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Experiments will be done to study the immune status in various models of specific unresponsiveness and to try to elucidate the mechanisms underlying the unresponsiveness. Both thymectomized and non-thymectomized mice treated with ALS and injected with low doses of donor bone marrow are unresponsive to donor antigens as shown by prolonged skin allograft survival. This unresponsiveness is a type of active enhancement since mice bearing perfect skin grafts exhibit cell-mediated immunity (CMI) against donor antigens and also have blocking factors present in their serum (SBF). These mice are not chimeras. In contrast, thymectomized ALS treated mice injected with large doses of hybrid lymph node-spleen cells are chimeras. They have no evidence of CMI against antigens. Experiments will compare these models with reference to (1) response to Cytoxan treatment before antigen is given (2) ability of host lymphocytes to form allorosettes with donor erythrocytes and (3) presence of suppressor cells in lymphoid organs and ability of these cells to transfer suppression to secondary syngeneic hosts. Other experiments will investigate the ability of platelets to induce unresponsiveness in ALS treated mice.

Young women are in urgent need of female-controlled HIV prevention strategies, including antiretroviral (ARV) drugs for topical or oral pre-exposure prophylaxis (PrEP). Two daily oral ARV PrEP studies and one vaginal gel study demonstrated efficacy in preventing HIV-1 infection in women. However, two other studies of daily oral PrEP and one of daily tenofovir (TFV) gel were stopped early for futility. Prior studies suggest differences in ARV metabolism and cervical immune cell populations between otherwise similar U.S. and sub-Saharan African women, but specific differences in the genital mucosal environment that may impact ARV pharmacokinetics (PK), pharmacodynamics (PD) and efficacy are understudied. We hypothesize that young age, bacterial vaginosis (BV), semen exposure, and depot medroxyprogesterone acetate (DMPA) use are associated with increased activation of CD4-i- T cells, changes in soluble immunity, and loss of vaginal lactobacilli, which increase HIV acquisition risk and alter topical ARV PK/PD. In Project 3, mechanisms by which known HIV risks impact PK/PD will be investigated in four clinical studies: women will be studied at BV diagnosis and after successful treatment, in the setting of unprotected vaginal sexual intercourse, before and after initiation of DMPA, and by comparing sexually active adolescents to adult women. The ultimate goal is to utilize knowledge of the mechanisms underlying HIV acquisition risk to advance selection of effective, female-controlled PrEP strategies. We also propose a pre-Phase 1 tenofovir disoproxil fumarate (TDF) intravaginal ring (IVR) study in U.S. and African women at risk for HIV to assess the hypothesis that changes in mucosal immunity related to hormonal effects and/or changes in vaginal microbiota modulate PK/PD of TDF. Drug levels will be measured in plasma, genital tract secretions and epithelial tissue following 14 days of TDF ring use (Core B). Pyrosequencing and species-specific quantitative PCR assays will be performed to examine changes in vaginal microbiota that may modulate PK/PD of TDF. These studies will utilize innovative approaches to inform the development of topical ARV PrEP strategies and will investigate the key hypothesis that distinct populations of high risk women may require differential preventative strategies

The process of polyadenylation is essential in the biogenesis of eukaryotic mRNA. The poly(A) tall is thought to participate in mRNA translation and stability. Defects in the formation of the poly(A) tail decrease the amount of mRNA available for translation into protein, and thus interfere with normal cell function. In addition, polyadenylation can play a role in the regulation of gene expression, especially in cases with alternative selection of poly(A) sites. In this way, it becomes part of a cell's response to stimuli governing growth, differentiation, and tissue-specific gene expression. The goals of this proposal are to determine the factors responsible for this processing and how they interact with each other to give an active processing complex. Understanding the basic mechanism of polyadenylation will make it feasible to ask how the process is regulated as the physiological. state of the cell changes, and how this regulation affects mRNA levels globally or specifically. This research will characterize the factors which recognize the signal sequences on polyadenylation precursor. It will focus on two proteins, pl55 and p68, which require the AAUAAA sequence to bind to precursor RNA, are found in polyadenylation-specific complexes, and dissociate from the RNA once it is cleaved and polyadenylated. These observations are consistent with a role for these proteins in the polyadenylation process, possibly in signal recognition and assembly of the processing complex. Their relationship to the enzymes responsible for cleavage and poly(A) addition will be determined by whether they chromatographically cofractionate. Other experiments using an in vitro system will explore how polyadenylation specific factors interact with each other and with precursor RNA and products during the polyadenylation reaction. Three approaches will be taken to clone polyadenylation-specific factors: a) screening of lambda gt11 human cDNA expression libraries with RNA probes containing polyadenylation signal sequences; b) use of ultraviolet-crosslinked ribonucleoprotein complexes as immunogens to produce antibodies to polyadenylation-specific proteins; and c) sufficient purification of these proteins so as to use them either as antigens for specific antibody production or to obtain protein sequence to generate DNA probes. Either reagent would then be used to screen expression libraries. Using an in vitro system, the 3' end processing of mRNAs from bovine leukemia virus, an oncogenic retravirus, and the role of viral specific factors in this processing will be examined. Finally, the role of polyadenylation in transcription termination will be investigated. Transcriptional templates which encode a self-cleaving RNA sequence and a termination site will be used to determine the role of cleavage of the nascent transcript on termination of RNA polymerase 11 transcription. This will be studied in vivo using transient expression assays, and if possible, with an in vitro system capable of transcription initiation and elongation, polyadenylation, and termination.

Oral ganciclovir may be a useful agent to prophylax against CMV disease in patients who are excessively immunosuppressed. This study will look at the efficacy and safety of oral ganciclovir in liver transplant patients. In addition, the pharmacokinetics of oral ganciclovir will be assessed to determine if cyclosporine alters the elimination of ganciclovir.

Modern treatment methods have led to major improvements in the survival rate of children with Wilms' tumor (WT). Combined therapy using surgery, radiation therapy and chemotherapy now cures 90% of patients with favorable histology WT. Those with renal tumors of unfavorable histology, especially those with metastases, either synchronous or metachronous, are at higher risk. Their survival is in the 50-65% range, and better treatments clearly are needed. At the same time, all therapies have their associated risks and complications, and some are costly and burdensome for the medical staff and the family. Refinements of therapy for children with a good outlook are therefore needed to increase efficiency, and reduce risks and costs. NWTS-4 is designed to achieve these objectives through better understanding of the renal tumors of childhood, their epidemiology and the late effects of successful therapy, while conducting clinical trials of novel therapies. These trials address the following question: Can single daily doses of effective chemotherapeutic agents given more frequently over a shorter interval improve the outlook while decreasing costs and family stress? The NWTS thus has undertaken a new task: the reduction of the socioeconomic burdens of therapy while continuing to strive for more cures and continuing its pioneering studies of the delayed consequences of Wilms' tumor treatments.

The diet is our sole source of nutrients, yet we are just beginning to understand how the human gut microbiota acts as an essential layer of metabolism. Resident microbes not only help to capture plant-derived carbohydrates from our diet, but also likely encounter (and modify) a steady stream of drug-like molecules from plants, a number of which are known to have important biological activity in humans (e.g. anti-inflammatory isoflavonoids and chemo preventative glucosinolates). We propose that these plant-derived small molecules mediate plant-microbe interactions not only in the rhizosphere, but also in the human gut. In this capacity, dietary compounds from plants might represent the most important environmental perturbation regularly experienced by the gut microbiota. Furthermore, we suggest that sensing and processing of dietary plant molecules by the gut microbiota generates compounds with enhanced biological activities, and therefore functions as a central mechanism for diet-based disease prevention. This project will characterize in molecular detail key plant-microbe interactions that take place in the human gut, and will elucidate the mechanisms by which gut microbes directly influence the absorption of 'plant drugs' from diet. Specific outcomes of this project will be insight into (1) how key dietary small molecules with known biological activity are processed, activated, and presented by gut microflora to the human host, and (2) how these metabolites impact microbial community dynamics in the gut. Our proposed experimental approach will draw on our expertise in chemical analysis, natural products biochemistry, and metabolic pathways. This work will be carried out in collaboration with the Sonnenburg lab in Microbiology and Immunology at Stanford who will contribute experience in gut microbiota community dynamics and gnotobiotic mouse models. We anticipate that the identification of how key dietary plant-derived molecules impact gut microbial species will enable the reprogramming of gut metabolism to maximize nutrient harvest and create a novel means of controlling a microbial niche that has a direct effect on human health and disease.

Diseases caused by kinetoplastid protozoa remain serious public health issues in many tropical and subtropical regions of the world. Recent work has revealed that these parasites exhibit novel mechanisms of gene expression and mRNA maturation. Clearly, these unique processes represent potential targets for chemotherapeutic intervention in treatment of the diseases caused by the kinetoplastids. The long term objective of this research project is to dissect the novel mechanisms of mRNA maturation in these orga- nisms. In the kinetoplastid protozoa, many protein coding genes are transcribed as long 'multicistronic' precursor mRNAs that are processed to maturity in a bi-molecular splicing event trans-splicing) in which a 39 nt spliced leader (SL) is ligated to the 5' terminus of each mature mRNA. The SL is transcribed as a short primary transcript-the SL-RNA-bearing the 39 nt SL at its 5' end. The SL-RNA is 5' capped prior to the trans-splicing event and therefore the cap on the mature mRNAs is derived from the cap on the SL-RNA. The 3' ends of mature mRNAs are defined by cleavage-polyadenylation events that are mediated by as yet undefined signals in the primary transcripts; no signal analogous to the 'AAUAAA' cleavage-polyadenylation signal of mammalian mRNAs has been identified. Experiments in this proposal are designed to elucidate the unique biochemical and molecular mechanisms of mRNA maturation in the kinetoplastid protozoa. Cell free nuclear extracts developed from T. cruzi and several other kinetoplastid protozoa are partially competent for several steps of mRNA processing; i.e., 5' capping, 3' polyadenylation, and several steps of trans-splicing. Using these extracts, another unique facet of mRNA maturation in the kinetoplastids has been discovered; i.e., the capping machinery in the nuclear extracts shows unprecedented specificity for certain RNAs. Thus, only the SL-RNA and the U-RNAs are capped in these extracts. Soluble capping enzymes from other eukaryotes show no specificity for any RNAs. This observation is particularly interesting in that the in vitro activity accurately reflects the in vivo capping requirements-since the mature mRNAs obtain their caps from the SL-RNA, the only RNAs that need be capped are the SL- and U-RNAs. Moreover, recent experiments have shown that the capping activity fractionates as a 15-20S RNA-protein complex on glycerol gradients or by large pore gel filtration chromatography. Experiments described herein are designed to identity the RNA primary sequence and/or secondary structure determinants of the specificity of the in vitro capping activity, and to identify, purify and characterize the trans-acting enzymes and factors in the capping complex.

Treatment with the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) has shown promising results for patients with lung cancer, the leading cause of cancer death in the United States. Activating mutations in the epidermal growth factor receptor tyrosine kinase gene, EGFR, occur frequently in lung adenocarcinoma and are associated with response to these kinase inhibitors. During the past year, we and others have identified several new classes of EGFR mutation in human tumors, as described in the "Preliminary Data" section. 1) A secondary kinase domain mutation, T790M, confers resistance to gefitinib and erlotinib in the setting of an activating, inhibitor-sensitive EGFR mutation. 2) One class of lung-cancer derived activating EGFR mutations, insertions within exon 20, are resistant to erlotinib and gefitinib. 3) Somatic mutations of the EGFR extracellular domain are found in glioblastoma. I now propose to conduct further studies to explore the mechanism of EGFR-dependent transformation and the mechanisms for resistance to EGFR inhibitors for tumor-derived EGFR mutations. The long-term goal of this work is to improve treatment of lung adenocarcinoma, glioblastoma, and other EGFR-dependent tumors. Specifically, I propose the following aims, tied to the recently discovered classes of EGFR mutants. Specific Aim 1. Test the inhibition of EGFR-dependent cell growth by specific small molecule EGFR inhibitors for a panel of transforming EGFR mutations. Specific Aim 2. Analyze the structural and biochemical determinants of EGFR-dependent transformation. Specific Aim 3. Identify secondary mutations in activated EGFR that cause resistance to EGFR inhibitors such as gefitinib. Specific Aim 4. Determine whether cancer-derived point mutations in the extracellular domain of EGFR are transforming and whether these mutants can be inhibited by small molecule kinase inhibitors. [unreadable] [unreadable] [unreadable] [unreadable]

Experimental allergic encephalomyelitis (EAE) serves as a model for the study of therapies for demyelinating disease of the central nervous system. Its similarity to multiple sclerosis (MS) lies not only in clinical features and pathology but, more importantly, like MS susceptibility to EAE is limited closely to IR genes of the major histocompatibility complex (MHC). It has been shown that treatment in vivo with monoclonal antibody directed against the cell surface of the susceptibility gene product the IA antigen can prevent the induction of acute EAE and reverse the course of chronic EAE. Similar observations have been made in other autoimmune diseases, wherein susceptibility was linked to the IR genes of the MHC. While the mechanism of action of anti IA antibody is not known, there is sufficient evidence to indicate that part of the suppression is induced by the generation of suppressor cells. This proposal attempts to characterize the cellular nature of suppression. Using adoptive transfer systems it hopes to define the nature of suppression of acute and chronic EAE following treatment in vitro and in vivo with monoclonal antibody directed towards IR gene products. In addition these experiments will study the genetic specificity of suppression using various inbred strains of mice and antigenic specificities using autoantigens and synthetic antigens whose immune responsiveness is under IR gene control. At the present time the mechanism by which IR genes confer susceptibility to autoimmune disease is not known. Understanding mechanisms by which antibody to IR gene products regulate autoimmune disease might lead to an improved knowledge of the pathogenesis of such diseases.

This is an application for a conference: Occupational Alcoholism Program Types and Program Outcomes: Linking Research and Practice. This conference will bring together 25 researchers and practitioners in the occupational alcoholism field to explicate a program typology and to differentiate program outcomes by program type. Practitioners and Researchers will prepare working papers prior to the conference and the conference itself will be used to arrive at a typology. A secondary goal is to open a dialogue on carrying out research in occupational settings focusing on the barriers which obstruct research and create mechanisms by which practice and theory can link around common research issues.

Depression and anxiety disorders are associated with hyperactivation of the amygdala, a brain area important in negative emotional processing. Many researchers propose that overactivation of the amygdala is responsible for some of the debilitating symptoms associated with mood disorders such as increased occurrence of intrusive negative thoughts and rumination over aversive experiences. Exposure to some stressors, particularly chronic or severe acute stressors, can sensitize amygdala responses and facilitate the formation of aversive memories. The cause(s) are not fully understood, but are of high clinical significance given that stress is a major risk factor for the development of mood and anxiety disorders. The goal of this proposal is to identify the cells and cellular mediators involved in stress-induced sensitization of the amygdala. This proposal centers on the possibility that microglia play a critical role in sensitizing amygdala responses. Microglia are the innate immune cells in the brain and while they are typically in a quiescent (resting) state, they can shift into a primed state of activation such as following stress exposure. In a primed state, microglia do not actively release inflammatory mediators but rather they upregulate their response capabilities such that if and when they become activated greater inflammatory responses are generated. We provide evidence that repeated stress exposure primes microglia responses to ?-adrenergic receptor (?-AR) activation and, in contrast to an immune stimulus, activation of ?-ARs cause microglia to release factors that facilitate learning and memory processes. Here, we test the hypothesis that primed microglia and their release of interleukin-1beta contribute to the heightened formation of fear memories in organisms exposed to chronic stress.

The specificity and potency of targeted gene knockdown through RNA interference (RNAi) has generated tremendous excitement for utilizing this ubiquitous pathway in the clinic. Our studies investigate RNAi as a potential microbicide to target sexually transmitted diseases including HSV-2 and HIV-1. HSV-2 infects ~ 20% of adults in the US and up to 90% in parts of sub-Saharan Africa. It is associated with high morbidity and there is no cure or prophylactic treatment. HSV-2 is also a major co-factor for HIV-1. Therefore, a microbicide to HSV-2 could also have a significant impact on slowing HIV-1 transmission. Using a mouse model of vaginal HSV-2 infection, we were the first group to show that vaginal application of siRNAs targeting HSV-2 viral genes or host-encoded viral entry receptor genes could protect mice from HSV-2 infection. These findings were encouraging, but we encountered issues that would need to be addressed for siRNAs to be developed for clinical use. Problems included toxicity of a cationic lipid used to complex the siRNAs, transient protection conferred by virus-specific siRNAs, and siRNA instability in vaginal washes. Each of these problems was addressed in a follow-up study. In this proposal we will undertake a comprehensive analysis of HSV-2 gene targeted siRNAs to maximize their specific silencing ability, and minimize any off-target effects. Using the murine HSV-2 infection model we will determine whether: (i) immune responses, induced either by the siRNA or through viral challenge affect siRNA-mediated protection; (ii) enhanced incorporation of siRNA into RISC can result in more potent or durable protection; and (iii) efficient siRNA uptake and intracellular localization in the vaginal mucosa requires the expression of a specific receptor.

Malnutrition, wasting of lean body mass, is a common development in HIV infection and may predate the development of the clinical complications of AIDS. The mechanism underlying protein wasting is unclear. Some studies have suggested that protein turnover is increased in HIV-infected individuals. This study seeks to determine rates of protein turnover in HIV patients and to compare those rates when subjects consume both normal and restricted dietary protein.

Osteoclast Activating Factor (OAF) is a lymphokine which may be involved in stimulation of localized bone destruction in periodontal disease or other diseases where chronic accumulation of lymphoid cells accompanies bone destruction. The objectives of this study are to characterize OAF chemically and biologically, to develop specific immunologic assays for OAF in biological samples, and to examine the sources and regulation of OAF production. OAF will be isolated from mitogen-activated human lymphocytes, and purified by established procedures. The isolated active species will be chemically characterized as to their structure and composition. The isolated fractions will also be used to immunize animals against OAF. The effects of OAF on isolated bone and bone cells with be studied to determine the mechanisms of action of OAF in producing bone destruction. In order to furnish monospecific antibodies for OAF, hybrid cell lines will be established and propagated. The antibodies so obtained will be used to design immunoassay and immunocytochemical methods for detection and quantitation of OAF in tissue and serum. Finally, the cellular source of OAF will be examined by fractionation of human lymphocytes into subpopulations by density gradient and affinity separation techniques. These studies will allow determination of the role of OAF in vivo in pathologic circumstances.

Solid tumors are frequently associated with increased bone resorption and hypercalcenia. The mechanism for increased bone resorption is production by the tumor cells of a humoral factor or factors which stimulate osteoclastic bone resorption. Recently, we and others have noted that tumors of the oral cavity which cause hypercalcenia are often associated with leukocytosis, concomitantly with the production of colony stimulating activity. In order to identify the factors responsible for hypercalcemia in these tumors, our approach will be to use an in vitro bioassay for bone resorption based on the release of previously incorporated 45Ca in organ culture to characterize the bone resorbing factors present in the tumor cell culture media, and determine the relationship between these factors and the tumor products responsible for leukocytosis which stimulate colony formation in cultures of mouse bone marrow mononuclear cells in methyl cellulose. We plan to evaluate the relationships between bone resorbing activity and colony stimulating activity by examining culture media from tumor cells derived from a patient with squamous cell carcinoma of the maxilla who manifested only leukocytosis, from a patient with squamous cell carcinoma of the maxilla who manifested only hypercalcemia, and from a patient with squamous cell carcinoma of the tongue who manifested both hypercalcemia and leukocytosis. These tumors have been carried in nude mice and preliminary results show that only those associated with colony stimulating activity production cause leukocytosis, and only those associated with bone resorbing activity production cause hypercalcemia. Since cells derived from these tumors are now maintained in culture, we plan to purify further the bone resorbing activity and the colony stimulating activity produced by these tumors, and determine the relationship of the colony stimulating activity to known colony stimulating factors (CSFs). Our hope is that these studies will clarify the mechanisms by which tumor cells increase osteoclastic bone resorption and cause leukocytosis, and may lead to identification of normal factors which influence both osteoclastic bone resorption and leukocyte differentiation.

It is the objective of the proposed research a) to advance knowledge on the biogenetic origin of a C7N unit which can be discerned in the mitomycin antibiotics and which presumbaly arises from an intermediate of the shikimic acid pathway; b) to further explore the role of D-glucosamine in the biosynthesis of a C6N unit discernible in the mitomycins; and c) to obtain information on the nature and on the formation of intermediates of mitomycin biosynthesis. These goals will be pursued in feeding experiments with isotopically labeled compounds, many of which will be synthesized, and they will be pursued in experiments with cell-free extracts of the mitomycin producing organism Streptomyces verticillatus. Cell-free studies will also be undertaken to gain information on the regulation of the formation and on the metabolism of essential precursors.

The long range goal is intervention to delay or prevent cognitive decline associated with unsuccessful aging, in order to improve the health and well-being of older Americans. The incidence of Alzheimer's disease is projected to increase dramatically, with the greatest prevalence in women. Synaptic loss contributes to memory impairments and estrogen (E2) promotes synaptogenesis and memory. Thus, E2 treatment could have a major impact on public health. However, the efficacy of E2 is greatly reduced if therapy occurs several years after the onset of menopause, suggesting a temporally limited therapeutic window. Evidence indicates estrogen receptor (ER) expression and ER polymorphisms contribute to a variety of hormone sensitive diseases, including cognitive decline. We hypothesize that differential expression of ER1 and ER2 interacts with the level of E2 to contribute to 1) the etiology of age-related memory deficits, 2) loss of E2 mediated synaptogenesis, and 3) the closing of the E2 therapeutic window. Aim 1 will combine aging ER1 and ER2 knockout mice with viral vectors to influence ER expression and systematically perform behavioral, molecular, and electrophysiological assays to test the hypothesis. Aim 2 will employ hippocampal viral delivery vectors to increase or decrease ER1 or ER2 in young, middle-age, and aged rats, and will use behavioral and molecular assays to test the hypothesis that shifting the ratio of ER1/ER2 expression rejuvenates hippocampal function. Aim 3 will employ viral vectors to alter the expression of ER1 or ER2, and will test the hypothesis that ER expression contributes to age-related changes in rapid E2 signaling. Knockout mice and viral vector gene delivery provide novel approaches to test the hypothesis that ER expression is a contributing factor for hippocampal aging. Together, these studies will determine whether altering the level of ER1 or ER2 expression is important for age-related memory decline, E2-induced synaptogenesis, and closing of the E2 therapeutic window, and will provide the groundwork for development of therapies to slow or prevent cognitive decline associated with aging and age-related diseases. PUBLIC HEALTH RELEVANCE: Research to reduce cognitive decline is crucial, given the burgeoning number of elderly, in which 3 in 10 will display serious cognitive decline due to age and Alzheimer's disease. Women exhibit greater risk for cognitive decline and comprise greater than 60% of the Alzheimer population. These studies will provide a scientific foundation for interventions to avert the impending wave of cognitively impaired seniors.

Objectives: The overall objective is to evaluate the feasibility of copper chelating strategies for the prevention and treatment of Alzheimer's disease. Specific aims: 1) To determine if copper complexing with zinc is an effective pathology-modifying therapy if initiated prior to the appearance of AD pathology. 2) To determine if zinc is effective if initiated after the onset of AD pathology. 3) To determine if copper loading accelerates the appearance of AD pathology. Plan: Copper complexing will be achieved with oral zinc sulfate, an established and safe intervention in animal models and human patients. The approach is based on experience with a more potent copper complexing agent (tetrathiomolybdate) which may be too toxic for use in human subjects. Methods: Transgenic mouse models of AD (Tg2576 and the Laferla triple transgenic mice) will be used for all of these experiments. Zinc and copper will be administered in the drinking water according to established protocols. Copper removal will be titrated by monitoring of plasma ceruloplasmin. At the end of the treatment period, spatial memory will be assessed by Morris Water Maze and brains then harvested and divided. Forebrain levels of soluble and insoluble amyloid species will be determined by ELISA and immunohistochemistry;oligomeric beta amyloid levels by western blot, copper levels by atomic absorption spectroscopy. Other proteins of interest will be measured by Western blot, and the expression of genes of interest by quantitative RT-PCR. Findings to date: 1) Brain copper and insoluble beta amyloid are reduced in a transgenic mouse model of AD when copper complexing therapy is initiated with tetrathiomolybdate (TM) prior to the age at which pathology typically appears. 2) TM treatment does not impair brain activity of copper dependent enzymes. 3) TM treatment does not impair behavioral function in mice treated for several months. 4) Zinc treatment lowers plasma ceruloplasmin and brain copper with less risk of over- chelation compared to TM. Clinical relevance: These data will be used to design clinical trials for prevention of AD in human subjects. PUBLIC HEALTH RELEVANCE: This proposal is relevant to a very important veterans'health care issue, namely the prevention and treatment of Alzheimer's disease. Alzheimer's disease is a major source of disability in late life, and afflicts vast numbers of veterans. The experiments proposed here use mice to test the possibility that therapies directed at lowering brain levels of copper will reduce the risk and perhaps slow the rate of progression of Alzheimer's disease. These studies are based on "test-tube" experiments which suggest that copper may interact with brain tissue to promote some of the changes that occur in Alzheimer's disease. Although these "test-tube" studies are convincing, it will not be possible to propose this type of treatment in human subjects unless there is confirmation of these results in an animal model, which more closely approximates the situation in human beings. We propose to use special mice which have been engineered to develop Alzheimer's-like changes in the brain. The mice will be treated with a simple, safe treatment which lowers brain levels of copper. The mouse memory and brain biochemistry will then be examined to determine if the treatment had its intended effect. If it works as expected, we will propose further studies in human subjects at risk of Alzheimer's disease. If it does not work, we will attempt to understand why not, in order to develop better treatment strategies for human patients, including aging veterans.

The proposed plan consists of three main projects. The first concerns the human X chromosome and consists of two parts. One is aimed at a deeper understanding of the structure and behavior of this chromosome by means of a very thorough cytological investigation of abnormal X chromosomes. The main source for these will be an ongoing survey of women with primary amenorrhea conducted in the Department of Gynecology and Obstetrics. A focus of interest in this work is the presumed inactivation center located in the proximal region of Xq. The second part of the X chromosome work will be an extensive statistical study of the XO syndrome and related conditions based mostly on a much more thorough evaluation of published cases than has been done before. This will have direct bearing on some aspects of the above mentioned cytological work. The second project covers various problems concerning chromosomes in general such as the behavior of inactivated centromeres in dicentric or even monocentric chromosomes. Another topic is the relation of quinacrine banding and base ratios. This will be investigated by means of autoradiographic grain counts after incorporation into DNA of one or the other of tritiated guanine or tritiated thymidine. Certain methodological problems seem to have been overcome so that clean results may be expected. The third project concerns cancer. One part is aimed ultimately at finding cancer drugs that cause maximal chromosome breakage in malignant cells but minimal harm in normal ones. The other part is an extensive study of mitotic anomalies in cancers of the female genital tract. These will be correlated with various clinical parameters. One purpose of this work is to evaluate the practical potential of the use of mitotic anomalies as a tool in cancer diagnosis as compared with the usual histological methods.

The psychological stress of bereavement, job insecurity, marital problems, and so forth, have been implicated as risk factors in cardiac sudden death. Our challenge has been to determine the higher cerebral mechanisms by which psychologically and physically stressful stimuli regulate the heart. Previous studies in our laboratory used tone-shock conditioning as the experimental model by which to investigate the effects of the two types of stress on cardiac responses. Electrophysiological recordings of extracellular slow potentials in response to the tone and shock stimuli and functional neural blockade, which abolished these responses as well as the conditioned cardiac response, have together delimited a cerebral system mediating the process. Current work is directed toward understanding the neurochemical transactions in the system (e.g. synaptic transmitters, intracellular mediators, etc.). Specifially we have found, using a new method of cryogenic fixation in vivo, that psychological and physical stress both reduce 3', 5'-adenosine monophosphate levels in parietal cortex. Slow potentials also evoked by the stressful stimuli were found to have a negative correlation (r equals -O.77, p less than or equal to .01) with the cyclic nucleotide level during the development of the response. Cryogenic blockade in the system was found to prevent the onset of ventricular filbrillation following coronary artery occlusion. BIBLIOGRAPHIC REFERENCES: Skinner, J.E. and Yingling, C.D. Central gating mechanisms that regulate event-related potentials and behavior: A neural model for attention. In J.E. Desmedt (Ed.), Progress in Clinical Neurophysiology. Karger, Basel, 1977. Vol. 1, pp 30-69. Yingling, C.D. and Skinner, J.E. Gating of thalamic input to cerebral cortex by nucleus reticularis thalami. In J.E. Desmedt (Ed), Progress in Clinical Neurophysiology. Karger, Basel, 1977. Vol. 1, pp 70-96.

A key component of inflammation is the adhesion of leukocytes to the endothelium in the fluid dynamic environment of the circulation. Unraveling the complex process of leukocyte adhesion to the endothelium will lead to an increased understanding of inflammation and will provide the foundation for the development of novel ways to treat a variety of pathological processes. We will investigate two hypotheses related to leukocyte adhesion to the endothelium. First, we have proposed and found evidence for the hypothesis that beta2 integrin (specifically Mac-1 (CDllb/CD18)) - E-selectin molecular interactions play a role in leukocyte adhesion to the endothelium. These results and our recent studies has led to the following hypothesis: As leukocytes adhere to the endothelium, leukocyte expressed Mac-1 can interact with the endothelium via two distinct mechanisms: one interaction occurring during leukocyte rolling and another interaction occurring during firm adhesion. We will probe this hypothesis by completing the following specific aim. Specific Aim I: To determine if leukocyte-sized microspheres coated with purified Mac-1 adhere to cytokine activated endothelial cells under physiologically relevant flow conditions through a multi-step process involving at least two distinct mechanisms. Our second hypothesis is that the number of E-selectin ligands expressed by a neutrophil is in significant excess of the number required to support a stable rolling adhesion on the endothelium via E-selectin. We will probe this hypothesis by completing the following specific aim. Specific Aim II: To determine if the E-selectin ligand density present on neutrophils is in significant excess of that required to support a stable rolling adhesion on cytokine activated human umbilical vein endothelial cells via E-selectin under physiologically relevant flow conditions.

The majority of adults with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) eventually relapse, and those with relapsed disease have a high likelihood of treatment failure with currently available agents. New drugs that more effectively treat these diseases are needed. This proposal describes two exciting phase I clinical trials in the targeting of aberrant signaling and survival pathways in leukemic cells. The first trial investigates a novel, pharmacokinetically driven schedule of flavopiridol in ALL or AML. Given new laboratory data demonstrating critical differences between drug-protein binding in fetal bovine serum (FBS) vs. human plasma, early negative clinical studies with flavopiridol based on in vitro studies in FBS may have failed to reach (or maintain) drug levels necessary for in vivo clinical activity. Pharmacokinetic modeling based on in vitro studies in acute leukemia cells cultured in human plasma suggested that administering flavopiridol by 30 minute intravenous (IV) bolus followed by 4 hour continuous IV infusion (CM) would achieve an in vivo plasma drug concentration similar to that necessary to induce apoptosis in vitro. Preliminary results of an ongoing phase I clinical study using this dosing strategy administered weekly in patients with refractory chronic lymphocytic leukemia (CLL) demonstrated impressive clinical responses. In addition, acute tumor lysis was observed as a dose limiting toxicity. The proposed trial administers flavopiridol in the manner described for three consecutive days every three weeks based on in vitro and animal studies. It aims to generate preliminary pharmacokinetic and phamacodynamic data to facilitate additional efficacy studies in ALL and AML. Investigations will then proceed to phase II trials and additional phase I trials in combination with other agents. The second trial in this proposal is a phase I combination study of the heat shock protein 90 (HspQO) inhibitor 17-allyamino-demethyoxygeldanamycin (17-AAG) with the proteasome inhibitor PS-341 (bortezomib, Velcade) in relapsed and refractory AML. Both agents act by altering normal cellular regulatory functions, 17-AAG by affecting chaperone function of Hsp90 and PS-341 by inhibiting the 20S proteasome. Due to inhibition of Hsp90 by 17-AAG, multiple client proteins including those important in survival pathways such as Akt are degraded. Inhibition of the 20S proteasome by PS-341 also has multiple effects, mainly due to loss of NF-KB activation (resulting from the inhibited proteasome's failure to degrade the NF-kB inhibitor IkB). Individually, both agents have been shown to have anti-leukemia efficacy. Treatment of leukemic cells with PS-341 results in a stress-response which includes upregulation of Hsp90 leading to resistance to apoptosis, and we hypothesize that treatment of AML patients with both 17- AAG and PS-341 will be an effective method to overcome treatment resistance. If the regimen is well tolerated, a phase II study will be performed, including patients with high risk, untreated AML. Both studies described above will serve as the foundation for the career development of Dr. William Blum, a promising young clinical investigator in the area of experimental therapeutics for ALL and AML. The mentors for the candidate's career development are Dr. Clara Bloomfield, Dr. John Byrd, and Dr. Guido Marcucci at The Ohio State University. They will be assisted in laboratory mentoring by Dr. Denis Guttridge and Dr. James Dalton. [unreadable] [unreadable] [unreadable]

Each year an estimated 1.5 million people sustain traumatic brain injury, presenting an enormous social and medical problem, with an economic burden exceeding $50 billion annually in the US. Head injury is one of the most important causes of acquired epilepsy; however, the mechanisms underlying post-traumatic epilepsy are not understood. Recently, we discovered that the GABAergic control of hippocampal pyramidal cells is subnetwork-specific (differential GABAergic inhibition exists for distinct pyramidal cell subpopulations) and temporally ordered (different interneuron subtypes fire in a particular temporal sequence during behaviorally relevant network oscillations). Here we propose to test the hypothesis that there is a significant disruption of the specialized, local GABAergic control o long-distance projecting excitatory pyramidal cells in post-traumatic epilepsy and that this compromised GABAergic inhibition constitutes a key mechanism underlying hyperexcitability and spontaneous seizures. The hypothesis will be tested in the controlled cortical impact model of traumatic brain injury during the chronic epilepsy phase, and the assessment will be carried out in the CA1 region of the mouse hippocampus with advanced in vitro and in vivo electrophysiological, immunocytochemical and optogenetic methods, complemented by data-driven, large-scale computational modeling approaches. The experiments of this proposal are designed to specifically target cellular-synaptic mechanisms underlying post-traumatic epilepsy and to test novel closed-loop optogenetic methods to stop chronic seizures in the post-traumatic brain. It is anticipated that defining the functional consequences of experimental post-traumatic epilepsy will aid in the future development of novel treatment strategies.

This competing renewal application seeks support for the continuation of a highly successful Physician-Scientist Program in Aging at Harvard Medical School's Division on Aging and its affiliated institutions. This joint effort involving investigators in basic and clinical departments has excelled in identifying and training outstanding clinicians for research careers in areas central to the mission of the National Institute on Aging. In the previous two finding periods, we have successfully selected 18 superb physician-scientists who are now well- directed toward gerontologic research careers. We have also successfully influenced their mentors/sponsors in pursuing aging-related issues in their respective scientific disciplines, thereby recruiting these renowned basic scientists to contemplate and address fundamental questions in the field of aging. The major goal in the next funding period will be to provide exciting opportunities for additional outstanding physicians to engage in intensive training experiences involving didactic and laboratory experiences in the basic and clinical sciences. Four research areas will be emphasized: (1) cardiovascular disease, (2) cell proliferation disorders, (3) neurodegenerative disease, (4) and endocrine/renal dysfunction. Enlarged scope and improved quality of the training program will ensure that the Physician-Scientist trainees will be introduced to the current biological concepts and technical aspects of basic science investigation (Phase I) and will apply such expertise toward the elucidation of mechanisms of aging and clinically relevant problems in the elderly (Phase II). A special feature of the present application is the continuation of a greatly expanded program of enrichment activities for the trainees that are identified not only with this program but with other NIA-related research training programs. The ultimate goal of the program is the development of Physician-Scientists who can advance knowledge and enhance the development of academic, research-intensive programs in the long-neglected areas of biomedical Gerontology and Geriatrics.

Transmission of Human Immunodeficiency Virus (HIV) occurs primarily via the mucosal routes, emphasizing HIV-1 vaccines must need to engender mucosal immune responses. However, mucosal immunization has been limited by the ability to deliver intact vaccine antigens across the mucosal barrier for induction of effective mucosal immunity. The long-term goal of this proposal is to determine whether the IgG transcellular pathway represents a novel delivery path for a subunit vaccine against infections of HIV and AIDS-related opportunistic pathogens. The goal of the project derives from the recent proof of concept that the neonatal Fc receptor (FcRn) mediates the bi-directional transport of IgG across polarized epithelial cells. FcRn was initially considered to transport maternal IgG to a fetus through the placenta or to newborns via the intestine. However, FcRn is expressed in a variety of tissues and cells in adult humans and animals; IgG is a predominant isotype of immunoglobulins in the lower respiratory and genital tract. Based on these evidences, we will test the hypothesis that using IgG transport pathway, FcRn can deliver HIV-1 antigen fused to an IgG-Fc across the mucosal barrier to the underlying mucosa-associated lymphoid tissue. The consequences of such transport could induce local immunity able to neutralize the virus at their port of entry and systemic immunity able to prevent systemic spread of the infection. HIV envelope glycoprotein gp120 will be used to probe immune responses to such immunization and to define protective immune responses. The specific aim of this proposal is to determine the ability of FcRn to deliver gp120-Fc antigen across the genital or the respiratory mucosal barrier to engender protective immunity against mucosallv-inoculated virus challenge. Data generated herein will provide valuable information not only for design of a HIV vaccine, but also for general vaccine strategy targeting AIDS-associated opportunistic pathogens or other pathogens, such as cytomegalovirus, herpes simplex virus, mycobacterium, chlamydia, influenza, etc., that infect at or invade across mucosal surfaces. [unreadable] [unreadable] [unreadable]

This competitive renewal proposal is directed at determining which cells from the mouse immune system express 6, g and K opioid receptors, and whether activation of these cells alters the amount of receptor expressed and the distribution of the multiple opioid receptors among the different lymphocyte populations. By using fluorescent opioids and flow cytometry, we have developed a novel indirect fluorescent method for detecting about: opioid receptors. This method is more sensitive than radioreceptor binding methodology. Double-labeling experiments have shown the highest level of about receptor expression on mouse thymocytes and macrophages, and on human microglial cells. The studies proposed in this application focus on the detection of 6 and/.t opioid receptors. Based on functional studies, the first hypothesis to be tested is that mouse thymocytes and CD4+ and CD8+ T cells express ;5 opioid receptors. To detect 6 receptors, two approaches will be compared. FITC-labeled derivatives of the 6-selective ligand, naltrindole, will be used in a manner similar to the labeling of the receptor with the FITC-conjugated K ligand. The second approach will use specific antibodies directed against the 6 receptor. The second hypothesis to be tested is that mouse peritoneal and T cells express the/,t opioid receptor. The g receptor will be labeled with either a FITC-labeled derivative of fentanyl or morphine, and/or antibodies directed against the la opioid receptor. Flow cytometry will be used to detect the labeled receptors. For both 6 and g receptors, the goal is to develop an optimal method for detecting the receptor, and then to use this method to study receptor expression. The third hypothesis is that activation of lymphocytes will increase the expression of opioid receptors since levels of receptor mRNA have been shown to increase with cell activation. Collectively, these studies will use an innovative approach and technique to determine which cells from the immune express about:, 6, and about opioid receptors and if receptor expression is altered with activation of the cells. These studies will lay the foundation for determining the cellular mechanisms by which opioids alter immune function and immunological responses to viral infection, including AIDS.

Social control theory proposed that our social relationships serve a regulatory function by discouraging us from engaging in deviant or risky behaviors. Applied to self-care, social control should promote health and longevity by encouraging engagement in healthy behaviors and avoidance of unhealthy behaviors. The goal of this project is to investigate the ways in which social control processes are associated with self-care in a sample of older adults. The aspect of self-care which will be investigated is engagement in health related behaviors such as exercise and appropriate medication use. The proposed research has three goals and will serve as the basis for future research on interventions to enhance self-care in older adults. The first goal is to develop reliable and valid measures of social control relevant to self-care which can be used in future research. The second goal is to investigate the ways in which social control processes within close relationships are associated with self-care in a sample of 2700 older men and women. A mediation model will be tested in which the associations between social control and self-care in older adults are expected to be mediated by emotional responses to the social control. Differences in the social control of health behaviors by various types of social ties will be investigated. The third goal is to empirically distinguish social control from other functions of social relationships, such as social support and companionship. Participants in this study will complete various measures of social control, social support, psychological well-being, self-care, and health status. In addition, reporter of the older adults' receipt of social control and engagement in self-care will be obtained from individuals who are identified by the participants as their primary social control agent.

The proposed study will identify subtypes of maritally violent men, to examine how and why different men are violent. Based upon previous batterer typologies, we hypothesize that three subtypes of maritally violent men will be identified (i.e., family only, dysphoric/borderline, and generally violent/antisocial) on the basis of three major descriptive dimensions-- severity of marital violence, generality of violence, and psychopathology/personality disorder. Based upon a review of previous typologies and our developmental model of marital violence, we hypothesize that these subgroups will also be found to differ on other theoretical variables of interest (i.e., variables hypothetically linked to the use of violence), including attachment, impulsivity, social skills, attitudes toward women, and attitudes toward violence. To increase the usefulness of our work to others, we will identify the smallest subset of measures needed to reliably distinguish the subtypes. The proposed study will address a number of limitations in the previous research on subtypes of batterers. We will recruit a wide variety of violent men, including not only men seeking treatment for marital violence, but also violent husbands from the community, to extend the findings of previous research to a broader sample of violent men. We will develop an initial typology, based on data from one sample of violent men, and then attempt to replicate the typology with a second sample of violent men. The stability, or test-retest reliability, of the typology will be examined by assessing one group of violent men at two points in time, separated by approximately 18 months. Two comparison groups of nonviolent men-- one maritally distressed and one nondistressed-- will be recruited, allowing us to compare each subtype of violent husband to the nonviolent groups. This will help us to better understand the different processes of violence among the subtypes and to distinguish men who are not severely violent from nonviolent men. Finally, we will not only gather information from the subjects, but also from their wives; we also will not only gather self-report information, but also behavioral measures (e.g., marital interactions, behavioral measures of impulsivity).

The Cell Culture and Physiology Core will be a centralized facility that will provide primary alveolar epithelial type 2 cells and secondary alveolar epithelial cell lines to each of the five projects within the program project, as well as conduct physiology experiments utilizing the isolated perfused lung model. Centralization of the cell culture facilities will ensure that a continuous supply of high quality and purity alveolar epithelial cells is available to each of the five projects. The core will characterize the primary and secondary alveolar epithelial cells used in this program project. The cell culture facility personnel have extensive experience in the isolation of alveolar epithelial type 2 cells, as well as general cell culture techniques. The consolidation of the cell culture facilities will provide an economical means of isolating and culturing alveolar epithelial cells. The core will also conduct physiology experiments to measure the rate of alveolar fluid reabsorption and active Na+ transport utilizing the isolated-perfused lung model. Centralization of the physiology experiments will reduce the cost of conducting these experiments within the individual projects and more importantly maintain the precision and accuracy in which these experiments are conducted.

Long-term objectives are to improve institutional treatment practices in public mental hospitals, intramural units of mental health centers, and community extended care facilities. A prerequisite for meaningful treatment evaluation in the latter settings is reliable and valid assessment of patient characteristics and behavior and of staff characteristics and treatment procedures. Similarly, a prerequisite for placing day-to-day clinical work on an applied-science basis is objective assessment for identification of patient problems and progress and for describing, monitoring, and training institutional staff in their treatment interactions with patients. In the course of a long-term comparative study of the treatment of chronic mental patients, two observational assessment instruments were developed which possessed exceptional reliability and validity within the original units and populations. These instruments offer exceptional promise of providing objective data for day-to-day clinical work, staff training and monitoring, while simultaneously providing higher level scores for use in evaluation research. The present application requests support for the concurrent collection of full week samples of observational data, along with standardized instruments for assessment of staff, patient, and ward activities, characteristics, and behavior over open-closed, chronic-acute placements and defined treatments programs in selected hospitals, mental health centers, and extended facilities. Descriptive and normative data on all instruments will then be established for future research and clinical work, while extensive reliability and validity analyses will be conducted on the new observational instruments on the same staff-patient populations. Target extended-care facilities for the latter data will be selected so as to obtain a second years follow-up on patients placed from the earlier comparative study. Generalizability studies will be conducted to determine parametric characteristics of observers and instruments through analyses of the latter data, data existing from the earlier comparative study and from special video-tape sequences of staff and patients established for this purpose. Self-contained training materials, consisting of manuals and video-taped sequences with scored protocols, will be developed and comparatively tested for effectiveness in observer training to insure diffusion and adoption of findings in practical clinical work and future research.

The etiological agent of Lyme disease (LD) is a tick-borne, hitherto unknown spirochete of the genus Borrelia. We are studying pathogenesis of LD, and to obtain useful reagents for this study as well as to understand the biology of the etiological agent, we have initiated a molecular approach. To date we have prepared a gene bank of LD spirochete DNA in Escherichia coli and have isolated three recombinant clones expressing LD spirochete antigens. One of the clones produces both of the major, outer-membrane associated proteins of LD spirochete B31. These and other clones will be examined in depth and will be used to assess the feasibility of immunoprophylaxis of LD.

We are studying the regulation of the MAL6 gene complex of yeast. Our long-term goal is to develop a complete description of all the components of this complex regulatory system. In this proposal we focus on the role of the major regulatory protein coded by the MAL63 gene. We intend to study its interaction with its promoter and other components of the regulatory apparatus, and ultimately to determine the mechanism by which it activates transcription. Initiation of mRNA synthesis is a fundamental control point in the regulation of gene expression. The frequency of initiation ultimately depends on the interaction of the proteins of the transcription apparatus with each other and with specific DNA sequences. While it is traditional to discuss separately transcriptional initiation in bacteria, the lower eukaryotes, and the higher eukaryotes, it has become increasingly clear that analogous mechanisms are involved. In this proposal we intend to use yeast as a model for transcriptional regulation in eukaryotes. In particular we intend to establish, using both biochemical and genetic techniques, the nature of the interaction between a yeast activator protein, its controlling sequences, and other components of the regulatory system.

The SIR provided (1) Selenocystine; 50mg [92-94% - 77 Se2]Selenocystine; 100mg (l)-tellurocystine; 150mg We have developed a bacterial expression system which allows the efficient substitution of cysteine residues in a protein by selenocysteine (see: Miller, S., Senn, H., Gsell, B., Vetter, W. and Bvck, A. (1994), Biochemistry 33, 3404-3412). It involves overexpression of the respective gene with the aid of the T7 promoter/polymerize system in a cysteine auxotroph strain. The system was applied to substitute the two cysteine residues in E.coli thioredoxin. (Se)2-Thioredoxin was isolated and biochemically characterized. Using this method, L-[3-77Se]cysteine provided by SIR will be incorporated into thioredoxin which will replace the disulfide bridge by a 77Se-enriched diselenide bridge. The isolated (77Se)2-Thioredoxin will be studied by 77Se NMR spectroscopy. The disuffide in thioredoxin serves as a two electron redox carrier for the reduction of nucleotides to deoxy nucleotides. We will develop a method directly determine the redox status of disulfides in proteins using 77Se NMR spectroscopy.

A common feature in all dementias associated with Lewy Bodies is the accumulation and aggregation of the small 14kD protein a-synuclein in the perikaryon and proximal neurites. This proximal accumulation of ct- synuclein in diseased states is very different from the physiologic situation, where the protein is predominantly localized to distant presynaptic sites. Thus pathologic conditions lead to a mis-localization of a-synuclein into proximal neuronal compartments, in addition to the accumulation/aggregation of the protein. While many previous studies have focused on the biochemical processes leading to the aggregation of o> synuclein into the insoluble fibrils that are seen in the end-stage Lewy bodies, much less is known about the initial mechanisms that lead to the proximal mis-localization of the protein. As a-synuclein is synthesized in the neuronal perikarya and is transported into axons, eventually targeting to synapses, our working hypothesis is that defects in the mechanisms of axonal transport and/or presynaptic targeting of a-synuclein is the basis for its mis-localization in pathologic states. To test this hypothesis, we have developed novel model-systems and imaging tools that allow us to directly visualize and precisely quantify axonal transport and presynaptic targeting of a-synuclein in axons and boutons of living neurons. Indeed defects in transport/targeting of pathologic forms of a-synuclein are seen in this system, supporting our hypothesis. Completion of the proposed project will provide insights into initial pathologic mechanisms in these dementias, and may also lead to novel early therapeutic targets. RELEVANCE (See instructions): Dementias associated with Lewy bodies is a common cause of dementia among the elderly, second only to Alzheimer's. To date, there is no known cure. Our best chance of treating this disease is to attack it at an early stage, however, early mechanisms leading to these dementias is poorly understood. In this project we will unravel such early events by determining how a key protein gets misplaced in neurons, causing disease.

The goal of this proposal is to develop a "vector-free" system for cloning PCR products which is efficient and which permits the direct transformation of bacterial cells with PCR products. The system will utilize the yeast FLP recombination system in vivo to clone PCR product into a recombinase target site located in a chromosomally integrated, rescuable plasmid vector. Subsequent generations of the target vector will include features which allow analysis and/or expression in eukaryotic systems (e.g. tissue culture cells, baculovirus expression vectors, yeast YAC vectors), facilitate large scale genome mapping when used in specialized prokaryotic cloning systems such as P1 and cosmid vectors, and allow high level expression and purification of the desired product. Application for cDNA library construction will also be explored. In light of the widespread use of PCR, and the relative inconsistency involved with cloning PCR products by conventional means, there is a broad general application for an efficient one-step PCR cloning system. As the current "state of the art" systems all require enzymatic and/or purification steps following PCR, but prior to transformation, FLP-mediated PCR cloning will offer a decided advantage in that no in vitro manipulation is needed beyond the PCR reaction.

1. The CACCC box is a DNA sequence that functions as a positive transcriptional regulatory element in the promoters of several genes. The rat malic enzyme (ME) gene promoter contains two CACCC boxes at a site containing a tandem repeat of the sequence CTCGCCACCC (malic promoter element 2 or MPE2, -74/-48) which functions as a cis-regulatory element. A sequence similar to a half-site of MPE2 is found in the human immunodeficiency virus long terminal repeat (HIV, LTR, -67/-42) and the human c-fos promoter also contains a sequence identified as a retinoblastoma control element (RCE, -100/-70) which is similar to MPE2. We have now obtained two proteins binding to this site: 1. By screening a rat liver cDNA expression library with the MEP2 binding site and 2. using HeMEP2 DNA as an affinity matrix in purifying a protein from a rat liver nuclear extracts. Both proteins bind specifically to this site. However they also interact specifically with the malic enzyme thyroid hormone response element. Thus the same or similar nuclear proteins bind to two separate transcriptional cis-regulatory elements in the ME gene promoter one of which also binds the T3 receptor. These liver nuclear proteins may play important roles in the ME gene transcriptional response to thyroid hormone. Further characterization of these proteins and DNA motifs to which they bind is being investigated. 2. The alpha and beta receptors are highly related to each other, bind the same ligand, and to the same TREs, but they give quantitatively different responses when activated by hormone suggesting that they do not have redundant functions. The T3 receptor alpha variant protein exerts distinct effects on transactivating functions of these receptors by a mechanism that is still unclear.

I am a clinical investigator with a research interest in the pathobiology of pulmonary infections. I perform molecular assays on clinical samples obtained by bronchoalveolar lavage and then conduct mechanistic studies in model systems. This approach has been productive and has been effective in training clinical investigators. My research is supported by 2 R01 grants. "HIV/TB Interaction in the Lung" investigates the hypothesis that resting alveolar macrophages express a set of transcriptional repressers that normally inhibit HIV replication in the lung. During tuberculosis (TB), inflammatory cells are recruited to the lung, contact macrophages, and derepress the HIV-1 LTR. Soluble factors from the inflammatory cells then activate transcription. This 2-step process is necessary and sufficient for the high-level replication of HIV-1 observed in persons with TB. A second recently funded R01-supported study, for which I am a co-Pi, is titled "Longitudinal Studies of HIV-Associated Bacterial Pneumonia." This study extends the work of the laboratory to investigate bacterial pneumonia. The hypothesis of this second study is that HIV-positive individuals receiving antiretroviral therapy will continue to develop bacterial pneumonia and other pulmonary infections that will lead to characteristic cellular recruitment and activation in the lung. This process will enhance HIV-1 replication and mutation, accelerating the course of AIDS. The longitudinal study is the subject of the research section of this grant. I currently spend 45% effort in clinical service. The K24 Midcareer Investigator Award in Patient-Oriented Research would enable me to reduce patient care responsibilities to 10% effort, so I can increase my focus on training the next generation of clinical investigators. I will be able to increase by 2-fold the number of physicians who I can adequately mentor in translational research. A second goal of the K24 award is to obtain training in infectious diseases epidemiology and matriculate in the MPH program at Columbia University. This didactic experience will enhance my collaboration with epidemiologists and will allow me to learn the quantitative techniques required to analyze large data sets. The support provide by the K24 award will enhance my potential to participate in translational research programs that develop effective novel strategies for the improved care of HIV-infected patients with secondary pulmonary infections. [unreadable] [unreadable] [unreadable]

The instrumental support core will design and construct all special purpose probes and coils required by the individual projects. This centralization of construction facilities will result in efficient design and construction and also assure the individual projects of optimum coils and probes for their particular application. The staff engineer will also fulfill an instructional role for the support staff.

Obesity and diabetes mellitus is the leading cause of cardiovascular and renal disease in the United States. This is of increasing concern since the incidence of obesity and insulin resistance is increasing and as many as 1 in 4 Americans are expected to have diabetes by the year 2030. In spite of all the beneficial interventions implemented in patients with diabetes, including tight glucose control, tight blood pressure control, angiotensin converting enzyme inhibition or angiotensin II receptor antagonism, renal disease progresses in most of these patients. Additional treatment modalities that modulate the pathogenesis pathways involved in obesity and diabetic nephropathy are therefore urgently needed to slow the progression of renal disease in patients with obesity and diabetes. Studies from our laboratory indicate that treatment of diabetic mice with a highly active and specific TGR5 agonist prevents the progression of diabetic nephropathy. The beneficial effects of TGR5 activation are associated with stimulation of estrogen-related receptor ? (ERR?) and sirtuin 3 (SIRT3). In this proposal we will test the hypotheses that: 1) TGR5 plays an important role in modulation of kidney disease in diabetes and obesity; 2) TGR5 inhibition accelerates and TGR5 activation prevents obesity and diabetic kidney disease; 3) the beneficial effects of TGR5 are mediated in part through stimulation of ERR?. In Specific Aim 1 we will determine the effects of kidney specific TGR5 deletion in kidney disease in obesity and diabetes. In Specific Aim 2 we will determine the effects of kidney specific TGR5 overexpression in kidney disease in obesity and diabetes. In Specific Aim 3 we will determine whether the effect of TGR5 in modulation of kidney disease in obesity and diabetes is dependent on kidney specific ERR? activation. Impact: The potential role of TGR5 and ERR? in modulating obesity and diabetic renal disease is very novel and will have major implications for the treatment of obesity and diabetes related renal complications. TGR5 and ERR? have distinct actions of regulating mitochondrial biogenesis, energy metabolism and energy expenditure: they are therefore quite distinct from other interventions used in treatment of diabetes and its complications where TGR5 can also induce weight loss and prevent obesity.

Myocardial infarction and stroke due to atherosclerosis are the leading causes of death in the United States. It has become clear that increases in reactive oxygen species (ROS) represent a common pathogenic mechanism for atherosclerosis. A particularly important mechanism for ROS-mediated cardiovascular disease appears to be stimulation of pro-inflammatory events. In the proposed research we will investigate the role of a newly discovered class of ROS mediators that we term SOXF for Secreted Oxidative stress induced Factors. Published data suggest that cyclophilin A (CyPA), a member of the immunophilin family is an important SOXF that contributes to inflammation and atherosclerosis. We found that CyPA is secreted from vascular smooth muscle cells (VSMC) in response to ROS, and stimulates ERK1/2 and JAK/STAT pathways, increases DNA synthesis, and inhibits apoptosis in VSMC. In contrast to the effects in VSMC, CyPA stimulates endothelial apoptosis and increases expression of adhesion molecules including E-selectin and VCAM-1. Studies of the CyPA knockout mice (CyPA-/-) show that CyPA regulates T helper cell Th1 and Th2 responses. Our published data show that overexpression of CyPA enhances intima formation after vascular injury associated with enhanced inflammation, while CyPA deficiency has the opposite effects. Preliminary data show that CyPA deficiency decreases atherosclerosis in the apoE-/- mouse. Thus CyPA is a novel cytokine that mediates vascular inflammation and remodeling. We propose the following major hypothesis: CyPA, increased by oxidative stress, promotes vascular dysfunction and atherosclerosis by altering VSMC, endothelial and white blood cell function. To prove this hypothesis 4 aims are proposed. Aim 1: Characterize the signal transduction mechanisms by which CyPA modulates VSMC expression of pro- and anti-inflammatory mediators. Aim 2: Determine the role of CyPA in atherosclerosis development and progression by evaluating the effect of CyPA overexpression and deficiency on pathology of the apoE-/- mouse. Aim 3: Evaluate the role of bone marrow- derived cells in CyPA-mediated atherosclerosis. Aim 4: Show that ROS-mediated atherosclerosis is CyPA- dependent. These studies will provide new insight into the mechanisms by which ROS regulate vascular function and the specific role of CyPA in atherosclerosis.

Systemic lupus erythematosus (SLE, lupus) is a chronic systemic autoimmune disease characterized by production and survival of autoreactive antibodies and deposition of immune complexes to various tissues, leading to organ damage. Lupus affects primarily women of reproductive age, with a female to male ratio of 9:1. Mortality in SLE is increased compared to the general population. Higher risk of death is associated with female sex, younger age, shorter SLE duration and African American race. Studies have shown that SLE is more severe among African American, Hispanic and women of other ethnic minorities. Over the last two decades, lupus mortality rates increased by 67.8% among African American women. Results of studies have suggested that this may be related to worse renal involvement and outcome in African American patients. Renal and other severe SLE manifestations respond poorly to current therapeutic modalities and often require replacement therapy. microRNAs (miRNAs) regulate a plethora of normal cellular and developmental processes and their aberrant expression and function is linked to human disease. miRNAs are aberrantly expressed in human and mouse SLE lymphocytes, however their specific function in lupus is poorly understood. We study the expression and function of miRNAs in SLE mouse models and our general hypothesis is that several miRNAs regulate pathways that contribute to the disordered immunoregulation in lupus. Our long-term objective is to characterize and interfere with miRNA-regulated pathways that are common in mouse and human SLE and to investigate the potential of miRNA inhibition as a novel therapeutic direction in lupus. Our preliminary studies showed that LNA antimiRs can be used to efficiently antagonize endogenous miRNAs in peripheral lymphocytes in vivo and that inhibition of a miR-21 using such compounds ameliorates the autoimmune disease phenotype of the B6.Sle123 model. Using the same novel methodology, we propose to study the effect of LNA antimiR inhibition in two of the most severe SLE manifestations: renal and lung disease. With experiments described in Aim I we will study the effect of in vivo miR-21 and miR-155 inhibition on renal disease manifestations in two mouse models of SLE, which recapitulate two different histological classes of human SLE nephritis. With experiments described in Aim II we will examine the effect of in vivo and in vitro miR-21 inhibition on interstitial lung disease in SLE using a mouse model and primary lung fibroblasts from SLE patients.

The long-term goal of this research is to understand the molecular mechanism of non-random chromatid segregation in mouse embryonic stem cells. The immortal strand hypothesis - posits that stem cells segregate the "oldest" DNA strands of all chromosomes asymmetrically to the self-renewing stem cells as a mechanism to prevent the stem cell from inheriting any errors in DNA replication that could lead to a cancerous state. Stem cells use this strategy on a chromosome by chromosome basis to direct the segregation of specific chromatids. Specifically, mouse embryonic stem cells selectively segregate chromatids of chromosome 7 and we propose to determine the mechanism. This proposal has two Aims. The first Aim is to test the hypothesis that non-random chromatid segregation depends on the replicative age of the DNA using chromosome oriented fluorescence in situ hybridization (CO-FISH). The second aim will test the hypothesis that LRD, a homolog of outer-arm 2-axonemal dynein heavy chain, functions as a molecular motor to direct non-random chromatid segregation. [unreadable] [unreadable] PUBLIC HEALTH REVELANCE: Stem cells are unique in having the ability to "self renew" and at the same time to be capable of differentiating into every cell in the body. It is critical that stem cells have mechanisms that protect their genetic integrity so that they may maintain their stem cell identity while faithfully executing the instructions needed to adopt many different cellular fates. One mechanism that stem cells use is to selectively segregate chromosomes after they are replicated so that specific copies are kept together. This is a mysterious process since we generally believe that replicated chromosomes are segregated randomly. Clearly, stem cells are different in this regard. The goal of this research is to understand the mechanism of selective chromosome segregation in stem cells. [unreadable] [unreadable] [unreadable]

DESCRIPTION: (Applicant's Abstract) This competing continuation seeks to extend an ongoing longitudinal project (RO1 DA-06025) examining relations among prenatal cocaine exposure, postnatal environmental instability, and the cognitive and social development of infants and children from birth to 36 months. The proposed study series aims to extend the follow-up of these children through age 7 years with a particular focus on the regulation of arousal and attention. Because of cocaine's effect on the developing monoaminergic system, prenatal cocaine-exposure may interfere with the developmental ontogeny of the ability to regulate states of arousal and thereby affect the normal development of capacities to regulate attentional states and to respond to stressful events. Inasmuch as the regulation of arousal-modulated attention is crucial to perceptual, cognitive, language, and social development, early disruption in the developmental ontogeny of arousal and attentional regulatory capacities may have effects that extend well into the school-aged years and alter the normal trajectory of cognitive, language, and social-emotional development. In the last 4 years, 411 children (233 prenatally cocaine-exposed and 178 non-cocaine-exposed) now ranging in age from 3 to 48 months have been recruited and maintained. Extensive data have been gathered on the associations of prenatal cocaine (and other drug) exposure with arousal and attention regulatory capacities, and cognitive, language, and adaptive behavior development, and the effects of maternal substance abuse on mother-child interactions. Findings from the first phase of follow-up suggest an association between cocaine exposure and impairments in the regulation of arousal and attentional states and also point to the disruptive effects of substance abuse on the parenting environment. The objective of this competing renewal is to continue through age 7 years, the follow-up of this cohort of cocaine-exposed and non-cocaine-exposed children with repeated assessments of: (1) arousal regulation operationalized behaviorally and neurophysiologically as the startle response and heart rate variability; (2) attention regulation operationalized as the ability to sustain attention, identify stimuli, and inhibit responses; (3) executive function (4) cognitive and language development, (5) adaptive and maladaptive behavior, (6) school achievement (7) social adjustment, (8) the incidence of childhood psychiatric disorders of attention, anxiety, and conduct and (9) social and environmental risks including continued maternal drug use, parental stress and dysfunction, and family disruption. The follow-up continues to build on an interactive model in which children with developmental risks are more or less vulnerable to poor outcomes depending on the severity of their environmental disruption and stress.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Excessive alcohol ingestion, occasionally or chronically, is co-morbid with medical disorders affecting the brain and behavior as well as other organ damage. Much of what is known about risk for and the consequence of heavy alcohol consumption, including mechanisms of organ damage, is derived from rodent studies or retrospective human accounts. The Monkey Alcohol Tissue Research Resource (MATRR) is a unique post-mortem tissue bank to ensure tissue from animals chronically drinking alcohol, as well as their related drinking and genetic information, is available to the wider alcohol research community insuring reduced numbers of animals used in research. The tissue is derived from a standard protocol of ethanol self-administration in three species of monkeys housed at ONPRC (rhesus and cynomolgus) and Wake Forest (vervet). This resource will provide novel data for hypothesis testing relating the risk for and consequences of alcohol consumption and serve to bi-directionally bridge the gap between rodent and human studies. The basis of the MATRR is that non human primates, specifically monkeys, show a range of drinking excessive amounts of alcohol (3.0 g/kg or a 12 drink equivalent/day) over long periods of time (12-30 months) with concomitant pathological changes in endocrine, hepatic and central nervous system (CNS) processes. These longitudinal designs span "stages of drinking" from ethanol-naive to early alcohol exposure to chronic abuse. The demand for, and the quality of, the tissues are already high as reflected in the number of requests and publications. Nevertheless, this resource needs further development in order to fulfill its potential as a cutting edge translational tool in alcoholism research.

The Inherited Neuropathy Consortium (INC) RDCRC is a network of clinical investigators dedicated to developing the infrastructure necessary to evaluate therapies for patients with heritable peripheral neuropathies, collectively known as Charcot-Marie-Tooth disease (CMT). Originally, the INC consisted of six sites. Supplemental funding from the Muscular Dystrophy Association (MDA) and Charcot Marie Tooth Association (CMTA) has allowed us to expand to 17 sites. CMT is caused by mutations in >70 genes. Mutations cause dominantly inherited demyelinating CMT (CMT1), dominantly inherited axonal CMT (CMT2), and recessively inherited CMT (CMT4). Despite scientific advances there are currently no medications to slow progression for any form. In part this is due to the lack of adequate natural history data, the lack of sensitive outcome measures and the lack of biomarkers for CMT. In addition, there has not been a coordinated international effort to share clinical data on patients. We have addressed these areas during our initial cycle of the INC. We have performed natural history studies, generated and tested outcome instruments for adults and children with CMT, and begun testing potential biomarkers. We have developed patient reported outcome (PRO) instruments. We have worked as an international group such that we will enroll > 5000 patients into our protocols by the end of our initial five year cycle. We have developed a Web Page that provides information to patients, families and investigators. It also has allowed us to directly interact with patients through our INC Contact Registry and have developed the CMT-lnternational Database (CMT-ID), that consists of national registries from around the world that use the same CMT Minimal Dataset that is used by the INC. Finally, we have trained a number of young investigators who are committed to a career investigating CMT. In our second cycle we propose Aims to extend our natural history data, to extend our Next Generation Sequencing data, to identify potential biomarkers and outcome measures, to perform clinical trials, and to provide information to patients, families and investigators through our INC Website.

The contractor will publish the proceedings of a seminar presented at the 31st Annual Meeting of the American Association for Laboratory Animal Science.

DESCRIPTION (Abstract reproduced verbatim): The goal of this project is to understand the role of nitric oxide (NO) as a regulator of cell proliferation and differentiation. Our studies have shown that NO acts as an anti proliferative agent during organism development, regulating the balance between cell proliferation and differentiation in the differentiating tissue, and, ultimately, controlling the shape and size of tissues and organs in the developing organism. Thus, NO emerges as an essential negative regulator of cell proliferation and a mediator of tissue differentiation. In this project, we will study the mechanisms of the anti proliferative action of NO during cell division and differentiation and identify the molecular targets of NO within the cell cycle machinery. In our first Specific Aim we will start by defining the phases of the cell cycle where the action of NO is most pronounced. Next we will dissect the crucial pathways within each of these phases and identify the stages that are affected by NO. After that, we will probe the individual potential targets of NO. In our second Specific Aim we will investigate whether cell cycle-related signaling by NO synthases is different from signaling induced by exogenous NO donors. Furthermore, we will study the signaling induced by each of the three NOS isoforms under similar conditions in order to identify the isoform-specific targets of NOS. In our third Specific Aim we will test the hypothesis that most of the antiproliferative activity of NGF during neuronal differentiation of PC12 cells is mediated by NOS. For this, we will determine the genes induced by NGF in PC12 cells and compare them to the genes induced by NO donors and NOS gene, will determine which of the genes induced by NGF are dependent on NOS activity for their activation, and will investigate whether NGF induced activation of p53 is dependent on NO production.

A major effort in contemporary biological research is to identify genetic loci that control developmental decisions. In addition, disturbance of the normal differentiation process can contribute to tumorigenesis. Determining the key regulatory mechanisms involved in generating and maintaining the differentiated state is important for understanding both normal development and disease. This proposal is designed to investigate the control of cell type determination and differentiation in mammalian cells. By utilizing myoblast cell lines grown in vitro, and a combined somatic cell and molecular genetic approach, the regulation of the MyoD family of muscle regulatory genes will be analyzed. Intertypic hybrids will be assayed for activation and repression of myogenesis. Genetic studies of this type have been used to identify positive and negative regulatory interactions in a number of experimental systems. The experiments proposed utilize somatic cell genetics to identify genetic alterations found in rhabdomyosarcomas that inhibit muscle differentiation. In contrast to standard molecular genetic approaches, somatic cell genetics has the ability to ascertain the functional significance of complex genetic lesions, such as gene amplifications, duplications, inversions and translocations, that occur in tumor cells. By utilizing this approach, my lab has determined that alteration of the ATR locus, by isochromosome formation, inhibits differentiation in rhabdomyosarcomas. This proposal is designed to determine the molecular mechanisms by which genetic alteration of ATR results in cell-cycle abnormalities and inhibition of muscle differentiation, as well as to determine whether the i(3q)s that are present at high frequency in other types of tumor result in similar phenotypic abnormalities.

DNA methylation represents an important epigenetic regulatory mechanism for repressing gene activity which ultimately operates by affecting chromatin structure. In order to understand how this works we have developed new approaches for deciphering how methyl moieties interact with the key components of the nucleosome and other features of higher order structure.In general, methylation patterns are not inherited from the parents but rather are generated anew in each individual, and this is carried out mainly through demethylation which is directed by local cis acting sequences. Using transgenic mice it will be possible to map these elements and then isolate trans acting protein factors that may be involved in facilitating demethylation. As a result of these studies it should be possible to devise strategies for selectively altering methylation patterns in vivo and thereby control gene expression.We have devised a unique system for studying the effects of global undermethylation on the regulation of genomic repression patterns and on cellular growth properties. These studies implicate DNA methylation as a cell division counting mechanism that may mediate senescence.One of the most important roles of DNA methylation is in the control of genomic imprinting where it serves to mark the parental alleles and direct the imprinted expression pattern. We have succeeded in mapping the central control elements which regulate imprinting and can now use transgenic mice to decipher the exact cis acting sequences and trans acting factors which mediate this epigenetic mechanism at different stages of development.

We propose to determine the quantitative relationships between the cellular oxygen concentration and the cellular metabolic state. The mitochondrial are responsible for most of the cellular oxygen consumption, utilizing the oxygen and cellular reducing power (substrates) to produce the require adenosine triphosphate as the energy supply. A mathematical description of these interrelationships has been developed which will be tested for its ability to fit the mitochondrial behavior both in isolated suspensions and in intact cells. This behavior will be tested for a wide range of oxygen concentrations and substrates in order to provide a basis for understanding tissue behaviour under low oxygen conditions. The properties and mechanism of the enzyme which directly reacts with molecular oxygen will also be examined in order to understand its central role in the control of the mitochondrial (and, therefore cellular) respiratory rate.

The overall aims of the Developmental Core (DC) are to encourage innovative preliminary work in neuroAIDS;to foster new investigators with targeted Developmental grant support to serve as a springboard for successful applications for independent NIH funding;and within the interdisciplinary context of the HNRC, to help train the next generation of scientists in collarborative research essential for advancement of the field, in the current funding cycle, the Developmental Core has awarded 15 developmental grants, all encompassing promising new directions in neuroAIDS research, to students, postdoctoral fellows, and junior faculty. In addition, the Core has provided training in neuroAIDS research methodology to 188 junior researchers, including 39 who completed the HNRC mentored investigator program. We will continue these programs in the new grant cycle, with several improvements to increase their effectiveness. In addition, we plan to submit in January 2011 an application to the NIH for a T-32 Training Program for a broad range of neuroAIDS researchers (including pre- and post- doctoral trainees, in both clinical and non-clinical fields). This grant will expand on the HNRC's long history of training neurobehavioral and neuromedical scientists. Key accomplishments during the past funding period include the expansion of the Developmental Core goals to include mentoring and pilot study support for international trainees and collaborators, recruitment of young investigators interested in transdisciplinary research (molecular neuroimmunology, genetics, epidemiology, imaging), and receiving an R25 award to support an interdisciplinary clinician-researcher fellowship in neuroAIDS (IRFN). The IRFN provides training to clinicians interested in careers in neuroAIDS research. The training emphasizes the interdisciplinary and translational nature of clinical problems in neuroAIDS in order to prepare clinician scientists to tackle emerging questions in the field. A new goal for the Core is to utilize novel information technology webcasting to facilitate virtual mentoring and journal clubs. The virtual mentoring will be implemented in collaboration locally with the CFAR and UCSD clinical research curriculum (CREST) programs, and extramurally with the AIDS Research Centers at JHU and UNMC. With its multidisciplinary research environment, talented and productive research staff, highly organized and integrated infrastructure, and rich longitudinal specimen and data banks, the HNRC is well-positioned to develop and support expanded opportunities for innovative research by fostering preliminary studies and mentoring of trainees from a wide array of disciplines.

Age-associated changes occur in T cell immunity, contributing to increased risk of infection and malignancy. Probably, the most intriguing alteration in CD8+ T cell immunity with aging is memory CD8+ T cell expansion although the mechanism(s) and the consequences of this phenomenon are largely unknown. Recently, my lab investigated whether the age-associated expansion of memory CD8+ T cells was secondary to increased expression of IL-7 receptor alpha chain (IL-7Ra) which is critical for CD8+ T cell survival. In this study, cells expressing IL-7Ra high and low were found in a subset of memory cells called CD45RA+ effector memory (EMCD45RA+, CD45RA+ CCR7-) CD8+ T cells. In contrast to the expectation, the elderly (age [unreadable]: 65) had expansion of IL-7Ra low EMCD45RA+ CD8+ T cells (25% of total CD8+ T cells) compared to the young (age [unreadable] 40). This finding raises several questions. How does such cell expansion occur? What is the immunologic consequence(s) of this cell expansion? The expansion of IL-7Ra low EMCD45RA+ CD8+ T cells could be secondary to chronic antigen stimulations by infections such as cytomegalovirus (CMV), which has been linked to the expansion of memory CD8+ T cells with aging. However, these cells poorly survive and proliferate in response to IL-7 and TCR triggering, respectively, indicating that neither IL-7 nor chronic TCR triggering alone is sufficient explanation for this phenomenon. Of interest, IL-7Ra low CD8+ T cells nicely proliferate and expand in response to IL-15 and a combination of IL-15 and TCR triggering, which suggests the role for IL-15 in expanding IL-7Ra low EMCD45RA+ CD8+ T cells. Thus, I hypothesize that IL-15 is critical for expanding IL-7Ra low EMCD45RA+ CD8+ T cells with aging via inducing antigen-independent (IL-15 alone) and -dependent (TCR triggering, possibly CMV, with IL-15) cell proliferation, and such expanded cells have altered functions. This hypothesis will be addressed with the following specific aims: 1) Investigate whether the expansion of IL-7Ra low EMCD45RA+ CD8+ T cells with aging is directly related to CMV infection; 2) Determine whether the expansion of IL-7Ra low EMCD45RA+ CD8+ T cells with aging steins from alterations in IL-15-mediated CD8+ T cell proliferation; and 3) Investigate whether expanded IL-7Ra low EMCD45RA+ CD8+ T cells in the elderly have altered function. The results of this study will advance our understanding about the effect of aging on the immune system, leading to better protection against infection and tumors. [unreadable] [unreadable] [unreadable]

This project focuses on exploring the psychological and neural mechanisms that underlie the interaction of motivation and cognitive control, and how this interaction might be influenced by different dimensions of individual difference. Motivation may provide a basic mechanism of cognitive self-regulation. The primary hypothesis tested in this project is that cognitive and behavioral goals are activated, prioritized, and maintained according to their motivational value to the individual. Moreover, different affective and motivational states may have distinct influences on cognitive processing and cognitive control. The current proposal provides an innovative and theoretically-driven cognitive neuroscience approach towards this question, by examining three different affective/motivational dimensions - other-oriented (social) vs. self-oriented, intrinsic vs. extrinsic, and positive affect vs. reward-based motivation - in terms of their influence on the neural mechanisms of cognitive control. Specifically, in a large-sample neuroimaging study we will explore how differences between these motivational states impact behavior and brain activity dynamics during performance of a cognitive task with high control demands (cued task-switching). A key component of the project will be to comprehensively and rigorously examine the moderating influence of individual differences in theoretically relevant motivational traits. Clear-cut and theoretically-guided testable hypotheses are provided regarding how each motivational dimension might impact components of cognitive control, and the associated neural circuitry. This project promises to have substantial significance by filling an important gap in knowledge regarding the role of motivation as a basic mechanism of cognitive self-regulation. As such, this work may have long-term impact on educational and workplace practices, and treatments for debilitating clinical disorders, such as schizophrenia, depression, ADHD, and substance abuse. PUBLIC HEALTH RELEVANCE: This project has high relevance for public health by advancing scientific knowledge regarding the psychological and biological basis by which motivation influences information processing and behavior in a range of areas, including problem-solving, decision-making, memory and attention. An improved understanding of these influences will be of critical importance for developing better educational and workplace practices, and improving treatments of clinical and psychiatric disorders, including substance abuse.

While the structure/function relationships of lens crystallins and their roles in protection of the lens from cataractogenic stresses (e.g., the chaperone-like antiaggregative activity of alpha-crystallin; possible antioxidative function of enzyme/crystallins) remain of great interest to this laboratory, the focus of the work has shifted toward certain noncrystallin proteins of the lens. During the past year, an enzyme has been isolated from the lens, which uses the reducing equivalents of NAD(P)H to inactivate free radical species such as the hydroxyl radical. Partial sequence has been obtained, which indicates a distant relationship to known oxidoreductases; however, at present it appears that the isolated protein is a novel enzyme. Dr. Chuan Qin is pursuing characterization of this enzyme, which we believe may be an important component of the antioxidant defenses of the lenses of primates and certain other species. Dr. P. Vasantha Rao is investigating the role of small GTPases in regulating critical cellular processes in the lens. He has demonstrated for the first time the presence in the lens of Ras, which is involved in regulating cell growth and differentiation, and Rho, which is involved in cytoskeletal organization. These signal transduction molecules are present in both the lens epithelial cells and lens fibers. He has also found GTPase-activating and exchange factors in the lens that are required for activity of the GTPases. There appear to be additional, possibly novel, GTP-binding proteins that are fiber cell specific and may be involved in terminal differentiation of lens fibers. Preliminary studies using human lens epithelial cell cultures and lens organ cultures demonstrate that inhibition of the activity of small GTPases leads to massive morphological changes in lens cells and to opacification of cultured lenses. Thus, these initial studies indicate that, as in other tissues, Ras family GTPases function in the lens as molecular switches regulating critical cellular processes and that dysfunction of these important signal transduction molecules could be involved in cataract development. The group is also involved with other investigators in the LMOD in studies on potential anticataract agents. The hydroxylamine reduction product of the nitroxide Tempol has proven very effective in prevention of cataract in an organ culture system. Studies are currently under way to develop effective means of administration of this agent to live animals so that its efficacy for cataract prevention in vivo can be determined.

Chronic hypoxia induces dramatic fibro-proliferative remodeling in the pulmonary artery (PA)adventitia that is especially impressive during the neonatal period and clearly contributes to the marked increase in pulmonary vascular resistance observed. One of the key mediators of these responses is protein kinase C (PKC) [unreadable] (zeta) which regulates hypoxia-inducible genes. To study the adventitial remodeling, we used PA adventitial fibroblasts cultured from neonatal control (Fib-C) and hypoxic pulmonary hypertensive calves (Fib-H) and compared the effects of hypoxia on PKC[unreadable]-related signaling pathways. We have observed that acute hypoxic exposure induces translocation of nuclear PKC[unreadable] into the nuclear membrane and the cytoplasm selectively in Fib-C, but not in Fib-H. In Fib-C, PKG;inhibition results in sustained activation of extracellular signal-regulated kinases, ERK1/2, whereas it blocks ERK1/2 activation in Fib-H. The mechanisms by which PKC[unreadable] can diversify its own biochemical properties in response to chronic hypoxia remain unexplored. Although PKC[unreadable] suppresses the hypoxia-induced proliferation of Fib-C, it translates the hypoxic signal into replicative responses in Fib-H. The molecular mechanisms that confer the ability of PKC[unreadable] to perform opposing functions, especially in proliferative responses, are unknown and will be examined here. We also found that PKC^ regulates MAP kinase phosphatase-1 (MKP-1) expression only in Fib-C, but not in Fib-H. The biochemical pathways responsible for uncoupling of PKQ;from the regulation of MKP-1 expression in Fib-H are not known. The aforementioned questions will be addressed by the following specific aims: 1) The development of hypoxic pulmonary hypertension leads to the appearance of fibroblasts with modified nuclear localization, activation and target specificity of PKC[unreadable];2) An altered functional role of PKC[unreadable] as a growth regulator, promotes enhanced proliferation of fibroblasts from chronically hypoxic animals;3) Nuclear PKC^-mediated growth-suppressing activity of MKP-1 is absent in fibroblasts isolated from the adventitial compartment of chronically hypoxic animals. Successful completion of the proposed experiments will provide new insights into the role of PKCf;and MKP-1 in pulmonary vascular diseases and could lead to the development of improved therapeutic strategies for the treatment of pulmonary diseases caused by chronic hypoxia.

This application describes a research program aimed at defining the mechanisms by which vitamin A is delivered to the mammary gland during lactation. It is known that vitamin A is transported in plasma in two well-defined lipid protein complexes: newly absorbed dietary retinol is transported in the lipid core of the chylomicron as retinyl esters, while vitamin A released from liver storage is transported by serum retinol-binding protein as retinol. The specific aims are: 1) to identify the roles of thoese two lipid-protein complexes in transporting vitamin A into the mammary gland and 2) to use the technique of autoradiography to identify the cell type(s) that recognize RBP and/or chylomicron and that transport vitamin A across this epithelial tissue. A combination of biochemical and morphological techniques are proposed. Chylomicrons labeled with (3H)retinyl esters will be employed to monitor transport of core lipids into the lactating mammary gland, liver and other tissues of the intact rat. Using the isolated perfused mammary gland, the uptake of (3H)retinol from RBP and (3H)retinyl esters from chylomicrons will be investigated. Light microscopic autoradiography will be used to localize the interaction of labeled RBP, or chylomicron remnants, with specific types of cells. A third specific aim asks whether the secretion of RBP and retinol from the isolated perfused rat liver is increased during lactation, and thus addresses the question of how peripheral utilization of this nutrient might affect its output from liver stores. This research plan addresses the transport of vitamin A between organs and blood, and is one part of a longer-range objective to understand the delivery of vitamin A to target organs, the intracellular metabolism of vitamin A in the breast, and regulation of the secretion of this nutrient into milk. These studies relate to the physiology of vitamin A transport, and the regulation of vitamin A nutrition during lactation.

The 500 Nations Haskell / KU Bridge program supports students from Haskell Indian Nations University who seek to transfer to other institutions;it works collaboratively with other MORE programs (IMSD, RISE, PREP, and IRACDA) to move American Indians into biomedical research careers. The Haskell / KU Bridge program has already achieved a transfer rate (65%) that approaches the requested NIGMS five year target (70%). In addition, Bridge participants who transfer to KU are retained and graduate at significantly higher rates than a paired group of non- Bridge Haskell transfers (retention: 65% versus 27%;graduation: 55% versus 13%). In this renewal application, we will build on these successes and provide innovative activities to meet stated goals. Promising Haskell students will be identified during their freshman or sophomore year, recruited into the Bridges program and provided information and academic counseling on majors and careers. If warranted based on assessment of math and English skills, developmental opportunities in math and composition will be provided by a highly successful Haskell Learning Center funded by the RISE program. Undergraduate research experiences will be preceded by an Orientation to Research / Responsible Conduct of Research seminar and an intensive Research Methods / Experimental Design lab workshop. Students will then move into KU research labs in a wide range of biomedical relevant disciplines. In preparation for transfer, Bridge students will be required to complete at least one course in their major at KU within the tuition free Haskell / KU Exchange program. In response to external evaluations of our current program, the renewed Bridge program provides significant support for the faculty and the science curriculum at Haskell. Faculty development activities will include participation in a flexible, professional development program that will support travel to national meetings, enrollment in off-campus classes, and support to develop new biomedical relevant courses and laboratories. Financial support for Bridge students following transfer to KU will be centered on a seamless continuation of support from the KU IMSD program. An external consultant will work with Bridge staff to track students, evaluate research performance and monitor faculty and student satisfaction. Evaluation procedures will allow for both regular adjustments of activities during the course of the program and an assessment of whether goals have been met. In summary, continuation of the Bridges program provides essential support to sustain the momentum and increase the success rate that Haskell and KU collaborators have achieved as they work toward increasing the representation of American Indians in the biomedical science community. Public Health Relevance: The proposed project will increase the diversity of the scientific community by enhancing the opportunity for American Indians to complete baccalaureate degrees and eventually enter careers in biomedical research. In the long term, the project will increase the size and diversity of the scientific workforce and stimulate interest in research on diseases that have a high incidence in minority populations thereby improving both health care and disease prevention in this country.

Transsphenoidal surgery (TSS) is the best treatment for corticotrophin adenomas causing Cushing's Disease (CD). Although caused by benign pituitary tumors, CD can drastically affect the lives of patients suffering from the disease. Increased adrenocorticotropic hormone (ACTH) and resultant hyper-cortisolemia can lead to obesity, hypertension, hyper coagulability, morphologic changes and death. Successful TSS can provide immediate cure from CD while preserving endocrine function in around 70 - 80% of patients. The patients that do no achieve remission with TSS eventually undergo many treatments including radiation, and life-long cortisol suppression therapy. Success in TSS is directly linked to the ability to accurately detect pituitary tumors before surgery. Routine pituitary magnetic resonance imaging (MRI) fails in up to 50% of cases of CD in detecting CA tumors, presumably due to small size or poor MRI contrast to noise. When tumors are identifiable on MRI before surgery, the cure rates can reach 90%. When adenoma is not identified on imaging before surgery, exploratory surgery is much less successful in curing the patient, and in many cases, eventually leads to radiation therapy and panhypopituitarism. Improved Imaging of the Pituitary Gland: We have developed a novel intracavitary MRI surface coil (the endosphenoidal coil or ESC) that is designed to be used during TSS. In pre-clinical cadaveric testing, we have demonstrated 10-20 fold increase in signal-to-noise ratio (SNR) when using the ESC compared to standard clinical imaging. High SNR allowed us to perform ultra high-resolution imaging of cadaveric pituitary gland to reveal features not normally visible on clinical imaging, including the pituitary capsule, the intercavernous sinus and microcalcifications in the pars intermedia. Based on the findings from the cadaveric study, we have designed a Phase 0 clinical trial to evaluate the feasibility of using ESC during TSS. The study is being submitted to the CNS IRB and FDA for approval. The study will be a non-randomized, pilot study of subjects requiring TSS for pituitary lesions. The study will evaluate the feasibility and effectiveness of using ESC as a clinical tool for imaging evaluation of pituitary tumors during TSS. Other strategies to detect pituitary adenomas can be developed that exploit the physiologic properties of these lesions. Specifically, fluorine-2-18-deoxyglucose (FDG) positron emission tomography (PET) imaging may help identify biologically active pituitary adenomas. Previous PET studies using FDG have reported locating tumors that were not visible with MRI, hinting at a potential use in detecting MR negative pituitary adenomas. In an ongoing laboratory effort, we have discovered that secretagogue stimulation can modulate their glucose uptake in a delayed fashion that is dependent on membranous translocation and transcriptional upregulation of glucose transporter 1 (GLUT1). Based on these findings, we have amended and continued an ongoing clinical trial to evaluate the effect of secretagogue stimulation on FDG-PET visualization of pituitary adenomas in CD. New therapeutic options for CD: In instances where TSS fails to cure CD patients, tumor cells are targeted by adjunctive therapies including radiation. Radiation is successful in 50% of patients, but it may take up to 2 3 years to achieve biochemical remission. During this time, hormonal suppression of the adrenal glands in necessary. Our recent laboratory investigations have shown that histone deacytelase (HDAC) inhibition with suberolyl anilide hydroxamic acid (SAHA) may achieve three goals that may lead to improved management of CD patients that have failed TSS. We have seen that SAHA leads to a reduction in ACTH output from corticotroph cells, and also causes a dose dependent reduction in tumor cell viability. It is also well known that HDAC inhibitors are potent radiation sensitizers. We are developing a clinical trial to investigate the effectiveness of SAHA (an FDA approved drug) in CD patients. We believe that SAHA will lead to improved management of CD as it may reduce the ACTH production in remnant tumors, cause tumor size reduction and sensitize the tumors to radiation therapy. Management of Cushings disease: We are also making efforts in improving the diagnosis and underlying causes of Cushings syndrome. We have made some advances in interpretation of hormone levels obtained immediately after surgery. We have found that these levels can predict whether the patient will be relieved of Cushings diseases within a few hours of surgery. Additional collaborative work with the NICHD colleagues is helping us figure out the underlying causes of Cushings disease.

The steroid hormone, progesterone, plays key roles including the growth and development of the mammary glands, uterus, and ovaries. The physiological effects of progesterone occur mainly through its interaction with the specific steroid receptor, progesterone receptor (PR). The target genes for PR have been identified in various tissues, including those implicated in breast cancer. Phosphorylation is an integral regulator of protein/protein interactions, which are required for steroid receptors (SRs), like PR, to recruit coactivators and interact with target genes. Understanding SR function requires the elucidation of how these complex interactions are regulated. The goal of this proposal is to understand how site-specific protein phosphorylation regulates the activity of the PR. The hypothesis is site-specific PR phosphorylation facilitates protein/protein interactions that are essential for the regulation of subsets of PR target genes. In addition, this phosphorylation is an important prerequisite for context and tissue-specific, physiological actions or PR in response to hormone. We have identified the Ser190 phosphorylation sites in human PR and the homologous site in mice, Ser191, and have extensive experience in studying PR actions and the effects of cell signaling on PR. The specific aims are: 1) to determine the mechanism by which Ser190 phosphorylation modulates PR function in human breast cancer cells, 2) to test the concept that selective elimination of a PR phosphorylation site will compromise PR function in vivo in mice, and 3) to perform limited gene expression studies in wild type and PRAIa191 mutant mice. This will be the first test for a role of any steroid receptor phosphorylation in vivo. It is expected that the site-specific phosphorylation of PR at Ser190 is essential for the regulation of a subset of PR target genes in response to hormone. The destruction of the homologous phosphorylation site in mice will verify its importance in vivo. Successful completion of these studies will have a major impact in demonstrating the importance of how PR modifications can affect its function. By increasing our understanding of how PR and other steroid receptors function, we can develop drugs that target certain functions of the specific steroid receptor to treat diseases like breast cancer and uterine cancer without causing harm to other parts of the body. [unreadable] [unreadable] [unreadable]

The recent report by Rasheed, Gardner and Huebner of a rat sarcoma virus derived in tissue culture by the in vitro co-cultivation of a chemically transformed rat cell and a spontaneously transformed rat cell producing rat helper virus, has led to an investigation of the relationship of this sarcoma virus to the Harvey and Kirsten sarcoma viruses. This project has involved both the genetic and biochemical characterization of this rat sarcoma virus.

Hmgi-c is a member of a novel, developmentally regulated gene family. It is responsible for the pygmy (pg) mutation in mouse and the human homologue is disrupted in a number of tumors. Therefore, this proposal is an attempt to understand the function of Hmgi-c and its role in the interdependent processes of proliferation and tumorigenesis. Based on the human tumor studies, the first specific aim will investigate the nature of the HMGI-C gene products required in tumorigenesis. Transgenic mice will be generated that harbor various HMGI-C transgenes whose structure is similar to that seen in human tumors. Transgenic mice which express wildtype HMGI-C in an inappropriate cell type will be analyzed for tumor formation and the tumors characterized for activation of the endogenous Hmgi-c alleles. Tissue culture studies imply that HMGI-C is necessary for tumorigenesis. Therefore, the second aim will examine the susceptibility of the pg mouse mutant to tumorigenesis by different oncogenic stimuli in various tissues. The third aim will analyze the effect of Hmgi-c expression on proliferation and the cell cycle. This will be performed on pg and wildtype embryonic fibroblasts grown under different culture conditions. Finally, in order to elucidate the molecular components of the Hmgi-c pathway, putative target genes will be identified by differential display. The long term objectives are to understand the role of Hmgi-c in growth and development. This will ultimately explain the phenotype of the pygmy mouse and how its disruption or deregulation leads to tumorigenesis including uterine leiomyoma, a major cause of hysterectomy in humans.

During the following year, we shall continue our work with Escherichia coli topoisomerases-enzymes that catalyze the concerted breakage and reunion of DNA. We will try to prepare large quantities of E. coli DNA gyrase for physical and immunologic analysis. There are two different covalent complexes of DNA to gyrase and we want to determine the nature of the bonds and if the complex represents an obligatory reaction intermediate. We are particularly interested in the mechanism of energy transduction. We have discovered a new E. coli topoisomerase and we will study its enzymatic, physical, and functional properties. Finally, we are continuing our search for still more E. coli topoisomerases.

Brain-derived neurotrophic factor (BDNF) and its TrkB receptor have been implicated in the control of energy balance in both humans and mice. Previous studies suggest that BDNF expressed in the ventromedial hypothalamus (VMH) plays a critical role in regulating energy balance. BDNF protein is synthesized in both neuronal somas and dendrites. In this application, we will examine the role of dendritic local BDNF synthesis in the control of energy balance. Aim 1 is to determine if leptin regulates local synthesis of BDNF in dendrites. We will employ green fluorescence protein reporter constructs and synaptoneurosome preparations to examine the effect of leptin on dendritic transport and translation of BDNF mRNA. Aim 2 is to determine if dendritically synthesized BDNF in the VMH regulates food intake and body weight. We will attempt to rescue the obese phenotype in a Bdnf mouse mutant which lacks dendritic local BDNF synthesis through viral expression of dendritically localized BDNF mRNA or somatically localized BDNF mRNA in the VMH. Aim 3 is to examine if lack of local BDNF synthesis alters morphologies of dendritic spines of TrkB-expressing hypothalamic neurons. There are TrkB-expressing neurons in the hypothalamic nuclei important for the control of energy balance, such as the arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH). We will examine spine morphologies of TrkB-expressing neurons in the ARC and DMH in the mouse mutant deficient in local BDNF synthesis. PUBLIC HEALTH RELEVANCE Identification and characterization of new molecules and signaling pathways that control energy balance will offer opportunities for designing improved obesity treatments. If successful, findings from this proposed project will suggest that mutations in proteins involved in transport and translation of dendritic mRNA will increase susceptibility to obesity. Therefore, these proteins could be potential drug targets for obesity treatment. [unreadable] [unreadable] [unreadable]

Activation of the renin-angiotensin system (RAS) is associated with increased cardiovascular death. A critical component of this system is angiotensin converting enzyme (ACE), which cleaves angiotensin I to angiotensin II (Angll). In humans, increased Angll levels are associated with an increased ventricular arrhythmic risk. Nevertheless, the reasons for the increased risk are unclear. One critical effect of RAS activation is Angll-induced oxidative stress mediated, in part, by increased NADPH oxidase activity. We hypothesized that oxidative stress caused by Angll induced cardiac arrhythmias. To investigate this, we developed a cardiac-restricted ACE overexpression mouse (ACE 8/8) that showed an increased risk of sudden death in the absence of heart failure or structural heart disease. Intracardiac recordings demonstrate poor conduction and various forms of AV nodal block. Ventricular pacing readily induced ventricular tachycardia. This phenotype is associated with reductions in cardiac sodium channels. We have developed preliminary data that Angll-mediated oxidative stress activates the transcription factor NFkB resulting in downregulation of the cardiac sodium channel. In this proposal, we hypothesize that increased Angll leads to oxidative stress which in-turn alters sodium channel transcription through NFkB activation. An altered ion channel level contributes to the ACE 8/8 mouse arrhythmic phenotype. This proposal is a plan to dissect the steps in this putative cascade and to identify which are proximate causes, which are upstream events, and which are associated but not causative steps. In each aim, we will establish to what extent measures of the sodium channel, NFkB activation, oxidative stress, and arrhythmic risk are altered by the disruptions in the proposed signaling cascade. Specific Objectives. Specific aim 1: To establish to what extent Angll-mediated signaling is responsible for the sodium channel regulation in our ACE overexpression model. Specific aim 2: To establish to what extent NADPH oxidase activation is responsible for the sodium channel regulation in our ACE overexpression model. Specific aim 3: To establish to what extent increased NFkB activation is responsible for the sodium channel regulation in our ACE overexpression model. This application presents a novel hypothesis about why RAS activation causes arrhythmias, contributing to atrial fibrillation (AF) and heart failure (HF)-associated sudden death.

The P.I. and collaborator's laboratories were the first to identify novel cytoplasmic foci known as GW bodies (GWBs), which are now known to be enriched with human autoantigens important in the effector arm of the RNA interference (RNAi) pathway. The initial characterization was achieved via expression cloning using a human autoimmune serum containing antibody to GW182, an 182KD protein autoantigen with unique glycine-tryptophan (GW) repeats that is specifically localized to GWBs. GW182 was shown to be associated with selected messenger RNAs (mRNAs) and the protein Ago2 which is the key enzyme in the RNAi mediated processing of mRNA. Our recent data showed that short interference RNAs (siRNAs) and microRNAs (miRNAs) are localized to GWBs and effective RNAi function requires intact GWBs. Taken together, the working hypothesis is that GWBs are novel sites regulating cytoplasmic mRNA levels via the siRNA/miRNA dependent RNAi process by maintaining stability and/or controlling degradation of mRNA. To date, human autoantibodies to GWBs are known to have overlapping subsets that recognize GW182, Ago2, and other autoantigens being characterized in the P.l.'s lab. Three Specific Aims have been designed to help further characterize the biological significance of GWBs in normal and in systemic rheumatic diseases including systemic lupus erythematosus (SLE) and Sjogren's syndrome (SjS) based on the preliminary data linking anti-GW182 autoantibody to these 2 autoimmune diseases. The interest in SjS is further developed with the observed overexpression of GW182 in certain foci of SjS minor salivary gland biopsies and the co- expression of anti-52kD SSA/Ro autoantibody in anti-GW182 positive autoimmune sera. The goal of the proposed study is to explore the biological functions of these novel subcellular foci which may shed some insight as to their involvement in the pathogenesis of SLE and SjS. Specific Aim 1 will address the biology of GW182, Ago2 and other closely associated autoantigens detected in GWBs. Specific Aim 2 will characterize anti-GWB antibodies in established mouse models of autoimmune diseases and test the hypothesis that autoimmune responses to GWBs are related to deregulation of GWB expression in target organs possibly resulting in defects in RNAi. Specific Aim 3 will characterize the significance of anti-GWB antibodies in human diseases and follow up on the preliminary data that GW182 and SS-B/La, a known major autoantigen in SjS and SLE, were overexpressed in foci of SjS salivary gland biopsies. The proposed studies will help define the biological role of GWBs in the pathogenesis of SLE and SjS. The animal models will provide the means to test hypotheses that are relevant to the induction of these autoantibodies targeting GWBs. Implication for mi RNA in cancer has been described recently and this proposed study will address whether there may be a role for miRNA deregulation in autoimmune diseases.

Human cytomegalovirus, the cause of serious infections in immunocompromised individuals, encodes several functions that block programed cell death. These functions are likely to play important roles in viral replication and pathogenesis by modulating the intrinsic impact of the virus on the host cell as well as by counteracting extrinsic signals of apoptosis that arise as a result of innate and adaptive clearance mechanisms mounted by the host. This continuation of research on CMV replication will focus on three genes encoding anti-apoptotic CMV functions: UL37, a viral mitochondrial-localized inhibitor (vMIA), UL36, a viral caspase 8 inhibitor (vlCA), and UL144, a TNF receptor homolog that may disrupt signalling. This project will investigate roles for these gene products preventing (1) intrinsic, stress-related induction of apoptosis that results from expression of viral functions in the course of productive replication, and (2) extrinsic signals such as those resulting from the immune response, including engagement of death domain-signalling receptors or introduction of pro-apoptotic granzymes. The two most potent viral inhibitors, pUL37x1/vMIA and gpUL36/vlCA, will be subjected to detailed mutagenesis to map specific domains that contact cellular targets. Minimal-size, fully active versions of each will be made and tested in the context of the virus for activity. The pro-apoptotic signals induced during infection will be evaluated to identify whether virion structural or immediate early gene products, such as the regulatory gene IE2-p86, trigger apoptosis. A role for UL37, UL36 or UL144 gene products in preventing apoptosis or other aspects of viral replication will be investigated by performing microarray analysis using viral mutants to assay changes in expression of 43,000 human cDNAs representing all human CMV genes. Evaluation of these functions will be carried out in cell culture with human CMV mutants and in infected mice using murine CMV, either by mutating the gene homolog or by replacing/adding the human CMV function to the murine CMV genome. The role of these functions in evading cytokine/death receptor or cell-mediated immune responses will be determined in strains of mice lacking relevant effector mechanisms. Through these efforts we expect to gain a complete understanding of the role of CMV genes that promote efficient replication or deflect the host immune response by preventing host cell apoptosis.

PROJECT SUMMARY The Department of Surgery at Washington University School of Medicine (WUSM) is one of the nation's leading academic surgery departments, with a strong and uncompromising commitment to training the next generation of academic surgeon-scientists. The Surgical Oncology Basic Science and Translational Research Training Program is a critical component of this overall training goal. NCI support will provide up to eight surgical trainees (i.e. postdoctoral trainees) from general surgery and other surgical subspecialties the opportunity to develop essential skill sets in basic science, translational, and public health research. The Surgical Oncology Research Training Program has evolved in parallel with a dynamic training environment at WUSM, and currently takes advantage of unique resources in the Department of Surgery, the Siteman Cancer Center, the Institute of Clinical and Translational Sciences, the Department of Surgery's Division of Public Health Sciences, and the Division of Biology and Biomedical Sciences to develop customized and highly structured formal didactic and mentored research experiences for individual surgical trainees. The success of the program is demonstrated by the important research accomplishments made by trainees working with program faculty, and the long-term success of trainees in academic medicine. 21/33 trainees (64%) who have completed the research training program and their clinical training in the last fifteen years remain in academic medicine, significantly better than published metrics (16-44%). The Surgical Oncology Research Training Program continues to evolve, and we restructured the program in 2014. Changes made at that time included additions to the program leadership, development of two distinct research tracks (basic science track, and translational research/public health/clinical effectiveness track), and reduction in the number of training slots to allow for an increase in the tuition budget. These changes have been very successful, and will ensure that the Surgical Oncology Research Training Program will continue to maintain excellence at the forefront of two different surgical oncology research paradigms, basic science research, and translational/public health/clinical effectiveness research. NCI support will allow WUSM to continue this highly successful research training program, providing the next generation of surgeon-scientists with the research training required to succeed in an increasingly competitive research environment.

Holoprosencephaly (HPE) is a developmental defect of the forebrain, which occurs in 1 out of 250 pregnancies. HPE is characterized by either forebrain truncation or failure of forebrain septation and various other midline defects. In the developing mouse embryo, the forebrain is first specified during gastrulation by the anterior visceral endoderm (AVE), an extra-embryonic tissue. The AVE specifies forebrain in the underlying embryonic tissue by blocking the signals from the primitive streak that provide posterior character to the tissue. As development proceeds, the AVE is displaced by the anterior definitive endoderm (ADE), which also has forebrain specification activity. Cripto, which is expressed in the posterior aspect of the gastrulating embryo, is critically involved in AVE migration and ADE specification. Cripto null embryos have severe gastrulation defects, whereas Cripto hypomorphs have HPE. I have isolated a mouse line, gonzo, that also displays gastrulation and HPE phenotypes. Furthermore, I have mapped the gene responsible for this mutation to a small region of chromosome 1, and have identified a mutation in Pig-N, a GPI-biosynthesis enzyme. Because the phenotypes of Cripto and gonzo mutants are strikingly similar, and because Cripto is a GPI-anchored protein, I will test the following hypotheses: First, HPE results from either an AVE migration defect or failure to specify the ADE. Second, the Pig-N mutation is responsible for the HPE phenotype. Third, aberrant GPI modification of Cripto disrupts Cripto signaling - and is the causative factor for the HPE phenotype. In this proposal I will complete the following aims: Aim 1: I will further characterize the HPE phenotype, and specifically look at specification of the AVE and ADE by examining molecular markers known to be involved in head morphogenesis. Aim 2: I will confirm that the mutation in Pig-N is responsible for the HPE phenotype and determine its effect on Pig-N stability, localization and activity. Aim 3: I will determine Cripto activity in gonzo embryos, and will attempt to rescue the phenotype by artificially targeting Cripto to its site of activation. Holoprosencephaly (which occurs in 1 out of 250 pregnancies) is characterized by forebrain defects;the most severe cases result in a single, cyclopic eye. I have identified a new gene in the mouse that results in holoprosencephaly when it is mutated. This proposal will identify the normal function of this gene thus leading to a better understanding of the causes of holoprosencephaly.

My specific aims are to examine the role of cancer stem cells and their niche microenvironment in malignant progression of subtypes of human breast premalignancy, ductal carcinoma in situ (DCIS). The objectives are to test whether premalignant cell lines, SUM225, MCF10ATDCIS.com, and subtypes of human DCIS contain distinct cancer stem cell subpopulations which exhibit unique cancer stem cell properties of increased self-renewal potential, quiescence, and tumorigenicity. My strategy is to use fluorescent activated cell sorting (FACS) and known surface markers to isolate stem/progenitor and differentiated subpopulations and to examine their cancer stem cell properties which include, 1) in vitro self-renewal and differentiation potential by standard mammosphere assay and colony formation in Matrigel, respectively, 2) in vivo long term and short term self-renewal by using a novel strategy of xenotransplantation into humanized stroma of mice fat pads, 3) tumorigenicity by growth rate and potential to form invasive lesions, and 4) quiescence by long term label retaining studies. Furthermore, the role of endothelial cells in establishing a cancer stem cell niche microenvironment will be examined by in vitro co-culture and in vivo co-transplantation studies. My rationale is that cancer stem cells and normal tissue stem cells express similar surface markers by which they may be identified and characterized. Once cancer stem cells are identified, efforts will be aimed at defining their unique gene profiles. These studies will provide a basis for discovery of molecular targets for risk assessment, prevention of recurrence, and malignant progression tailored to subtypes of DCIS. My long term career goal is to lead a research group focused towards prevention of human breast cancer malignant progression in a translational research environment. I served as an academic and clinical pharmacologist for seven years before obtaining a Ph.D. and post-doctoral training in basic sciences in cellular signaling and mouse mammary gland stem cell biology. The Career Development Award is an excellent opportunity to gain additional experience in research in human breast cancer in collaboration with the Breast Cancer Center at Baylor College of Medicine. Baylor College of Medicine houses exceptional research centers and facilities and provides an ample opportunity for scientific collaboration, and education. Relevance: Breast pre-cancer lesions may contain a rare population of cancer stem cells. Cancer stem cells may be unique to different types of pre-cancer lesions and may explain why some patients will experience recurrences or develop invasive cancers while others do not. My goal is to examine the role of cancer stem cells in invasive progression of subtypes of pre-cancer lesions and to develop individualized strategies for risk assessment and prevention of invasive breast cancer.

Optimal management of patients with congenital heart disease often depends on the ability to monitor pulmonary hemodynamics and assess pulmonary vascular impairment. Need for improved techniques has prompted investigations of relationships between abnormal pulmonary circulations and pulmonary blood velocity patterns, which can be observed noninvasively with pulsed Doppler ultrasound. Features commonly associated with pulmonary hypertension are increased flow reversal in the main pulmonary artery and decreased acceleration time (time from onset of systole to peak velocity). However, recent studies indicate that acceleration time is primarily a function of arterial compliance; flow reversal is directly related to the curvature in the pulmonary outflow tract and/or peripheral vascular resistance. These findings imply that idealized relationships with pressure may exist only in the presence of expected, but not guaranteed, structural changes. Pulmonary vascular impairment is commonly associated with lack of response to vasodilators: however, changes in velocity profiles and the associated pulmonary input impedance spectra may be better indicators of responsiveness. This study will address these issues through experiments designed to identify and quantitate the determinants of the velocity profiles in animal models developed to produce the pulmonary vascular changes associated with either elevated pressure or flow from infancy through childhood. Newborn lambs will be chronically instrumented to measure pulmonary pressures, velocity profiles and axial dimensions from birth to 6 months of age. Elevated pulmonary artery pressure or blood flow will be produced via monocrotaline pyrrole injections or arteriovenous shunts, respectively. Acute interventions designed to assess changes in vascular reactivity will be made during data collection sessions. After natural histories are determined, the ability of long-term pharmacological interventions to alter the high pressure model history will be assessed. Data collected will be used to determine 2- and 3- dimensional velocity profiles, resistance, compliance and shape changes and input impedance spectra. After the terminal hemodynamic study, casts of the right ventricle and proximal pulmonary arteries will be made in situ under hemodynamically matched systolic pressure. The casts will be CT scanned and their structure committed to computer memory, from which 2- and 3-dimensional images can be reconstructed. Algorithms will be developed for quantifying curvature and torque of the main trunk, flow divider offset, taper, bifurcation angles, and segment dimensions. Experiments designed to test instrumentation and to determine the effects of changes in peripheral vascular resistance as a result of selective embolization of individual lungs will be conducted in acute preparations. Techniques for measuring the pulmonary input impedance spectrum and extracting its salient features will be developed using simple models of the pulmonary vascular bed. Finally, results obtained in the animal models will be compared to available clinical data, thus testing the belief that better understanding of the determinants of the velocity profile will lead to improved patient diagnosis and care.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Complex carbohydrates on cell surface and extracellular proteins play essential roles in molecular recognition events critical to cell growth and differentiation. In humans, the emerging congenital disorders of glycosylation (CDGs) include severe multisystemic syndromes linked to abnormal protein glycosylation;most of the identified CDGs reflect disruptions of glycan biosynthetic pathways. Recent studies have revealed that glycosylation abnormalities and CDG clinical presentations can also be caused by defects in intracellular protein trafficking. Genetically tractable simple organisms provide an opportunity to study the regulatory mechanisms of these conserved pathways. We have identified three genes in the nematode Caenorhabditis elegans which, when mutated, elicit binding of the lectin Wheat Germ Agglutinin on the cuticle surface, suggesting a role in the modulation of one or more glycosylation pathways. These mutations inactivate members of a conserved protein family (p24) implicated in cargo selectivity of endoplasmic reticulum to Golgi transport. Interestingly, these mutants exhibit specific neuronal abnormalities (our preliminary results), offering the opportunity to explore the role of p24 activity on the development of the nervous system. We use genetic and biochemical approaches in C. elegans to pursue the following aims: 1) examine the relationship between p24 activity and protein glycosylation status, by analysis of metabolically labeled glycoproteins from wild type and mutant embryonic cells and lectin purified fractions;2) explore extracellular signaling pathways as downstream targets of p24 activity, using double mutant analysis;and 3) exploit abnormal lectin binding to the cuticle surface to identify additional genes involved in surface protein glycosylation and trafficking.

Adoptive cellular immunotherapy (ACI) treats disseminated cancers with ex-vivo expanded tumor-reactive lymphoblasts. These cells traffick poorly into target lesions contributing to the mixed results observed in therapeutic trials. This grant focuses on the mechanisms and clinical consequences of leukocyte recruitment into sites of metastic disease during ACI with vaccine-primed, ex-vivo expanded lymph node cells. New preliminary studies show that a single-dose of TNF-alpha, administered intraperitoneally 4 hours prior to infusion, increased the recruitment of infused, vaccine-primed cells into lungs with established micrometastases. This treatment promoted inhibition of tumor-growth by these cells regardless of the protocol used for ex-vivo expansion. Pretreatment with TNF-alpha alone was inactive at the concentration used (one mu g/animal, single dose) and did not produce visible symptoms. New preliminary studies also suggest that the vaccine-primed cells responsible for inhibition of tumor growth (the Tc/h-1 subsets) can be distinguished from those that suppress this response (the Tc/h-2 subsets) based on high levels of selectin-ligand expression. The PI proposed that selectin-mediated recruitment of the Tc/h-1 cells into sites of metastatic disease is essential for inhibition of tumor growth. Consequently, transient induction of selectins on the vasculature associated with metastases as well as enrichment of the Tc/h-1 subset in the population of infused cells should improve clinical outcome. Furthermore, selectin-based fractionation of vaccine-primed and expanded cells should both enrich the Tc/h-1 subsets and deplete the Tc/h-2 subsets. Specific Aim 1 investigates the adhesion receptors required for the recruitment of tumor-draining lymph node cells (TDLN) and host leukocytes into lungs with 3-day established pulmonary micrometastases. TDLNs labeled with stable fluorescent dyes or congenic markers, receptor-specific antibodies and mice with disrupted selectin-genes will be used in these experiments. The relative contributions of TDLN and host cell recruitment to clinical outcome will also be determined. Specific aim 2 investigates the adhesion receptor phenotype of TDLN and VPLN (human-counterparts currently in clinical trials) that secrete high levels of IFN-gamma and GM-CSF (Tc/h-1) and high levels of IL-4 and IL-10(Tc/h-2). In addition, selectin-based fractionation procedures will be developed that separate the Tc/h-1 and Tc/h-2 subsets. The fractionated populations will be compared to the starting population and each other for activity against malignant cells in vitro and in vivo. Specific aim 3 determines whether induction of endothelial adhesion receptors on tumor-associated vessels augments TDLN recruitment and inhibits tumor-growth. The new preliminary studies cited above support for this hypothesis. Proinflammatory cytokines, such as TNF-alpha, will be administered prior to infusion of TDLNs. The impact of these preparative regimens on the behavior of standard and fractionated TDLN (Tc/Th-1 and Tc/Th-2 subsets; high and low selectin-ligand expression) will be established. The impact on TDLN activity against poorly immunogenic or large neoplasms, on the number of cells required for inhibition of metastases and on the dose or duration of systemic IL-2 therapy will be assessed. The role of adhesion receptors and recruitment (TDLN and host cells) will then be determined for preparative regimens that improve clinical effectiveness. The extensively revised grant rectifies the weakness detected by the initial review. The new preliminary studies demonstrate the potential for direct and immediate benefits in ACI. Many of the reagents and technologies used in the grant are recently developed by the PI and not currently available elsewhere. The project complements the funded clinical trial of ACI at the University of Michigan (CA69102) and will enhance the scientific return from this project. Finally, the collaboration between the PI and co-PI will enhance productivity and ensure the rapid transfer of information from a pre-clinical to a clinical setting.

The reactions catalyzed by farnesyl pyrophosphate synthetase and squalene synthetase are the fundemental building steps in the cholesterol biosynthetic pathway. This proposal addresses mechanistic and topological questions for substrates and reactive intermediates in these two transformations. In both instances, molecules will be synthesized which covalently link partners present during catalysis but which are not normally covalently linked. A novel class of bisubstrate analogs with join homoallylic and allylic substrates for farnesyl pyrophosphate synthetase is being synthesized. These molecules are designed so the range of topologies which can bind and react is restricted. A study of the kinetic properties of the analogs and elucidation of products and stereochemistries should permit us to deduce permissible topologies for binding and provide information concerning the location of active-site functionality in the enzyme. The conversion of presqualene pyrophosphate to squalene is thought to be directed by the pyrophosphate anion during rearrangement of the presqualene cyclopropylcarbinyl cation. The substrate, presqualene pyrophosphate, is bound and the components of the ion pair are generated during catalysis. Kinetic studies show that squalene synthetase is inhibited when inorganic pyrophosphate and ammonium analogs of the carbocation are present in the buffer. It should be possible to eliminate entropic considerations in the binding of the two species by covalently linking the components of the ammonium-pyrophosphate ion pair. Ion pair analogs should be potent inhibitors of squalene synthetase. Squalene synthetase is an attractive target for inhibition of cholesterol biosynthesis because the enzyme lies at the branchpoint to sterols and regulation at that point permits expression of the other branches of the isoprene pathway. The research described in the proposal combines the traditional disciplines of enzymology (purification, kinetic methods) and organic chemistry (synthesis, isolation-identification) to study farnesyl pyrophosphate synthetase and squalene synthetase.

Myotonic dystrophy type 1 (DM1) is a relatively common form of muscular dystrophy. The genetic basis is an expansion of CTG repeats in the 3' untranslated region of DMPK, a gene encoding a protein kinase expressed in skeletal, cardiac, and smooth muscle, and in neurons. This unusual mutation gives rise to RNA dominance, in which expression of RNA containing an expanded CUG (CUG^'') repeat leads cell dysfunction. The mutant RNA accumulates in nuclear foci and initiates a complex cascade of downstream events, such as, defects in the regulation of RNA splicing. An attractive therapeutic approach, therefore, is to attack the problem at its root cause, by accelerating the clearance of the toxic RNA. To this end, we are proposing to develop RNase H-active antisense oligonucleotides (ASOs) targeting the mutant human DMPK {mut-hDMPK) mRNA. Previous studies have indicated that biodistribution and activity of ASOs in skeletal and candiac muscle is low. In contrast, we have found that systemically-delivered ASOs are highly active in muscle of wild-type mice, when targeted against a transcript that is retained in the nucleus - presumably because this is the compartment in which RNase H is active. We therefore postulated that CUG-expanded transcripts may also show efficient knockdown in muscle, because they also are retained in the nucleus. Consistent with this idea, subcutaneous administration of ASO for four weeks caused highly effective knockdown of CUG(R)'' transcripts in muscle, reversal of RNA splicing derangements, and rescue of myotonia in transgenic mice. We seek now to develop optimally-effective, systemically-active ASOs that target mut- hDMPK transcripts for cleavage in skeletal and cardiac muscle. Development plans call for identification of ASOs that are highly active in cells, selection of ASOs that show optimal mut-hDMPK knockdown in transgenic mice, elimination of ASOs that show unacceptable toxicity in rodents and monkeys, and then IND- enabling toxicology/pharmacology studies using GMP-manufactured drug. To accomplish these goals we have formed an academic-commercial collaboration that includes all of the scientific, clinical, regulatory, and manufacturing expertise that is needed to bring a drug treatment for DM1 to the clinic. PUBLIC HEALTH RELEVANCE: Myotonic dystrophy is an inherited disease that causes progressive disability and premature death. No treatment is currently available that can improve the symptoms or slow the disease progression. The goal of this project is to develop a drug treatment for myotonic dystrophy.

The objectives are to continue our studies to: 1) identify, isolate and characterize intracellular pools of membrane bound glycoconjugates undergoing fast axonal transport, 2) to characterize different functional compartments of the neuronal cytoskeleton related particularly to phosphorylation of component and associated polypeptides of cytoskeletal organelles. Immunocytochemical analysis will be used to study the distribution of glycoprotein antigens in cerebellum whose biochemical properties mimic the cytochemical properties of specialized smooth membrane systems in Purkinje cell axons and dendrites. We will use specific radioisotope labeling together with pharmacological manipulations of oligosaccharide processing and axonal transport to identify the origin and fate of newly synthesized glycoconjugates destined for fast axonal transport in retinal photoreceptor and ganglion cells. Enzyme-linked cytochemical, autoradiographic and biochemical analysis will be used to begin to characterize oligosaccharide chains of transported glycoconjugates compared to those that have reached the presynaptic terminal of retinal neurons. We will use immunocytochemical and immuno-blot analysis of tissue sections, cultured neurons and isolated fractions of cytoskeletal organelles to study the intracellular compartmentalization of the phospholipid/Ca2+-dependent protein kinase (PKC) and its substrates in axons. Endogenous and exogenous PKC will be used in in vitro phosphorylation experiments for comparison with in vivo labeling patterns to elucidate specific substrates for the enzyme among the cytoskeletal polypeptides. Retinal tissue slices and cultures of retinal and sympathetic ganglion neurons will be used to ask the question whether or not PKC translocates from the cytosol to the plasma membrane of neurons in response to physiological stimulation or pharmacological activation of PKC. We will use non-hydrolyzable analogs of ATP to probe microtubule deficent axons of Myxicola infundibulum for the presence of "kinesin" type translocator molecules. Kinesin preparations from rat brain will be used to test for the presence of axonal polypeptides associated with the cytoskeleton, particularly the tau proteins and PKC.

Histologic and ultrastructural studies disclosed a layered structure, resembling that of left ventricular outflow tract endocardium, in fibrous rings excised at operation from patients with discrete subaortic stenosis.

Significant strides have been made in our understanding of the initial events of sensory transduction in taste cells during the last two decades, yet little is known about the mechanisms of intercellular com- munication within the taste bud. Only one-fifth of the taste cells in a mouse taste bud havemorphologi- cally identifiable "classical" synapses from the taste cell onto an afferent nerve fiber. Little, however, is known about the molecular features of neurotransmitter release at these sites in taste cells. It is significant that not all taste cells thought to play a role in sensory transduction have classical synapses. Taste cells that display gustducin immunoreactivity respond to bitter compounds, yet our preliminary data suggest that they do not have classical synapses onto nerve processes. Howthen does information pass from these bitter-sensing cells to the central nervous system?We have observed taste cells in the mouse that have unusual contacts with nerve processes in which subsurface cisternae (SSC) of smooth endoplasmic reticulum or large, atypical mitochondria with tubular cristae are located only at the close appositions with nerve processes. This proposal explores the possibility that these zones of close apposition may serve as nonclassical synapses, i.e., zones where information is trans- mitted from taste cells to nerve fibers via mechanisms other than the classical synapses. The goalof this grant application is to test the hypothesis that both classical synapses andnonclas- sical taste cell-nerve fiber contacts are important in transmission of gustatory information from taste cell to nerve fiber. During the next funding period we will utilize immunocytochemistry, in situ hybridization and electron microscopy to investigate both classical and novel contacts between taste cells and nerve fibers to accomplish the following aims: Specific Aim 1. What molecular machinery is present at morphologically "classical" synapses? Specific Aim 2, What is the nature of nontraditional taste cell-nerve fiber contacts?

An important aspect of brain aging is the increased glutamate (Glu) release and accumulation in the extracellular space of neurons. Age-associated increases in extracellular Glu occur because of partial loss of activity of Glu transporters. Essentially all neurons in the central nervous system (CNS) are exposed to elevated extracellular Glu, yet not all brain regions suffer equally. The sensitivity of certain neurons to the toxic effects of Glu produced through Ca2+- and oxidative stress-mediated processes, increases with age. The reasons for differential vulnerability of certain neurons to Glu are still not known. Also, no animal model of age-associated increases in Glu release in CNS is available to determine how excess Glu produces its effect on aging neurons. We have generated transgenic (Tg) mice that have extra copies of the gene for Glu dehydrogenase 1 (GLUD1), a mitochondrial enzyme considered to be a rate-limiting, step in the biosynthesis of Glu as a transmitter. The GLUD1 transgene, introduced under the control of a neuron-specific promoter, is expressed only in neurons. GLUD1 mice have higher levels of depolarization-induced Glu release than wild type (wt) and suffer losses in specific neuronal populations. GLUD1 mice also have a shortened life span without exhibiting severe neurological dysfunction. The hypothesis being tested is that excess extracellular Glu in aging brain initiates events that lead to altered metabolic states in CNS, damage to select populations of neurons, an imbalance between damage and recovery, metabolic stress in peripheral tissues, and decreased longevity. Short-term objectives are: a) To quantify changes in longevity and protein/ DNA oxidation in brain and other tissues of hyper-glutamatergic and wt mice;b) To determine changes in metabolism, gene expression, and morphology of vulnerable and resistant neurons;and c) To characterize a signaling protein complex involved in active neurite remodeling and define how Glu hyperactivity and aging affect this complex. The long-term objectives are to understand the molecular and cellular processes that link increased Glu activity in CNS to age-dependent changes in neuronal structure and function, but without neurological disease, and to identify potential targets for therapeutic intervention. To test the hypothesis, we developed the following specific aims: 1) Assess longevity and protein and DNA oxidation levels in brain and other tissues of wt and Tg mice during aging;2) Determine the effects of neuronal GLUD1 overexpression and of aging on structural, metabolic and gene expression changes in vulnerable and resistant neurons;and 3) Determine the age-dependent changes in expression, composition and activity of a Ca2+-sensitive, dendrite-growth controlling complex in GLUD1 and wt mice. These studies make use of a novel animal model of age-associated hyperglutamatergic states to probe mechanisms of differential neuronal vulnerability and novel molecular mechanisms of neuronal recovery from stress.

This is an application for a 2nd NIMH K02 award. The applicant's research has dealt with the psychobiology of stress in young children, emphasizing activity of the hypothalamic-pituitary-adrenocortical (HPA) system an adequate understanding of the psychobiology of stress in childhood, however, requires integration of our rich understanding of psychosocial processes in human development, with our emerging understanding of multiple stress-sensitive physiological systems. Much of the first K02 period has been spend developing collaborative relations and beginning expertise that has allowed me to obtain funding for a 5-year period to test an integrative (temperament-coping resources-stress) psychobiologic model of stress in childhood. According to this model, a "stress reactive" temperament (the biological components of which are assessed through baseline EEG asymmetry, vagal tone, basal HPA activity, and positive late components of the ERP) influences the child's likelihood of perceiving events as potentially threatening. However, activation of physiological stress reactions (cardiac, HPA, and immune) are presumed to follow from expectations that the threat will be realized. Mediating these reactions are the coping resources (person and situation factors) available to the child. The proposed studies are designed to integrate 3 major literatures: (1) the physiology of infant/child temperament, (2) socioemotional development, including attachment research, and (3) psychobiologic studies of infant/child stress. This Career Plan emphasizes the need to extend this research to the study of child health (e.g. stress-immune functioning-infectious disease) issues in pediatric research (i.e., pain regulation, colic), high risk populations (i.e., maltreated children, early deprivation(, and to include other systems important in the regulation of stress (i.e., sleep as a process variable). Preliminary work, collaborative relations (with T. Boyce, R. Barr, R. Dahl, S. Suomi, M. Carlson, D. Cicchetti), and pilot data are described to support need for release time to enhance training and research into these related areas. The Institute of Child Development and the University of Minnesota provide rich resources for the candidate's career development. The institute is fully committed to this award as evidenced in the last K02 period. The candidate's would continue to be able to commit 80 percent time to research, with a minimal teaching load and additional support for travel and the development of collaborations within and without the university.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent developments in MR data acquisition technology, in part driven by the advances in the types of phased-arrays developed by Project 2, are starting to yield images that show anatomical features of the hippocampal formation at an unprecedented level of detail, providing the basis for hippocampal subfield measurement. Because of the role of the hippocampus in human memory and its implication in a variety of disorders and conditions, the ability to reliably and efficiently quantify hippocampal subfields through in vivo neuroimaging is of great interest to both basic neuroscience and clinical researchers. In the current cycle of the P41 we are in the process of developing a fully-automated method for segmenting the hippocampal subfields from ultra-high resolution MRI data. Using a Bayesian approach, we build a computational model of how images around the hippocampus are generated, and use this model to obtain automated segmentations. We validate the proposed technique by comparing our segmentation results with corresponding manual delineations in ultra-high resolution MRI scans of five individuals.

The aim of this protocol is to determine the clinical safety and efficacy of the excimer laser system in patients with coronary artery disease. Specific goals are 1)to determine the adequacy of the procedure alone in reducing obstructive atherosclerotic lesions of the coronary arteries as determined by repeat angiography at six months, and 2)to determine the adequacy of excimer laser angioplasty in bypass graft recanalization.

The experiments in this proposal investigate how two guidance cues, nerve growth factor (NGF) and Semaphorin3A (Sema3A), regulate the migration of the growing tips (growth cones) of sensory (DRG) axons. NGF is produced in targets of sensory neurons, like skin. Immature axons navigate to the skin, then, invade and branch to form sensory endings. However, if nerve growth factor is absent in skin, sensory innervation does not occur. Similar innervation deficits occur if neurotrophins, NGF, BDNF or NT3, are absent from other target organs. Sema3A is expressed in central and peripheral tissues that sensory growth cones avoid, and animals with defective functions of Sema3A or its receptors have defasciculated and misrouted axons. Knowledge of how NGF and Sema3A regulate the motility of sensory axonal growth cones can be applied to understanding developmental deficits in innervation and neural circuits. In addition, neurotrophins can promote axonal regeneration in the central and peripheral nervous systems, and Sema3A contributes to the inhibitory environment that blocks axonal regeneration. Thus, our findings will be generally relevant in repairing diseased and damaged neural circuits. The long term goal of our project is to understand how guidance cues, like NGF and Sema3A regulate growth cone migration, as sensory axons innervate skin targets. At the tip of a growing axon, protrusive motility explores the environment, makes adhesive contacts to promote axonal growth and initiates responses that direct axonal pathfinding. Activation of trkA receptors by NGF triggers polymerization of actin filaments and protrusion of the growth cone margin. Sema3A activates receptors to inhibit protrusion and keep growing axons from straying on their way to peripheral and central target zones. We will test the hypothesis that NGF and Sema3A act through three actin-associated proteins to mediate the motility of growth cones. The aims of this proposal are to investigate the roles and necessity of ADF/cofilin, the Arp2/3 complex, and ERM proteins in the regulation of growth cone behaviors. These proposed experiments involve in vitro and in vivo studies, using DRG sensory neurons from chicken embryos. Cellular and molecular methods, time lapse microscopy, immunocytochemistry, image analysis, and neuronal transfection will be used in three aims. The first aim investigates the roles of ADF/cofilin, Arp2/3 and ERM proteins in mediating the responses of sensory neuronal growth cones to NGF. ADF/cofilin sever actin filaments to generate new barbed ends for actin polymerization, the Arp2/3 complex may nucleate actin filaments and ERM proteins link actin filaments to the plasma membrane. The second aim will investigate whether inhibition of these activities mediates the loss of protrusive motility and avoidance responses that Sema3A induces in growth cones. The third aim involves studies to investigate the roles of these proteins in mediating in vivo responses of sensory axons to NGF and Sema3A.

The broad, long-term objective of this proposal is to evaluate the role of education in the incidence of diabetic complications, in a large, well-characterized population, within an integrated, managed care setting Kaiser Permanente (KP). This study will take advantage of a cohort begun in 1994 of approximately 78,000 survey respondents (83% response rate) for whom self-reported educational attainment data was collected. Using the survey date as baseline, follow-up data (up to 14 years) will be collected on mediating variables and the following outcomes: incident complications, including myocardial infarction, stroke, congestive heart failure, end-stage renal disease, lower-extremity amputation, proliferative retinopathy, acute metabolic events, costs and death. Second, a new survey will be conducted in a stratified random sample of approximately l 5,000 type 2 diabetic patients, ages 50-75, with equal representation of African American, Caucasian and Latinos. This new survey will provide a rich source of data on education and potentially modifiable factors that explain the link between education and complications of diabetes. Primary aims include collaborating with other RFA grantees to evaluate different measures of education; estimating the educational gradient in complications; evaluating modifiable factors that mediate the educational gradient (e.g., health behaviors, diabetes knowledge, psychosocial factors); and differentiating the direct effects of education from those mediated through income. Secondary aims include evaluating the following: ethnic differences in the educational gradient; perceptions regarding barriers to care; the impact of education on patient-provider relationships and adherence; whether rates of referral to specialty care differ by educational attainment; and educational differences in health care costs. Our preliminary studies showed that less educated diabetic patients were in poorer health, had poorer self-care skills, underutilized specialty care, but had higher complication rates, were more likely to be hospitalized or to be seen in the ER, and incurred greater health care costs. Uniform health coverage in this study population avoids residual confounding common to population-based samples, which often suffer from inadequate adjustment for disparities in health care access and/or quality. Modifiable factors that mediate the pathway between education and complications pose candidate targets for interventions aiming to reduce educational disparities. A focus on diabetic complications is particularly compelling given observations of a substantial education differential in health care costs and the existence of efficacious interventions for modifying behavioral and clinical risk factors.

The California Teachers Study (CTS) is a prospective study of 133,479 California teachers initiated in 1995 by a consortium of California research centers with expertise in epidemiology and cancer etiology. The study was designed to understand better the etiology and prevention of breast cancer and of other major women's health problems; as such, the initiation of the cohort was supported by California tobacco taxes targeted to breast cancer research. Members of the cohort were identified through their participation in the California State Teachers Retirement System (STRS) to which all public school teachers and administrators must belong. An early observation from the CTS cohort is the extraordinarily high breast cancer rates experienced by California teachers. Incidence rates for invasive breast cancer are approximately 51% higher than those expected among women of comparable age and race in California. Moreover we have reported that the CTS cohort has very high rates of endometrial and ovarian cancers as well as melanoma. Among the strengths of the CTS cohort are its size and the detailed exposure and lifestyle assessment made at baseline and through subsequent contacts with cohort members. We were able to gather more detailed data from participants in the CTS than are typically collected from members of other large healthy cohorts, primarily because of the uniformly high education levels of the CTS membership. The scientific specific aims for the next five years of the CTS are focused around a small series of detailed exposure characteristics, including various patterns of hormone therapy (HT) use, dietary intake of isothiocyanates, alcohol consumption, and physical activity/energy balance. Because of the size and excellent follow-up of the CTS, we will expand the cancer outcomes of interest for selected hypotheses to include not just breast cancer as in the previous funding period, but also colorectal cancer, endometrial cancer, ovarian cancer, and lung cancer. We also propose to target 2,500 incident breast cancer patients and an equal sample of healthy CTS participants for blood collection and DNA extraction to evaluate a series of hypotheses regarding genetically-based modification of or interactions with certain exposure/breast cancer relationships. Finally, we propose to maintain the sophisticated infrastructure already in place, to continue detailed follow-up and periodic recontact of CTS members, and to process new data and continue analysis of existing data on CTS participants.

The long-term goal of this proposal is to define the molecular mechanisms that promote axonal degeneration following injury or disease. Axonal degeneration is a common feature of many neurological diseases. Neuropathies due to axonal degeneration are a hallmark of disorders such as diabetes, glaucoma, and chemotherapy-induced neurotoxicity and axonal loss is an early feature of debilitating neurodegenerative diseases. The great length of many axons makes them particularly vulnerable to mechanical injury, and axonal degeneration following trauma is a major cause of disability. Recent studies demonstrate that axonal degeneration is an active and highly regulated process, yet the intrinsic, neuronal mechanism promoting degeneration is poorly understood. This proposal investigates what causes axons to degenerate, and how this can be prevented. Axonal degeneration is an active process of self-destruction that appears to be naturally primed and waiting for a triggering stimulus that activates the execution phase. It proceeds as a stepwise process that begins with microtubule destabilization, followed by rapid blebbing of the axonal membrane, axonal fragmentation, cytoskeletal degradation and eventual engulfment by glial and/or phagocytic cells. We now demonstrate that the DLK pathway functions in the intrinsic neuronal pathway that promotes axonal degeneration following injury. Identifying and characterizing the function of components of the intrinsic axonal degeneration pathway will provide insights into its mechanism as well as potential therapeutic targets for the many neurological diseases characterized by axonal degeneration. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because it will improve our understanding of the mechanism of axonal degeneration following injury and disease. Axonal degeneration is a prominent component of many neurological disorders including neuropathies associated with trauma, diabetes, glaucoma, chemotherapy-induced neurotoxicity, and neurodegenerative diseases. Identifying components of the pathway in axons that promote degeneration will provide insights into the fundamental mechanism underlying axonal degeneration as well as potential therapeutic targets for the many neurological diseases characterized by axonal degeneration.

Tight regulation of the spindle microtubule (MT) dynamics is vital for the success and fidelity of mitosis, but the mechanism for regulating spindle MT dynamics remains unknown. Without this knowledge, a complete understanding of spindle regulation during mitosis is impossible. In recent years, a number of MT regulatory proteins have been identified, but little is known of how they interact with each other to collectively manipulate spindle MT dynamics. The first endeavor along this direction is the recent identification of a network of five regulatory proteins (KLP59C, KLP67A, Mast, EB1 and Msps) that governs kinetochore MT (kMT) plus-end dynamics during metaphase. This network utilizes a complex balance between MT polymerases and depolymerases (instead of polymerases alone) to induce net polymerization at kMT plus-ends, which counteracts constant depolymerization at minus-ends to maintain the metaphase kMTs in a steady state. The long-term goal is to elucidate the molecular events that drive the assembly and function of the mitotic spindle. The objective of this application is to determine how the actions of only a handful of MT regulatory proteins give rise to the broad range of dynamics at spindle MT plus-ends from prometaphase through anaphase. The central hypothesis is: the regulatory networks controlling spindle MT dynamics at other mitotic stages can be attained by shifting the balance among the components of the metaphase network. Guided by strong preliminary data, this hypothesis will be tested through the pursuit of three specific aims: (1) Determine the changes to the kMT regulatory network that transform the plus-end dynamics from net polymerization (metaphase) to net depolymerization (anaphase A). (2) Determine the kMT regulatory network that generates the plus-end dynamics driving chromosome congression during prometaphase. (3) Determine the regulatory networks governing non-kinetochore MT plus-end dynamics to establish/maintain a bipolar spindle during pre- anaphase and to promote spindle elongation during anaphase B. These aims will be achieved using complementary computer simulation, a custom-developed automatic image tracking method, live cell imaging and RNAi-based protein knockdowns. By bridging hypothesized molecular interactions with cellular-scale experimental observables quantitatively and rigorously, simulations allow us to discriminate alternative molecular mechanisms that experiments alone cannot due to lack of necessary spatial and temporal resolution. The innovation of this plan stems from both the novelty of its hypotheses and the broad and unique array of tools it wields to test them. The proposed research is significant because it will provide a systems-level understanding of an essential module of the spindle machinery, which will fill a severe gap in the current knowledge of mitosis. Moreover, the knowledge thus gained will deepen the understanding of the mechanisms of aneuploidy--the underlying cause of many forms of cancers. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under-investigated area of mitosis that has potential applicability to understanding the mechanisms of aneuploidy--the underlying cause of many forms of cancers. The proposed research has relevance to public health, because the fundamental mechanisms to be investigated are expected to be conserved across the phyla. Thus, the findings are ultimately expected to be applicable to the health of human beings.

In the last period we have worked on PRKN, PINK, APP, PS1, PS2, PRNP, PGRN and ATXN2/3 mutations in varied neurological diseases. We have continued our work in spinocerebellar ataxias by establishing the frequency of SCA15 mutations identified by us in a large cohort and by follow up of as yet unpublished SCA loci identified by us using autozygosity mapping. In addition we continue to work with our collaborators at NINDS and NHGRI to establish which of our patients have LRRK2 mutations and can be enrolled in our collaborative study following these patients and their as yet unaffected family members prospectively. In addition this year we have performed an assessment of APOE variability in Parkinson's disease, as all previous studies provided somewhat unclear results. This work involved analysis of the common coding variants (epsilon types) in APOE in a very large cohort of PD patients (this work is currently under review). We have also performed an assessment of candidate loci for recessive ataxia in a series of patients from Tunisia who have ataxia. This work allows us to parse such families into those with known mutations, and those that should be prioritized for further genetic work aimed at finding new genetic causes of disease.

The goal of this program is to coordinate and augment a cancer education and training program directed to pre-doctoral dental students. The program includes the biological, preventive, diagnostic, therapeutic and rehabilitative aspects of cancer, especially of the head and neck. The various aspects of the present program in cancer education is improved and augmented by a multiphasic additional program involving a multidisciplinary task force from: the N.Y.U.C.D. Departments of: Pathology, Oral Surgery, Oral Diagnosis and Prosthetics; the N.Y.U. School of Medicine Departments of: Medical Oncology and Dermatology; the N.Y. Veterans Hospital, Dental Service. The program provides a coordinated didactic program in cancer biology and a series of clinical pathological conferences on head and neck cancer cases with student participation. A clinical program in cancer detection and management is implemented in the N.Y.U.C.D. Department of Oral Diagnosis by the Clinical Coordinator of the Cancer Program. This effort involves the 12,000 new patients which register in the clinic each year as well as the patients under the treatment who constitute the 80,000 patient visits per year. Additional clinical instruction occurs during the one week half-time instructional period at Memorial Hospital in the Dental Service and Head and Neck Service, where the students in small groups participate in the head and neck conferences and clinics, observe in radiation therapy and surgery, and participate in the dental rehabilitation aspects of head and neck cancer. The Tumor Service and the Dermatology Service of N.Y.U. College of Medicine each provide a half-day session for all students in small groups where they gain additional clinical training in head and neck tumors and skin cancers.

Diffusion tensor MRI (DTI) is a powerful in vivo technique that is sensitive to deep brain tissue water microdynamics and microstructure. DTI-derived orientation and scalar maps have the unique potential to provide objective and specific measures of the Multiple Sclerosis pathology. The hallmarks of MS pathology may include inflammation, demyelination, gliosis, direct axonal loss directly or indirectly through Wallerian degeneration (WD). WD can cause axonal loss distal from the initial demyelinating lesion and its signature in MS has not been elucidated using a comprehensive DTI and conventional MRI approach. The Corpus callosum (CC), pyramidal, corticospinal tracts coursing through the internal capsule (IC) are important structures that are implicated in neurological deficit in MS. Unfortunately, the limited published literature on DTI of MS in these structures is often inconsistent and sometimes contradictory. Based on our preliminary studies, these inconsistencies and contradictions, at least in part, could be attributed to sub-optimal acquisition schemes, arbitrary region of interest placement, failure to recognize the regional heterogeneity of these structures, the age and gender dependence of DTI measure. In order to overcome some of these limitations, we propose to acquire DTI data at 3.0 T using parallel imaging at different age groups on normal males and females. In addition, MRI data will also be acquired on MS subjects. The DTI data will be acquired from the whole brain using optimized Icosa21 scheme that is shown to be balanced and unbiased. Specifically we will concentrate on the CC, pyramidal and CST tracts which are implicated in MS, to identify WD signature in MS. We will divide the corpus callosum into seven functionally distinct sub regions. Similarly the internal capsule will be divided into four quadrants and its temporal-spatial correlations will be followed longitudinally. DTI values will be derived from each one of these individual structures. A robust DTI analysis tool will be developed for automatic analysis. This tool will also help in the fusion of multi-modal MRI data for a robust segmentation of the subregions of CC and 1C. The DTI measures, after accounting for the age and gender dependence will be correlated with the clinical measures. [unreadable] [unreadable] [unreadable]

The objective of the proposed research is to understand the on-line operation of intermediate cerebellar networks during the motor control and learning of protective eyelid movements. This will be studied using the model of classically conditioned eyeblinks. A series of studies will be performed in rabbits to characterize the function of the inferior olive in movement control and in the acquisition of anticipatory protective reflexes. The proposed research is based on the general hypothesis that the cerebellum, together with extra-cerebellar circuits, is responsible for recognizing a threatening environmental situation and generating adaptive anticipatory behavior. The unique feature of these proposed experiments is their use of an innovative method for manipulating specific synaptic inputs to central neurons without affecting spontaneous tonic activity of investigated neural networks. The first experiments will examine the specific role of sensory signals, carried from the inferior olive to the cerebellum, in the expression and retention of learned movements. In the second series of experiments, the role of the cerebellar inferior olivary input in conditioned response acquisition will be examined. This latter research represents a fundamental test of the popular cerebellar learning hypothesis. The results of this research are important for understanding the mechanisms related to the acquisition and retention of anticipatory defensive behaviors, and also for improving the rehabilitation of patients with deficits in sensorimotor integration through the understanding of the mechanisms underlying motor learning and recovery of motor function.

The PRMS office is responsible for overseeing the protocol review process which is undertaken by the PRMS Committee. These responsibilities include administrative and record keeping tasks associated with this process. The PRMS office is in addition responsible for reviews of accrual and scientific progress of all approved and activated protocols. This process requires a yearly audit for accrual and scientific progress and comparison with planned accrual, review of these findings with the Associate Director for Clinical Research and the PRMS Committee, and evaluation of the replies from the Pi's with the Associate Director for Clinical Research. The PRMS Committee - The Protocol Review and Monitoring System operational in the UAB Cancer Center utilizes the PRMS Committee to provide peer-review and approval-disapproval recommendations. The PRMS Committee provides scientific evaluation of proposed clinical trials, establishment of research priorities appropriate to the Center's scientific resources and patient populations and monitors the status and progress of all active protocols. Our clinical research activities are broad in scope, but priority is given to Cancer Center faculty initiated trials and innovative translational research protocols. The formal charge to the PRMS Committee is summarized as follows: Review all cancer related protocols which will be undertaken at the University of Alabama at Birmingham, and make recommendations regarding scientific quality, feasibility, consistency of the protocol with Cancer Center objectives, and existence of competing studies. Based upon the above considerations provide recommendations regarding approval (with or without modifications) or disapproval. Progress Report - In this funding period the number of protocols reviewed each year has increased by 42%. To facilitate review of this increased number of studies the PRMS Committee meets twice rather than once monthly in two Boards. In the last reporting period (3/1/03 to 2/28/04), 74 protocols were reviewed for scientific merit and 25 for priority only (National Protocols); 21 protocols were institutional, 5 other peer-reviewed and 47 were pharmaceutical. Of the protocols reviewed for scientific merit, 26% were approved as written, 46% required minor revision, 26% major revision and 2% were disapproved. In the last reporting period, we undertook 51 reviews for accrual and scientific progress: 68% were approved for continuation, 20% were placed on probation and 12% were closed for inadequate progress.

This revised proposal focuses on the genetic and cellular events leading to pulmonary smooth muscle proliferation in lymphangiomyomatosis (LAM), a progressive and often-fatal lung disease affecting almost exclusively women. LAM can occur in women with tuberous sclerosis complex (TSC) or in women who have no signs of TSC (sporadic LAM). Renal angiomyolipomas occur in most TSC patients and in 50% of sporadic LAM patients. In work funded by this grant, we found TSC2 mutations in angiomyolipomas and pulmonary LAM cells from women with sporadic LAM, but not in normal kidney or lung, which strongly suggests that LAM and angiomyolipoma cells are derived from the same cell. This raises the possibility of an unusual disease mechanism for sporadic LAM: the migration of histologically benign smooth muscle cells between angiomyolipoma and lung. Recent work by another group has demonstrated that hamartin (the TSC1 gene product) interacts with the moesin-ezrin-radixin family of cytoskeletal proteins, activates the small GTPase Rho, and regulates cellular adhesion. Our preliminary data indicate that tuberin (the TSC2 gene product) also activates Rho and regulates cell adhesion and migration. Therefore, the central hypothesis of this proposal is that mutational inactivation of either TSC1 or TSC2 leads to LAM because of aberrant signaling in cellular pathways including Rho. To address this hypothesis, we propose the following specific aims: Aim 1: To define the role of TSC1 and TSC2 mutations in LAM. Aim 2: To determine whether LAM cells and angiomyolipoma cells have identical clonality patterns. Aim 3: To determine the mechanisms through which TSC1 and TSC2 affect cell migration. Our long-term goal is to define the cellular pathways through which mutations in the TSC1 and TSC2 genes lead to smooth muscle cell proliferation and LAM. A natural corollary of this goal is to test the hypothesis that LAM and angiomyolipoma cells are derived from the same precursor cell. We are optimistic that this project will contribute to the development of targeted therapeutic strategies for LAM and TSC patients, for whom there are currently limited treatment options. Hamartin and tuberin are expressed in most human tissues, and the manifestations of TSC include tumors of the brain, heart, and kidney, as well as mental retardation, seizures, and autism. We strongly believe, therefore, that the pathways in which hamartin and tuberin participate have broad medical and biological significance.

Human and animal studies indicate that bilirubin monoglucuronide (BMG) may play a central role in the formation of gallstones (GS). Significantly higher gallbladder concentrations of BMG have been found in patients with GS than in patients without GS. Disorders associated with an increased BMG production (e.g. hemolytic anemias or glucuronyl transferase deficiency) are associated with an increased excretion of BMG and an increased incidence of GS. The guinea pig, hamster, deer mouse and prairie dog, all models for GS formation, excrete BMG as the major pigment in bile. Unconjugated bilirubin and BMG have been associated with biliary sludge, perhaps the first step in GS formation. Three mechanisms may account for these relationships. Firstly, because of the presence of only one polar glucuronic acid, BMG may precipitate in bile because it is less soluble than the major pigment of human bile, bilirubin diglucuronide (BDG). Secondly, BMG may undergo spontaneous hydrolysis in alkaline bile to form UCB and glucuronic acid. Thirdly, BMG easily undergoes molecular rearrangement to BDG and very insoluble unconjugated bilirubin (UCB) -- the nidus for most cholesterol GS and the major tetrapyrrolic component in many pigment GS. To test these hypotheses, I will systematically study in inert polyethylene eppendorf test tubes the stability and solubility of pure BMG (purified from rat bile by the HPLC method of Spivak and Carey) over a range of physiologic pH in various physiologic concentrations of bile salts, cholesterol, phospholipids and proteins at 37 C. A novel 10 minute HPLC method will allow us to serially measure concentrations, of BMG in the supernate and precipitate and to detect the formation of BDG and III-Alpha, XIII-Alpha and IX-Alpha isomers of UCB from BMG. Since the IX-Alpha isomer of BMG is the only pigment present initially, formation of predominantly IX-Alpha UCB will indicate BMG hydrolysis while formation of the III-Alpha and XIII-Alpha isomers of UCB and BDG will indicate molecular rearrangement of BMG. These solubility, hydrolysis and rearrangement studies of BMG should yield crucial information about the physical-chemical phenomena in gallbladder pigment precipitation.

In the current fiscal year we have advanced our knowledge on novel proteins in saliva of blood feeding ticks and mosquitoes. Specifically, we discovered a novel protein found in saliva of mosquitoes that prevents collagen from triggering platelet adhesion and aggregation by different receptor interactions (1), characterized a second cystatin from the tick vector of Lyme disease with pharmacological potential (2) and identified prostaglandin E2 as the major salivary constituent of Ixodes scapularis (the main vector of Lyme disease in the US) that prevents antigen presentation by dendritic cells, and thus is a major immunosuppressive agent in tick saliva. [unreadable] [unreadable] We have also increased our salivary transcriptomes database by exploring the salivary transcriptome of vector species so far undescribed, such as the vector of human plague, the flea Xenopsylla cheopis (4), the mosquitoes Aedes albopictus (5) and Anopheles funestus (6), and the vector of Chagas disease, Triatoma brasiliensis (7). We have also expanded the EST coverage of the salivary transcriptome of Aedes aegypti (8), the main vector of Dengue in the World. To better understand the evolution of gene families associated with salivary function, we also sequenced and analyzed a salivary transcriptome of the seed feeding bug, Oncopeltus fasciatus (9), which is closely related to blood sucking bugs. Each transcriptome uncovers dozens of novel protein families which represent a rich resource for mining pharmacologically active and antimicrobial compounds.[unreadable] [unreadable] Our bioinformatic capability allowed us to collaborate with other research groups inside and outside the NIAID, leading on this fiscal year to a publication with the group of Dr. X. Su (LMVR/NIAID) on gene predictions within the malaria parasite genome of Plasmodium falciparum (10), and with Dr. Nora Besansky's group at Notre Dame on the genome structure of Anopheles gambiae.

The study is based on the hypothesis that HIVIG will decrease viral burdens in children with moderately advanced HIV infection. Its primary objectives include evaluating HIVIG's (a) safety & tolerance; (b) dosage & pharmacokinetics by serial measurements of immunoglobulins, anti-p24 antibody & rubella antibody titers, and (c) anti-viral activity at 3 dosage levels by assays of viral burden. Its secondary goal will be to collect lab & clinical data by monitoring growth/weight, CD4 counts, incidence of opportun. infections, & survival rates.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has rapidly emerged as an effective treatment in medically refractory Parkinson's disease (PD). However, our understanding of the effects of DBS is limited and future attempts at optimizing electrode design or stimulation parameters will depend on defining the mechanisms by which DBS achieves its therapeutic effect. The central hypothesis of the planned work is that DBS generates heterogeneous patterns of activation and inhibition in the different neuron types (local cells, fibers of passage, afferent inputs) that surround the electrode and these stimulation effects are transmitted throughout the interconnected nuclei of the basal ganglia. We further hypothesize that therapeutic and non-therapeutic stimulation result in different patterns of activation in the different neuron types and we can modulate the activation of specific neuron types via alterations in the stimulation parameters. In turn, our goal is to assess the therapeutic importance of different neural activation patterns on the behavioral and neurophysiological effects of DBS. To accomplish this goal we will develop a systems level model of DBS that will be coupled to ongoing experimental work using DBS of the parkinsonian macaque (R01-NS37019, PI: J. Vitek). The first specific aim will generate realistic field-neuron models of the electric field generated by DBS, and the different neuron types influenced by stimulation. The second and third specific aims will couple the field-neuron model to hypothesis driven research experiments with quantitative predictions of the effects of DBS as a function of the stimulation parameters. This work will provide fundamental knowledge necessary for the advancement of DBS technology for treatment of PD in the human, and may also enhance the application of DBS to other disorders such as dystonia, epilepsy, obsessive-compulsive disorder, and major depression.

Dysfunction of the vascular endothelium contributes to most cardiovascular diseases. This is not surprising, as the endothelium is the central regulator of hemostasis, vascular development, angiogenesis, vasomotor tone, and the response to vascular injury and inflammation. We have used several complimentary approaches during the past funding cycle--taking advantage of previous work from our lab and others indicating an early role for the vascular endothelial growth factor receptor VEGFR-2 in blood vessel growth-- to fill in the gaps in our understanding of how endothelial cell destiny and phenotypic diversity are determined. We have recently defined essential roles for members of the homeobox and bone morphogenetic protein (BMP) families as critical regulators of endothelial cell differentiation and phenotypic modulation. In particular, we have identified the homeodomain protein HoxB5 as the first transcription factor yet described to be sufficient to elicit differentiation of endothelial cells from mesoderm-derived precursors. We have also characterized BMPER, a novel BMP-binding protein, as an angioblast-specific regulator of endothelial growth and differentiation. The present proposal exploits well-characterized aspects of molecular biology, genomics, and cell biology as tools to explore further the earliest steps in blood vessels formation. These tools will allow us to determine how HoxB5 commits precursor cells to assume an endothelial phenotype in AIM #1. The consequences of HoxB5-dependent gene regulation on angiogenesis and endothelial progenitor function will be dissected in AIM #2. In Aim #3, we will delineate the role of BMP family members in regulation of vascular developmental and we will specifically test the role of BMPER in endothelial precursor commitment decisions. Our studies are informed by the general hypothesis that developmental and physiologic vascular processes share fundamental similarities at the molecular level. The scope of the this proposal is intended to address relevant biological and physiological questions using state-of-the-art molecular biology techniques. Knowledge gained from this proposal should provide crucial information about endothelial cell development from multipotent precursors, blood vessel formation in health and disease, and endothelial cell-type specific gene expression. In addition, mechanisms for targeted gene delivery to endothelial cells and for disruption of angiogenesis in its pathologic forms may be revealed. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Background: Depression is a widely diagnosed and highly debilitating disorder. Cognitive theories of depression posit that depressed individuals are characterized by cognitive biases favoring in the processing of negative affective information. An information-processing bias frequently associated with major depression is increased memory sensitivity for negative information and, often, decreased memory sensitivity for positive material. Importantly, the amygdala has been centrally implicated in encoding and memory for emotionally valenced material and has been found to show elevated resting state activity in depression. The primary goal of this study is to assess the role of amygdalar activation in the negative memory bias associated with depression. Methods and Results: Ten participants meeting criteria for major depressive disorder in the absence of other Axis-I disorders and ten age and sex-matched healthy control participants served as participants in this study. Functional magnetic resonance imaging (fMRI) was used to measure neural activation during presentation and affective evaluation of positive, neutral and negative pictures. One week following scanning, participants engaged in an out-of-scanner recognition memory task during which they were presented with pictures that they saw in the scanner and with never- seen pictures, and were asked to rate the familiarity of each picture. Memory sensitivity for negative pictures was significantly higher in depressed than in control participants;the two groups of participants did not differ in memory sensitivity for positive pictures. Corresponding to this finding, activity in the left amygdala was significantly higher during encoding of subsequently remembered negative pictures in depressed than in control participants, but not during encoding of neutral or positive pictures. To read about other projects ongoing at the Lucas Center, please visit http://rsl.stanford.edu/ (Lucas Annual Report and ISMRM 2011 Abstracts)

This is a cross-sectional study examining behavioral and neurophysiologic changes associated with learning Mandarin Chinese as a second language (L2) by native English-speaking adult learners (ages 18 years and up). It will focus on the acquisition of lexical tones, which are pitch patterns that are used to contrast word meaning in Mandarin, and is not a linguistic feature of English. At the behavioral level, various studies investigating the learning of isolated non-native consonants in non-lexical and non-communicative contexts show that with the appropriate form and degree of training, adults can learn to perceive and/or produce sounds that do not occur in their native language. However, there have been few studies that investigated the learning of lexical tones or suprasegmentals, and fewer yet that considered the use of these non-native sounds in actual communicative contexts such as words. In recent years, several studies examined neural changes associated with speech and word learning; but they are either confined to laboratory training of speech sounds, or the learning of words without considering the contribution of phonetic features, especially suprasegmentals. While cross-linguistic research has been done comparing behavioral and neurophysiologic responses of native Mandarin-speaking and native English-speaking adults perceiving Mandarin tones, none was conducted to describe changes in non-Mandarin (English) speaking subjects as they become proficient in Mandarin. The current study will involve behavioral and fMRI experiments on university students learning Mandarin Chinese at four different stages (years 1 to 4 of the university curriculum). The general hypothesis is that increased L2 proficiency will result in behavioral and neurophysiologic responses closer to those in native speakers. An understanding of language development is important for developing the most efficacious language rehabilitation programs in cases of injuries such as strokes. For the rehabilitation of adults, it is especially important to understand how adults learn. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY/ABSTRACT Each year 500,000 patients undergo cardiac surgery in the United States, and acute kidney injury (AKI) complicates recovery in 25% of patients. AKI is associated with subsequent postoperative arrhythmias, wound infections, and sepsis, and independently predicts a 5-fold increase in death at 30 days. Despite advancements in surgical technique and perioperative patient management, cardiac surgery-associated AKI (CSA-AKI) remains a major problem and no therapies have been shown to improve clinical outcomes. While there are many reasons that previous efforts to identify and validate new therapeutic targets for AKI have been unsuccessful, a key feature is that discovery efforts were not primarily driven by human data. Leveraging BioVU (Vanderbilt's large- scale DNA biobank), we performed a Phenome-Wide Association Study (PheWAS) based on ICD billing code and genotype data in a disease-agnostic cohort of ~36,000 patients and identified novel genotype-phenotype associations between single nucleotide polymorphisms in the gene that encodes the protein target of montelukast (CYSTLR1) and AKI phenotypes. Montelukast is an anti-inflammatory leukotriene receptor antagonist that is FDA approved to treat asthma and allergic rhinitis, and inflammation is a mechanism of AKI. In additional preliminary studies montelukast reduces renal injury in preclinical models, urinary concentrations of leukotrienes increase significantly during cardiac surgery and more so in patients who develop AKI, and patients taking montelukast have a 38% reduction in AKI over time compared to patients not taking montelukast. To determine if montelukast can be repurposed to prevent CSA-AKI and potentially other forms of AKI in subsequent initiatives, we will first perform a phase II trial to measure the effect of montelukast on CSA-AKI and assess any safety events. To properly design and execute this phase II trial we assembled a multidisciplinary team of physician scientists and staff with the relevant expertise and experience to accomplish four specific aims: (1) Determine study cohort availability and baseline characteristics by simulating study cohorts using VUMC's Synthetic Derivative; (2) Determine optimal montelukast dosing regimen and refine the trial's mechanistic studies by measuring LTC4, LTD4, and LTE4 leukotriene subtypes in urine of patients who did and did not develop AKI in a previous study; (3) Optimize study design to most efficiently recruit patients and test the hypothesis; and (4) Complete all study startup tasks. The successful completion of this project will allow us to commence the clinical trial immediately. Proving that a safe, affordable, generic drug can be used to prevent CSA-AKI is a major priority. In addition, demonstration that the published and publicly available computational PheWAS algorithm is an effective tool for drug repurposing will lead to additional medical treatments.