results in the formation of a pyridinium ion. It has now become possible to evaluate the role of methylation in the formation of certain hepatocarcinogenic amines. Hepatocarcinogenicity for compounds such as benzidine, N- methylazobenzene and some other aminobiphenyls is well established. Based on work with purified enzymes, N-methyltransferase and a microsomal flavincontaining monooxygenase, it was concluded that carcinogenicity of such aromatic amines required methylation followed by N-oxidation of the resultant N-methyl derivative; the cytochrome P-450 system did not appear to be involved. Thus ben and 4-aminobiphenyl are both methylated and carcinogenic. 4- Aminoazobenzene is neither methylated nor carcinogenic, although the methylated species, 4-methylaminoazobenzene is carcinogenic. All three carcinogenic monomethyl arylamines yield N-oxidized products with the flavin oxygenase. 2-Aminobiphenyl is neither a substrate for the transferase nor does it act as a tumor inducer. It would appear that N-methylation, followed by oxidation with the flavoprotein system, is a means for producing carcinogenic derivatives of the aminobiphenyls.


Reducing Duration of Untreated Psychosis (DUP) is a primary goal for improving long-term outcomes in young people with a first episode of psychosis (FEP). The standard of FEP care within the US focuses on targeted provider education1 regarding signs and symptoms of early psychosis to motivate patient referrals to FEP services, followed by initiation of services within largely clinic-based settings 2. Experience at the Early Diagnosis and Preventive Treatment (EDAPT) FEP specialty program at U.C. Davis in Sacramento has identified two important bottlenecks to reducing DUP, consistent with reports in the literature from other FEP clinics. These are 1) delays in the identification of psychotic symptoms by referral sources, and 2) delays or disruptions of patient engagement in specialty FEP care. Building upon a comprehensive and established referral network of 20 sites across the Sacramento area (schools/universities, ER/inpatient hospitals, outpatient mental health, primary care), we will address delays in patient identification and engagement using a two- phase, cluster randomized design. We will consecutively test the impact of two interventions to reduce DUP, defined in this RFA as time from first onset of psychotic symptoms to engagement in FEP specialty care. To address identification delays, we will examine the use of standard targeted provider education plus novel technology-enhanced screening compared to standard targeted provider education alone, testing the hypothesis that the education plus technology-enhanced screening will identify more patients, earlier in their illness. To address engagement delays, we will compare the use of a mobile community-based, telepsychiatry- enhanced engagement team to standard clinic-based procedures for intake, engagement and initiation of treatment, to test the hypothesis that the mobile approach facilitates earlier and more stable engagement, thereby reducing DUP. The proposed work will provide new specific evidence-based practices for reducing DUP and improving outcomes through specialty care of individuals with a first episode of psychosis.


The Mouse Genetics Facility was established in 1999 in response to the increasing need of Cancer Center investigators for analysis of gene function in mouse models, and has received Cancer Center support since 2002. Since the last renewal, the Facility has changed leadership, with Dr. Anthony Capobianco now the scientific director of the Facility and Dr. Ping Jiang the facility director. In this funding period, the Institute has invested $167,450 for new and upgraded equipment and renovated laboratory and animal facility space. The Facility provides highly efficient production of both transgenic and knock-out/knock-in (KO/KI) mice, plus it provides a unique resource and expertise in embryonic stem (ES) cell cultivation and engineering, which benefits Cancer Center investigators generating both in vivo and in vitro murine models of cancer. Current services are aligned with the research objectives of the Cancer Center programs and include: (1) production of transgenic mice by pronuclear microinjection of DNA transgene constructs;(2) generation of genetargeted 129 and C57BL mouse ES cell clones;(3) production of chimeric mice derived from microinjection of gene-targeted ES cells into blastocysts;(4) rederivation of mouse lines by embryo transfer;and (5) ES cell cultivation and engineering. Since 2006, the Facility has produced 15 lines of transgenic mice, successfully generated 11 lines of KO/KI mouse ES cell clones, and produced 8 lines of chimeric KO/KI mice. In addition, the Facility successfully rederived a line of an engineered mouse strain and will offer a mouse embryo cryopreservation service in 2008. The Facility is actively collaborating with Cancer Center investigators to utilize its mouse ES cell technology, with the aim of using human ES cells as a vehicle to derive mutant or disease specific cell lineages as a new human disease model system. Goals for the Facility over the next funding period include developing lentiviral mediated transgenesis for some strains of mice that are difficult to produce with DNA microinjection, developing use of tetraploid complementation as an efficient approach to produce chimeras from injections of ES cells of C57BL origin into blastocysts, and establishing human ES cell cultivation and engineering as a routine service.


Rigorous biochemical analyses of pulmonary surfactant show that it is comprised of disaturated phosphatidylcholine, monoenoic phosphatidylcholine, phosphatidylglycerol, a unique apoprotein, and small amounts of neutral lipids, other phospholipids, and carbohydrate. The principal physiological function of pulmonary surfactant is well-known -- it lowers the surface tension of the alveolar interface to less than 15 dynes/cm. It has been determined that the disaturated phosphatidylcholine is capable of this process. Still unknown, however, are the physiological roles of the other lipids, and the apoprotein. This project will explore how the composition of these lipids may affect the physical properties of the entire complex, and how this may relate to respiratory mechanics. Significant emphasis will be given to the interaction of the apoprotein of surfactant with well-defined mixtures of lipids, and the effect of this interaction on the physical properties of the recombinant complex. An attempt will be made to find which of the lipid and/or protein constituents are needed to form tubular myelin, and to correlate morphologic structure with physicochemical properties.


PROJECT SUMMARY Social interaction and communication begin in early infancy, and, although these are fundamental human functions, little is known about the underlying neural mechanisms that regulate them particularly in Autism Spectrum Disorder (ASD). ASD is a neurodevelopmental disorder characterized by significant disabilities in language and social skills, and the specific neural mechanisms that lead to these disabilities remain active topics for investigation. Emerging theoretical directions converge on problems with eye-contact as a salient component of these communication and social disabilities. Technical limitations, however, associated with imaging of two or more individuals during natural communication and mutual eye contact have been a primary obstacle to these investigations. To overcome this technical impasse, we employ a rapidly developing brain imaging technology, functional near-infrared spectroscopy (fNIRS) allowing simultaneous neural imaging of two individuals during valid interactions to observe the neural effects of eye-to-eye contact and actual dialogue. Functional NIRS detects active neural tissue based on the blood-oxygen-level-dependent (BOLD) signal by measuring variations in the absorption spectra associated with oxyhemoglobin and deoxyhemoglobin. Because detectors and emitters are surface mounted on the head, they are relatively insensitive to head movement, and, as such, fNIRS is well suited for investigations of neural events engaged during active interpersonal interactions between two participants. The neural mechanisms that underlie atypical interpersonal interactions and eye contact in adult ASD are the focus of this proposal. Pilot studies confirm the feasibility of all aspects of this research project. Dyads consisting of a confederate and a participants with typical development (TYP) or ASD will be compared during neuroimaging while engaged in natural interaction and communication. We introduce a computational approach based on wavelet analysis to quantify regional cross-brain coherence between the two participants and hypothesize that cross-brain coherence associated with speech and eye contact will be reduced in ASD relative to the TYP cohort. Cross-brain computations also form the basis for a model of dynamic neural processes based on neural ?send and receive? functions during communication. We hypothesize that these dynamic ?cross-brain communication? systems unify and coordinate the roles of language production and reception (Broca's and Wernicke's Areas), respectively, with visual reception involving face specializations (fusiform gyrus). Computational comparison of cross-brain connectivity effects as well as conventional functional connectivity and segregation/contrast effects during live communication both with and without direct eye contact provides a transformational technical, empirical, computational, and theoretical advance toward understanding the dynamic neural mechanisms associated with social and communication disabilities in ASD.


DESCRIPTION (Applicant's abstract verbatim): Noble gas isotopes that can be nuclear spin-polarized are very attractive imaging agents because of their high detection sensitivity for nuclear magnetic resonance. In particular, spin-polarized 3He enables fast, gas-space imaging of the lungs and airways for clinical diagnosis and physiological studies. Unfortunately, the limited availability of 3He severely limits the number of high-resolution lung imaging studies that can be performed. We propose to develop a system to recover and recycle the rare helium isotope so that high-resolution lung imaging can become common and inexpensive. The innovation is a cryogenic gas separation system that extracts and purifies the 3He from a patient's exhalation stream and saves it for re-polarization and reuse in other patients. In Phase I we proved the feasibility of the 3He recovery system by (1) performing a proof-of-concept test that demonstrates the feasibility of efficiently separating, purifying, and sterilizing the 3He from exhalation gases, and (2) producing a conceptual design of a complete system to recover and recycle 3He. In Phase II we will build and demonstrate a complete recovery and recycling system. PROPOSED COMMERCIAL APPLICATION: Not Available


There are a variety of molecular mechanisms by which organisms can regulate gene expression during growth and development. One such mechanism is observed in B. subtilis during the process of sporulation. In response to nutrient limitations a well-defined developmental program is elicited. This developmental pathway which culminates in the dormant cell type known as an endospore is controlled, in part, by regulatory proteins (sigma factors) that alter the transcriptional specificity of RNA polymerase. The core RNA polymerase can interact with at least four different sigma factors each of which confers distinct transcriptional specificity. The central issue which we intend to address concerns the mechanism by which the sigma factors alter the template specificity of DNA-dependent RNA polymerase. In one model it has been postulated that each sigma factor induces a unique conformation in the core RNA polymerase, thereby allowing it to preferentially interact with a particular canonical promotor sequence. We plan to test this hypothesis by introducing chromophores into core RNA polymerase and spectroscopically monitoring putative structural altervations that may occur in the care enzyme when each individual sigma factor binds. Our approach involves replacing the endogenous, spectroscopically inert ZN(II) in the Beta' subunit of RNA polymerase (oligomeric structrure: Alpha2 Beta Beta'Sigma) with either Ni(II) or Co(II), thereby introducing chromophores at the metal binding sites. Similar metal ion substitutions in other systems such as E. coli RNA polymerase have been shown to be innocuous. To establish this for our system, we will examine the enzymic and physical properties of the metal-substituted derivatives. Next we will use spectroscopie techniques such as circular dichroism and difference spectorscopy to monitor putative conformational changes, upon ligand binding, in the subunit of RNA polymerase which has been shown to extensively interact with the DNA template. Similar techniques have been used to monitor ligand-induced conformational changes in E. coli Co(II)-RNA polymerase. Thus, with this approach, we should be able to correlate the transcriptional specificity of each sigma factor with its effect on the conformation of the core enzyme. These results will have important implications in regard to the mechanisms of gene regulation which may be operative in mammalian systems.


Delivery to nerve terminals of cytoskeletal proteins synthesized in the cell body is crucial for growth, maintenance and regeneration of neurons. Despite its importance, little is known about what motors are responsible for slow transport or in what forms these proteins are transported. We have shown that tubulin moves down the squid giant axon as an unpolymerized protein by both diffusion and slow axonal transport. Additionally, it appears as if the soluble motor that transports tubulin is loosely coupled to its cargo, since separation of the axon from the cell body leads to the rapid loss of slow transport, but not tubulin. To test this hypothesis directly, we are now measuring the binding coefficients of proteins within the transported complex. We have also developed a new 2-photon technique for studying slow transport by selectively disrupting individual cytoskeletal tracts. Moreover, we have shown that myosin, one of the candidate motors for slow transport, is also responsible for the purse-string constriction of the axon following transection. Finally, we have sequenced and characterized a number of the different myosins in the squid giant axon, and are now developing antibodies to determine the functional role of myosins in slow axonal transport.


The Cystic fibrosis transmembrane conductance regulator (CFTR) is the membrane protein product of the gene known to be defective in cystic fibrosis patients. CFTR functions by transporting chloride ions through a transmembrane (TM) domain believed to consist of two sets of six tightly packed and assembled membrane spanning helical segments (TM1-6, TM7- 12, ca, 20 amino acids each). However, channel remain to be deduced in molecular terms. (1) Since packing of TM domains appears to be directed by specific recognition elements contained within the individual TM segments, and the N-terminal portion of CFTR (TM1-6) is known to form a functional channel, we propose to prepare a series of peptides based on the native sequences of the CFTR TM domain segments 1-6 by solid-phase synthesis, and to determine the affinities of the individual TM segments for each other. Individual TM segments will be combined in combinations (e.g., TM5-6, TM6-1, TM1-2, etc.).Affinities would be quantitated by fluorescence energy transfer, circular dichroism spectroscopy, and gel electrophoresis. These experiments will be supported by molecular modeling to identify favorably- packed helical dimers of CFTR helices, and by channel conductance measurements performed on CFTR TM peptides individually and in combinations. Pairs found to have affinity of dimerization would likely be in direct 3-D contact in the native channel. (2) An increasing number o missense mutations within the CFTR TM domain are now being associated with CF disease. We reason that certain 'high-information' residues impair channel function by interfering with helix-helix packing. Peptides prioritized to key TM-domain mutations observed in genotyped CF patients from the in-house database at the Hospital for Sick Children (e.g., G85E, R347H, R347W) ill be prepared and studied as above in combinations with wild type partners. From the combined information thus obtained, we will construct a working model for the molecular geometry of the CFTR channel, and use the epidemiological data to generate hypotheses concerning possible relationships between severity of CF disease and molecular defects in the corresponding mutant CFTR's.


In this proposal we will attempt to further define the mechanistic pathways involved in enhancing tumor immunity using epigenetic agents and improved strategies for enhancing their effectiveness. The long-term goal is to transfer this technology to clinical application. Our specific aims are to determine: 1) the role of enhanced antigen presentation by histone deacetylase inhibitor (HDACi) treated tumor cells in a B16 melanoma vaccine model; 2) the contribution of NK cells to immunity in the epigenetic model; 3) the efficacy of these models in combinations of HDACi with hyperthermia and radiation; 4) the effects of systemic HDACi therapy in combination with other modalities. An additional major aim is to characterize the role of Dicer and microRNA in immune gene regulation. Our studies have identified miRNAs which target several miRNA machinery components including Dicer. We also defined several stresses that downregulate Dicer (ROS, anoxia, dsRNA) and will determine the pathways by which expression of Dicer is regulated. MiRNAs and siRNAs targeting Dicer will be employed to de-repress silenced genes and to determine whether this enhances immune gene expression in combination with HDACi which are currently in clinical trials. The proposed studies are pre-clinical and exploit the B16 melanoma, colon26, as well as the SCC head and neck cancer models. Our studies will employ in vitro immunological assays, standard molecular biology techniques and microarrays for characterization of gene and miRNA expression. Fresh H&N tissue, removed at surgery, will be studied in preparation for a proposed clinical trial. PUBLIC HEALTH RELEVANCE: This application focuses on pre-clinical and mechanistic studies of a new class of agents (histone deacetylase inhibitors) now currently being evaluated in clinical trials. These studies address a novel mechanism by which these agents may improve cancer therapy with low toxicity (compared to standard chemotherapeutics). [unreadable] [unreadable] [unreadable]


The laboratories and offices of the COBRE PI, Dr. Igor Roninson, COBRE target faculty Dr. Michael Shtutman, the Co-Directors of the COBRE Microscopy/Flow Cytometry Core, Dr Chang-uk Lim and Dr. Gary Schools, are scattered in various inadequate rooms and laboratories between floors 3 and 7 of the Coker Life Sciences Building (CLS) on the Columbia Campus of USC in space that was temporarily provided to Pharmacy from the Department of Biology. This lack of cohesive modern molecular biology laboratory space is disruptive, inefficient, and raises many obstacles for Dr. Roninson's research program and the CTT COBRE. This A&R request is for $300,000 of support toward a $1,150,000 renovation project on the 7th floor of the Pharmacy wing of CLS that will result in 4,857 s.f. of new modern office and research space for the CTT COBRE and Dr. Roninson's entire program. Specific Aim 1. To renovate space on the 7th floor of the Pharmacy Wing of the Coker Life Sciences Building on the USC Columbia campus to house the proposed CTT COBRE. This space will provide modern office and laboratory space for (1) the COBRE PI, (2) COBRE target faculty Michael Shtutman, (3) the COBRE Microscopy/Flow Cytometry Core and its Co-directors Dr. Gary Schools and Dr. Chang-uk Lim, and (4) part of the COBRE Functional Genomics Core. Additionally, this space will house the Administrative Coordinator for the COBRE Center for Targeted Therapeutics, a Conference Room, and a Break Room.


Trifolitoxin (TFX) is a peptide bacteriocin produced by Rhizobium leguminosarum bv. trifoli T24 which is bacteriostatic toward most strains of R. leguminosarum and R. fredii. Sequence analysis of the TFX production and resistance genes strongly suggests that this peptide is produced ribosomally (Breil, et al. 1993, J. Bacteriol., in press). The DNA sequence of the tfxA structural gene and Edman degradation of the purified peptide show that the amino acid sequence of the peptide precursor of TFX is DIGGSRQGCVA. This peptide sequence is post-translationally modified by the tfx genes such that the glutamine residue is cyclized to a UV absorbing chromophore. The purpose of this work was to determine the structure of this chromophore. Two isomers of TFX were isolated by reverse phase HPLC. The mass of the molecules was confirmed by low resolution FAB-MS to be 1037.4 for both molecules. The structure of the chromophore was determined by a combination of modern NMR techniques, such as COSY, TOCSY and HMQC, to be a 2-pyridone derivative which tautomerizes to give TFX isomers. Both isomers of TFX also contain a thiazoline ring which is the result of ring closure between the sulfhydryl group of cysteine and the carbonyl of glycine.


Eye diseases with immunologic properties are common ophthalmic problems. A study of these problems is aided by work on the basic ocular immune mechanisms. Though both cell and antibody mediated mechanisms are important, we have chosen to pursue mostly the role of antibody dependant mechanisms in protecting and in other instances harming the eye, especially the cornea. In the proposed grant period we plan to study by the fluorescent antibody and immunoglobulin quantitation methods 1) the amount, type, distribution and source of immunoglobulins in the cornea; 2) types of plasma cells in the main and accessory lacrimal glands as an indication of the source of immunoglobulin in the tears; and 3) relationship of tear and serum immunoglobulins to serum as a possible source of a part of tear immunoglobulins. Our interest in diseases with immune properties includes study of 1) the possible local production of IgE in vernal conjunctivitis; 2) tear immunoglobulin levels in anterior and posterior uveitis. A continuation of our interest in antibody dependent eye diseases is our planned study of a human model of ocular atopy. The model is produced by dropping small amounts of water soluble pollen antigen into the cul de sacs of persons allergic to this antigen. The amount of reaction, its signs, duration, variation day to day, and variation between subjects is to be observed. The effect of various drug preparations on the model is to be studied. Our interest in clinical diseases with immunologic properties includes the role of tissue antigen in corneal transplantation. We plan to evaluate 1) the role of HL-A typing in corneal transplantation; 2) the role of anti HL-A antibodies before and after grafting; 3) the role of ABO mismatching; and 4) the role of IgM and IgG anti-ABO antibodies. BIBLIOGRAPHIC REFERENCES: Older, J.J. and Allansmith, M.R. Penetrating keratoplasty in a patient with 75 percent third degree burns. Annals of Ophthal., 7:309, 1975; Allansmith, M.R., Drell, D.W., Kajiyama, G. and Fine, M. ABO blood groups and corneal transplantation. Amer. J. Ophthal., 79:493-501, 1975.


Inflamm-aging model is attractive because it derives from the knowledge that aged humans develop low-grade inflammatory state. It is conjectured that the state of low-grade inflammation is a consequence of multiple protective immune responses that develop in order to combat pathogenic infections throughout life. Whereas the notion that chronic inflammatory state could result from multiple immune responses is credible it remains to be demonstrated experimentally. Once this is demonstrated experimentally it will be of interest to demonstrate that experimentally induced immune responses over a period of time correlate with accelerated aging of the whole organism. Long term goals of the experiments proposed in this report aim to establish the role of multiple immune responses in generating a chronic inflamed state, which in turn contributes to accelerated aging in mice. In the short term we will determine if macrophages from young and old mice respond to a common inducer, lipopolysaccharide (LPS), respond differently by measuring waves of gene expression patterns. These studies will set the stage for assaying effects of induced inflamed state in the inflamm-aging model on innate immunity.


Antigen presenting cells (APCs) form the critical link between innate and adaptive immune responses in mammals. The mechanisms of lymphocyte activation in lower vertebrates, however, are largely unknown. Our preliminary studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from zebrafish to mammals. In mammals, dendritic cells (DCs) are the most potent cells in priming an adaptive response through direct activation of lymphocytes. We have identified DC-like cells in the lymphoid organs of zebrafish and initiated studies to enable their prospective isolation and functional testing. We will begin our proposed studies in the adult to determine the cellular effectors of antigen presentation. We will develop functional assays to determine which cell types can traffic foreign antigen to secondary lymphoid organs and stimulate proliferation of antigen-specific T lymphocytes. To complement these functional approaches, we will identify markers capable of enriching APCs from hematolymphoid tissues. These basic studies will set the stage for an assessment of the ontogeny of immunity in the zebrafish embryo. We will make use of novel and existing transgenic lines to fluorescently label APCs and the major branches of the hematolymphoid system, and leverage the unique attributes of zebrafish to image the immune response to pathogen in real time in living animals. These studies will help us determine the predominant sites of immune cell interactions. With the ability to specifically identify each of the APC subsets, we will utilize this information to determine the cellular architecture of secondary lymphoid organs under steady-state conditions. We will subsequently ascertain the changes that result in these tissues following immune challenge. Finally, we will ablate APCs using a conditional transgenic toxin to determine the effects upon lymphoid architecture and the overall immune response. Taken together, these studies will provide a new level of precision in the study of the zebrafish immune response, and will lead to a better understanding of the role of APCs in bridging the ancient and acquired arms of the vertebrate immune system. In addition, these efforts will provide unprecedented, real-time study of APC behavior in living animals. Ultimately, this work should lead to new discoveries regarding antigen presenting cells by utilizing the unique imaging and genetic advantages of the zebrafish.